Asian Citrus Psyllid: Biology, Ecology and Management of the Huanglongbing Vector 2019046501, 2019046502, 9781786394088, 9781786394095, 9781786394101

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Asian Citrus Psyllid

Biology, Ecology and Management of the Huanglongbing Vector

Asian Citrus Psyllid Biology, Ecology and Management of the Huanglongbing Vector

Edited by

Jawwad A. Qureshi and Philip A. Stansly† University of Florida, USA

CABI is a trading name of CAB International CABI Nosworthy Way Wallingford Oxfordshire OX10 8DE UK Tel: +44 (0)1491 832111 Fax: +44 (0)1491 833508 E-mail: [email protected] Website: www.cabi.org

CABI WeWork One Lincoln St 24th Floor Boston, MA 02111 USA T: +1 (617)682-9015 E-mail: [email protected]

© CAB International 2020. All rights reserved. No part of this publication may be reproduced in any form or by any means, electronically, mechanically, by photocopying, recording or otherwise, without the prior permission of the copyright owners. A catalogue record for this book is available from the British Library, London, UK. Library of Congress Cataloging-in-Publication Data Names: Qureshi, Jawwad A., editor. | Stansly, Philip A. (Philip Anzolut),  editor. Title: Asian citrus psyllid : biology, ecology and management of the   Huanglongbing vector / [edited by] Jawwad A. Qureshi and Philip A.  Stansly. Description: Oxfordshire, UK ; Boston, MA : CAB International, [2020] |   Includes bibliographical references and index. | Summary: “This is the   first book to be published which specifically focuses on Asian Citrus   Psyllid and the intractable disease it spreads in citrus crops   (Huanglongbing disease)”-- Provided by publisher. Identifiers: LCCN 2019046501 (print) | LCCN 2019046502 (ebook) | ISBN   9781786394088 (hardback) | ISBN 9781786394095 (ebook) | ISBN   9781786394101 (epub) Subjects: LCSH: Citrus--Diseases and pests. Classification: LCC SB608.C5 (print) | LCC SB608.C5 (ebook) | DDC  634/.3--dc23 LC record available at https://lccn.loc.gov/2019046501 LC ebook record available at https://lccn.loc.gov/2019046502 References to Internet websites (URLs) were accurate at the time of writing. ISBN-13: 9781786394088 (hardback) 9781786394095 (ePDF) 9781786394101 (ePub) Commissioning Editor: Ward Cooper Editorial Assistant: Emma McCann Production Editor: Marta Patiño Typeset by SPi, Pondicherry, India Printed and bound in the UK by Severn, Gloucester

Preface

The Asian citrus psyllid, Diaphorina citri Kuwayama, is an economically important insect pest of citrus worldwide, mainly due to its role as vector of ‘Candidatus liberibacter’ pathogens which cause huanglongbing, or citrus greening disease. The Asian citrus psyllid is now reported from 26 countries in Asia, four in Africa, two in North America, 14 in Central America and Caribbean, six in South America, and five in Oceania. It is the primary vector of ‘Candidatus liberibacter asiaticus’, the bacterium that causes Asian huanglongbing, one of the world’s most devastating diseases of citrus. Asian huanglongbing is reported from more than 50 countries, and there are only a few places where both psyllid and huanglongbing are not present together. Studies have shown that this psyllid is also capable of vectoring ‘Candidatus liberibacter africanus’, the bacterium that causes the African huanglongbing common in South Africa and some other countries, spread primarily by the African citrus psyllid, Trioza erytreae Del Guercio. The Asian citrus psyllid is a sap-sucking insect pest that attacks all types of citrus and some other rutaceous plants, particularly species of the genus Murraya, an ornamental widely known as orange jasmine. The psyllid is found in viable and abandoned commercial citrus plantings, and in urban environments where citrus and other hosts are present. Feeding by psyllid immatures (nymphs) and adults on young citrus shoots causes reduced and distorted leaf development. Of much greater concern is the ability of the psyllid to transmit the pathogens responsible for causing huanglongbing. Both nymphs and adults have been shown to acquire the disease pathogen from an infected plant and transmit it. Nymphs are more efficient at acquiring the pathogen, whereas the adults, which are capable of flying, spread the pathogen from tree to tree over medium and long distances. The psyllid has a high reproductive rate and eggs are laid in newly developing buds or shoots of the host plant, where the nymphs develop in cohorts. The repeated feeding events increase the titer of the pathogen in the plant, and therefore control of the vector is critical to reduce re-inoculation of the pathogen and disease severity. This book was long overdue, considering the significance of this vector pathogen complex, its threat to the world’s citrus, and the growth of knowledge developed during the past century, particularly over the past two decades. In 2012, Phil Stansly and I organized the symposium ‘Asian Citrus Psyllid and “Huanglonbing”: Devastating Pest–Disease Complex and Threat to Citrus Production Worldwide’ at the 60th Annual Meeting of the Entomological Society of America, followed by another symposium on the same subject in 2016 at the XXV International Congress of Entomology. Additional sessions on this vector–pathogen system at the International Research Conference on Huanglongbing further emphasized the need for a book. The idea was discussed with the Centre for Agriculture and Bioscience International (CABI), the organization where I started my professional v

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Preface 

career and where I worked for several years to develop integrated and sustainable pest management systems in multiple crops. This is the first book that describes the Asian citrus psyllid’s biology, behavior and ecology with discussions on cultural, biological, and chemical methods of its control in different regions. It also includes chapters on vector interactions with symbionts and pathogens, its acquisition and transmission of ‘Candidatus liberibacter asiaticus’, and advanced genetic tools for managing the vector and the pathogen. The biology and behavior of the psyllid are discussed in Chapters 1–4, followed by its ecology and host relations in Chapters 5 and 6, vector relations in Chapters 7–9, and management in Chapters 10–17. Ultimately, the development of huanglongbing-tolerant/resistant citrus varieties will be an important addition to the management programs for this vector–pathogen complex. Meanwhile, citrus growers in affected areas are largely dependent on psyllid suppression and horticultural practices, which continue to evolve to sustain tree health and productivity. I would like to extend my special thanks to Dr Phil Stansly for all the valuable contributions he made toward citrus pest management, particularly the target vector psyllid and this book. He was actively involved in co-editing the chapters with me and providing suggestions for improvement. Unfortunately, he passed away in 2018. He will always be remembered by all the contributing authors and others with whom he worked. I would also like to commend all the authors for their contributions, which made this book possible. Also, I would like to thank the CABI staff, including Ward Cooper, Marta Patiño, Emma McCann and several others who worked behind the scenes in bringing this book to production. It was a great experience for me to bring to fruition such an enormous source of knowledge for researchers of entomology and related disciplines, students, agricultural scientists and professionals, extension agents, and pest management consultants. Jawwad A. Qureshi Entomology and Nematology University of Florida, USA

Contents

List of Abbreviations List of Contributors

ix xiii

Prefacev 1.  Asian Citrus Psyllid Life Cycle and Developmental Biology1 David G. Hall 2.  Functional Anatomy of the Asian Citrus Psyllid12 Joseph M. Cicero 3.  Mating Behavior of the Asian Citrus Psyllid30 Richard W. Mankin and Barukh Rohde 4.  Visually and Chemically Guided Behavior of the Asian Citrus Psyllid43 Sandra A. Allan 5.  Hosts of the Asian Citrus Psyllid67 George A.C. Beattie 6.  Abiotic and Biotic Regulators of the Asian Citrus Psyllid Populations88 Jawwad A. Qureshi 7.  Symbionts and Pathogens of the Asian Citrus Psyllid101 Kirsten S. Pelz-Stelinski 8. Huanglongbing Pathogens: Acquisition, Transmission and Vector Interactions113 El-Desouky Ammar, Robert G. Shatters Jr and Michelle Heck 9. Epidemiology of Huanglongbing: Implications of Infective Colonization Events140 Susan Halbert and Burton Singer 10.  Sampling and Economic Thresholds for Asian Citrus Psyllid156 Cesar Monzo and Philip A. Stansly vii

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11. Management Objectives and Integration of Strategies for the Asian Citrus Psyllid166 Philip A. Stansly and Jawwad A. Qureshi 12.  Management of the Asian Citrus Psyllid in Asia179 George A.C. Beattie 13.  Asian Citrus Psyllid Management in São Paulo, Brazil210 Marcelo Pedreira Miranda and Antonio Juliano Ayres 14. Integrated Management of Asian Citrus Psyllid and Huanglongbing in Florida: Past, Present and Future222 Philip A. Stansly and Jawwad A. Qureshi 15.  Area-wide Management of Asian Citrus Psyllid in Texas234 Mamoudou Sétamou 16.  Management of Asian Citrus Psyllid in California250 Elizabeth E. Grafton-Cardwell 17. Advances in RNA Suppression of the Asian Citrus Psyllid Vector and Bacteria (Huanglongbing Pathosystem)258 Wayne B. Hunter, Sasha-Kay V. Clarke, Andres F. Sandoval Mojica, Thomson M. Paris, Godfrey Miles, Jackie L. Metz, Chris S. Holland, Greg McCollum, Jawwad A. Qureshi, John M. Tomich, Michael J. Boyle, Liliana Cano, Sidney Altman, Kirsten S. Pelz-Stelinski Index285

List of Abbreviations

Abbreviation

Full

AAP ACP AGO AIMS ak-dsRNA APHIS ARS BAPC BLS Ca. Liberibacter spp. Cas CHMA CHRP CI CLaf CLam CLas CLso CoA COPF CPP CREC CRISPR CTV CUPS CVC D. citri DMDS DPI dsRNA EAG EDT EIL EPG

acquisition access period Asian citrus psyllid agricultural mineral oil area-wide integrated management system arginine kinase double-stranded ribonucleic acid (USDA) Animal and Plant Health Inspection Service Agricultural Research Service branched amphiphilic peptide capsule bacterium-like structure Candidatus Liberibacter species CRISPR-associated protein Citrus Health Management Area Citrus Health Response Program (or Plan) cytoplasmic incompatability ‘Candidatus Liberibacter africanus’ ‘Candidatus Liberibacter americanus’ ‘Candidatus Liberibacter asiaticus’ ‘Candidatus Liberibacter solanacearum’ coenzyme A citrus orchards with perimeter fencing cell-penetrating peptide Citrus Research and Education Center (University of Florida) clustered regularly interspaced short palindrome repeats citrus tristeza virus citrus under protective screens (or structures) citrus variegated chlorosis Diaphorina citri dimethyl disulfide (FDACS) Division of Plant Industries double-stranded RNA electroantennogram early detection technique economic injury level electrical penetrating graph, electropenetrography

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x

List of Abbreviations

Abbreviation

Full

ET FDACS FISH GAIN GLOBALG.A.P. GMO HLB HMO IAP IFAS IMD IOCV IPM IRAC IRCHLB Laf Lam Las LC50 LCV LED LRGV METI MLST MoA MRL mtCOI NAA NAS NCBI NIFA NMNH NRC nt ORN PAS PCA PCD PFT PGRP PIC PMA PMO PNA PoP PPMO PPQ RH RNAi RR SDI SEM siRNA SPIF

economic threshold Florida Department of Agriculture and Consumer Services fluorescent in situ hybridization Global Agricultural Information Network Global Good Agricultural Practices genetically modified organism huanglongbing horticultural mineral oil inoculation access period (University of Florida) Institute of Food and Agricultural Sciences immune deficiency International Organization of Citrus Virologists integrated pest management Insecticide Resistance Action Committee International Research Conference on Huanglongbing Liberibacter africanus Liberibacter americanus Liberibacter asiaticus lethal concentration 50% Liberibacter-containing vacuoles light-emitting diode Lower Rio Grande Valley mitochondrial electron transport inhibitor multi-locus sequence typing model of action maximum residue level mitochondrial cytochrome oxidase I naphthalene acetic acid National Academy of Science National Center for Biotechnology Information (USDA) National Institute for Food and Agriculture (US) National Museum of Natural History National Research Council nucleotide olefactory receptor neuron Phytosanitary Alert System Pest Control Advisor Pest Control District pathogen-free tree peptidoglycan recognition protein Produção Integrada dos Citros psyllid management area (or Psyllid Management Area) phosphorodiamidate morpholino oligomer polynucleic acid potato psyllid (cell-penetrating) peptide phosphorodiamidate morpholino Plant Protection and Quarantine relative humidity RNA interference resistance ratio Saturation Deficit Index scanning electron microscopy short interfering RNA Sistema de Pulverização Integrado do Fundecitrus



List of Abbreviations

Abbreviation

Full

SPLAT SPS ssRNA TALEN TCA TCPDMC TDA TEM TRV TRX UCANR UF ULV UR USDA UV WDi YST

Specialized Pheromone and Lure Application Technology São Paulo State single-stranded RNA TAL effector nuclease tricarboxylic acid Texas Citrus Pest and Disease Management Corporation Texas Department of Agriculture transmission electron microscopy tree-row-volume thioredoxin University of California Agriculture and Natural Resources University of Florida ultra-low volume unsulfonatable residue United States Department of Agriculture ultraviolet Wolbachia–Diaphorina citri yellow sticky trap

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List of Contributors

Sandra A. Allan (corresponding author), United States Department of Agriculture, Agriculture ­Research Service, Center for Medical, Agricultural, and Veterinary Entomology, 1700 SW 23 Dr, Gainesville, FL 32608, USA. Email: [email protected] Sidney Altman, Yale University, New Haven, CT, USA Antonio Juliano Ayres, Fundo de Defesa da Citricultura – Fundecitrus, Av. Adhemar Pereira de ­Barros, 201, CEP: 14807-040 - Vila Melhado, Araraquara, São Paulo, Brazil El-Desouky Ammar (corresponding author), USDA-ARS, US Horticultural Research Laboratory, Fort Pierce, FL 34945, USA. Email: [email protected] George A.C. Beattie (corresponding author), School of Science, Western Sydney University, Locked Bag 1797, Penrith NSW 2751, Australia. Email: [email protected] Michael J. Boyle, Smithsonian Marine Station at Fort Pierce, FL 34939, USA Liliana Cano, University of Florida, Institute of Food and Agricultural Sciences, Indian River ­Research and Education Center, 2199 South Rock Road, Fort Pierce, FL 34945, USA Joseph M. Cicero (corresponding author), Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611, USA. Email: [email protected] Sasha-Kay V. Clarke, University of the West Indies, Department of Basic Medical Sciences, Biochemistry Section, Kingston, Jamaica; University of Florida, Institute of Food and Agricultural Sciences, Indian River Research and Education Center, 2199 South Rock Road, Fort Pierce, FL 34945, USA Elizabeth E. Grafton-Cardwell (corresponding author), Department of Entomology, University of California, Riverside, CA 92521, USA. Email: [email protected] Susan Halbert (corresponding author), Florida Department of Agriculture and Consumer Services, Division of Plant Industry, Gainesville, FL 32608 USA. Email: [email protected] David G. Hall (corresponding author), US Department of Agriculture – Agricultural Research ­Service, US Horticultural Research Laboratory, 2001 South Rock Road, Fort Pierce, FL 34945 (Retired). Email: [email protected] Michelle Heck, Boyce Thompson Institute, Ithaca, NY, USA; USDA-ARS Emerging Pests and Pathogens Research Unit, Robert W. Holley Center for Agriculture and Health, Ithaca, NY, USA; Plant Pathology and Plant Microbe Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, USA Chris S. Holland, Maverick Biologicals, Inc., 2400 S. Ocean Drive, Fort Pierce, FL 34945, USA Wayne B. Hunter (corresponding author), USDA, ARS, US Horticultural Research Laboratory, 2001 South Rock Road, Fort Pierce, FL 34945, USA. Email: [email protected]

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List of Contributors

Richard W. Mankin (corresponding author), US Department of Agriculture, Agricultural Research Service, Center for Medical, Agricultural, and Veterinary Entomology, Gainesville, FL 32608, USA. Email: [email protected] Greg McCollum, USDA, ARS, US Horticultural Research Laboratory, 2001 South Rock Road, Fort Pierce, FL 34945, USA Jackie L. Metz, University of Florida, Institute of Food and Agricultural Sciences, Indian River ­Research and Education Center, 2199 South Rock Road, Fort Pierce, Florida 34945, USA Godfrey Miles, USDA, ARS, US Horticultural Research Laboratory, 2001 South Rock Road, Fort Pierce, Florida 34945, USA; Florida Atlantic University Harbor Branch, 5600 US 1, Fort Pierce, FL 34945, USA Marcelo Pedreira Miranda (corresponding author), Fundo de Defesa da Citricultura – Fundecitrus, Av. Adhemar Pereira de Barros, 201, CEP: 14807-040 - Vila Melhado, Araraquara - São Paulo, Brazil. Email: [email protected] Cesar Monzo (corresponding author), Instituo Valenciano de Investigaciones Agrarias, Centro de Protección Vegetal y Biotecnología, Unidad de Entomología, CV-315 Km 10, 7, Valencia, Spain 46113. Email: [email protected] Thomson M. Paris, University of Florida, Institute of Food and Agricultural Sciences, Indian River Research and Education Center, 2199 South Rock Road, Fort Pierce, FL 34945, USA. Kirsten S. Pelz-Stelinski (corresponding author), Department of Entomology and Nematology, Citrus ­Research and Education Center, University of Florida, Lake Alfred, Florida, USA. Email: pelzstelinski@ ufl.edu Jawwad A. Qureshi (corresponding author), University of Florida, Department of Entomology and Nematology, Institute of Food and Agricultural Sciences, Southwest Florida Research and Education Center, 2685 State Road 29 North, Immokalee, FL 34142, USA. Email: [email protected] Barukh Rohde, Department of Electrical and Computer Engineering, University of Florida, Gainesville, FL 32611, USA Andres F. Sandoval Mojica, Florida Atlantic University Harbor Branch, 5600 US 1, Fort Pierce, FL 34946, USA Mamoudou Sétamou (corresponding author), Texas A&M University-Kingsville, Citrus Center, 312 N International Blvd, Weslaco, TX 78596, USA. Email: [email protected] Robert G. Shatters Jr, USDA-ARS, US Horticultural Research Laboratory, Fort Pierce, FL 34945, USA Burton Singer, University of Florida, Emerging Pathogens Institute, Gainesville, FL 32611, USA Philip A. Stansly, University of Florida, Department of Entomology and Nematology, Institute of Food and Agricultural Sciences, Southwest Florida Research and Education Center, FL, USA John M. Tomich, Kansas State University, Department of Biochemistry and Molecular Biophysics, Manhattan, KS 66502, USA

1 

Asian Citrus Psyllid Life Cycle and Developmental Biology

David G. Hall* US Department of Agriculture – Agricultural Research Service, Fort Pierce, Florida, USA (Retired)

Considerable research and review information has been published on the biology of the Asian citrus psyllid (ACP), Diaphorina citri. Hussain and Nath (1927) laid the foundation for present knowledge of ACP biology in an early comprehensive review upon which many advances have been made. Presented here are highlights of the ACP life cycle and developmental biology. ACP favors tropical/subtropical climates and hot, coastal zones (Catling, 1970; Hodkinson, 2009; Jenkins et al., 2015). Typical of all Hemiptera, ACP undergoes simple (incomplete) metamorphosis with the three typical life stages: egg, nymph and adult. Citrus is regarded as a primary host plant of the psyllid and the most important from an economic standpoint, but a number of other species within the plant family Rutaceae, subfamily Aurantioideae, are utilized by the psyllid for food and reproduction. The psyllid’s reproductive biology is closely synchronized with the production of shoots of new leaf growth (flush), as oviposition occurs exclusively on emergent leaves (often called feather flush), sometimes including young leaves associated with emergent floral shoots (Hall et al., 2008a), and young, unhardened leaves are required for the development of nymphs. While immatures of some species within the Psylloidea, including the African citrus psyllid Trioza

erytreae, develop in pit-like deformations or galls induced on leaves, the Asian citrus psyllid does not and thus is free-living throughout its development on a flush shoot. However, many eggs and early instar nymphs are usually protected or hidden from view within unexpanded leaves or clusters of young leaves.

1.1  Adult Reproductive Biology, Life Characteristics and Polymorphisms The ACP is a bisexual species, with equal numbers of females and males observed in some populations (Aubert and Quilici, 1988; Tsai and Liu, 2000; Nava et al., 2007) and a predominance of females in others (Pande, 1971; Hodkinson, 1974; Alves et al., 2014). Temporal emergence patterns of males and females are similar, with no evidence of protandry or protogyny (Wenninger and Hall, 2007; Hall and Hentz, 2016). Adults are small (2.7–3.3 mm long) with mottled brown wings (Fig. 1.1A). The end of the male’s abdomen bends upward while the end of the female’s abdomen is straight and pointed (Husain and Nath, 1927) (Fig. 1.1B). Adults rest or feed on plants with their bodies

*  Email: [email protected] © CAB International 2020. Asian Citrus Psyllid: Biology, Ecology and Management of the Huanglongbing Vector (eds. J.A. Qureshi and P.A. Stansly)

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(a)

D.G. Hall

(b)

(c)

(d)

Fig. 1.1.  (A) An adult Asian citrus psyllid, Diaphorina citri. (B) Backlight illumination can be used to distinguish females from males based on the tip of their abdomens. The upper adult is a female, the lower adult is a male. (C) Asian citrus psyllid eggs. (D) The five nymphal instars of the Asian citrus psyllid.

characteristically held at a ~45° angle (range 30–60°) to the plant surface. Adults feed on young stems and on leaves of all stages of development but preferentially move to newly developing flush to feed, mate and oviposit. Mating may primarily take place on flush shoots where females feed and lay eggs, but both sexes frequently walk on limbs and branches in the interior of a tree where they can sometimes be found mating. Adult males and females locate mates, in part, using substrate-borne vibrational sounds (Wenninger et al., 2009a). These sounds cannot be detected by the human ear, but individuals calling mates can be seen rapidly vibrating or beating their wings for short periods of time. Behavioral evidence indicated that females emit a sex pheromone (Wenninger et al., 2008), and recently Zanardi et al. (2018) identified acetic acid as possibly being involved. In addition, female-produced cuticular hydrocarbons may function as sex pheromones when males are in close proximity (Mann et al., 2013; Martini

et al., 2014a; Moghbeli et al., 2014). During copulation, a male and female are positioned side by side with their heads facing the same direction, the male bending the tip of his abdomen down to the female. He uses his legs on the side next to the female to hold her while supporting himself on the plant surface with his legs on the other side (Hussain and Nath, 1927). Wenninger and Hall (2007) reported that copulation lasts from 20 to 100 min and occurs predominantly during daylight hours. Pande (1971) reported that mating takes place at any time during the day or night. Females maintain optimum reproductive output by mating multiple times with the same or different partners (Wenninger and Hall, 2008a). Adults exhibit three relatively distinct abdominal colors: gray/brown, blue/green and orange/yellow (Husain and Nath, 1927; Wenninger and Hall, 2008b). Most individuals within Florida populations are blue/green, while gray/brown individuals are rarest (Hall and



Life Cycle and Developmental Biology

Hentz, 2016). Age-related shifts may occur over time in an individual’s color, but are not seasonal. The biological significance of these polymorphisms is slowly being unraveled. Husain and Nath (1927) reported that the abdomen of gravid females turns distinctively orange, particularly during spring. Wenninger and Hall (2008b) reported that abdominal color has little value as an indicator of sexual maturity and only limited value for discerning female mating status. The orange/yellow color in females reflects the presence of eggs in the abdomen; in males, it seems to derive from the color of the internal reproductive organs and this color is generally only expressed in older males. Females may associate male color with reproductive success, as they avoid blue males after previous experience (Stockton et al., 2017). Orange males mate more frequently than blue males and appear to be more sexually aggressive in mating attempts (Stockton et al., 2017). Interestingly, females that mated with orange males laid twice as many eggs as those mated to blue males (Stockton et al., 2017). There is evidence that blue/green individuals are more apt for long-distance dispersal (Martini et al., 2014b), which could be related to their larger size (Paris et al., 2016). Differences have been reported among color morphs with respect to insecticide resistance (Boina and Bloomquist, 2015). Hemocyanin may in part be responsible for the blue/green morph (Ramsey et al., 2017), but it is not known why some psyllids might produce more hemocyanin than others. Husain and Nath (1927) reported that new adults in the Punjab region of what is now Pakistan began copulating soon after emergence and females began laying eggs on citrus soon afterwards following a pre-oviposition period of 1–3 days. In nearby Rajasthan (India), Pande (1971) reported that adults copulated 12–60 h after emergence and that oviposition commenced 8–20 h later, indicating a pre-oviposition period of 0.8–3.3 days. Wenninger and Hall (2007) reported that newly emerged adults in Florida at 26°C on orange jasmine (Murraya paniculata) mated within 2–3 days with oviposition beginning 1 day after mating for a pre-oviposition period of 3–4 days. Contrasting observations in Brazil by Alves et al. (2014) and Nava et al. (2007) indicated a pre-oviposition period of 8.5–10.9 days at 24–25°C on orange jasmine. Clearly, there are some discrepancies among

3

pre-oviposition periods reported for ACP that may be due to environmental and host plant factors. Based on data from Yang (1989), changes in photoperiod and light intensity can influence the pre-oviposition period. Uechi and Iwanami (2012) noted that maturation of a female’s ovaries was faster when new adults fed on younger leaves. Females lay eggs throughout their lives, provided that tender flush is available. Adult females typically lay 500–800 or more eggs over their lifetime (Husain and Nath, 1927; Tsai and Liu, 2000; Nava et al., 2007), with a reported maximum of 1378 (Tsai and Liu, 2000; a maximum of 1900 referenced by Tsai and Liu was apparently an error). The number of eggs laid by an individual female may vary depending on factors such as temperature and host plant species (Liu and Tsai, 2000; Tsai and Liu, 2000; Nava et al., 2007; Westbrook et al., 2011; Alves et al., 2014; Hall and Hentz, 2016; Hall et al., 2017a). Adult females held at 25°C were reported to lay an average of 858 eggs on grapefruit (Citrus paradisi) compared with 572, 613 and 626 on rough lemon (Citrus jambhiri), sour orange (Citrus aurantium) and orange jasmine, respectively (Tsai and Liu, 2000). Differences in oviposition rates reported on different host plant species (e.g. Tsai and Liu, 2000; Nava et al., 2007; Westbrook et al., 2011; Hall et al., 2017a) may be attributed to differences in plant volatiles, secondary plant compounds, nutritional quality and other factors but not in the abundance of simple foliar trichomes (Hall et al., 2017b). At 25°C, oviposition rates per female steadily increased over the first 10 days after mating, reaching a peak within 12–18 days, after which rates steadily declined on orange jasmine and grapefruit. In contrast, oviposition rates on lemon and sour orange peaked at about the same time but then remained somewhat steady through 60 or more days (Tsai and Liu, 2000). Nava et al. (2007) reported that females laid the majority of their eggs during the first 10 days after oviposition commenced. There can be substantial day-to-day variation among individual females in numbers of eggs laid (Husain and Nath, 1927). Such variation can be a result of mate availability, male color morph, age and possibly other biotic or abiotic factors. Based on Nava et al. (2007), differences in fecundity may also have a genetic basis.

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D.G. Hall

A number of factors may influence longevity of adult ACP, including temperature, relative humidity, host plant species, food availability and reproductive status (Tsai and Liu, 2000; McFarland and Hoy, 2001; Nava et al., 2007; Hall et al., 2008b). Husain and Nath (1927) reported that adults live as long as 2 months or more, during which females may continually lay eggs. These authors reported that one adult lived for 189 days. Pande (1971) indicated that female ACP generally live longer (mean 45.0 days) than males (mean 40.5 days). Nava et al. (2007) reported that at 24°C adult males lived an average of 21–25 days and females lived an average of 31–32 days on lime (Citrus limonia), Sunki mandarin (Citrus sunki) and orange jasmine. Longevity was one of several factors that collectively suggested a greater importance of females compared with males in the epidemiology of huanglongbing (Hall, 2018). Females held at 25°C lived an average of 40–48 days on rough lemon, sour orange, grapefruit and orange jasmine (Tsai and Liu, 2000). Adult psyllids can survive without food for at least several days, depending on environmental conditions (Hall and McCollum, 2011). They may sustain themselves for 3–8 days or more on some plant species outside of the Rutaceae such as cotton (Gossypium hirsutum), guava (Psidium guajava) or tomato (Solanum lycopersicum) (Hall et al., 2008b).

1.2  Development of Eggs and Nymphs Eggs are oval, clear to light yellow when freshly deposited and bright yellow-orange with two distinct red eye spots at maturity (Fig. 1.1C). Eggs are laid on terminal growth of newly developing plant tissue, including leaf folds, petioles, axillary buds, upper and lower surfaces of young leaves and tender stems (Tsai and Liu, 2000). The average size of an egg was reported by Tsai and Liu (2000) to be 0.31 mm long and 0.14 mm in diameter. Eggs are anchored to young flush shoots with a tapered basal stalk (pedicel) at the posterior/lower end averaging 0.038 mm in length (Husain and Nath, 1927). Once embedded in plant tissue, the pedicel facilitates water exchange with the plant, which is essential for egg development (Burckhardt, 1994).

Means of 16–27 eggs per shoot have been reported along with a maximum of 777 eggs on one single flush shoot (Hall et al., 2008a). If young flush is not available, gravid females may discharge eggs, singly or in groups, on to the surface of a plant without imbedding the stalk. These eggs fail to develop. The ACP develops through five nymphal instars (Fig. 1.1D). Early instars are largely sedentary and move only when disturbed or overcrowded (Tsai and Liu, 2000), whereas older nymphs are more mobile. Nymphs feed on young leaves and stems, continually secreting copious amounts of honeydew in conjunction with a white wax-like material (Tsai and Liu, 2000; Ammar et al., 2013). Adult females also produce a similar white excretory substance, but males only produce clear sticky droplets (Hall et al., 2010). Honeydew associated with large infestations of nymphs collects below infested flush on which black sooty mold can develop. Husain and Nath (1927) and Tsai and Liu (2000) presented information on distinguishing the five instars. The first-instar nymph is 0.3 mm in length and 0.17 mm in width with a light pink body and red compound eyes. The second instar is 0.45 mm in length and 0.25 mm in width; rudimentary wing pads are visible on the dorsum of the second instar’s thorax. The third instar averages 0.74 mm long and 0.43 mm wide, with well-developed wing pads and evidence of antennal segmentation. Third-instar nymphs have a single seta on each antenna. Fourth instars average 1.01 mm in length and 0.7 mm in width, with mesothoracic wing pads extended to the compound eyes and metathoracic wing pads extended to the third abdominal segment. The fourth instar has two setae on each antenna. Fifth instars average 1.6 mm long and 1.02 mm wide, with the mesothoracic wing pads extended toward the front of the compound eyes and the metathoracic wing pads reaching the fourth abdominal segment. Three setae are found on each antenna of fifth-instar nymphs. In some mature nymphs, the abdominal color turned bluish green while in others the abdomen turned pale orange (Tsai and Liu, 2000), but the significance of these color changes is not known. The ACP is a multivoltine species and so the number of generations produced each year varies depending on temperature and other environmental factors in conjunction with the



Life Cycle and Developmental Biology

availability of young flush for oviposition. Husain and Nath (1927) and Pande (1971) reported nine to ten generations annually, with as many as 16 generations observed by Atwal et al. (1968). Young trees may continually produce at least some new growth over much of the growing season, supporting many generations each year. Older trees usually flush at certain times of the year and, after a flush, adults may be stimulated to disperse from these trees in search of new growth to support reproduction. Population levels of the ACP in Florida are usually highest during late spring or early summer before the rainy season and lowest during winter. However, large populations of eggs, nymphs and adults can occur at any time of year, depending on environmental conditions and the presence of young flush (Tsai et al., 2002; Hall et al., 2008a). Diapause has not been reported in ACP (Burckhardt, 1994).

1.3  Temperature Effects The effects of temperature on ACP longevity, reproduction and development have been studied in detail under laboratory conditions (Liu and Tsai, 2000; Mizuno et al., 2004; Fung and Chen, 2006; Nakata, 2006; Nava et al., 2007; Hall et  al., 2011; Hall and Hentz, 2014). Table 1.1

5

presents a summary of published information pertinent to the biology of the psyllid reared on orange jasmine at temperatures of 15–33°C. Nava et al. (2007) reported female longevity on ‘Rangpur’ lime to range from a mean of 88.3 days at 15°C to 28.7 days at 33°C. Liu and Tsai (2000) reported that maximum female longevity ranged from 117 days at 15°C to 51 days at 30°C. Data from Wenninger and Hall (2007) suggested mating activities paused in a greenhouse during mid-afternoon when air temperatures reached 40°C. Lower and upper temperature thresholds for oviposition were reported as 16.0°C and 41.6°C, respectively, with 29.6°C estimated as optimal (Hall et al., 2011). Skelley and Hoy (2004) reported a cessation of oviposition following an air-conditioning failure causing the temperature in a rearing room to remain at 34°C for 5 days. Oviposition gradually resumed over 2–3 weeks once temperatures went back to normal. Fecundity, survival from egg to adult and development time vary with temperature and among host plant species (Tsai and Liu, 2000; Alves et al., 2014). Lower and upper temperature thresholds for development were reported to be 10.9–11.7°C and < 33°C, respectively (Liu and Tsai, 2000). The optimal temperature range for ACP development on orange jasmine is generally around 24–28°C based on fecundity, survivorship and speed of development and closer to 28°C based on intrinsic rates of increase

Table 1.1.  Biology of Diaphorina citri on orange jasmine at different constant temperatures. Duration (days)

10°Ca 15°Ca 20°Ca 24°Cb 25°Cc 25°Ca 28°Ca 30°Ca 33°Ca

Longevity (days) females

Number eggs per female Egg

– 88.3±4.3 50.6±1.6 32.4±3.2 34.3±1.9 39.7±1.4 34.7±1.1 33.5±1.1 28.7±1.4

– 171±25 494±51 348±56 381±35 626±22 748±35 316±31  67±10

– 9.7 7.0 3.6 4.6 4.2 3.5 3.3 –

1st

2nd

3rd

4th

5th

– 6.4 3.7

– 5.5 2.7

– 6.5 3.4

– 7.3 5.1

– 13.8  7.0

– 2.0 1.6 1.7 –

– 1.6 1.4 1.5 –

– 1.7 1.9 1.8 –

– 2.4 2.3 2.5 –

–  5.2  3.4  5.4 –

Liu and Tsai, 2000; 75–80% relative humidity, 13 h light phase. Nava et al. 2007; 70% relative humidity, 14 h light phase. c Alves et al. 2014; 60% relative humidity, 14 h light phase. d Extrapolated from Nava et al. (2007). a b

Egg to adult

Survival (%) egg to adult

Intrinsic rate of increase (Rm)

– 49.3±0.4 28.8±0.5 17.7±0.8d 17.3±0.3 17.0±0.2 14.1±0.2 16.3±0.3 –

 0.0 61.9 69.8 78.5±6.0d 64.3±7.1 75.4 83.9 73.7  0.0

– 0.036±0.003 0.092±0.002 – – 0.162±0.002 0.199±0.002 0.130±0.003 –

Nymphal instar

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D.G. Hall

(Rm) (Table 1.1). However, only 75–84% of immatures survived to the adult stage within this optimum temperature range, even under fairly ideal laboratory conditions (Liu and Tsai, 2000). The majority of immatures failing to reach the adult stage died during the egg and first nymphal instar stages. Linear regressions relating developmental rates of immatures to air temperature have facilitated estimates of developmental thresholds and degree-day growth models for the psyllid (Liu and Tsai, 2000; Fung and Chen, 2006; Nava et al., 2007; Milosavljević et al., 2018). Information has been published on life-table parameters for the psyllid, including intrinsic rates of increase, net reproductive rates, mean generation times and population doubling times at different temperatures and on different host plants (Tsai and Liu, 2000; Fung and Chen, 2006; Nava et al., 2007; Alves et al., 2014). ACPs occur in some of the hottest climates in which citrus is grown and are known to survive at least short exposures to temperatures as high as 45°C in arid climates (Aubert, 1988, 1990). Mortality due to high temperatures may be intensified when it occurs in conjunction with low humidity producing a high saturation deficit index (SDI) (Hodkinson, 2009). Research on other psyllid species has shown that high SDI may result in mortality, reduced fecundity and slower rates of development. However, Hall and Hentz (2014) found that adult ACPs were less tolerant of high temperatures (45°C) when humidity was moderate (75%) than when it was low (23%). Heat acclimation helps explain why the psyllid can survive in some geographical areas where afternoon air temperatures sometimes exceed 40°C for several hours. Atwal et al. (1968) reported that 5th-instar nymphs and adults withstood 45°C for up to 4 h and adults withstood 40°C for up to 12 h. Heat shock proteins probably help psyllids survive high temperature extremes (Marutani-Hert et al., 2010). ACPs are common and abundant in Pakistan and yet daily air temperatures during the summer may exceed 40°C for 7–8 h, with peaks as high as 44°C with 33–39% relative humidity (RH) (Hall and Hentz, 2014). Levels of mortality or adverse biological effects from such temperature extremes are not known, but ACP populations remain prevalent in the Punjab region of Pakistan (Hoddle and Hoddle, 2013). Some eggs and nymphs of the

ACP may escape high lethal air temperature events because they are protected within clusters of young flush leaves. In subtropical areas with seasonal changes in climate, psyllids infesting citrus may sometimes be subjected to freezing temperatures for various periods of time. ACPs are known to survive at least short exposures to temperature extremes as low as –7°C in subtropical wet areas (Aubert, 1988, 1990). Atwal et al. (1968) reported that fifth-instar nymphs and adults exposed to 0°C died within 6–8 h. Working with psyllids reared under greenhouse conditions at ~25°C, 66–48% of adults survived exposure for 2–3 h to temperatures of –5 to –6°C; greater than 60% of nymphs survived 8–10 h of exposure to temperatures as low as –4°C; and around 50% or more eggs remained viable after being exposed for 2–3 h to temperatures as low as –8°C (Hall et al., 2011). The psyllid can acclimate to colder temperatures and subsequently survive some freeze events that would otherwise be lethal (Hall et al., 2011). According to Hodkinson (2009) and others, insects can avoid lethal freezes by lowering the ‘super cooling point’ of their body tissues. It is likely that some adult psyllids may survive a freeze by finding cold protection in bark crevices or within ground litter. Some freeze events might not be severe enough to kill a large percentage of psyllids but may indirectly cause mass mortality of eggs and nymphs by killing young flush. Young citrus flush shoots have been noted as being fairly tolerant of a 3 h freeze at –1.7°C or a 30 min freeze at –2.2°C, but large percentages of new shoots may die following freezes of –2.2°C for 2–3 h or following a –3.3°C freeze for 30 min (Oswalt, 2008).

1.4  Humidity, Rain and Sunlight Most laboratory investigations on ACP biology have been conducted at constant relative humidity levels in the 60–80% range. In Florida, where high population levels of the psyllid are known to occur, humidity during the night and early hours after daybreak usually approaches 90% or higher. Skelley and Hoy (2004) reported on a rearing procedure for the psyllid in which RH in an air-conditioned rearing room varied seasonally from 35% to 65%, noting that females produced fewer eggs when the



Life Cycle and Developmental Biology

RH in the room dropped below 40% (information was not provided on humidity levels in the canopies of rearing plants). McFarland and Hoy (2001) reported that, in the absence of a host plant, adult longevity decreased as RH was incrementally decreased from 97% to 7% at either 25°C or 30°C. The effect was more pronounced at 30°C, at which about 80% adults survived for 20 h at 97% humidity, compared with 0% survival at 7% humidity. With respect to rain, Aubert (1987) speculated that monthly rainfall in excess of 15 cm was generally associated with low populations of the psyllid due to eggs and young nymphs being washed off plant surfaces. However, according to Husain and Nath (1927), because eggs are anchored into plant tissue by their stalks, they cannot be washed off by rain. Population levels of eggs, nymphs and adults did not appear to be negatively influenced by rain in two Florida orchards where rainfall sometimes exceeded 9 cm a month (Hall et al., 2008a). Adult activity (flight, walking, feeding, mating and oviposition) is prompted by daylight. Flight activity varies with changes in sunlight (Hall, 2009) and is pronounced during warm, sunny afternoon hours (Aubert and Hua, 1990; Sétamou et al., 2012; Paris et al., 2015). Flight initiation by ACP is influenced by changes in barometric pressure and increases in air temperature, but changes in humidity do not affect its dispersal (Martini and Stelinski, 2017). The psyllid is positively phototaxic (Husain and Nath, 1927; Aubert and Hua, 1990) and its behavior is strongly influenced by sunlight (see Hall et al., 2008b, 2018). Paris et al. (2017) showed that positive phototaxis by walking psyllids was associated with short-wavelength ultraviolet (UV) light (350–405 nm), while little or no walking responses were observed at longer wavelengths in the visible spectrum from green to yellow to orange (500–620 nm). Increased UV light (high spectral irradiance in the 250– 400 nm range) was one of several factors speculated as possibly being responsible for lower population levels of the psyllid at higher altitudes (Jenkins et al., 2015), but the effects of high UV on psyllid reproduction, development and longevity are not known. Published research findings on ACP biology under laboratory conditions are usually accompanied by information on photoperiod, which is

7

usually in the range of 13–16 h of daily illumination. However, irradiation and light intensity are generally not reported, although light type and position sometimes are. For example, Skelley and Hoy (2004) reared the psyllid in rooms illuminated only by fluorescent lamps (5000–6000 lux output) suspended 2.5 cm above rearing cages. Hall et al. (2016) illuminated ACP-infested plants in the laboratory using light-emitting diode lamps (PAR38 22º, 17 W, white 3000K floodlights) positioned 45–60 cm above the plants, and in a walk-in chamber using 400 W mercury vapor lamps positioned 23 cm above rearing cages. Wenninger et al. (2009b) reported working at a light intensity of about 3200 lux (provided by fluorescent lamps) recorded just above rearing vials. Good comparisons remain to be made of ACP biology under fluorescent lamps, mercury lamps, light-emitting diodes, halogen and tungsten lamps versus sunlight. Hall and Hentz (2016) considered cooler temperatures during winter as primarily responsible for slower development and reduced productivity of ACP reared on different host plant species, although shorter photoperiods and reduced solar radiation/light intensity may also have been factors. Data presented by Yang (1989) indicated that fecundity increased with light intensity (1200– 15,000 lux) and photoperiod (6–18 h daily illumination), and that these parameters influenced the pre-oviposition period. Information on solar radiation as it relates to ACP reproduction potential and intrinsic rates of increase is needed. Massproducing of ACP would benefit from expanded information on the influence of light on the reproductive biology of the psyllid. To this end, Hall and Hentz (2019) reported that photoperiod, irradiance and illuminance were positively correlated with ACP reproductive rates.

1.5  Expanding the Knowledge Base Newly emerging research interests are continually expanding our knowledge of the biology of the ACP. Endosymbionts such as Wolbachia, Profftella and Carsonella may affect certain parameters of psyllid reproduction and biology (Hoffmann et al., 2014; Ramsey et al. 2017). Much remains to be discovered regarding the developmental biology of ACP infected by ‘Candidatus Liberibacter asiaticus’ (CLas), the bacterial

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D.G. Hall

pathogen responsible for huanglongbing. Evidence has already been presented that this pathogen increases ACP dispersal potential (Martini et al., 2015) and susceptibility to insecticides and entomopathogens (Tiwari et al., 2011; Orduño-Cruz et al., 2016). Ren et al. (2016) reported that the bacterium had obvious effects on the biology of the psyllid, including increased fecundity and longevity of females. Pelz-Stelinski et al. (2010) and Pelz-Stelinski and Killiny (2016) found that females carrying the pathogen were more fecund than uninfected counterparts and that developmental rates of

infected nymphs were reduced, but that longevity of infected adults was reduced. Electropenetrography showed that ACP infected by the pathogen foraged more often than healthy ACP (Killiny et al., 2017). Transcriptome and proteome analyses on ACP infected by CLas showed an upregulation of transcripts and proteins involved in defense and immunity (Kruse et al., 2017; Ramsey et al., 2017). Further advances in our knowledge of ACP biology will be beneficial with respect to finding novel methods of managing vector populations and thus huanglongbing.

References Alves, G.R., Diniz, A.J.F. and Parra, J.R.P. (2014) Biology of the huanglongbing vector Diaphorina citri (Hemiptera: Liviidae) on different host plants. Journal of Economic Entomology 107, 691–696. Ammar, E.D., Alessandro, R., Shatters, R.G. Jr and Hall, D.G. (2013) Behavioral, ultrastructural and chemical studies on the honeydew and waxy secretions by nymphs and adults of the Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae). PLOS ONE 8(6), e64938. Atwal, A.S., Chaudhary, J.P. and Ramzan, M. (1968) Studies on the development and field population of citrus psylla, Diaphorina citri Kuwayama (Psyllidae: Homoptera). Journal of Research Punjab Agricultural University 7, 333–338. Aubert, B. (1987) Trioza erytreae Del Guercio and Diaphorina citri Kuwayama (Homoptera: Psylloidea), the two vectors of citrus greening disease: biological aspects and possible control strategies. Fruits 42, 149–162. Aubert, B. (1988) Management of the citrus greening disease in Asian orchards. In: Regional Workshop on Citrus Greening Huanglongbing Disease, Fuzhou, China, 6–12 December 1987. FAO-UNDP, Rome, pp. 51–52. Aubert, B. (1990) Integrated activities for the control of huanglongbing-greening and its vector Diaphorina citri Kuwayama in Asia. In: Aubert, B., Tontyaporn, S. and Buangsuwon, D. (eds) Proceedings of the Fourth International Asia Pacific Conference on Citrus Rehabilitation, Chiang Mai, Thailand, 4–10 February 1990. FAO-UNDP RAS/86/022 Regional Project, FAO-UNDP, Rome, pp. 133-144. Aubert, B. and Hua, X.Y. (1990) Monitoring flight activity of Diaphorina citri on citrus and Murraya canopies. In: Aubert, B., Tontyaporn, S., and Buangsuwon, D., (eds) Proceedings of the Fourth International Asia Pacific Conference on Citrus Rehabilitation, Chiang Mai, Thailand, 4–10 February 1990. FAO-UNDP RAS/86/022 Regional Project, FAO-UNDP, Rome, pp. 181–187. Aubert, B. and Quilici, S. (1988) Monitoring adult psyllas on yellow traps in Reunion Island. In: Timmer, L.W., Garnsey, S.M. and Navarro, L. (eds) Proceedings of the 10th Conference of the International Organization of Citrus Virologists, 17–21 November 1986, Valencia, Spain. International Organization of Citrus Virologists, University of California, Riverside, California, pp. 249–254. Boina, D.R. and Bloomquist, J.R. (2015) Chemical control of the Asian citrus psyllid and of huanglongbing disease in citrus. Pest Management Science 71, 808–823. doi: 10.1002/ps.3957 Burckhardt, D. (1994) Psylloid pests of temperate and subtropical crop and ornamental plants (Hemiptera: Psylloidea): a review. Trends in Agricultural Sciences (Entomology) 2, 173–186. Catling, H.D. (1970) Distribution of the psyllid vectors of the citrus greening disease, with notes on the biology and bionomics of Diaphorina citri. FAO Plant Protection Bulletin 18, 8–15. Fung, Y.-C. and Chen, C.-N. (2006) Effects of temperature and host plant on population parameters of the citrus psyllid (Diaphorina citri Kuwayama). Formosan Entomologist 26, 109–123. Hall, D.G. (2009) An assessment of yellow sticky card traps as indicators of the relative abundance of adult Diaphorina citri in citrus. Journal of Economic Entomology 102, 446–452. Hall, D.G. (2018) Incidence of ‘Candidatus Liberibacter asiaticus’ in a Florida population of Asian citrus psyllid. Journal of Applied Entomology 142, 97–103. doi: 10.1111/jen.12466.



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Hall, D.G. and Hentz, M.G. (2014) Asian citrus psyllid (Diaphorina citri) tolerance to heat. Annals of the Entomological Society of America 107, 641–649. Hall, D.G. and Hentz, M.G. (2016) An evaluation of plant genotypes for rearing Asian citrus psyllid (Hemiptera: Liviidae). Florida Entomologist 99, 471–480. Hall, D.G. and Hentz, M.G. (2019) Influence of light on reproductive rates of Asian citrus psyllid (Hemiptera: Liviidae). Journal of Insect Science 19(1), 1–7. doi: 10.1093/jisesa/iey141. Hall, D.G. and McCollum, G. (2011) Survival of adult Asian citrus psyllid on harvested citrus fruit and leaves. Florida Entomologist 94, 1094–1096. Hall, D.G., Hentz, M.G. and Adair, R.C. Jr (2008a) Population ecology and phenology of Diaphorina citri (Hemiptera: Psyllidae) in two Florida citrus groves. Environmental Entomology 37, 914–924. Hall, D.G., Gottwald, T.R., Chau, N.M., Ichinose, K., Dien, L.Q. and Beattie, G.A.C. (2008b) Greenhouse investigations on the effect of guava on infestations of Asian citrus psyllid in citrus. Proceedings of the Florida State Horticultural Society 121, 104–109. Hall, D.G., Shatters, R.G. Jr, Carpenter, J.E. and Shapiro, J.P. (2010) Progress toward an artificial diet for adult Asian citrus psyllid. Annals of the Entomological Society of America 103, 611–617. Hall, D.G., Wenninger, E.J. and Hentz, M.G. (2011) Temperature studies with the Asian citrus psyllid, Diaphorina citri Kuwayama: cold hardiness and temperature thresholds for oviposition. Journal of Insect Science 11, 1–15. Hall, D.G., Albrecht, U. and Bowman, K.D. (2016) Transmission rates of ‘Ca. Liberibacter asiaticus’ by Asian citrus psyllid are enhanced by the presence and developmental stage of citrus flush. Journal of Economic Entomology 109, 558–563. Hall, D.G., Hentz, M.G. and Stover, E. (2017a) Field survey of Asian citrus psyllid (Hemiptera: Liviidae) infestations associated with six cultivars of Poncirus trifoliata. Florida Entomologist 100, 667–668. Hall, D.G., Ammar, E.D., Bowman, K.D. and Stover, E.W. (2017b) Epifluorescence and stereomicroscopy of trichomes associated with resistant and susceptible host plant genotypes of the Asian citrus psyllid (Hemiptera: Liviidae), vector of citrus greening disease. Journal of Microscopy and Ultrastructure. doi: 10.1016/j.jmau.2017.04.002. Hall, D.G., Borovsky, D., Chauhan, K.R. and Shatters, R.G. (2018) An evaluation of mosquito repellents and essential plant oils as deterrents of Asian citrus psyllid. Crop Protection 108, 87–94. Hoddle, M.S. and Hoddle, C.D. (2013) Classical biological control of Asian citrus psyllid with Tamarixia radiata in urban Southern California. Citrograph 4, 52–58. Hodkinson, I.D. (1974) The biology of the Psylloidea (Homoptera): a review. Bulletin of Entomological Research 64, 325–339. Hodkinson, I.D. (2009) Life cycle variation and adaptation in jumping plant lice (Insecta: Hemiptera: Psylloidea): a global synthesis. Journal of Natural History 43, 65–179. Hoffmann, M., Coy, M.R., Kingdom Gibbard, H.N. and Pels-Stelinski, K.S. (2014) Wolbachia infection density in populations of the Asian citrus psyllid (Hemiptera: Liviidae). Environmental Entomology 43, 1215–1222. Husain, M.A. and Nath, D. (1927) The citrus psylla (Diaphorina citri, Kuw.) [Psyllidae: Homoptera]. Memoirs of the Department of Agriculture in India, Entomological Series 10, 1–27. Jenkins, D.A., Hall, D.G. and Goenaga, R. (2015) Diaphorina citri (Hemiptera: Liviidae) abundance in Puerto Rico declines with elevation. Journal of Economic Entomology 108, 252–258. Killiny, N., Hijaz, F., Ebert, T.A. and Rogers, M.E. (2017) A plant bacterial pathogen manipulates its insect vector’s energy metabolism. Applied and Environmental Microbiology 83, e03005–16. doi: 10.1128/ Kruse, A., Fattah-Hosseini, S., Saha, S., Johnson, R., Warwick, E., Sturgeon, K., Mueller, L., MacCoss, M.J., Shatters, R.G. Jr and Heck, M.C. (2017) Combining 'omics and microscopy to visualize interactions between the Asian citrus psyllid vector and the Huanglongbing pathogen Candidatus Liberibacter asiaticus in the insect gut. PLOS ONE 12, e0179531. doi: 10.1371/journal.pone.0179531. Liu, Y.H. and Tsai, J.H. (2000) Effects of temperature on biology and life table parameters of the Asian citrus psyllid, Diaphorina citri Kuwayama (Homoptera: Psyllidae). Annals of Applied Biology 137, 201–206. Mann, R.S., Rouseff, R.L., Smoot, J., Rao, N., Meyer, W.L., Lapointe, S.L., Robbins, P.S., Cha, D., Linn, C.E., Webster, F.X. et al. (2013) Chemical and behavioral analysis of the cuticular hydrocarbons from Asian citrus psyllid, Diaphorina citri. Insect Science 20, 367–378. Martini, X. and Stelinski, L.L. (2017) Influence of abiotic factors on flight initiation by Asian citrus psyllid (Hemiptera: Liviidae). Environmental Entomology 46, 369-375.

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Martini, X., Kuhns, E.H., Hoyte, A. and Stelinski, L.L. (2014a) Plant volatiles and density-dependent conspecific female odors are used by Asian citrus psyllid to evaluate host suitability on a spatial scale. Arthropod-Plant Interactions 8, 453–460. Martini, X., Hoyte, A. and Stelinski, L.L. (2014b) Abdominal color of the Asian citrus psyllid (Hemiptera: Liviidae) is associated with flight capabilities. Annals of the Entomological Society of America 107, 842–847. Martini, X., Hoffmann, M., Coy, M.R., Stelinski, L.L. and Pelz-Stelinski, K.S. (2015) Infection of an insect vector with a bacterial plant pathogen increases its propensity for dispersal. PLOS ONE 10(6), e0129373. doi: 10.1371/journal.pone.0129373. Marutani-Hert, M., Hunter, W.B. and Hall, D.G. (2010) Gene response to stress in the Asian citrus psyllid (Hemiptera: Psyllidae). Florida Entomologist 93, 519–525. McFarland, C.D. and Hoy, M.A. (2001) Survival of Diaphorina citri (Homoptera: Psyllidae) and its two parasitoids, Tamarixia radiata (Hymenoptera: Eulophidae) and Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae), under different relative humidities and temperature regimes. Florida Entomologist 84, 227–233. Milosavljević, I., Amrich, R., Strode, V. and Hoddle, M.S. (2018) Modeling the phenology of Asian citrus psyllid (Hemiptera: Liviidae) in urban southern California: effects of environment, habitat, and natural enemies. Environmental Entomology, nvx206. doi: 10.1093/ee/nvx206. Mizuno, T., Yoneda, M., Mizuniwa, S.I. and Dohino, T. (2004) Studies on cold hardiness and fecundity of the Asian citrus psylla Diaphorina citri Kuwayama (Homoptera: Psyllidae) – possibility of over-wintering of the Asian citrus psylla in the southern part of Kyushu. Research Bulletin of Plant Protection Japan 40, 89–93. Moghbeli, G.A., Ziaaddini, M., Jalali, M.A. and Michaud, J.P. (2014) Sex-specific responses of Asian citrus psyllid to volatiles of conspecific and host-plant origin. Journal of Applied Entomology 138, 500–509. Nakata, T. (2006) Temperature-dependent development of the citrus psyllid, Diaphorina citri (Homoptera: Psylloidea), and the predicted limit of its spread based on overwintering in the nymphal stage in temperate regions of Japan. Applied Entomology and Zoology 41, 383–387. Nava, D.E., Torres, M.L.G., Rodrigues, M.D.L., Bento, J.M.S. and Parra, J.R.P. (2007) Biology of Diaphorina citri (Hem., Psyllidae) on different hosts and at different temperatures. Journal of Applied Entomology 131, 709–715. Orduño-Cruz, N., Guzmán-Franco, A.W. and Rodríguez-Leyva, E. (2016) Diaphorina citri populations carrying the bacterial plant pathogen Candidatus Liberibacter asiaticus are more susceptible to infection by entomopathogenic fungi than bacteria-free populations. Agricultural and Forest Entomology 18, 95–98. doi: 10.1111/afe.12138/epdf. Oswalt, C. (2008) 2008–09 Winter Weather Watch Manual. Polk County Cooperative Extension Service. University of Florida Institute of Food and Agricultural Sciences, Gainesville, Florida, pp. 56–60. Pande, Y.D. (1971) Biology of citrus psylla, Diaphorina citri Kuw. (Hemiptera: Psyllidae). Israel Journal of Entomology 6, 307–311. Paris, T.M., Croxton, S.D., Stansly, P.A. and Allan, S.A. (2015) Temporal response and attraction of Diaphorina citri to visual stimuli. Entomologia Experimentalis et Applicata 155, 137–147. Paris, T.M., Allan, S.A., Hall, D.G., Hentz, M.G., Croxton, S.D., Ainpudi, N. and Stansly, P.A. (2016) Effects of temperature, photoperiod, and rainfall on morphometric variation of Diaphorina citri (Hemiptera: Liviidae). Environmental Entomology 46, 143–158. Paris, T.M., Allan, S.A., Udell, B.J. and Stansly, P.A. (2017) Wavelength and polarization affect phototaxis of the Asian citrus psyllid. Insects 8, 88. Pelz-Stelinski, K.S. and Killiny, N. (2016) Better together: association with ‘Candidatus Liberibacter asiaticus’ increases the reproductive fitness of its insect vector, Diaphorina citri (Hemiptera: Liviidae). Annals of the Entomological Society of America 109, 371–376. Pelz-Stelinski, K.S., Brlansky, R.H., Ebert, T.A. and Rogers, M.E. (2010) Transmission parameters for Candidatus Liberibacter asiaticus by Asian citrus psyllid (Hemiptera: Psyllidae). Journal of Economic Entomology 130, 1531–1541. Ramsey, J.S., Chavez, J.D., Johnson, R., Hosseinzadeh, S., Mahoney, J., Mohr, J., Robison, F., Zhong, X., Hall, D.G., MacCoss, M. et al. (2017) Protein interaction networks at the host–microbe interface in Diaphorina citri, the insect vector of the citrus greening pathogen. Royal Society Open Science 4, 160545. doi: 10.1098/rsos.160545. Ren, S.-L., Li, Y.-H., Zhou, Y.-T., Xu, W.-M., Cuthbertson, A.G.S, Guo, Y.-J. and Qiu, B.-L. (2016) Effects of Candidatus Liberibacter asiaticus on the fitness of the vector Diaphorina citri. Journal of Applied Microbiology 121, 1718–1726.



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Sétamou, M., Sanchez, A., Patt, J.M., Nelson, S.D., Jifon, J. and Louzada, E.S. (2012) Diurnal patterns of flight activity and effects of light on host finding behavior of the Asian citrus psyllid. Journal of Insect Behavior 25, 264–276. doi: 10.1007/s10905-011-9295-3. Skelley, L.H. and Hoy, M.A. (2004) A synchronous rearing method for the Asian citrus psyllid and its parasitoids in quarantine. Biological Control 29, 14–23. Stockton, D.G., Pescitelli, L.E., Martini, X. and Stelinski, L.L. (2017) Female mate preference in an invasive phytopathogen vector: how learning may influence mate choice and fecundity in Diaphorina citri. Entomologia Experimentalis et Applicata 164, 16–26. Tiwari, S., Pelz-Stelinski, K. and Stelinski, L.L. (2011) Effect of Candidatus Liberibacter asiaticus infection on susceptibility of Asian citrus psyllid, Diaphorina citri, to selected insecticides. Pest Management Science 67, 94–99. Tsai, J.H. and Liu, Y.H. (2000) Biology of Diaphorina citri (Homoptera: Psyllidae) on four host plants. Journal of Economic Entomology 93, 1721–1725. Tsai, J.H., Wang, J.J. and Liu, Y.H. (2002) Seasonal abundance of the Asian citrus psyllid, Diaphorina citri (Homoptera: Psyllidae) in southern Florida. Florida Entomologist 85, 446–451. Uechi, N. and Iwanami, T. (2012) Comparison of the ovarian development in Diaphorina citri Kuwayama (Hemiptera: Psyllidae) in relation to the leaf age of orange jasmine, Murraya paniculata (L.) Jack. Bulletin of the National Institute of Fruit Tree Science 13, 39–42. Wenninger, E.J. and Hall, D.G. (2007) Daily timing of and age at mating in the Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae). Florida Entomologist 90, 715–722. Wenninger, E.J. and Hall, D.G. (2008a) Importance of multiple mating to female reproductive output in Diaphorina citri. Physiological Entomology 33, 316–321. Wenninger, E.J. and Hall, D.G. (2008b) Daily and seasonal dynamics in abdomen color in Diaphorina citri (Hemiptera: Psyllidae). Annals of the Entomological Society of America 101, 585–592. Wenninger, E.J., Stelinski, L.L. and Hall, D.G. (2008) Behavioral evidence for a female-produced sex attractant in Diaphorina citri (Hemiptera: Psyllidae). Entomologia Experimentalis et Applicata 128, 450–459. Wenninger, E.J., Hall, D.G. and Mankin, R.W. (2009a) Vibrational communication between the sexes in Diaphorina citri (Hemiptera: Psyllidae). Annals of the Entomological Society of America 102, 547–555. Wenninger, E.J., Stelinski, L.L. and Hall, D.G. (2009b) Relationships between adult abdominal color and reproductive potential in Diaphorina citri (Hemiptera: Psyllidae). Annals of the Entomological Society of America 102, 476-483. Westbrook, C.J., Hall, D.G., Stover, E.W., Duan, Y.P. and Lee, R.F. (2011) Colonization of Citrus and Citrus–related germplasm by Diaphorina citri (Hemiptera: Psyllidae). HortScience 46, 997–1005. Yang, Y.B. (1989) Effects of light, temperature and humidity on the development, reproduction and survival of citrus psylla. Acta Ecologica Sinica 9, 348–354. Zanardi, O.Z., Volpe, H.X.L., Favaris, A.P., Silva, W.D., Luvizotto, R.A.G., Magnani, R.F., Esperança, V., Delfino, J.Y., de Freitas, R., Miranda, M.P., Parra, J.R.P., Bento, J.M.S. and Leal, W.S. (2018) Putative sex pheromone of the Asian citrus psyllid, Diaphorina citri, breaks down into an attractant. Scientific Reports 8, 455. doi: 10.1038/s41598-017-18986-4.

2 

Functional Anatomy of the Asian Citrus Psyllid Joseph M. Cicero* Department of Entomology and Nematology, University of Florida, Gainesville, USA

2.1 Introduction Classical authors made huge and valuable baseline contributions to understanding homopteran anatomy for the 80 or so years before quantum improvements in technique (Karnovsky, 1965; Friedrich et  al., 2014) and resolution of microscopy (Palucka, 2002) arrived. However, many ­aspects of their works, along with their crude sketches and interpretive skills, have been grandfathered into modern literature without validation using modern instruments, techniques or rigor. Further, a homological school of thought presided for use in determining identifications, functions and evolution of anatomical components. Understandably, this school persists today, even though advancements in cell biology with special reference to cytological and histological miniaturization, reduction and simplification (Beutel and Haas, 1998; O’Malley et  al., 2016; Randolf et  al., 2017) make it very difficult to maintain the school while at the same time ushering in a new era that caters to protein chemistry, molecular biology, endocrinology and pest control. In fact, review of the classical homopteran literature led to the suggestion that the difficulty with maintaining the school is because the task of correlating certain anatomies from one taxon to another is actually a phylocytological problem

(Cicero and Brown, 2012). Nevertheless, on ­review of the ACP literature, it is clear that a modern working system of descriptive, structural terminology for homoptera is still necessary and in dire need. An example solution to this problem would be a homopteran anatomy ontology modeled from the successful Hymenoptera Anatomy Ontology Project (Yoder et al., 2010). As of this writing, a search of prominent databases for articles using ‘Diaphorina’ and ‘citri’ as Boolean keywords yielded surprising results (Table 2.1). Excluding the taxonomic and pre1970 literature, only ten monographic publications elucidating aspects of external anatomy (morphometrics, five) and seven elucidating aspects of internal anatomy were garnered from the total number of hits. The rest were mostly insect pest management, ecology, pathology and molecular. It is clear from these statistics that anatomy of ACP falls far short of critical need. In fact, most of what can be assembled regarding ACP functional anatomy is transferred, or inferred, from studies of other species, especially the potato psyllid, Bactericera cockerelli (Sulc) (PoP). As a summary chapter of ACP anatomy has already been published (Brown et al., 2016), and monographs of ACP anatomy are few, this chapter’s sections will summarize core knowledge of  select anatomical components and, where

*  Email: [email protected]

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© CAB International 2020. Asian Citrus Psyllid: Biology, Ecology and Management of the Huanglongbing Vector (eds. J.A. Qureshi and P.A. Stansly)



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Table 2.1.  Hits from the Boolean keyword search ‘Diaphorina’ AND ‘citri’ using three prominent US databases (last accessed: September, 2018). Database

No. of hits

Academic Search Premier (EBSCO, Ipswich, Maine) Agricola (ProQuest, National Agricultural Library, Beltsville, Maryland) PubMed (National Institutes of Health, Bethesda, Maryland)

rostrum

396 420 labium

461

­ ossible, extend them into a prospectus of ideas p that critically need consideration during the eventual movement to Gupta’s (1994) ‘molecular morphology’.

2.2 Embryology No modern studies of ACP embryology (Fig. 2.1) are currently available. Therefore, the general aspects of psyllid embryonic development are only inferential from those determined by modern techniques for relatives such as aphids, Oncopeltus, Rhodnius and others. Of special interest in ACP embryology is the biogenesis of the stylets (Section 2.3.5) and biogenesis of the filter chamber (Section 2.4.2). Embryonic biogenesis of the configuration of hypodermal cells needed to secrete and house presumptive (new) stylets of the true first instar (psyllids may have pre-eclosion instars with their own molting events) should have marked differences from that of consecutive pharate instars insofar as the first embryonic process probably does not involve an end-cap, and almost certainly does not involve an embedded matrix. Concordantly, the filter chamber is mostly made up of midgut tissue and, reasonably assuming that first instars (‘crawlers’) have one, then in theory embryogenesis of the midgut should, to a certain extent, follow the primitive motif of initiation between the stomodeal and proctodeal invaginations, then depart from that motif to allow co-location, convolution and ­ensheathing of its opposing ends to form one. This process would be in contrast to the direct inheritance of the filter chamber from one instar to the next. The departure is of great interest

Fig. 2.1.  Post-katatreptic embryo of ACP. Prepared by phase partition fixation (Zalokar and Erk, 1977) using 8% aqueous formaldehyde and heptane. Abdominal segmentation is not well differentiated. Line = 50 μm.

­ ecause it is the ontogenetic stage in which b study of filter chamber biogenesis and evolution is best undertaken.

2.3  Oral Region The oral region of PoP was defined as internal and external anatomy occurring between the anterior margin of the ‘rostrum’, a ventral, tearshaped sclerite, and the profurcasternum, located at the base of the labium (Cicero et  al., 2015) (Figs 2.1, 2.2). The ACP oral region is consistent with this PoP model. In both, the ­labium is shaft-like and evolutionarily posteriorized to where it is now directed ventrally ­between, or slightly posterior to, the procoxae, a condition considered to be derived for Sternorrhyncha (e.g. scales, larval whiteflies, psyllids) (Hennig, 1981:248; Grimaldi, 2003:340). This posteriorization coevolved with, and facilitated, the reflexing of oral region components primitively dorsal to it (maxillae, mandibles, labrum and clypeus) to their current position along the

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J.M. Cicero

stylet bundle inside crumena tentorial arms broken away crumena rostrum

location of profurcastenum

auricle

posterior v e n t r a l

auricle inner lateral surface of mandibular

postoral section (tentorium)

preoral section labium

loading sleeve

exposed stylet epicuticle hollow between loading sleeve and stylet auricle

Fig. 2.2.  Oral region of adult ACP. The rostrum is ventral to structures shown and fully out of view. Tentorial arms, or simply called ‘side arms’, are graphically drawn in place (*) since they were broken away to access the mandibular and show its inner lateral surface (Section 2.3.5). Profurcasternal sclerites were torn away during extirpation. The crumena redirects the stylet bundle from a posterior direction to a ventral direction for direct address into the host plant. The auricle is an inner lateral extension of the ‘flat’ or basal terminus (cf. Fig. 2.3). Line = 100 μm. Inset: magnification of mandibular showing that all but the distal end of the loading sleeve was torn away during extirpation. The cushion collects at a critically significant space inside the hollow, at the inner lateral surface of the proximal base of each stylet (encircled). This position is directly posterior to the auricle (cf. Fig. 2.5C and Section 2.3.5). Line = 10 μm.

ventral face of the cranium so that the mandibular stylets (‘mandibulars’) are ventral to the maxillary stylets (‘maxillars’) (Fig. 2.3). In the primitive condition, the outer lateral mandibular and maxillar surfaces were exposed to the outside. However, this reflexing or ‘ventralization’ (‘opisthognathy’ or ‘opisthorrhynchy’), included closure of the exposure by modification of external lateral head structures (e.g. the gena, lora, maxillary and mandibular plates of various authors) such that only a minute opening, the ‘preoral orifice’, remained (Fig. 2.3). It also included modification of the precursors of the stylets into very long, highly specialized feeding devices that pass through this tightly fitting orifice. In simplest terms, this explains how the ­stylet bases and the true mouth were deeply

i­ nternalized. With this lateral closure, the preoral orifice became the only continuity between the complex of airspaces (‘hollows’) inside the head and the air outside the body. Diagnosis of the identities and evolutionary origins of the lateral head structures involved in this closure has gone through a tortuous history (Spooner, 1938:16; Parsons, 1974), with no resolution, and the most appreciable explanation for this is that they were cell biological events that probably occurred in different ways by different taxa to accomplish the same functional end. For this reason, the large, lateral, cuticle-­ bound aspects of the adult PoP oral ­region that provide holstering for the stylets are simply called ‘lateral tissue blocks’ (Cicero et  al., 2015:749, f.5Bd). According to Weber’s (1929:75, abb.5a)



Functional Anatomy of the Asian Citrus Psyllid

15

eye

crumena distal base

base location of holsters

proximal base location of loading sleeves

stylet bundle

dorsal surface

auricles the ‘flat’ xs

shaft

most bendable axis

hollow rotation

xs start maxillar interlock outer lateral surface

preoral orifice

mandibulars are dorsoventral to maxillars

rostrum

mandibulars are laterolateral to maxillars

mandibulars are ventral to maxillars

Fig. 2.3.  Adult ACP. Schematic showing the routing of stylets from their basal termini to exit from the crumena. The stylet ‘base’ is the length inside the head. The stylet ‘shaft’ is the length outside the head. Not drawn to scale – maxillar interlock and mandibular rerouting actually occur very near exit of the stylet bundle through the preoral orifice. The stylet basal terminus is the ‘flat’. Its auricle protrudes in an interior direction (cf. Fig. 2.2). Cross-section (xs) indicates two loci representing the intersected edges of the basal terminus or ‘flat’ of the stylet (cf. Fig. 2.5C). With the head reflexed, the mandibulars are ventral to the maxillars at their basal termini, but they are rerouted to a position lateral to the maxillars, then the stylet bundle as a whole is rotated 90°.

scheme for the adult apple sucker, Cacopsylla (= Psylla) mali Schmidberger, these lateral blocks correlate to the ‘laminae maxillaris’. Weber’s adult ‘laminae mandibularis’ correlate to a smaller block in PoP (Cicero et  al., 2015:749, f.5Bt) which does not appear to participate in adult stylet functionality. Weber indicated that, in the general (primitive) Hemipteran head capsule, these structures were external, and that in C. mali adults they were internalized (p. 67, abb.1).

2.3.1  Larval stylet bundle docking In larval ACP (Fig. 2.4A), C. mali (Pollard, 1970), Boreoglycaspis melaleucae Moore (Ammar and Hall, 2013), and PoP (Brown et  al., 2016:405, f.28.2), the stylet bundle is held externally in a bilaterally asymmetrical configuration; it exits

the preoral orifice and loops around the perimeter of the rostrum before it enters the labial groove. The first two references specifically indicate that, in dorsal view, exit is from the left side and entry is from the right. Psyllid larvae have not been examined for possible inter- and intraspecific reversal of this asymmetry. For C. mali larvae, Weber (1929:97, abb.14) placed the laminae maxillaris and the dorsal half of the laminae mandibularis inside the head, and depicted the ventral half of the laminae mandibularis extending out to the exterior, where they curve anteriorly along the lateral aspect of the rostrum and serve to hold the larval stylet bundle loop in place. Pollard (1970:297, f.1,2, ‘lamina’) also identified the exterior location and stylet-holding function of the laminae mandibularis in larvae of C. mali. Ammar and Hall (2013) used scanning electron microscopy (SEM) to bring this structure into sharp focus for ACP larvae,

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J.M. Cicero

calling it a ‘stylet-­holding organ’ (Fig. 2.4A,B). In PoP, cuticle in the analogous position consists of an accordion-like arrangement of arthrodial membrane pleats (Brown et al., 2016:406, f.28.3). The modern interpretation of this structure as an ‘arthrodial membrane’ is sound, since any homological identity it may have had to primitive indurate sclerites has been lost. On further analysis, the ACP stylet-­holding tab is clearly an organ of the integumentary system. It is functionally more organized than in PoP, and in fact, at high magnification (Fig. 2.4B), distinct pleats are not well defined. In PoP, it is ­believed that the pleats function in the pharate ­instars by allowing the preoral orifice of the ecdysing insect to open widely during a molt so that the stationary ecdysial tentorium can pass through it. The ecdysial tentorium, while much wider than the pharate preoral orifice, would be malleable and compressible after predigestion, also facilitating passage. In ACP, the organ probably serves this function also, but since it does not have distinct pleats for expansion of the preoral orifice diameter, this suggestion needs verification. As above, the organ also serves to anchor the stylet bundle loop close to the body whether it is docked in its sulcus or not, so that snagging on host plant parts or other obstructions is minimized. It would be interesting to determine whether muscles are involved in holding the organ down firmly. Snagging would impede ambulation and may even draw the loop out of its anchored position, but in fifth-instar C. mali at least, loss of anchoring is not fatal. Pollard (1970) determined the means by which they are able to restore the bundle to its docked position when it is experimentally pulled out of the labium and right stylet-holding organ and left to sway in an anterior direction. Pollard indicated that a critical feature allowing for restoration is that the right stylet-­holding organ overlaps the left. The ACP and PoP stylet-holding organs do not overlap, and restoration has not yet been demonstrated.

clypeolabral demarcation is evident (Grimaldi, 2003:332) as well as a postclypeus, anteclypeus and labrum when all three are recorded as present (Singh, 1971:297 for ACP, possibly Indian specimens). Ammar and Hall (2013) looked specifically for a possible demarcation at the acute end of this structure that might represent a minute labrum in Floridian larval ACP, but reported its absence. Such multiple choices for identifying a structure can create problems for elucidation of anatomy because, evolutionarily, sclerites can subdivide, adjacent sclerites can increase and decrease in size relative to each other, and dividing sutures can obsolesce. Therefore, Cicero et  al. (2015) used ‘rostrum’ as a generic term to simplify descriptions and avoid the uncertainty inherent in attempts to homologize. Another example of a psyllid structure with uncertain homology is the epipharynx, which is supposedly derived from the labrum or the structure underneath the labrum when both are present (Snodgrass, 1935).

2.3.3 Tentorium Directly dorsal to the rostrum is a crate-shaped endoskeletal structure consisting of two sections, ‘preoral’ and ‘postoral’ (Fig. 2.2). They are very different from each other in structure and function. It is clear that the postoral section is derived from the primitive tentorium and is therefore the modern ‘tentorium’, but the preoral section was not, as a whole, assigned a singular terminological name because it was derived from consolidation of several head structures, namely the epipharynx, hypopharynx and lateral tissue blocks. Because of the consolidation, one surface of a ­cuticular invagination can belong to one named structure while the opposing surface of the same cuticular invagination can belong to another (Fig.  2.4C). In resolution, Cicero et  al. (2015) gave generic names to each preoral endoskeletal surface of PoP. All invaginations except the esophagus and stylet loading sleeves end in a crease. Refer to ­Cicero et al. (2015) for extended details.

2.3.2 Rostrum 2.3.4 Stylets The rostrum mentioned above has been variously labeled postclypeus, anteclypeus, clypeolabrum, and, separately, clypeus and labrum when a

The adult ACP stylet base is the length inside the head (Fig. 2.3). A terminological distinction



Functional Anatomy of the Asian Citrus Psyllid

17

docking sulcus

stylet holding organ

stylet holding organ enter from right

exit from left

labial clamp

labial apex

stylet bundle

(B)

(A)

double-walled, cuticular, panelshaped invagination

hollow of panelshaped invagination* salivary pump

cuticle of lateral tissue block

cuticle of hypopharynx thinned cuticle of lateral tissue block

hollow of maxillar core

crease is anterior to this locus

maxillar ridges holster hollow* holster cuticle

crease is posterior to this locus

lumen of cibarium

invagination corridor* crease is anterior to this locus crease is posterior to this locus cuticle of epipharynx mandibular

(C)

loading sleeve hollow* loading sleeve cuticle

Fig. 2.4.  Larval and adult ACP. (A) Light micrograph of third instar larva, ventral view. Stylet bundle is out of its docking sulcus. Its configuration naturally occurs in bilateral asymmetry. It is herein annotated in dorsal view as exiting the preoral orifice from the left, and entering the labial groove from the right (n=1). Line = 100 μm. (B) Ventrofrontal view of a last instar ACP exuviae. The paired stylet holding organ serves to constrain the stylet bundle such that it maintains a looped configuration and is closely appressed to the body, minimizing possible snagging on anything passing underneath the insect during ambulation.

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J.M. Cicero

­ etween ‘proximal’ base and ‘distal’ base is used b to describe its characteristics. The stylet base is surrounded by the ‘loading sleeve’ hollow proximally (Figs 2.2 (inset) and 2.4C) and the ‘holster’ hollow distally (Fig. 2.4C). Both hollows are continuous with each other, and thence with the airspace outside the body. These two generic terms were coined to reflect their function during installation of the presumptive (new) stylets that replace ecdysial (old) stylets during a molt (Cicero et al., 2015) (Section 2.3.5). The hollow of the maxillar core (Fig. 2.4C) extends to near the preoral orifice only, but the hollow of the mandibular core extends into the shaft (Fig. 2.5A) and ends near the apical terminus. The hypodermal cells of both fill these hollows to near the preoral orifice, but thereafter in the mandibular, only dendrites continue the cellular aspect of the core hollow down the shaft. It is not known whether dendrites enter into the maxillar core hollow. Two dendrites are seen in all cross-sectional images of psyllid mandibulars including ACP (Garzo et  al., 2012), but transmission electron microscopy (TEM) processing techniques have not yet been used to bring out ultrastructural details attained for other homoptera (Wensler, 1974; Foster et  al., 1983). Infiltration of the dendrites from the CNS into the mandibular core hollow has not been tracked for any homopteran. Techniques are available for this task (Strausfeld and Miller, 1980), as well as whether they represent unipolar or bipolar neurons, and whether there are one or two dendrites on entrance. As a consequence of opisthognathy (Fig. 2.3), the stylets are widely spaced from each other at their basal termini so that muscles can attach to their loading sleeves for manipulation.

They gradually converge until the maxillars interlock inside the preoral section. In ACP and PoP, maxillar interlock involves three ridges on the dorsal half of the inner lateral aspect and two on the ventral side. The middle ridge of one stylet is the ‘ridgelet’ (Fig. 2.5A, locant 2*), shorter than the others and functioning to close the salivary canal. The ridgelet is not developed inside the head (Fig. 2.4C) and, in PoP, it does not close the salivary canal in the labium. It has not yet been determined whether the ridgelet occurs on the left or right maxillar in bilateral asymmetry with the opposing maxillar.

2.3.5  Stylet replacement Stylet replacement involves three basic processes: biogenesis; despooling; and fitting into functional positions. Figure 2.5 (B–D) shows that ACP has enough of the basic features of the PoP stylet replacement apparatus (Cicero, 2017) to assert that its processes are comparable to the PoP model. 2.3.5.1 Biogenesis Basic principles of molting assert that, going backward in time from the adult, the apical membranes of cells adhering to the walls of each stylet hollow of all intrastadial instars identify those cells as the hypodermis that secreted that stylet in each prior pharate stage. Said another way, going forward in time, the cells adhering to the walls of each stylet hollow of all intrastadial instars (except the adult) will secrete the new stylet for the next instar as well. These cells continue out of their hollow to form a hemispherical mass called the ‘end-cap

Fig. 2.4.  Continued Line = 10 μm. (C) Frontal section of an adult ACP head showing cross-sections of some endocuticular invaginations of the oral region. The outer wall of the double-walled panel is cuticle of the lateral tissue block, and its inner wall is cuticle of the hypopharynx. Its outer wall undergoes a secondary invagination to shape the holster with a very thin cuticle for flexibility during stylet manipulations. Panel and holster hollows, and others marked with (*), are continuous with each other and trace to the airspace outside the body. The double-walled panel ends in a crease anterior to the plane of this cross-section, but the holster hollow and holster cuticle continue beyond the crease as a tubular ‘loading sleeve’ that connects directly to the basal terminus of the stylet within. Similarly, the lateral epipharyngeal and hypopharyngeal cuticles of the cibarium end in a crease anterior to the plane of this cross-section also, but the mesal cuticles and lumen continue beyond the crease as a tubular pharynx and esophagus (cf. Cicero et al., 2015). Note that maxillar ridges are undeveloped inside the head. Line = 5 μm.



Functional Anatomy of the Asian Citrus Psyllid

19

expansion pleats molting space-2

molting space-1

sleeves

1 1

3 2*

dendrites in mandibular hollow

salivary canal food canal

2 3

rim of outer wind

1 1

2 2 intima crumenal hollow

hub

crumenal cuticle composition unknown crumenal cells

(A)

(B)

hub

incipient maxillar of inner wind crowded nucleus

matrix

cushion pleats tapered cytoplasm

loci of basal terminus stylet

stylet core nucleus

loading sleeve hollow

(C)

loading sleeve

(D)

Fig. 2.5.  Some pharate and stasis configurations of ACP stylet anatomy. (A) Adult stylet bundle inside the crumenal hollow. An electron transparent space, composition unknown, occurs between the clearly indicated crumenal cuticle and its cells. This space appears consistently enough in micrographs to suggest that it is not artifactual. Its transparency suggests that it may, along with the pleats, facilitate stretching during retraction of the bundle. Numbers indicate three ridges on the dorsal half, and two on the ventral half, of both maxillars. (2*) locates the ‘ridgelet’. The maxillar core does not extend this far distally. Line = 1 μm. (B) Pharate 3rd instar. Maxillar rim and partial aspect of its extension into the hub. Cross-section passes through two winds of the same maxillar. Since synthesis is ongoing at the end of the innermost wind, the outermost maxillar cross-section has elaborated further toward completion than the innermost one. Molting space-1, -2, (cf. section 2.3.5.2). Line = 1 μm. (C) Diagonal section through the basal terminus of an adult stylet. The intersection of the basal terminus cuticle and the plane of the section consists of two loci as indicated (cf. Fig. 2.3 xs). Nuclei of core cells occur in the end-cap (not shown in this section) and only shallowly into the core. They do not extend further into the core because of size constraints. As modeled, the ‘cushion’ is the cuticular intima in (B) that was secreted to line the atrium of the prior pharate stage. It gathers into a bolus of pleats where indicated. Line = 2 μm. (D) Diagonal section through the head of a pharate adult passing through a large lobate structure in the head, tentatively identified as a hub aggregate. Each of the four hub aggregates consists of characteristically elongate, ­‘tadpole’-shaped cells (crowded nuclei and elongate, tapered cytoplasm (inset)). Each aggregate is believed to be a consolidation of the bases of cells that form the hub of the atrium of each of the four independent stylet replacement apparatuses (Cicero, 2017:655, f.11A). Line = 2 μm.

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J.M. Cicero

aggregate’, somewhat resembling a golf ball on a tee. The nuclei of these cells reside in the endcap and extend somewhat into the proximal base of the hollow, but thereafter only cytoplasm continues distally into the narrower reaches (Pesson, 1951:1396; Cicero, 2017:648). The end-cap contains a matrix of tightly folded cells (Fig. 2.5C) in its interior which, in PoP at least, narrowly extends to its periphery, where it connects to another mass called the ‘companion aggregate’ (Cicero, 2017:648). This is the ‘­stasis’ or ‘intrastadial’ configuration. As modeled, on apolysis, the end-cap (and probably also the companion aggregate) ‘deconstructs’ to release the matrix, allowing it to expand into a large, flat, coiled, snail shellshaped structure called the ‘atrium’, which houses the presumptive stylet as it is secreted. A cuticular intima is secreted on expansion to line the atrial hollow (Fig. 2.5B) (see animation, Cicero et al., 2018a). In the cross-section of the ACP crumena (Fig. 2.5A), the cuticle of all four stylets is seen to be entire except for ‘sleeves’ of failed cuticle deposition at the dorsal and ventral aspects. Superficial irregularities in deposition appear elsewhere in the stylet, and are especially prominent in Bemisia tabaci (Cicero and Brown, 2016:218, f.15.3). In light of the presence of multiple hollows in Auchenorrhyncha, these authors suggested that they may be residual indications of multiple hollows in ancestral Sternorrhyncha. In addition to the end-cap aggregate and companion aggregate, two other aggregates have been characterized in PoP, the ‘rim aggregate’ and ‘hub aggregate’ (Cicero, 2017). Both are histologically distinctive, and the hub aggregate has a truncate-lobate end (Cicero, 2017:655, f.11A). This truncate-lobate end was found in ACP also (Fig. 2.5D), but correlation to that of PoP is tentative, since both rim and hub aggregates are three-dimensional structures identified from only a few cross-sections. 2.3.5.2  Despooling and fitting It is believed that the atrium is preconfigured into the snail-shell shape (animation: Cicero, 2017, supp. fig. 5). That is, in association with despooling of the stylet into its functional position, the atrium retains its coiled shape as it c­ ollapses back down into the stasis configuration to await the

next molt. Analysis is based on comparison of TEM micrographs of adults, pharate adults and last-instar larval exuviae. These fall considerably short of enabling in-depth elucidation of this complex process because, it is believed, expansion of the matrix into the atrium and contraction back into the stasis configuration are accomplished by water transport proteins (aquaporins) and would occur very rapidly. Apolysis of the atrial intima is initiated to allow for despooling. According to the model, as a stylet despools, the free intima (Fig. 2.5B) is ‘scraped’ down with it, and it accumulates into a bolus called the ‘cushion’ (Fig. 2.5C) (Wensler, 1974, pl.2, f.5cm; Cicero et  al., 2018a) in the critically significant space underneath the loading sleeve and at the inner lateral surface of the proximal base made by the outwardly projecting auricle (Fig. 2.2, inset). The walls of the adult mandibular holsters are modified such that, as the mandibulars despool through them, they are rerouted from their ventral position to a position laterolateral to the maxillar interlock, forming a ‘stylet bundle’ (Fig. 2.3). Modification of the holster walls continues anteriorly such that they then route the bundle into a 90° rotation about their long axis before exit through the preoral orifice. On exit, the bundled stylets are dorsoventral to each other and can bend along their ‘most bendable axis’ into the next structure in their pathway – the ‘crumena’ (see Section 2.3.6). Without this rotation, the bundle would bend contrary to the ridge-groove interlock, resulting in poor sliding of stylets against each other during drilling, and possibly causing the maxillars to disengage from each other.

2.3.6 Crumena Notable among Sternorrhyncha is the presence of an inverted (endocuticular) sac or pouch, the ‘crumena’, that retains part of the total length of the stylet bundle in a looped configuration in its interior. This ‘unused’ portion, as it is sometimes referred to, is actually highly functional. In PoP and ACP, the crumena reroutes the bundle from a posterior direction to a ventral direction on issuance from the preoral orifice so that direct (perpendicular) management of pressure



Functional Anatomy of the Asian Citrus Psyllid

during drilling can be exerted on the ‘unused portion’ (Cicero et al., 2015; Alba-Tercedor et al., 2017:275–6, f.5b, 6). The crumena may also be involved in forward thrust during penetration by actively contracting, but this has not yet been determined. Two anatomical features appear to facilitate passive expansion and contraction: pleats; and an electron transparent space between cuticle and cells (Fig. 2.5A). This latter space resembles a molting space (cf. Fig. 2.7). Weber (1929:93–95) figured larval C. mali as having both a crumena and a rostral sulcus (Section 2.3.1). As sketched, the stylet bundle bypasses the crumena and enters the labial groove directly from the rostral sulcus. This oddity has yet to be confirmed, since it is not known how the bundle is routed into these adult and larval loops on ecdysis.

2.3.7 Labium Garzo et  al. (2012) observed three labial segments in adult ACP, while Ammar and Hall (2013) observed only two in the larva. The apex of the adult labium has a short, ventral slit which, in association with the dorsal groove, divides the apex into two valves (Garzo et  al. 2012:81, f.1B; Fig. 2.6A). Pleated cuticles were observed within (Ammar and Hall, 2013:265, f.1D) that may correlate to the cuticle that wraps around the stylet bundle apical terminus when the latter is fully retracted to that level (Garzo et  al., 2012:84, f.3A). This wrap may function with the valves in facilitating angular flexing of the exiting bundle.

2.3.8  Salivary glands The salivary glands (also called labial glands) of ACP consist of one pair of primary salivary glands and one pair of accessory salivary glands tethered to an ectodermal ducal system (Cicero et  al., 2009). In PoP, two other paired organs, one smooth walled and the other rough, are located next to the salivary glands (Cicero et  al., 2017:42, f.4C). One or both may be corpora, but they remain unidentified. These are not yet known to occur in ACP, but when present, they may complicate identification of the salivary

21

glands by TEM if they are also made up of secretory cells. Four different sections are recognized in the primary ACP salivary gland: three apical and one basal (Cicero et  al., 2009:658). In one of the three, cells are filled with secretory storage spherules that are a highly organized spherically or cylindrically concentric array of densely packed, squamose, bead-shaped subunits. In a second of the three, the spherules appear to be partially ­depleted, and in the third the spherules are ­electron transparent. The basal section is uncharacterized. Several important issues surfaced with first analysis of this information. The first two configurations suggest the possibility that storage is tapped from one section while being ­recharged in another. It might also be that the ­different cell profiles correspond to homologous or analogous acini of a branched configuration (Wayadande et  al., 1997) that may have been primitive to the psyllid evolutionary line. In this case, the ‘partially depleted’ spherules may actually represent different salivary ingredients. Ammar et  al., (2017:12, f.3D) discovered spherules in ACP that had a similar appearance to the first of the three described above but were coalesced. Also striking in their study were the different patterns of unit packing. At least one is suggestive of widely concentric radial packing, others are suggestive of narrowly concentric radial packing, and still others of parallel packing. If concentric and parallel arrays are different cross-sections of the same packing configuration, then the crystalline structure can be interpreted as ‘stacked’ (Fig. 2.6C). The large dark staining areas of certain spherules in Ammar et  al. (2017), Brown et  al. (2016:410, f.28.7) and Fig. 2.6C, and the small punctate areas in Fig. 2.6D are interpreted as representing native biochemistry with strong affinity for post-stain. 2.3.8.1  Salivary gland ducal system The known ducal system of ACP consists of a salivary pump and an afferent, or median, duct which branches into two lateral ducts. The presumed function of the first appliance in pumping saliva has not been demonstrated, and other functions are possible (Cicero and Brown, 2012). An efferent duct is not yet identified in psyllids. The lateral ducts of ACP do not branch further to service separate primary and accessory glands

22

J.M. Cicero

microvilli? corona sinuosa?

lumen intima

slit outer lateral surface of one mandibular of exiting stylet bundle

(B)

(A) stacked

(C)

(D)

Fig. 2.6.  Aspects of adult and larval ACP. (A) Adult stylet bundle exiting through the labial apex, ventral view. Note that mandibulars and maxillars are dorsoventrally opposed to each other as they exit, indicating that the stylet bundle has rotated 90° (cf. Fig. 2.3). Line = 10 μm. (B) Adult salivary duct. Radially arranged membranes may actually be laminar rather than digitate (cf. section 2.3.8, Salivary gland ducal system). Line = 0.5 μm. (C-D) Salivary gland spherules of a third instar larva infected with HLB. (C) A spherule with an apparent ‘stacked’ configuration. Other spherules present large electron opaque areas. (D) Spherules with small, punctate, electron opaque areas. Both opaque areas in (C) and (D) appear to have high post-stain specificity. Subunits are not organized into stacked or concentric arrays. Lines = 1 μm.

as is the case in B. tabaci (Cicero and Brown, 2012). Instead, each lateral duct, along with its intima, enters directly into its primary gland as a ductule, and the accessory gland is an eccentric appendage of the primary gland. The intima is a homogeneous wall surrounding the lumen and is thought to be cuticular in construction. Dissections of exuviae have not yet been performed to determine if this putative cuticle can be recovered; it is possible that the intima is continued

internally by a non-cuticular substance (Brown et al., 2016:409 and see below). Both the duct and ductule are surrounded by a complex array of laminar pleats (Fig. 2.6B). These were variously identified by TEM as microvilli, ‘outfoldings’ or ‘infoldings’ of cell apical membranes by contemporary authors (e.g. Gildow, 1982; Ammar, 1986; Cicero and Brown, 2012). However, Moericke and Wohlfarth-Bottermann (1960:41) studied the Myzus persicae (Sulzer)



Functional Anatomy of the Asian Citrus Psyllid

salivary gland ductules by TEM at much higher resolution. Their conclusions, compiled into a 3D sketch, bring these identifications into question for aphids and possibly also psyllids (e.g. Brown et  al., 2016; Ammar et  al., 2017) and other homopterans as discussed below. Moericke and Wohlfarth-Bottermann (1960) recognized that there are no circular cross-­ sectional renderings of the pleats that would be evincive of microvillae. This and their serial sections conclusively show that the pleats are actually cellular laminae alternating with intercellular space. They are radially oriented relative to the canal’s axis, and, in contrast to microvillae, longitudinally elongate in association with the canal’s length. The authors appropriately named the array the ‘corona sinuosa’. Further, their high magnifications of the accessory gland indicate that the pleats (‘coronal folds’ or ‘microsinuses’) do not contact the canal (1960:41, Kanalnaher Interzellularraum zwischen Sekretkanalwandung und Faltenkranz). That is, the canal, with its homogeneous wall (intima), is suspended in intercellular space. Plaques (Locke and Huie, 1979) were not indicated. The authors’ interpretations imply that this intercellular space may also traffic fluids in addition to the ductule lumen. Lastly, in contrast to the homogeneous ducal intima elsewhere, the luminal wall near the accessory gland apex consists of five double layers of membranes from which the coronal folds apparently elaborate. Four can be seen in Gildow (1982:1291). The ACP ductule exhibits an exosomal transport system about the secretory cell storage spherules (Cicero et al., 2009:659) and the ductule lumina (Brown et al., 2016:410). It appears that secretion is stored not only in the spherules but also in the ductule intima and the corona sinuosa for exosomal trafficking along lateral membranes. These interpretations do not oppose those of Moericke and Wohlfarth-Bottermann. The volume of literature on exosomal transport is burgeoning (Vlassov et al., 2012), and elucidation of the actual cytological configuration of the apical cell membrane complex in ACP, as it occurs between the secretory cell cytosol and the luminal intima, is imperative. Given the complexity discussed above, it is clear that the basic concept of bacterial pathogen trafficking through a ‘loadable’ section of the ductule (Cicero and Brown, 2012) and thence along the flow route of aqueous secretions is woefully

23

inadequate. In fact, one of the biggest gaps in understanding ACP salivary glands, especially in the context of trafficking (see Vector Relations in Chapter 8), is the cytology involved in passage of Liberibacter across barriers between the hemolymph and the lumen of the ductule: specifically, across the gland basal lamina, then, as shown for PoP (Cicero et al., 2017:47) along secretory cell pericellular space, then through the ductule cells, then through the laminar array, then through the intima.

2.4  Alimentary Canal Technically, the alimentary canal begins with the primitive mouth, but only putative demarcations of a mouth are reported for psyllids: opposed tanidia (Cicero et al., 2015:775, f.10Ab) and the location of stylet basal termini (2015:758). Because of this and other indications of evolutionary complexation of the oral region (Section 2.3), new and alternative terminologies have been proposed for simplicity and flexibility. Examples are: extension, by definition, of the anterior end of the alimentary canal to occur at the preoral orifice (Ammar et al., 1994), reference to the distinct, consecutive oral region alimentary lumina as the ‘primary oral invagination’ (Cicero et  al., 2015) and itemization of each cuticular surface of the oral region (Harris et  al., 1996:291, t.27.1; Cicero et al., 2015). Whether any will be vetted in the long term is an important question in light of the need to apply proteomics and transcriptomics to specific anatomical loci. This need critically outweighs the need to maintain a homologically based terminology. The esophagus (Section 2.4.1), and, to a much greater extent, the filter chamber and midgut loop (Section 2.4.2), have been given paramount attention in ACP because of their role in disease transmission (see Vector Relations in Chapter 8), but the hindgut has been mostly overlooked. The esophagus and hindgut have an identifying cuticular intima because they are an invaginated aspect of the exoskeleton, but the midgut, its appendages (Section 2.4.3) and filter chamber do not. When pressed against each other, some of the components of these latter three may be difficult to identify in cross-section, because they have not been distinguished cytologically. Without

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J.M. Cicero

this criterion, relative differences in girth are observable, but unreliable as identifiers at different cross-sectional depths, especially for components that transition gradually from one to another. When extirpated PoP and ACP alimentary canals are allowed to float freely in buffer, they take on a characteristically ‘relaxed’ habitus wherein the midgut arm (see Fig. 2.8, locant 3) assumes a horizontal attitude before entering the filter chamber, and the outer hindgut (locant 4) assumes a horizontal attitude on exit from the filter chamber.

molting space (presumptive lumen)

presumptive intima

ecdysial intima ecdysial lumen discontinuities

2.4.1 Esophagus Epicuticle is absent on the esophageal intima among aphids (Ponsen, 1990), but the figures cited in this section indicate that it is present in ACP. One of the distinct characteristics of the PoP and ACP esophagus is the buckled appearance of its intima when imaged by TEM. Buckling of the intima of intrastadial specimens (Cicero et al., 2017:46, f.8D) can be interpreted as being caused by the thinness or low-density areas, and apparently discontinuous areas, of the epicuticle. Buckling of the ecdysial (old) intima of intrapharate specimens (Ammar et  al., 2017:14, f.5A) can be interpreted as being caused by thin or low-density areas of the epicuticle also, as well as predigestion (Fig. 2.7). Buckling of the presumptive (new) intima of intrapharate specimens, weakly seen in Fig. 2.7 and especially strong in Ammar et al. (2017:14, f.5A), can be interpreted as being caused by design: as with all other cuticles, the presumptive esophagus needs to expand to a wider diameter after ecdysis. The weak buckling in Fig. 2.7 suggests that secretion of presumptive intima was incipient when the specimen was fixed.

2.4.2  Filter chamber and midgut loop Evolutionary changes to primitive alimentary ­canals, from which filter chambers of certain homopteran clades were derived, involved anteriorization of the posterior end of the midgut, ­together with the anterior end of the hindgut, to meet with, and complex with, the anterior end of the midgut. In ACP, the complex is sheathed, and

Fig. 2.7.  Pharate 3rd instar esophagus. Several discontinuities in the epicuticle of the ecdysial intima, as well as its predigestion, are believed to be associated with the crenulate configuration of the ecdysial lumen. When emergence occurs and the ecdysial cuticles are abandoned as exuviae, the molting space becomes the functional lumen of the next instar. Line = 1 μm.

connected to the rest of the canal in four places (Fig. 2.8): (1) entrance of the esophagus; (2) exit to the ‘second ventriculus’ (V2); (3) re-entry of the ‘midgut arm’; and (4) final exit to the outer hindgut. The stretch of canal from (1) to (2) is the ‘inner midgut’ (V1), and the stretch from exit (2) to re-entry (3) is called the ‘midgut loop’ or V2+V3. The alimentary tract inside the filter chamber of PoP and ACP consists of two sections that are connate to each other and have luminal flows moving in antiparallel (also called ‘counter-­ current’ by Hamilton (2015)) directions (see animation, Cicero et al., 2018b). One section (V1) extends from entrance (1) to first exit (2), and the other from re-entry (3) to final exit (4). Excess water ingested through (1) is filtered through this ‘zone of contact’ and disposed directly into the stretch from (3) to (4) so that only retentate continues through (2) to V2. The section from entrance to first exit, (1) to (2), can reasonably be identified as derived from the primitive midgut using orthodox homological comparisons to other taxa. However, cytological technique has not been deployed to characterize the section of the tract between re-entry and final exit, (3) to (4). As no demarcation has been identified in psyllids



Functional Anatomy of the Asian Citrus Psyllid

25

esophagus

third ventriculus (V3) apparently seamless transition

midgut appendage IV

1

filter chamber (first ventriculus (V1 ) is inside)

outer hindgut 4

2

3

second ventriculus (V2 ) V2 / V3 transitional region midgut appendage I

midgut appendages II and III

Fig. 2.8.  Habitus of a relaxed, extirpated, free-floating adult ACP gut, fixed and processed for SEM. Externally, the connection between esophagus and filter chamber is apparently seamless. Arrows indicate direction of food flow. The esophagus enters the filter chamber vertically (1) to become the inner midgut (V1). V1 exits from inside the filter chamber (2), transitioning into a much larger V2. V2 gradually narrows in girth, transitioning into V3. V3 ascends back to the filter chamber, transitioning into the ‘midgut arm’ which reenters (3). V2 + V3 + ‘midgut arm’ is the midgut loop. Once inside again, it behaves as a ‘functional hindgut’ because it adheres to V1 in a counter-current direction and accepts filtrate from it. It then exits horizontally, e.g. at a 90° angle to the esophagus, as the outer hindgut (the skewed rectangle is meant to represent the horizontal plane tangent to the filter chamber at the exit point of the inner hindgut). It then continues posteriorly to the anus (4). The midgut appendages, short and wide in girth (I to IV, anterior to posterior), occur on the midgut loop. II and III are characteristically closely set in ACP and PoP. The four are arguably considered Malpighian tubules by some authors (cf. Section 2.4.3). Line = 100 μm.

that would indicate the division between midgut and hindgut, the section is provisionally and functionally termed ‘inner hindgut’ since it accepts excess water from V1 and waste from V3 (Cicero et  al., 2009:652). The section is also termed the ‘posterior midgut’ in keeping with classical authors who base their identification on configurations seen in other families. However, it should be noted that identification of Malpighian tubules, an identifier of the midgut/

hindgut interface, is, for psyllids, in controversy (Section 2.4.3). Further, among aphids, some or all of the hindgut is endodermal (Ponsen, 1990). V2 is a large dilation of the outer midgut and probably serves as a canteen of condensed foodstuff from the processing of imbibed sap by the filter chamber. Without it, the insect would have to subsist on the relatively low ongoing volume of fresh foodstuff from filtration and the relatively low volume moving through V3.

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J.M. Cicero

2.4.3  Excretory system

2.5  Reproductive System

The most conspicuous feature of the ACP excretory system is the ‘curtaining’ effect of the long plumb lines of waxy secretions from numerous larvae on the same host plant branch. These are generated through a complex array of circumanal pores and slits, elucidated by Ammar et al. (2013). The historical literature on the germ layer origin of Malpighian tubules is fraught with disagreement. Some early authors concluded that they are endodermal, some ectodermal (Snodgrass, 1935). Snodgrass discussed examples of species wherein tubule attachment is too close to the midgut–hindgut interface to determine which section sponsors them. Goodchild (1966) asserted, without data, that the tubules open primitively into the extreme posterior end of the midgut in Hemiptera, and are absent in Aphidoidea. In ACP and PoP, there are only four tubular appendages spaced out along the midgut loop (Fig. 2.8). These are very short and of wide girth in contrast to those of related families that sweep the hemolymph for waste. Therefore, their identification as Malpighian (Brittain, 1923 for C. mali; Ammar et al., 2011 for ACP) has recently been brought into question (Brown et  al., 2016). However, advancements in genetics have broadened the range of possible explanations beyond the generally accepted principles of comparative morphological homology. It is now conceivable that in these psyllids the most parsimonious justification for identifying the psyllid tubules as Malpighian is that genes for excretory tubules are activated in mesal midgut cells regardless of whether they were primitively located at the distal midgut or the distal hindgut. As a first step in reconciling this apparent autapomorphy, the metabolic function of these ACP midgut appendages needs to be demonstrated. This can be approached using classical cytochemistry (Wigglesworth and Salpeter, 1962) as well as assays for hemolymphic waste, performed during, and directly after, a feeding session. Correlation of the ACP midgut transcriptome to the Drosophila and Aphis Malpighian tubule gene set (Jing et al., 2015), and TEM colloidal gold in situ hybridization upon ultrathin sections of ACP appendages could also be revealing.

As of this writing, male and female ACP genitalia have not been satisfactorily detailed. A sizeable ­literature has been built from studies of other ­genera, but, as with other anatomical systems, ­recourse to them is handicapped by their crude sketches and confusing interpretations. The male ACP reproductive system is currently in the process of comprehensive, monographic review and is therefore omitted in this chapter. Microphotographs and descriptions of Dossi (2008) and ­Carter (1961) offer potential characters for sex ­determination of late-instar psyllid larvae.

2.5.1 Female The female reproductive system consists of mesodermal ovaries grafted to an ectodermal ­luminal complex that provides nutritive glandular secretions (the accessory glands), reservoir space (the spermatheca), egg-laying accommodation (colleterial glands) and interluminal transport (vagina, ducts and ductules) for the media of these organs, as well as sperm and eggs. The above order of these organs, from anterior to posterior in terms of ducal flow, has been carefully laid out for ACP by Dossi and ­Consoli (2014). ACP, and apparently all other psyllids investigated (Buning, 1985; Dossi and Consoli, 2014), have meroistic telotrophic ovarioles arranged in a rosette at the apex of each oviduct. In simplest terms, ‘meroistic’ refers to the presence of nurse cells and ‘telotrophic’ refers to a configuration wherein nurse cells are chambered in a tropharium, apical to, and separate from, the germ cells chambered in a vitellarium. Similarities and differences between D. citri ovaries and those of other psyllids, the broader Hemiptera and members of other Orders have been detailed by Dossi and Consoli (2014). Most notable were the large number of ovarioles, the variation in location of the accessory glands, and reported absence of microfilaments that are known to be present in the nutritive cords of other psyllids. Also, ‘nuage-like’ electron-dense agglomerates were located in a perinuclear arrangement, although not illustrated with ­ micrographs. Nuage are perinuclear structures



Functional Anatomy of the Asian Citrus Psyllid

identified in Drosophila to function as repressors of selfish genes (Lim and Kai, 2007). An intima lines the lumen of the oviducts and spermatheca.

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Some of this ectoderm is reported to be surrounded by mesoderm that is grafted on to its exterior (Dossi and Consoli, 2014).

References Alba-Tercedor, J., Hunter, W.B., Cicero, J.M., Sainz-Bariain, M. and Brown, S.J. (2017) Use of micro-CT to elucidate details of the anatomy and feeding of the Asian citrus psyllid Diaphorina citri Kuwayama, 1908 (Insecta: Hemiptera, Liviidae). In: Bruker Micro-CT Users Meeting. Bruker Corporation, Kontich, Belgium, pp. 270-285. Ammar, E.-D. (1986) Ultrastructure of the salivary glands of the planthopper, Peregrinus maidis (Ashmead) (Homoptera: Delphacidae). International Journal of Insect Morphology and Embryology 15(5/6), 417– 428. doi: 10.1016/0020-7322(86)90034-6. Ammar, E.-D. and Hall, D.G. (2013) Retracted stylets in nymphs of the Asian citrus psyllid are held externally against the clypeus by a special paired organ not found in the adults. Florida Entomologist 96(1), 264–267. doi: 10.1653/024.096.0142. Ammar, E.-D., Järlfors, U. and Pirone, T.P. (1994) Association of potyvirus helper component protein with virions and the cuticle lining the maxillary food canal and foregut of an aphid vector. Phytopathology 84, 1054–1060. doi: 10.1094/Phyto-84-1054. Ammar, E.-D., Shatters, R.G. Jr, Lynch, C. and Hall, D.G. (2011) Detection and relative titer of Candidatus Liberibacter asiaticus in the salivary glands and alimentary canal of Diaphorina citri (Hemiptera: Psyllidae) vector of Citrus huanglongbing disease. Annals of the Entomological Society of America 104(3), 526–533. doi: 10.1603/an10134. Ammar, E.-D., Alessandro, R., Shatters, R.G. and Hall, D.G. (2013) Behavioral, ultrastructural and chemical studies on the honeydew and waxy secretions by nymphs and adults of the Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae). PLOS ONE 8(6), e64938. doi: 10.1371/journal.pone.0064938. Ammar, E.-D., Hall, D.G. and Shatters, R.G. Jr (2017) Ultrastructure of the salivary glands, alimentary canal and bacteria-like organisms in the Asian citrus psyllid, vector of citrus huanglongbing disease bacteria. Journal of Microscopy and Ultrastructure 5, 9–20. doi: 10.1016/j.jmau.2016.01.005. Beutel, R.G. and Haas, A. (1998) Larval head morphology of Hydroscapha natans (Coleoptera, Myxophaga) with reference to miniaturization and the systematic position of Hydroscaphidae. Zoomorphology 118, 103–116. doi: 10.1007/s004350050061. Brittain, W.H. (1923) The morphology and synonymy of Psyllia mali Schmidberger. Proceedings of the Acadian Entomological Society 7, 23–42. Brown, J.K., Cicero, J.M. and Fisher, T.W. (2016) Psyllid-transmitted Candidatus Liberibacter species infecting citrus and solanaceous hosts. In: Brown, J.K. (ed.) Vector-mediated Transmission of Plant Pathogens. American Phytopathological Society, St Paul, Minnesota, pp. 399–422. doi: 10.1094/9780890545355.028. Buning, J. (1985) Morphology, ultrastructure, and germ cell cluster formation in ovarioles of aphids. Journal of Morphology 186, 209–221. doi: 10.1002/jmor.1051860206. Carter, R.D. (1961) Distinguishing sexes in nymphs of the tomato psyllid, Paratrioza cockerelli. Annals of the Entomological Society of America 54, 464–465. doi: 10.1093/aesa/54.3.464. Cicero, J.M. (2017) Stylet biogenesis in Bactericera cockerelli. Arthropod Structure and Development 46, 644–661. doi: 10.1016/j.asd.2016.12.007. Cicero, J.M. and Brown, J.K. (2012) Ultrastructural studies of the salivary duct system in the whitefly vector Bemisia tabaci (Aleyrodidae: Hemiptera). Annals of the Entomological Society of America 105(5), 701–717. doi: 10.1603/AN12030. Cicero, J.M. and Brown, J.K. (2016) Bemisia tabaci-mediated transmission of Begomoviruses: History and anatomical, biological, and cellular interactions. In: Brown, J.K. (ed.) Vector-mediated Transmission of Plant Pathogens. American Phytopathological Society, St Paul, Minnesota, pp. 211–230. doi: 10.1094/9780890545355.015. Cicero, J.M., Brown, J.K., Roberts, P.D. and Stansly, P.A. (2009) The digestive system of Diaphorina citri and Bactericera cockerelli (Hemiptera: Psyllidae). Annals of the Entomological Society of America 102(4), 650–665. doi: 10.1603/008.102.0410.

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Cicero, J.M., Stansly, P.A. and Brown, J.K. (2015) Functional anatomy of the oral region of the potato p ­ syllid (Hemiptera: Psylloidea: Triozidae). Annals of the Entomological Society of America 108(5), 743–761. doi: 10.1093/aesa/sav059. Cicero, J.M., Fisher, T.W., Qureshi, J.A., Stansly, P.A. and Brown, J.K. (2017) Colonization and intrusive invasion of the potato psyllid by ‘Candidatus Liberibacter solanacearum’. Phytopathology 107, 36–49. doi: 10.1094/PHYTO-03-16-0149-R. Cicero, J.M., Alba-Tercedor, J., Hunter, W.B., Cano, L.M., Saha, S. et  al. (2018a) An animated correspondence of Asian citrus psyllid stylets to the model for biogenesis of potato psyllid stylets. Available at: https://citrusgreening.org/microtomography/cicero_stylet_2018 (accessed 28 June 2019). Cicero, J.M., Hunter, W.B., Cano, L.M., Saha, S., Mueller, L.A. et al. (2018b) An animated detailing of the alimentary canal of the Asian citrus psyllid, with special reference to the configuration and function of the filter chamber. Available at: https://citrusgreening.org/microtomography/cicero_alimentarycanal_ 2018 (accessed 28 June 2019). Dossi, F.C.A. (2008) Ultraestrutura do aparelho reprodutor feminino e mecanismos de transmissão transovariana de endossimbiontes de Diaphorina citri Kuwayama, 1908 (Hemiptera: Psyllidae). MSc dissertation. University of São Paulo, Brazil. Dossi, F.C.A. and Consoli, F.L. (2014) Gross morphology and ultrastructure of the female reproductive system of Diaphorina citri (Hemiptera: Liviidae). Zoologia 31(2), 162–169. doi: 10.1590/S198446702014000200007. Foster, S., Goodman, L.J. and Duckett, J.G. (1983) Sensory receptors associated with the stylets and ­cibarium of the rice brown planthopper, Nilapavarta lugens. Cell and Tissue Research 232, 111–119. doi: 10.1007/bf00222377. Friedrich, F., Matsumura, Y., Pohl, H., Bai, M., Hörnschemeyer, T. et al. (2014) Insect morphology in the age of phylogenomics: innovative techniques and its future role in systematics. Entomological Science 17, 1–24. doi: 10.1111/ens.12053. Garzo, E., Bonani, J.P., Lopes, J.R.S. and Fereres, A. (2012) Morphological description of the mouthparts of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae). Arthropod Structure and Development 41, 79–86. doi: 10.1016/j.asd.2011.07.005. Gildow, F.E. (1982) Coated-vesicle transport of luteoviruses through salivary glands of Myzus persicae. Phytopathology 72, 1289–1296. doi: 10.1094/phyto-72-1289. Goodchild, J.P. (1966) Evolution of the alimentary canal in the Hemiptera. Biological Reviews (­Cambridge) 41, 97–140. doi: 10.1111/j.1469-185X.1966.tb01540.x. Grimaldi, D.A. (2003) First amber fossils of the extinct family Protopsyllidiidae, and their phylogenetic ­significance among Hemiptera. Insect Systematics and Evolution 34(3), 329–344. doi: 10.1163/ 187631203788964746. Gupta, A.P. (1994) Insect anatomy – morphology: Quo vadis? Annals of the Entomological Society of America 87(2), 147–156. doi: 10.1093/aesa/87.2.147. Hamilton, K.G.A. (2015) Anatomy: the poor cousin of morphology? American Entomologist 61(2), 88–95. doi: 10.1093/ae/tmv003. Harris, K.F., Pesic-Van Esbroeck, Z. and Duffus, J.E. (1996) Anatomy of a virus vector. In: Stansly, P.A. and Naranjo, S.E. (eds) Bemisia: Bionomics and Management of a Global Pest. Springer, Dordrecht, The Netherlands, pp. 289–318. Hennig, W. (1981) Insect Phylogeny. John Wiley & Sons, Chichester, England. Jing, X., White, T.A., Yang, X. and Douglas, A.E. (2015) The molecular correlates of organ loss: the case of insect Malpighian tubules. Biology Letters 11, 0154. doi: 10.1098/rsbl.2015.0154. Karnovsky, M.J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron-­ microscopy. Journal of Cell Biology 27 (abstract #270), 137A–138A. Lim, A.K. and Kai, T. (2007) Unique germ-line organelle, nuage, functions to repress selfish genetic elements in Drosophila melanogaster. Proceedings of the National Academy of Sciences USA 104(16), 6714–6719. doi: 10.1073_pnas.0701920104. Locke, M. and Huie, P. (1979) Apolysis and the turnover of plasma membrane plaques during cuticle ­formation in an insect. Tissue and Cell 11(2), 277–291. doi: 10.1016/0040-8166(79)90042-9. Moericke, V. and Wohlfarth-Bottermann, K.E. (1960) Zur funktionellen morphologie der speicheldrüsen von Homopteren IV. Zeitschrift für Zellforschung und Mikroskopische Anatomie 53, 25–49. doi: 10.1007/bf00319340. O’Malley, M.A., Wideman, J.G. and Ruiz-Trillo, I. (2016) Losing complexity: The role of simplification in macroevolution. Trends in Ecology and Evolution 31(8), 608-621. doi: 10.1016/j.tree.2016.04.004.



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Palucka, T. (2002) Overview of electron microscopy. History of recent science and technology. Available at: http://authors.library.caltech.edu/5456/1/hrst.mit.edu/hrs/materials/public/ElectronMicroscope/ EM_HistOverview.htm (accessed 28 June 2019). Parsons, M.C. (1974) The morphology and possible origin of the hemipteran loral lobes. Canadian Journal of Zoology 52, 189–202. https://doi.org/10.1139/z74-023. Pesson, P. (1951) Ordre des Homopteres (Homoptera Leach, 1815). In: Grasse, P. (ed.) Traité de Zoologie, Tome X: anatomie, systématique, biologie. 2: Insectes supériores. Masson, Paris, pp. 1390–1656. Pollard, D.G. (1970) The mechanism of stylet movement in Psylla mali Schmidberger (Homoptera: Psyllidae). Zoological Journal of the Linnean Society 49, 295–307. doi: 10.1111/j.1096-3642.1970.tb00743.x. Ponsen, M.B. (1990) Phylogenetic implications of the structure of the alimentary tract of the Aphidoidea. Part II. The Aphis group. Wageningen Agricultural University Papers 90(4), 22–52. Randolf, S., Zimmermann, D. and Aspöck, U. (2017) Head anatomy of adult Coniopteryx pygmaea Enderlein, 1906: Effects of miniaturization and the systematic position of Coniopterygidae (Insecta: Neuroptera). Arthropod Structure and Development 46, 304–322. doi: 10.1016/j.asd.2016.12.004. Singh, S. (1971) Morphology of the head of Homoptera. Research Bulletin (N.S.) of the Panjab University 22, 261–316. Snodgrass, R.E. (1935) Principles of Insect Morphology. McGraw-Hill, New York. Spooner, C.S. (1938) The phylogeny of the Hemiptera based on a study of the head capsule. Illinois Biological Monographs 16(3), 1–102. doi: 10.5962/bhl.title.50343. Strausfeld, N.J. and Miller, T.A. (1980) Neuroanatomical Techniques. Insect Nervous System. Springer Verlag, New York. doi: 10.1007/978-1-4612-6018-9. Vlassov, A.V., Magdaleno, S., Setterquist, R. and Conrad, R. (2012) Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochimica et Biophysica Acta 1820, 940–948. doi: 10.1016/j.bbagen.2012.03.017. Wayadande, A.C., Baker, G.R. and Fletcher, J. (1997) Comparative ultrastructure of the salivary glands of two phytopathogen vectors, the beet leafhopper, Circulifer tenellus (Baker), and the corn leafhopper, Dalbulus maidis DeLong and Wolcott (Homoptera: Cicadellidae). International Journal of Insect Morphology and Embryology 26(2), 113–120. doi: 10.1016/s0020-7322(97)00009-3. Weber, H. (1929) Kopf und thorax von Psylla mali Schmidb. (Hemiptera-Homoptera). Eine morphogenetische studie. Zeitschrift für Morphologie und Ökologie der Tiere 14, 59–165. doi: 10.1007/bf00419345. Wensler, R.J.D. (1974) Sensory innervation monitoring movement and position in the mandibular stylets of the aphid, Brevicoryne brassicae. Journal of Morphology 143, 349–364. doi: 10.1002/jmor.1051430307. Wigglesworth, V.B. and Salpeter, M.M. (1962) Histology of the Malpighian tubules in Rhodnius prolixus Stal (Hemiptera). Journal of Insect Physiology 8, 229–307. doi: 10.1016/0022-1910(62)90033-1. Yoder, M.J., Miko, I., Seltmann, K.C., Bertone, M.A. and Deans, A.R. (2010) A gross anatomy ontology for Hymenoptera. PLOS ONE 5(12), e15991. doi: 10.1371/journal.pone.0015991. Zalokar, M. and Erk, I. (1977) Phase-partition fixation and staining of Drosophila eggs. Stain Technology 52(2), 89–95.

3 

Mating Behavior of the Asian Citrus Psyllid

Richard W. Mankin1* and Barukh Rohde2 US Department of Agriculture, Agricultural Research Service, Center for Medical, Agricultural, and Veterinary Entomology, Gainesville, Florida, USA; 2 Department of Electrical and Computer Engineering, University of Florida, Gainesville, Florida, USA 1

Many aspects of the mating behavior of Diaphorina citri Kuwayama (Hemiptera: Liviidae), the Asian citrus psyllid (ACP) are shared by other members of the Psylloidea. Adults are reproductively mature within about 2 days post-eclosion, and both sexes mate multiple times during their lifetime. Typically, courtship is mediated by short-range, substrate-­borne vibrational communication and semiochemicals. In citrus orchards, D. citri courtship is facilitated by host-seeking and foraging behavior, as both sexes are attracted to green and yellow colors, as well as to volatiles of young flush shoots on the host tree, and short-range communication is sufficient for finding mates in aggregations that develop soon after the flush opens. Courtship behavior includes a series of duets, in which a searching male produces vibrational calls that elicit rapid replies from receptive females, enabling him to focus on willing partners. Both sexes produce vibrational communication signals by extending and fanning their wings while their legs hold on to the plant. The signal is transmitted to the host plant structures and then detected by vibration-­sensitive, chordotonal organs in the legs of the receiving conspecific. During the duetting bouts, male D. citri call intermittently, with an interval of 9 ± 1.4 s (mean ± standard error) between calls, and females reply within 0.95 ± 0.09 s. Males

produce signals ranging approximately 150– 500 ms in duration, and females 331–680 ms. The spectra of communication signals produced by D. citri have prominent frequencies that are multiples (harmonics) of the 170–250 Hz wingbeat frequency, and both sexes respond behaviorally to synthetic signals containing three or more wingbeat harmonics. When the male finds the female, he moves alongside with their heads pointing in the same direction and grasps her with his adjacent legs, bringing his abdomen from underneath to meet the opening of her genital segment. They remain in copulation for about 48 min. Dispersal and mating behavior of D. citri is influenced by abiotic factors including light, temperature, storms and barometric pressure, and by biotic factors including host plant flush, host plant structure, aggregation behaviors and learning behaviors. Opportunities exist to co-opt D. citri mating behavior for purposes of detecting and managing populations, enabling reductions in the incidence and spread of the bacteria causing huanglongbing, a devastating disease of citrus. This chapter describes details of what is currently known about D. citri mating behavior and how such knowledge has been applied in development of methods that apply vibrational communication to disrupt mating or trap males.

*  Email: [email protected]

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Mating Behavior of the Asian Citrus Psyllid 31

3.1 Introduction Diaphorina citri Kuwayama (Hemiptera: Liviidae) native to India (Hollis, 1987), initially was reported as a significant psylloid pest of Citrus spp. in China, Taiwan, Japan and the Philippines in the early 20th century (Crawford, 1912); however, little was reported about its ecology, life cycle and mating behavior (Husain and Nath, 1927; Catling, 1970; Pande, 1971) until it became important as a worldwide v ­ ector of huanglongbing (HLB), a devastating bacterial disease in citrus orchards (Halbert and M ­ anjunath, 2004; Gottwald et  al., 2007). The bacterium Candidatus Liberibacter spp. (Alphaproteobacteria) resides in symbiotic bacteriomes within the D. citri hemocoel that also contain nutritional and defensive symbionts in mutually i­ndispensable associations, all of which are transmitted transovarially (Dan et al., 2017). Economic damage caused by HLB has stimulated interest in development of knowledge about D. citri biology and mating behavior that could be used for an integrated, multidisciplinary approach to management of D. citri and thereby HLB (Aubert, 1990; Grafton-­ Cardwell et  al., 2013; Hall et  al., 2013), as has ­occurred also with other economically important insect pests (Mankin, 2012; Benelli et  al., 2014; Takanashi et al., 2019). In this chapter, we focus on aspects of mating behavior that can be co-opted to reduce D. citri populations in citrus and other hosts in the Aurantioideae subfamily of ­Rutaceae (Halbert and Manjunath, 2004; Hall et al., 2017). The life stages of D. citri on rough lemon Citrus jambhiri Lush, sour orange C. aurantium L., grapefruit C. paradisi Macfadyen, and orange jessamine Murraya paniculata (L.) Jack were described by Tsai and Liu (2000). These and other host species have a range of different physical and structural characteristics that, as discussed further below, strongly affect vibrational signal amplitudes and the efficacy of mate-seeking behaviors (Cocroft et al., 2006; Mankin et al., 2018).

3.2  General Aspects of Mating Behavior in Diaphorina citri and Other Psylloids Members of the Psylloidea, a group of about 3850 phloem-feeding Sternorrhynchan species

(Burckhardt and Ouvrard, 2012; Martoni et al., 2017), share numerous aspects of reproductive biology and mating behavior (Lubanga et  al., 2014, 2016a). Psylloids reproduce only sexually, unlike some hemipterans (Kennedy and ­Stroyan, 1959). Many male psylloids reach reproductive maturity within about 2 days post-­ eclosion, and both sexes mate several times (Burts and Fischer, 1967; Van den Berg et  al., 1991; Wenninger and Hall, 2007; Guédot et al., 2012; Lubanga et al., 2018) during a typical lifetime of 49 days or longer (Wenninger and Hall, 2008). Refractory periods have been reported for females of some psyllid species (Lubanga et  al., 2016a) but have not been reported in D. citri. Newly emerged D. citri females have i­mmature ovaries that remain without mature eggs until mating occurs (Dossi and Cônsoli, 2010). Mating stimulates vitellogenesis and rapid development of oocytes, and females often begin laying eggs on the day of mating (Wenninger and Hall, 2007). Because oocyte maturation is metachronous, with only one oocyte developing per oogenic cycle, the stimulatory effects of mating (Dossi and Cônsoli, 2010) may contribute to the polyandry (Wenninger and Hall, 2008) observed in this species. General aspects of several different male reproductive systems in the Psylloidea are described in Schlee (1969), Macharashvili and Kuznetsova (1997) and Kuznetsova et al. (1997); and the male D. citri genitalia and reproductive system were described in Stockton et al. (2017b) and Alba-Alejandre et al. (2018). Dossi and Cônsoli (2014) and Stockton et  al. (2017b) described the D. citri female reproductive organs. Short-range semiochemicals have been demonstrated to play a role in mate-finding of several (Wenninger et  al., 2008; Brown et  al., 2009; Guédot et  al., 2009, 2010; Mann et  al., 2013) but not all psylloids (Lubanga et al., 2016b). Wu et al. (2016) investigated the antennal and abdominal transcriptomes of male and female D. citri to consider whether chemosensory proteins could be identified for development of attractants or repellents. It was found that a large proportion of chemosensory genes were similar in male and female antennae and terminal ­abdominal tissues, but two were expressed at higher levels in male than female antennae (Wu et al., 2016), which is consistent with a potential role of antennal chemosensilla in D.  citri

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mate-finding or species identification (Onagbola et al., 2008). In addition, substrate-borne vibrational communication is an important mechanism for mate location in many psylloids (Ossiannilsson, 1950; Virant-Doberlet and Čokl, 2004; Tishechkin, 2005; Percy et  al., 2006; Lubanga et  al., 2014, 2016b; Eben et al., 2015; Liao and Yang, 2015, 2017; Liao et al., 2016), as well as other Hemiptera (Cocroft and Rodríguez, 2005) including the Auchenorrhyncha (Percy and Day, 2005) and the Cicadellidae (Gordon et  al., 2017). The capability to detect and produce vibrations is essentially ubiquitous in terrestrial invertebrates, which attests to the importance of vibrational cues for reproduction and predator avoidance (Cocroft and Rodríguez, 2005; Pollack, 2017; Takanashi et  al., 2019). In many Hemiptera, sexual communication involves a duet, in which a searching male will call and a sedentary female will reply, which facilitates searching behavior as well as mate recognition (Bailey 2003; Derlink et al., 2014). Unlike in air, a relatively uniform substrate, the physically non-uniform characteristics of the interiors and interfaces within plant structures strongly affect propagation of vibrations from their sources to the sensing insect (Michelsen et al., 1982). Reflections from surface edges, frequencydependent attenuation and background noise make it difficult to locate or estimate the distance to a vibration source precisely (Michelson et al., 1982; Mankin et al., 2011, 2018; Dent, 2017; Gordon et  al., 2019). In addition, the small size of D. citri (and many other psylloids) as well as ­frequency-dependent attenuation observed in plant structures essentially reduces the ‘active space’, i.e. the maximum communication distance, of ­vibrational communication signals to 1–2 m (Ichikawa 1979; Michelsen et al., 1982; Mazzoni et al., 2014). Consequently, the information that vibrational communication provides is typically transmitted over only a short range. Males of Nezara viridula (L.) and other large hemipteran species have been reported to use the time delay between vibrational signals that reach two different legs as a directional cue for locating the female (Čokl et al., 1999). The distances between the legs of psylloids, however, may be too small to use this delay effectively in search behavior (Tishechkin, 2007). Virant-­ Doberlet and Čokl (2004) suggested that even

small insects can stretch their legs between branches at bifurcations, thereby increasing the time delay, which could provide directional cues at bifurcation points. Nevertheless, background noise from wind or other loud sound or vibration sources masks the weak signals produced by small insects and interferes with precise identification of the direction of a replying female psyllid (Tishechkin, 2013). To accommodate directional uncertainties and signal masking, many hemipterans augment their searching behavior with a ‘call-fly’ strategy under which a searching male produces substrate-borne vibrational calls spontaneously when it first lands on a host plant and then moves upward and/or towards a receptive female who has produced a duetting reply in response to his call (Hunt and Nault, 1991). Such a strategy is beneficial when the females aggregate towards the upper perimeter of the plant, as is discussed in Section 3.4 below.

3.3  Substrate-borne Communication in Diaphorina citri Substrate-borne communications associated with mating behavior of virgin male and female ACP, 5–7 days post-eclosion, were characterized by Wenninger et al. (2009a). Males and females both produce low-amplitude vibrational signals, 140–700 ms in duration, by extending and flapping their wings rapidly while holding on to the substrate with their legs, as in many other psylloids (Tishechkin, 1989, 2005). The signal then is transmitted through the legs to the substrate and connected structures. The communication signal spectral frequencies are harmonics of the 170–250 Hz wingbeat frequency (i.e. the fundamental frequency) (Mankin et al., 2016), which is negatively correlated with body mass (Wenninger et al., 2009a). ACP production of sounds consisting of wingbeat fundamental and harmonic frequencies is similar to that observed in many flying insects such as mosquitoes and bumblebees, the aerodynamics of which are described in Bae and Moon (2008). Due to its small size, ACP produces only faint sounds and the signal is carried primarily through vibrations transmitted through the leaf or twig surfaces (Michelsen et  al., 1982), which have signal attenuation and other structural characteristics



Mating Behavior of the Asian Citrus Psyllid 33

that vary considerably among the different plants that are ACP hosts (Mazzoni et al., 2014; Ebert et al., 2018). Several other psylloids have rows of teeth on the axillary cords of the wing mesoscutellum and metascutellum which can serve as a stridulatory organ for sound production (Heslop-Harrison, 1960; Taylor, 1985a; Liao et al., 2019). However, stridulatory organs usually produce chirps with high-amplitude fundamental frequencies and weak harmonics (e.g. Mankin et al., 2009; Grant et al., 2014) caused by the friction of the pars stridens scraping over the plectrum. Because such spectral patterns are not observed in its signals (Mankin et al., 2016), ACP possibly uses only wing-­flapping and not stridulation as a sound-production mechanism. ACP searching and mating activity on citrus occurs primarily on new leaf flush between 10:00 am and 3:00 pm (Wenninger and Hall, 2007). As with Cacopsylla pyri (L.) (Eben et  al., 2015), male D. citri usually produce calls spontaneously within about 15 min after landing on a host plant, except during extreme changes in weather or barometric pressure (Zagvazdina et al., 2015). Receptive females produce a duetting reply within 0.95 ± 0.09 s (mean ± standard error) after the male call (Wenninger et al., 2009a). The female remains sedentary while the male moves intermittently towards the female. Durations of male

1

Fr

Fr

Fr

Mc

Relative amplitude

calls range from approximately 148 to 544 ms, and female replies from 331 to 680 ms (Wenninger et  al., 2009a). After detecting a female reply, a male that has begun searching on a leaf or branch of a citrus tree typically moves in the direction of the reply for a few seconds until it reaches a bifurcation or other visually identifiable transition point. There it pauses, calls again, and then continues or returns back along the original path. Males call intermittently during the duetting bouts, with an interval of 9 ± 1.4 s between calls (Lujo et al., 2016). Typical movement speeds of males involved in searching behavior are > 9 mm/min (­Zagvazdina et al., 2015). Several instances of reciprocating behavior may occur before the male finds the female or ceases searching. Lujo et  al. (2016) reported that, in 17 mating tests on small citrus trees, a male placed on a separate branch from a female searched for 15.9 ± 2.4 min before finding the female. An example of a 45 s section of a duetting bout recorded from a small tree in an anechoic chamber is shown in Fig. 3.1. It should be noted that only virgin D. citri males and females were tested in the Mankin et al. (2013), Rohde et  al. (2013), Lujo et  al. (2016), and Hartman et  al. (2017) studies, and there is evidence that males learn female-produced olfactory cues and associated environmental odors

Mc

Mc

0

–1 10

20

30

40

Time (s) Fig. 3.1.  A 45 s period of a D. citri duetting bout recorded on a small tree in an anechoic chamber. Solid oval (Mc) designates male call; dashed oval (Fr) designates female reply.

34

R.W. Mankin and B. Rohde

during their first mating encounter and subsequently are attracted to such odors (Stockton et  al., 2017a). Similar learning responses have been observed in mice (Remedios et  al., 2017). Previously mated D. citri males may be more ­responsive than virgins to female odor and associated environmental odor cues, as well as to vibrational reply cues when searching for females. In addition, there is evidence that females can learn cues associating male color with subsequent ­reproductive success and thereby increase fecundity (Stockton et al., 2017b). Learning may be particularly beneficial (Dukas et  al., 2006) when multiple movement biases or other factors result in aggregations with high encounter rates between males and females, the topic of the next section. There is evidence that male cuticular ­hydrocarbons, including dodecanoic acid (Mann et al., 2013), degradation products of citrus volatiles (George et  al., 2016; Lapointe et  al., 2016; Zanardi et al., 2018), and combinations of yellow, green and ultraviolet light (Paris et al., 2017a, b) may serve as significant cues in male searches for mates and host plants, as well as in female searches for host plants. Once the male finds the female, he moves to her side with their heads pointing in the same direction, similarly as in Trioza erytreae (Del Guercio) (Van Den Berg et al., 1991). If she is receptive, he holds on to her abdomen with his nearest legs, bending his upward-pointing genital segment down to meet the opening of the ­female segment, while supporting himself on the substrate with his remaining legs, and begins copulation (Husain and Nath, 1927). They copulate while the male holds the female with legs on one side of his body and supports himself on the plant with his remaining legs (Husain and Nath, 1927). The mean duration in copula has been measured as 48.3 ± 8.4 min, ranging from 15.2 min to 98 min (Wenninger and Hall, 2007). The female usually begins ovipositing on the day of mating (Wenninger and Hall, 2007) and may lay up to 800 eggs over a lifetime of 2 months (Husain and Nath, 1927). It has not been established whether differences among the temporal or spectral components of female replies affect ACP male mating preference although preference has been observed in fulgorid males (Mazzoni et al., 2015). It may be relevant, however, that ACP females infected with the CLas pathogen are more fecund

and therefore have greater reproductive fitness than uninfected females, which may facilitate the spread of HLB (Pelz-Stelinski and Killiny, 2016). It is not known whether mating behavior itself is affected by CLas infection but increased fecundity could result from multiple matings with high-fertility partners, from changes in hormonal regulation of immune function and metabolic allocation (Harshman and Zera, 2007) or from increased movement leading the female to healthier flush (Martini et al., 2015). In addition, there are numerous abiotic factors that could interfere with different aspects of communication, physical activity or physiological processes associated with mating behavior. These include weather extremes and barometric pressure extremes (Zagvazdina et  al., 2015; Martini and Stelinski, 2017; Udell et al., 2017; Martini et al., 2018), high altitude (Jenkins et al., 2015) and high levels of wind and other interfering vibrational background noise (Tishech­ kin, 2013).

3.4  Movement Bias towards Light and Flush: Impacts on Mating Behavior As with many psylloids, D. citri females and males exhibit phototaxis, both when flying (Sétamou et  al., 2011; Anco and Gottwald, 2015; Paris et al., 2015) and walking (Pregmon et  al., 2016; Paris et  al., 2017a). They are attracted to green and yellow colors (Paris et  al., 2015); yellow colors are known to induce settling of many hemipteran herbivores on host plants (Döring, 2014). There is evidence that citrus tree volatiles play a role in attraction to the host (Wenninger et  al., 2009b). In addition, D.  citri are attracted to new leaf flush (Catling, 1970; Hall and Albrigo, 2007; Patt and Sétamou, 2010; Sule et  al., 2012; Sétamou et  al., 2016; Hall and Hentz, 2016, Stelinski, 2019) on which eggs are laid and nymphs develop. Nutrient availability (Steinbauer, 2013), ability to easily probe the thinner structure of citrus leaf veins in young flush (Ammar et  al., 2013) and phagostimulants (George et  al., 2016; Lapointe et al. 2016) may play a role in such attraction. Oviposition cues detected by sensilla on the legs and female ovipositor also may play a role in



Mating Behavior of the Asian Citrus Psyllid 35

flush attraction (Zhang et al., 2019). Such movement biases can result in both-sex aggregations on upper canopies (Soemargono et  al., 2008), border trees (Sétamou and Bartels, 2015) and flush (Tsai et  al., 2000; Sétamou et  al., 2008; Hall et al., 2015). Aggregation and social behaviors also have been documented in numerous other hemipterans (e.g. Kennedy et  al., 1967; Way and Cammell, 1970; Lin, 2006). The occurrence of aggregations may reduce predation, partly by increased tending by ants (e.g. Navarrete et al., 2013) which would lead to more rapid increases of D. citri populations. Given that the volume of upper-canopy flush is generally only a fraction of the volume of the complete canopy, mate-seeking males that move towards light and flush will, on average, have less distance to cover in searching for a female than males that search at random. Consequently, such biases are reproductively advantageous. Likewise, when the density of nymphs and adults increases to levels that reduce flush healthiness, it is reproductively advantageous to migrate to areas with lower ACP populations (Martini et  al., 2015; Martini and Stelinski, 2017), as has been observed frequently in auchenorrhynchans (Taylor, 1985b).

A relatively unstudied impact of ACP aggregations is whether the presence of nearby conspecifics of both sexes may affect mating behavior. Until now, social interactions involving vibroacoustic communication have been studied primarily in social insects (Hunt and Richard, 2013) but interactions also have been documented in Delphacidae (Ott, 1994) and Cicadellidae (Hunt and Morton, 2001), and acoustic interactions are well documented in mating swarms of mosquitoes and midges (Mankin, 2012; Simões et  al., 2016; Jakhete et  al., 2017). Recordings from D. citri in infested orchard trees (Mankin et  al., 2016) as well as in greenhouse trees with D. citri maintained for behavioral bioassays (Paris et al., 2013, and unpublished), suggest that social interactions in which multiple males and females take part in duets occur frequently in aggregations during the time of day when mating typically occurs, an example of which is shown in Fig. 3.2. The notable differences in the amplitudes and fundamental frequencies of the signals in Fig. 3.2 suggest that two different males and three different females had participated in the duets. Unpublished studies suggest that the duetting frequency per number of aggregated individuals decreases relative to the F3-r

M2-c

M1-c 1

F1-r

Relative amplitude

F2-r

0

–1 1

3

5

Time (s) Fig. 3.2.  A 6 s period of duetting recorded from a tree in a D. citri colony reared for behavioral ­bioassays: solid ovals, M1-c and M2-c, indicate male calls; dashed ovals, F1-r, F2-r, and F3-r, indicate female replies. Differences in the amplitudes and fundamental frequencies of the signals suggest that two male calls and three female replies were produced by different individuals.

36

R.W. Mankin and B. Rohde

duetting frequency of isolated pairs, possibly because males can locate females in the aggregation readily by random movement without ­calling. Also, it has been observed that previously mated ACP males are less likely than virgin males to begin calling spontaneously (Wenninger et al., 2009a), which may reduce the rate of calling in aggregations.

3.5  Potential for Mimicking or Interfering with Vibrational Communication Signals to Trap Males or Disrupt Mating Soon after D. citri vibrational signals were first characterized in 2009, interest developed in the possibility of devices that mimicked the female reply signal to attract and capture males or disrupt mating. An understanding of D. citri population densities and spatial distributions in citrus groves is important for development and timing of management decisions (Sétamou et  al., 2008), but commonly used stem-tap and sweep-net sampling methods (Hall et  al., 2013; Monzo et  al., 2015) have limited efficiency at low population densities, and sampling with yellow sticky traps is costly and requires a relatively large time commitment (Hall and Hentz, 2010; Hall et al., 2010; Monzo et al., 2015). Mating disruption seemed feasible, having been demonstrated previously by Saxena and ­Kumar (1980) on cotton leafhopper Amrasca devastans Dist. and rice brown planthopper Nilaparvata lugens Stål. Disruption of substrate-based communication is a natural competitive practice in the leafhopper Scaphoideus titanus Ball (Mazzoni et al., 2009). Therefore, a series of investigations was conducted to develop methods to trap and/or disrupt D. citri mating behavior. Initially, recordings of duets (Rohde et  al., 2013) as well as synthetic mimics (Mankin et al., 2013) were bioassayed for their potential to elicit female replies and male searching behavior in citrus trees. The bioassays demonstrated that males were attracted to the recorded replies as well as to synthetic mimics in which three or more harmonics of the fundamental frequency were present (Mankin et al., 2016). Such knowledge of the spectral and temporal patterns needed for D. citri species recognition and male attraction thereafter led to development of

prototype signal-mimicking devices that disrupted mating (Lujo et al., 2016) and attracted male D. citri to traps (Mankin et al., 2016; Hartman et al., 2017). A potentially useful result of the male D. citri trapping study (Hartman et al., 2017) was that males were found to be variably responsive to searching cues. Also, Zagvazdina et  al. (2015) had previously reported variability in male searching behavior, demonstrating that changes in barometric pressure affected the proportions of males who moved either > 9 mm/min or 5 weeks (Koizumi et al., 1996) Nymphs (Halbert and Manjunath, 2004; Halbert, pers. comm.) Eggs (Halbert, pers. comm.); nymphs (Halbert, and Manjunath, 2004; Halbert, pers. comm.) Adult feeding but oviposition and development not known (Aubert, 1987, 1990, 1992), the latter based on comments by Zhao Xueyuan as reported by Barkley et al., 1980; as ‘missionis’ (citation by Garnier and Bove (1993) was based on Barkley et al. (1980) and Aubert (1988); marked increase in populations (Koizumi et al., 1996) Normal development in laboratory (Xu et al., 1988); unsuitable for development (Felisberto et al., 2018) Survival for > 7 weeks (Koizumi et al., 1996; Hung et al., 2000; Halbert and Manjunath, 2004) 7 of 10 adults observed on caged plant on third of 3 days (Beattie et al., 2010) Adult observed in survey (Halbert and Manjunath, 2004; Halbert, pers. comm.) 4 of 10 adults observed on caged plant on third of 3 days (Beattie et al., 2010) Survival for > 7 weeks (Koizumi et al., 1996) Moderate or good host (Capoor et al., 1967; Chavan and Summanwar, 1993; Chavan, 2004) Normal development (Halbert and Manjunath, 2004); high populations prevented bud development (Halbert, pers. comm.) Eggs (Halbert, pers. comm.)

G.A.C. Beattie

Species or hybrid

70

Table 5.1.  Summary of records related to Diaphorina citri on genotypes of Rutaceae within the Aurantieae.

Common or cultivar name cited

Cited as

Observation

Citrus australasica F. Muell.

Microcitrus australasica (F.J. Muell.) Swingle

Australian finger-lime

Citrus australis (Mudie) Planch.

Microcitrus australis (Planch.) Swingle Citrus ichangensis Swingle

Australian round lime, dooja Ichang (Yichang) papeda

Normal development in laboratory cages (Aubert, 1987, 1990, 1992); survival for 5 weeks (Koizumi et al., 1996); damage evident, adults only present (Halbert and Manjunath, 2004) Damage evident, adults only present (Halbert and Manjunath, 2004; Halbert, pers. comm.) Slight damage observed (Halbert, pers. comm.)

Eremocitrus glauca (Lindley) Swingle Eremocitrus hybrid

Australian desert lime

Rapid death, survival < 3 days (Koizumi et al., 1996)

Citrus hystrix DC.

leech lime, limau purut, limau hantu, kaffir lime, ‘Mauritius’ papeda Russell River lime, largeleaf Australian wild lime kumquat (‘Meiwa’)



Species or hybrid

Citrus cavaleriei H. Léveillé ex Cavalerie Citrus glauca (Lindl.) Burkill Citrus glauca × Shakura Citrus reticulata Citrus hystrix DC.

Citrus japonica Thunb.

Fortunella crassifolia Swingle

Hong Kong kumquat

‘Citrus limonia’ Osbeck

Fortunella hindsii (Champ. ex Benth.) Swingle Fortunella japonica (Thunb.) Swingle Fortunella margarita (Lour.) Swingle Fortunella polyandra (Ridley) Tanaka Fortunella sp. Citrus limonia Osbeck

Citrus maxima (Burm.) Merr.

Pomelo

pomelo, pummelo

Citrus decumana L. Citrus grandis (L.) Osbeck

pomelo, pummelo pomelo, pummelo

kumquat kumquat kumquat lemon

Normal development (Halbert and Manjunath, 2004; Halbert, pers. comm.) Some adults and damage (Halbert and Manjunath, 2004); host plant (Li et al., 2007) Host plant (Li et al., 2007) Few nymphs and adults, little damage (Halbert, pers. comm.) Nymphs (Halbert and Manjunath, 2004; Halbert, pers. comm.)

71

Eggs, nymphs and adults (Halbert and Manjunath 2004; Halbert, pers. comm.) Occasional host in cage studies (Aubert, 1987, 1990, 1992) Most favored of 6 hosts (Hoffmann, 1936); highly suitable (Felisberto et al., 2018) Usually a minor pest, sometimes occurring in large numbers and doing considerable damage (Fletcher, 1919) Attacked (Husain and Nath, 1927) Least favoured of 6 hosts (Hoffmann, 1936); occasional host (Aubert, 1987, 1992); moderate incidence (Rao and Pathak, 2001) Continued

Hosts of the Asian Citrus Psyllid

Citrus inodora F. M. Bailey

Damage and eggs (Halbert and Manjunath, 2004; Halbert, pers. comm.) Occasional host (Aubert, 1987, 1992; common host (Aubert, 1990); commonly infested, important alternative (Lim et al., 1990a; Osman and Lim, 1992) Slight damage (Halbert, pers. comm.)

72

Table 5.1.  Continued. Common or cultivar name cited

Citrus maxima (Burm.) Merr.

pomelo, pummelo

Citrus obovoidea Hort. ex Tanaka cv ‘Kinkoji’ Citrus medica L.

pomelo kinkoji

Citrus medica medica Citrus deliciosa Tenore Citrus depressa Hayata Citrus nobilis Loureiro Citrus nobilis Loureiro var. deliciosa (Ten.) Swingle2

citron mandarin flat lemon kam kat, ‘Kinnow’

Citrus reticulata Blanco

mandarin

Citrus reshni Hort. ex Tan.

‘Cleopatra’ mandarin

Citrus trifoliata L.

Poncirus trifoliata (L.) Raf.

trifoliate orange

Citrus wintersii Mabb.

Microcitrus papuana H.F. Winters

Brown River finger lime

Citrus sp.

Citrus limmettoides ‘Microcitrus’ sp. Citrus spp.

sweet lime

Citrus medica L.

Citrus reticulata Blanco

Citrus spp.

citron

Observation Attacked (Catling, 1968); occasional host (Aubert, 1990); 2–4 of 10 adults observed on 2 caged plants on third of 3 days (Beattie et al., 2010) Survey in Florida (Halbert and Manjunath, 2004) Common host (Aubert, 1987, 1990, 1992); some on ‘Gandharaj’ (Rao and Pathak, 2001) Attacked (Husain and Nath, 1927) Common host (Aubert, 1987, 1990, 1992) Host (Yasuda et al., 2005) Third most favored of 6 hosts (Hoffmann, 1936) Fourth most favored of 6 hosts (Hoffmann, 1936); nymphal development longer than on sweet orange, shorter than on rough lemon and Kagzi lime (Nehru et al., 2004) Survival for > 7 weeks but no increase in numbers on ‘Som-pan’ (Kuwayama, 1908; Koizumi et al., 1996); common host (Aubert, 1987, 1990); attacked (Catling, 1968); good on ‘Khasi’ (Rao and Pathak, 2001); common (Halbert and Manjunath, 2004); 5 of 10 adults observed on caged plant on third of 3 days (Beattie et al., 2010) Oviposition, survival and development rate < on sour orange (Tsagkarakis and Rogers, 2010) Survival for > 7 weeks but no increase in numbers (Koizumi et al., 1996); eggs, but no nymphal development (Aubert, 1987, 1990, 1992); low to intermediate suitability (Felisberto et al., 2018) Damage, nymphs and adults (Halbert and Manjunath, 2004; Halbert, pers. comm.); low to intermediate suitability (Felisberto et al., 2018) Moderate host (Rao and Pathak, 2001) Unsuitable for development (Felisberto et al., 2018) Host (Crawford, 1912, 1913; Lal, 1917, 1918); uncommon host (Miyatake, 1965); Xu et al., 1988; common host (Aubert, 1990; Viraktamath and Bhumannavar, 2001; Halbert and Manjunath, 2004)

G.A.C. Beattie

Cited as

Species or hybrid

Cited as

Common or cultivar name cited

Citrus ×aurantiifolia (Christm.) Swingle

Lime

lime

Citrus aurantifolia (Christm.) Swingle

lime, kagzi lime

Citrus pennivesiculata (Lush.) Tanaka Orange

moi

Citrus aurantium L.

sour orange, karun jamir

Citrus aurantium L.

‘Chinotto’

Citrus maxima var racemosa Citrus nobilis Lour. Citrus × nobilis Lour. Citrus × paradisi Macfad.

not cited not cited grapefruit

Observation

Species or hybrid

Citrus ×aurantium L.

Citrus sinensis (L.) Osbeck

sweet orange (navel and ‘Valencia’); soh nariang

Citrus sulcata hort. ex I. Takahashi Citrus tamurana hort. ex Tanaka

sanbokan

Usually a minor pest, sometimes occurring in large numbers and doing considerable damage (Fletcher, 1917, 1919) Moderate (Rao and Pathak, 2001); oviposition, survival and development rate > on ‘Cleopatra’ mandarin (Tsagkarakis and Rogers, 2010) Damage, eggs, nymphs and adults (Halbert and Manjunath, 2004; Halbert pers. comm.) Occasional host (Aubert, 1990) Common host (Aubert, 1987, 1990, 1992) Common host (Halbert and Manjunath, 2004) Occasional host (Aubert, 1987, 1992); best host in laboratory (Tsai and Liu, 2000); common, preferred (Halbert and Manjunath, 2004) Attacked (Husain and Nath, 1927); fifth most favored of 6 hosts (Hoffmann, 1936); attacked (Catling, 1968); common host (Aubert, 1987, 1990, 1992); nymphal development shorter than on Kinnow mandarin, rough lemon and Kagzi lime (Nehru et al., 2004); moderate (Rao and Pathak, 2001); common (Halbert and Manjunath, 2004); highly suitable (Felisberto et al., 2018) Heavy psyllid (Halbert, pers. comm.)

Hyuganatsu pomelo yuzu

Host (Halbert, pers. comm.) Some damage (Halbert, pers. comm.)

Hosts of the Asian Citrus Psyllid 73

Citrus × junos Siebold ex Tanaka (possibly a C. cavaleriei H. Léveillé ex Cavalerie (syn. C. ichangensis Swingle) × Citrus reticulata Blanco hybrid)

orange

Usually a minor pest, sometimes occurring in large numbers and doing considerable damage (Fletcher, 1919) Attacked (Catling, 1968); preferred host (Aubert, 1987, 1990, 1992); zero population (Rao and Pathak, 2001; Halbert and Manjunath, 2004); heavy psyllid damage (Halbert, pers. comm.); nymphal development shorter than on sweet orange, Kinnow mandarin and rough lemon (Nehru et al., 2004) Moderate damage (Halbert, pers. comm.)

Continued

74

Table 5.1.  Continued. Species or hybrid

Cited as

Common or cultivar name cited

Citrus × limon (L.) Osbeck

Lemon

lemon

Citrus assamensis S. Dutta & S.C. Bhattach Citrus lemon Citrus limon (L.) Burm. f.

adajamir

sour lime, khatta sweet lime, mitha lemon, limu

Attacked (Husain and Nath, 1927) Attacked (Husain and Nath, 1927)

Citrus × microcarpa Bunge

Citrus medica var. acida Hook. f. Citrus medica var. limetta Citrus medica var limonum Hook. f. Citrus meyeri U. Tan Citrus madurensis Lour.

Common host (Aubert, 1987, 1990, 1992) Attacked (Catling, 1968); 3 of 10 adults observed on caged plant on third of 3 days (Beattie et al., 2010) Attacked (Husain and Nath, 1927)

‘Meyer’ lemon calamondin

Citrus × taitensis Risso

Citrus jambhiri Lushington

rough lemon

Citrus × virgata Mabb.

Microcitrus sp. ‘Sydney’

‘Sydney’ hybrid

Limonia acidissima L.

Limonia acidissima L.

Merrillia caloxylon (Ridl.) Swingle Murraya elongata

Merrillia caloxylon (Ridley) Swingle Murraya elongata

Indian wood apple, elephant apple, wood apple kamuning, katinga, ketengah, Malay lemon

Survey (Halbert and Manjunath, 2004) Common host plant in Malaysia (Aubert, 1990); important alternative host (Osman and Lim, 1992) Some to moderate (Rao and Pathak, 2001); nymphal development longer than on sweet orange, Kinnow mandarin, shorter than on Kagzi lime (Nehru et al., 2004); surveys (Halbert and Manjunath, 2004) Damage, eggs and nymphs (Halbert and Manjunath, 2004; Halbert, pers. comm.) All stages present (Khan and Borle, 1989); marked increase in populations (Koizumi et al., 1996); suitable host (Hung et al., 2000)

lemon

Observation Usually a minor pest, sometimes occurring in large numbers and doing considerable damage (Fletcher, 1919) Good host (Rao and Pathak, 2001)

0 of 10 adults observed on caged plant over 3 days (Beattie et al., 2010); host, first record on species of Murraya native to South Asia (Om, 2017)

G.A.C. Beattie

Cage in laboratory (Lim et al., 1990b)

Common or cultivar name cited

Cited as

Murraya paniculata (L.) Jack

Murraya exotica L.

jasmine orange, orange-jessamine

Murraya paniculata (L.) Jack

jasmine orange, orange-jessamine

Murraya paniculata var. ovatifoliolata

jasmine orange, orange-jessamine

Observation

Species or hybrid

Swinglea glutinosa (Blanco) Merr.

Triphasia trifolia (Burm. f.) P. Wilson

hesperethusa

tabog

Swinglea glutinosa (Blanco) Merr.

tabog

Triphasia trifoliata

limeberry, triphasia

Triphasia trifolia (Burm. f.) P. Wilson

limeberry, triphasia

But possibly C. × aurantium ‘King’ orange, often called, incorrectly, ‘King’ mandarin.

Eggs, nymphs and adults observed (Halbert and Manjunath, 2004, Halbert, pers. comm.) Feeding in laboratory cage studies (Aubert, 1990; Waterhouse, 1998) Successful feeding and transmission (Tirtawidjaja, 1981); damage, eggs and nymphs observed (Halbert and Manjunath, 2004); feeding, oviposition and development (Beattie et al., 2010); low to intermediate suitability (Felisberto et al., 2018) Survival for 5 weeks (Koizumi et al., 1996); complete development in laboratory, occasional field host (Aubert, 1987, 1990, 1992); complete development in cages (Osman and Quilici, 1991) Adult survival for several weeks (Koizumi et al., 1996); ­occasional host (Aubert, 1987); damage, adults plentiful, all stages present (Halbert and Manjunath, 2004)

75

2

Murraya paniculata Naringi crenulata (Roxb.) Nicolson Atalantia missionis (Wall. ex Wight) Oliv. Pamburus missionis (Wall. ex Wight) Swingle Swinglea glutinosa

Hosts of the Asian Citrus Psyllid

Murraya sumatrana Roxb. Naringi crenulata (Roxb.) Nicolson Pamburus missionis (Wight) Swingle

Feeding in laboratory cages (Aubert, 1990); common or preferred host (Catling 1968; Khan and Borle, 1989; Koizumi et al., 1996; Yasuda et al., 2005; Yang et al., 2006; Beattie et al., 2010); feeding and transmission of ‘CLas’ (Damsteegt et al., 2010) Host (Maki, 1915; Kuwayama, 1931; He (= Hoffmann) and Zhou, 1935; Miyatake, 1965; Cheema and Kapur, 1975; Singh and Nimbalkar, 1977; Catling et al., 1978; Tirtawidjaja, 1981; Aubert and Quilici, 1984; Tsai et al., 1984; Aubert, 1987, 1990, 1992; Osman and Lim, 1992; Koizumi et al., 1996; Tsai et al., 2000, 2002; Halbert and Manjunath, 2004; Li et al., 2007); feeding and transmission of ‘CLas’ (Damsteegt et al., 2010); highly susceptible (Felisberto et al., 2018) Westbrook et al. (2011): misidentified as Murraya paniculata var. ovatifoliolata in huanglongbing tolerance evaluations reported by Ramadugu et al. (2016) and Miles et al. (2017) Beattie et al. (2010) Heavy infestations (Halbert and Manjunath, 2004; Halbert, pers. comm.) Successful feeding and transmission (Tirtawidjaja, 1981)

Cited as

Common name

Development

Bergera koenigii L.

Murraya euchrestifolia Hayata Murraya koenigii (L.) Spreng.

curry leaf

Hung et al. (2000)

curry leaf

Alternative food plant (Fletcher, 1917, 1919; Husain and Nath, 1927); found on shoots (Fletcher, 1919); when given a choice all adult psyllids migrated from citrus seedlings to curry leaf seedlings on which successful breeding colonies were established and maintained (Chakraborty et al., 1976); preferred host (Singh and Nimbalkar, 1977); variable host with no or limited development (Aubert, 1987); a good host (Aubert, 1990); an important alternative host (Osman and Lim, 1992); an important alternative host (Lim et al., 1990a); good population growth (Osman and Quilic, 1991); infests and breeds throughout the year (Chavan and Summanwar, 1993); some increase in population (Koizumi et al., 1996); not an excellent host but will support a small population (Halbert and Manjunath, 2004); highly suitable (Felisberto et al., 2018) Feeding, seemingly more attractive than Murraya paniculata, but no oviposition in cage tests (Gavarra and Mercado, 1988; Aubert, 1990) Complete development (Aubert, 1990); important alternative host (Lim et al., 1990a; Osman and Lim, 1992); 0 of 10 adults observed on caged plant over 3 days (Beattie et al., 2010) A successful feeding and transmission (Tirtawidjaja, 1981); 0 of 10 adults observed on caged plant over 3 days (Beattie et al., 2010) Second most favored of 6 hosts (Hoffmann, 1936); survives and propagates normally (Xu et al., 1988); variable adult survival but no multiplication (Koizumi et al., 1996); complete development and a common field host in Asia (Aubert, 1990); eggs, nymphs and adults, populations highly variable (Halbert and Manjunath, 2004; Halbert, pers. comm.); 0-1 of 10 adults observed on caged plant over 3 days (Beattie et al., 2010); low to intermediate suitability (Felisberto et al., 2018) 0 of 10 adults observed on caged plant over 3 days (Beattie et al., 2010) 0 of 10 adults observed on 2 caged plant over 3 days (Beattie et al., 2010)

Clausena anisum-olens Merr. Clausena excavata Burm f.

Clausena anisum-olens Merrill Clausena excavata Burm. f.

Clausena indica Oliv.

Clausena indica Oliver

Clausena lansium (Lour.) Skeels

Clausena lansium (Lour.) Skeels

Clausena sp. Glycosmis lucida Wall. ex C.C. Huang Glycosmis parviflora (Sims) Little Glycosmis pentaphylla (Retz.) DC. Micromelum hirsutum Oliv.

Clausena sp Glycosmis lucida Wallich ex C.C. Huang Glycosmis parviflora (Sims) Little Glycosmis pentaphylla (Retz.) DC.

kayumanis

wong p’ei, wampee, huangpi

0 of 10 adults observed on caged plant over 3 days (Beattie et al., 2010) orangeberry, gin berry

Non-host, non-refuge (Felisberto et al., 2018) 0 of 10 adults observed on caged plant over 3 days (Beattie et al., 2010)

G.A.C. Beattie

Species or hybrid

76

Table 5.2.  Summary of records related to Diaphorina citri on species of Rutaceae within the Clauseneae.

Species or hybrid

Cited as

Common name

Observations

Amyris madrensis S. Wats.

Amyris madrensis

Adult survival < 15 days, partial development (Sétamou et al. 2016)

Amyris texana (Buckley) P. Wilson Balfourodendron riedelianum (Engl.) Engl. Casimiroa edulis Llave Casimiroa greggii (S. Watson) F. Chiang Casimiroa tetrameria Millsp.

Amyris texana

mountain torchwood Texas torchwood marfin

Non-host, non-refuge (Felisberto et al., 2018)

Balfourodendron riedelianum (Engl.) Engl. Casimiroa edulis Llave

Choisya arizanica

Choisya ternata

Esenbeckia berlandieri Baill. Esenbeckia febrifuga (A.St.-Hil.) A.Juss. ex Mart. Esenbeckia leiocarpa Engl. Helietta apiculata Benth.

Esenbeckia berlandieri Esenbeckia febrifuga (A.St.-Hil.) A.Juss. ex Mart. Esenbeckia leiocarpa Engl. Helietta apiculata Benth.

Helietta parvifolia (A.Gray) Benth Melicope pteleifolia (Champ. ex. Benth.) T.G. Hartley Ptelea trifoliata L.

Helietta parvifolia Melicope pteleifolia (Champ. ex. Benth.) T.G. Hartley Ptelea trifoliata

Ravenia spectabilis (Lindl.) Planch. ex Griseb. (syn. Limonia spectabilis Lindl.) Tetradium ruticarpum (A. Juss.) T. G. Hartley Toddalia asiatica (L.) Lam.

Evodia rutaecarpa (A. Juss.) Benth. Toddalia asiatica (L.) Lam.

white sapote yellow chapote

Non-host, non-refuge (Felisberto et al., 2018) Adult feeding, no development (Mamoudou Sétamou, Texas A&M University-Kingsville, pers. comm. with Pat Barkley) matasano, woolly- Adult survival < 10 days, oviposition, no hatching (Sétamou et al. 2016) leaved sapote Arizona orange Feeding, development to adults (Sétamou et al. 2016)

Mexican orange blossom Berlandier's jopoy quina-do-mato

Adult survival < 14 days, partial development (Sétamou et al. 2016) Adult survival < 11 days, no oviposition (Sétamou et al. 2016) Non-host, non-refuge (Felisberto et al., 2018)

Brazilian boxwood Non-host, non-refuge (Felisberto et al., 2018) canela de venado Unsuitable host, a few misshapen unviable eggs, 40% of adults alive 30 days after confinement (Felisberto et al., 2018) baretta Adult survival to 14 days, development to adults (Sétamou et al. 2016) 0 of 10 adults observed on caged plant over 3 days (Beattie et al., 2010) hop tree

Adult survival < 9 days, oviposition, partial development (Sétamou et al. 2016) lemonia, limonia, Adults relatively easy to find on plants in Fairchild Botanic Gardens, pink ravenia Miami, Florida (Richard Lee and Susan Halbert, pers. comm., November 2008) evodia, wu zhu yu Record not verified and Evodia misspelt as Euodia: He (2000) cited by Yang et al. (2006) orange-climber, Feeding but no oviposition in cage studies (Aubert, 1987, 1990, 1992) forest pepper Continued

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Choisya dumosa (Torr.) A. Gray var. arizonica (Standl.) L. Benson Choisya ternata Kunth

Adult survival < 9 days, oviposition, no hatching (Sétamou et al. 2016)

Hosts of the Asian Citrus Psyllid

Casimiroa tetrameria



Table 5.3.  Summary of records related to Diaphorina citri on species of Rutaceae within the subfamily Amyridoideae (formerly Rutoideae or Toddalioideae).

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Table 5.3.  Continued. Species or hybrid

Cited as

Common name

Observations

Vepris lanceolata (Lam.) G. Don Zanthoxylum avicennae (Lam.) Candolle Zanthoxylum cucullatipetalum Guillaumin Zanthoxylum fagara (L.) Sarg.

Vepris lanceolata G. Don Zanthoxylum avicennae (Lamarck) Candolle Zanthoxylum cucullatipetalum Guillaumin Zanthoxylum fagara (L.) Sarg.

white ironwood

Feeding but no oviposition in cage studies (Aubert, 1987, 1990, 1992) 0 of 10 adults observed on caged plant over 3 days (Beattie et al., 2010)

Zanthoxylum nitidum (Roxb.) DC. Zanthoxylum nitidum (Roxburgh) Candolle Zanthoxylum rhoifolium L.

shiny-leaf prickly-ash, liang mian zhen tambataru, prickly Non-host, non-refuge (Felisberto et al., 2018) ash

G.A.C. Beattie

Zanthoxylum rhoifolium Lam.

lime prickly-ash

1 of 10 adults observed on caged plant on third day of 3 days (Beattie et al., 2010) Plenty of suitable new shoots in Florida arboretum survey, very few D. citri found, possible non-host (Halbert and Manjunath, 2004); adult survival < 15 days, oviposition, partial development (Sétamou et al. 2016) 1 of 10 adults observed on caged plant on third day of 3 days (Beattie et al., 2010)



Hosts of the Asian Citrus Psyllid

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Table 5.4.  Summary of records related to Diaphorina citri on species of Rutaceae within the subfamily Cneoroideae (formerly Rutoideae or Toddalioideae). Common name

Species or hybrid

Cited as

Dictyoloma vandellianum A. Juss. Metrodorea stipularis Mart.

Dictyoloma vandellianum A. Juss. Metrodorea stipularis Mart.

Batra et  al. (1970) evaluated the susceptibility of 24 genotypes (23 Citrus and Atalantia buxifolia) to colonization by D. citri between mid-autumn (October) 1968 and early spring (March) 1979 at Abohar in the Indian Punjab. Assessments were based on the presence of nymphs. Susceptibility of the immature flush growth (up to 5 cm in length) of 24 genotypes to colonization under natural conditions was based on five categories: (1) completely ‘resistant’: no nymphs observed (0% infestation), (2) commercially ‘resistant’: nymphs very rarely observed (0.10% infestation), (3) slightly ‘resistant’: incidence of nymphs very low (10–20%), (4) moderately susceptible: moderate infestations (20–40%), and (5) highly susceptible: severe infestations (40–100%). No genotypes were completely resistant. ‘Cleopatra’ mandarin, and ‘Rubidoux’ trifoliate orange were commercially ‘resistant’. ‘Kara’, ‘Honey’, ‘Wilking’ and ‘Kinnow’ mandarins, ‘Orlando’ tangelo, Rangpur lime, and Coorg lime (Citrus × aurantiifolia) were slightly resistant. A. buxifolia, ‘Blood Red’, ‘Campbell Valencia’ and ‘King’ oranges, sweet lime (Citrus × limon, ‘limettioides’), ‘Pearl’ tangelo, ‘Dancy’ mandarin, rough lemon, and two citranges (‘Carrizo’ and ‘Savage’) were moderately susceptible, and ‘Minneola’ tangelo, ‘Frost March’ grapefruit, two citranges, including ‘Troyer’ citrange, and ‘Kharna Khatta’ sour orange were highly susceptible. Patil et  al. (1972) screened 87 3-year-old Citrus and other genotypes for their susceptibility of attack by citrus psylla in a field trial ­between late autumn and mid-spring in Maharashtra, India, when incidence of the psyllid was high. Assessments were based on the presence of nymphs on immature flush up to 5 cm in length. Susceptibility of trees to colonization was based on Batra et  al. (1970). ‘Gajnimma Rusk’ citrange, Mexican lime, ‘Coorg’ citron, ‘Lisbon’ lemon and ‘Agle’ were completely resistant.

pau-marfim

Observations Non-host, non-refuge (Felisberto et al., 2018) Non-host, non-refuge (Felisberto et al., 2018)

Casimiroa edulis, trifoliate orange, citrange cultivars such as ‘Carrizo’, ‘Morton’, ‘Rusk’ and ‘Rusk Morocco’, grapefruit cultivars including ‘Deshndo’, ‘Foster’ and ‘Thompson’, mandarin cultivars such as ‘Kinnow’, ‘Orange-Michal’ and ‘Satsuma-micon’, and lemon cultivars such as ‘Coorg’, ‘Kodur’ and ‘Nakur’ were commercially resistant. Very susceptible genotypes included rough lemon, sour orange, and lime cultivars such as ‘Baramasi’, ‘Karna’, ‘Philippine-red’, and sweet lime. Chakravarthi et  al. (1998) evaluated 77 Citrus cultivars. Presence of adults and nymphs on immature growth was assessed during the peak period of psyllid incidence. Susceptibility of trees to colonization was based on Batra et  al. (1970). No cultivars were highly resistant. Thirteen cultivars were highly resistant: Enterprise 8718 sweet orange, sour orange 8751, ‘Temple’ orange, Rangpur lime (from California), ‘Rangtra’ and ‘Cleopatra’ mandarins, ‘Umatilla’ tangelo, calamondin, Citrus hystrix, ‘Citromelos 4475’, Ichang lemon (possibly Citrus × junos), ‘Karna’ lime and ‘Trifista trifoliata’ (likely Triphasia trifolia). Sixteen cultivars were highly susceptible: ‘Jaffa 8761’ sweet orange, ‘Campbell Valencia’, ‘Valencia’, ‘Valencia late’ and ‘Paper’ oranges, Citrus × aurantium (as Citrus taiwanica Tanaka & Shimada: ‘Nanshô Daidai’ sour orange), ‘Sours dig’, ‘Rajkaipuli’ sour orange, acid and sweet limes, ‘Lisbon’ and ‘Nakoor’ lemons, ‘Kunembo’ mandarin, ‘Marsh’ white grapefruit, C. × aurantiifolia (‘Moi’), and ‘Alemow’ (C. × macrophylla Wester, ‘incertae sedis’). Numbers of psyllids ranged from 25 to 67 per flush. The remaining cultivars were moderately resistant or susceptible to colonization with populations ranging from 10 to 24 per flush. Westbrook et al. (2011) compared 87 Rutaceae seedling genotypes in Florida as hosts of D. citri in a free-choice field experiment. No eggs, nymphs or adults

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G.A.C. Beattie

were observed on Casimiroa edulis (Amyridoideae). Very low levels of D. citri were found on two C. trifoliata seedlings of ‘Simmon’s Trifoliate’ and ‘Little-Leaf ’. The plants most frequently colonized by adults were Citrus reticulata, B. koenigii, M. paniculata, C. maxima, and C. × macrophylla. Taxa least favored for colonization comprised Citrus australasica, Citrus glauca, Citrus halimii B.C. Stone, Citrus inodora, C. × aurantium (standard sour orange), Koji orange (Citrus × leiocarpa hort. ex Tan., ‘incertae ­sedis’), and two C. trifoliata genotypes, Citrus × virgata (‘Sydney’ hybrid, Citrus australis × C. australasica) (Aurantieae), Glycosmis pentaphylla (Clauseneae), and Casimoroa edulis and Zanthoxylum ailanthoides (Amyridoideae) (Westbrook et al., 2011). Taxa most favored as hosts for oviposition included Afraegle paniculata, B. koenigii, M. paniculata (including the accession incorrectly cited as Murraya paniculata var. ovatifoliolata), genotypes of C. maxima, Citrus medica, C. reticulata, and a range of hybrids including Citrus × amblycarpa (Hassk.) Osche, C. × aurantiifolia, C. × aurantium sour orange, C. × limon, ‘Citrus limonia’, and C. × virgata. Those least favored for oviposition were Aegle marmelos, C. × aurantium sour orange genotype, C. inodora, a C. glauca hybrid, a C. medica genotype, and a C. trifoliata genotype (Aurantieae), Clausena harmandiana, Glycosmis pentaphylla (Clauseneae), and C. edulis and Z. ailanthoides (Amyridoideae) (Westbrook et al., 2011). Taxa most favored for development of nymphs included B. koenigii, genotypes of C. maxima, C. medica, C. reticulata, and a range of hybrids including C. × aurantiifolia, C. × aurantium, C. × limon, ‘C. limonia’. Plants least favored for development of nymphs included Atalantia buxifolia, C. australasica, two C. trifoliata genotypes (Autantieae), Clausena harmandiana, Glycosmis pentaphylla (Clauseneae), and C. edulis and Z. ailanthoides (Amyridoideae) (Westbrook et al., 2011). Richardson and Hall (2013) compared ‘Alemow’ and 81 accessions of C. trifoliata and its hybrids and other Citrus spp. as hosts of ACP in no-choice tests. Oviposition was higher on ‘Alemow’ than on nearly all accessions of C. trifoliata, and no eggs were laid on 36% of the accessions. Additionally, more eggs were laid on ‘Alemow’ than on 10 of 34 C. × insitorum cultivars. Lifespans of adults were approximately 2.5 to 5 times longer on 11 of the 17 C. trifoliata cultivars.

Borgoni et al. (2014) evaluated ‘resistance’ of ‘Pêra’, ‘Natal’, and ‘Washington Navel’ (‘Baia’) oranges, ‘Marsh Seedless’ grapefruit, C. trifoliata ‘Rubidoux’, kumquat (‘margarita’), ‘Swingle’ citrumelo (C. × insitorum) and ‘Troyer’ citrange grafted on to ‘Rangpur’ lime to D. citri in voile cages in greenhouses. Preferences for oviposition and development in a no-choice test, and the ­effect of genotype, were evaluated. C. trifoliata ‘­Rubidoux’ was the least preferred genotype for oviposition; reduced number of eggs were also found to occur on ‘Troyer’ citrange; and ‘Marsh Seedless’ was the genotype with the most eggs. There was no significant variation in the duration of the embryonic development, but egg viability was variable and lowest on ‘Swingle’. Kumquat and ‘Marsh Seedless’ genotypes were correlated with increased durations of nymphal development. There was no difference in the survival of nymphs. Numbers of eggs laid on ‘Troyer’, ‘Swingle’, and kumquat were reduced. It was concluded that sweet orange cultivars were the most favourable host plant genotypes in the study, and that for oviposition, C. trifoliata ‘­ Rubidoux’ exhibited ‘resistance’ of the antixenosis type. Hall et  al. (2015) reported variable ‘resistance’ in C. trifoliata accessions to oviposition by D. citri. They attributed this variability to possible influences of environmental conditions, plant age and physiology, proximity of plants to susceptible germplasm, and the presence or ­absence of mature leaves. Alves et al. (2018) evaluated the influence of different combinations of scion and rootstock citrus varieties in Brazil on the development and feeding of D. citri. The following citrus varieties were used: ‘Hamlin’, ‘Pêra’ and ‘Valencia’ sweet oranges, ‘Ponkan’ mandarin, and ‘Sicilian’ lemon grafted on ‘Rangpur’ lime, citrange, or Sunki mandarin rootstocks. Survival rates for eggs were highest (88%) on ‘Valencia’ orange and ‘­Sicilian’ lemon, both grafted on ‘Sunki’ mandarin, and lowest (68%) on ‘Hamlin’ orange on ‘Rangpur’ lime. The lowest levels of both nymphal and total viability (egg to adult) were obtained on ‘Hamlin’, regardless of the rootstock used. The total development time (egg–adult) ranged from 17.9 to 19.3 days for the ‘Pêra’ on ‘Sunki’ and the ‘Hamlin’ on ‘Swingle’ combinations, respectively. Cluster analysis separated the hosts into two groups, the first consisting of the combinations of the ‘Hamlin’, and the second group formed by



Hosts of the Asian Citrus Psyllid

the other varieties. The highest food value (­assessed by the area of honeydew produced) was observed for the orange scion varieties, and among these, the highest value was observed on ‘Valencia’ (0.9 cm2); the smallest honeydew area was obtained on ‘Ponkan’ mandarin (0.27 cm2). The rootstocks did not affect the feeding behavior.

5.3  Records other than Rutaceae Aubert (1987, 1992) erroneously reported ‘Coriea’ and ‘Corica’ as hosts. This error stemmed from a typographical error or misrecording of comments by Zhou Xueyuan recorded by G.A.C. Beattie in Barkley et al. (1980). Atalantia sp. was noted as host (Barkley et  al. (1980). José Francisco Corrêa da Serra is the author of the genus Atalantia. Peña et  al. (2006) undertook field surveys and no-choice greenhouse tests in Florida to determine if jackfruit (Artocarpus heterophyllus (Rosales: Moraceae)) is a host of D. citri. They concluded that jackfruit it is not an acceptable host and that a report by Shivankar et al. (2000) of the psyllid attacking jackfruit in India was ­erroneous. In contrast to jackfruit, Thomas and De Leon (2011) showed that the psyllid can develop on the common fig (Ficus carica L. (Rosales: Moraceae)) and concluded that it may be an adventitious breeding host for the psyllid. Thomas (2011) reported the presence of D. citri on hackberry (Celtis spp. (Rosales: Ulmaceae)) and, in large numbers, in potato (Solanum tuberosum L. (Solanales: Solanaceae)) fields. Fan et al. (2011) reported detection of CLas in Archidendron ­lucidum (Benth.) I.C. Nielsen

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(syn. Pithecellobium lucidum Benth.), a leguminous tree (Fabales: Leguminosae). Yellow shoot symptoms mimicking those of huanglongbing were evident in leaves of the tree, which was growing in a citrus orchard in which citrus trees with severe symptoms of huanglongbing were present. Infection was attributed to transmission of the pathogen during opportunistic feeding by D. citri adults harboring the pathogen. Following trapping of D. citri in the Lake Kissimmee State Park forest in Florida, Martini et al. (2013) evaluated the three most common non-rutaceous plants, gallberry (Ilex glabra (L.) Gray (Aquifoliales: Aquifoliaceae)), Darrow’s blueberry (Vaccinium darrowii Camp (Ericales: Ericaceae)), and redbay (Persea borbonia (L.) Spreng. (Laurales: Lauraceae)), within the ­forest as host of the psyllid. Survival of adults on each species was similar to that observed on  ‘Valencia’ orange for 3 days but declined significantly by the fourth day. There was ­ some survival on the plants evaluated for up to 7 days. Johnston (2018) compared adult D. citri suitability of and preference for M. paniculata to three common weed species found in Florida citrus groves (Bidens alba (L.) DC. (Asterales: Asteraceae), Ludwigia octovalvis (Jacq.) P.H. Raven (Myrtales: Onagraceae), and Eupatorium capillifolium (Lam.) Small (Asterales: Asteraceae)). Both B. alba and E. capillifolium increased survivorship of adults twofold compared with starvation conditions with only water available. Choice trials revealed no difference in initial ­selection between M. paniculata and the weed hosts; although M.  paniculata was favored over time. This latter result suggested that host selection was initially by sight, and only later by taste and/or smell.

References Alves, G.R., Beloti, V.H., Faggioni-Floriano, K.M., de Carvalho, S.A., Moral, R. de A., Demétrio, C.G.B. and Parra, J.R.P. (2018) Does the scion or rootstock of Citrus sp. affect the feeding and biology of Diaphorina citri Kuwayama (Hemiptera: Liviidae)? Arthropod-Plant Interactions 12, 77–84. Ammar, E.-D., Richardson, M.L., Abdo, Z., Hall, D.G. and Shatters, R.G. Jr (2014) Differences in stylet sheath occurrence and the fibrous ring (sclerenchyma) between ×Citroncirus plants relatively resistant or susceptible to adults of the Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae). PLOS ONE 9(10), e110919.1–10. Atwal, A.S., Chaudhary, J.P. and Ramzan, M. (1970) Studies on the development and field population of citrus psylla, Diaphorina citri Kuwayama (Psyllidae: Homoptera). Journal of Research 7(3), 333–338.

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Aubert, B. (1987) Trioza erytreae Del Guercio and Diaphorina citri Kuwayama (Homoptera: Psylloidea), the two vectors of citrus greening disease: biological aspects and possible control strategies. Fruits 42, 149–162. Aubert, B. (1988) Management of the citrus greening disease in Asian orchards. In: Aubert, B., Ke, C. and Gonzales, C. (eds) Proceedings of the Second Asian/Pacific Regional Workshop on citrus greening, Lipa, Philippines, 20–26 November 1988. UNDP-FAO, Rome, pp. 51–52 + literature. Aubert, B. (1990) Integrated activities for the control of huanglongbing-greening and its vector Diaphorina citri Kuwayama in Asia. In: Aubert, B., Tontyaporn, S. and Buangsuwon, D. (eds) Proceedings of the Fourth International Asia Pacific Conference on Citrus Rehabilitation, Chiang Mai, Thailand, 4–10 February 1990. FAO UNDP, Rome, pp. 133–144. Aubert, B. (1992) Malaysian citriculture report of visits October 19th–22nd 1987 and February 23rd–28th 1989. In: Setyobudi, L., Bahar, F.A., Winarno, M. and Whittle, A.M. (eds) Proceedings of Asian Citrus Rehabilitation Conference, Malang, Indonesia, 4–14 July 1989. Ministry of Agriculture, Republic of Indonesia Agency for Agricultural Research and Development. FAO UNDP INS/84/007, pp. 16–28. Aubert, B. and Quilici, S. (1984) Biological control of the African and Asian citrus psyllids (Homoptera: Psylloidea), through eulophid and encyrtid parasitoids (Hymenoptera: Chalcidoidea) in Réunion ­Island. In: Garnsey, S.M., Timmer, L.W. and Dodds, J.A. (eds) Proceedings of the Ninth Conference of the International Organization of Citrus Virologists, Puerto Iguaçu, Misiones, Argentina, 9–13 May 1983. International Organization of Citrus Virologists, University of California, Riverside, California, pp. 100–108. Barkley (Broadbent), P., Beattie, G.A.C., Van Velsen, R.J. and Freeman, B. (1980) Report on the Visit to the People’s Republic of China – 2nd–29th November 1979. Department of Agriculture New South Wales. D West, Government Printer, New South Wales. Batra, R.C., Uppal, D.K. and Sohi, B.S. (1970) Indexing the genetic stock of different species of citrus against citrus leaf miner and citrus psylla. Indian Journal of Horticulture 27, 76–79. Beattie, G.A.C. and Barkley, P. (2009) Huanglongbing and its Vectors: a pest-specific contingency plan for the citrus and nursery and garden industries (Version 2), February 2009. Horticulture Australia, Sydney. Beattie, G.A.C., Holford, P. and Haigh, T. (2010) Huanglongbing Management for Indonesia, Vietnam and Australia. ACIAR HORT/2000/043 Final Report. Australian Centre for International Agricultural ­Research, Canberra. Borgoni, P.C., Vendramim, J.D., Lourencão, A.L. and Machado, M.A. (2014) Resistance of Citrus and related genera to Diaphorina citri Kuwayama (Hemiptera: Liviidae). Neotropical Entomology 43, 465–469. Burckhardt, D., Ouvrard, D., Queiroz, D. and Percy, D. (2014) Psyllid host-plants (Hemiptera: Psylloidea): resolving a semantic problem. Florida Entomologist 97, 242–246. But, P.P.H., Kong, Y.C., No, K.H., Chang, H.T, Li, Q., Yu, S.X. and Waterman, P.G. (1986) A chemotaxonomic study of Murraya (Rutaceae) in China. Acta Phytotaxonomica Sinica 24, 186–192. But, P.P.H., Kong, Y.C., Li, Q., Chang, H.T., Chang, K.L., Wong, K.M., Gray, A.I. and Waterman, P.G. (1988) Chemotaxonomic relationship between Murraya and Merrillia (Rutaceae). Acta Phytotaxonomica Sinica 26, 205–210. Capoor, S.P., Rao, D.G. and Viswanath, S.M. (1967) Diaphorina citri Kuway., a vector of the greening disease of citrus in India. The Indian Journal of Agricultural Science 37, 572–576. Catling, H.D. (1968) Report to the Government of the Philippines on the distribution and biology of Diaphorina citri, the insect vector of leaf mottling (greening) disease of citrus. United Nations Development Programme TA 2589. FAO, Rome. Catling, H.D., Garnier, M. and Bové, J.M. (1978) Presence of citrus greening disease in Bangladesh and a new method for rapid diagnosis. FAO Plant Protection Bulletin 26(1), 16–18. Cen, Y.J., Yang, C.L., Holford, P., Beattie, G.A.C., Spooner-Hart, R.N., Liang, G.W. and Deng, X.L. (2012) Feeding behaviour of the Asiatic citrus psyllid (Diaphorina citri Kuwayama) on healthy and huanglongbing-infected citrus. Entomologia Experimentalis et Applicata 143, 13–22. Chakraborty, N.K., Pandey, P.K., Chatterjee, S.N. and Singh, A.B. (1976) Host preference in Diaphorina citri Kuwayama, vector of greening disease of citrus in India. Indian Journal of Entomology 38, 196–197. Chakravarthi, V.P., Savithri, P., Prasad, P.R. and Naidu, V.G. (1998) Relative susceptibility of citrus germplasm to citrus psylla, Diaphorina citri Kuwayama (Homoptera: Psyllidae). In: Reddy, P.P., Kumar, N.K.K. and Verghese, A. (eds) Advances in IPM for Horticultural Crops. Proceedings of the First National Symposium on Pest Management in Horticultural Crops: environmental implications and thrusts, Bangalore, India, 15–17 October 1997, pp. 30–31.



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Hall, D.G., Hentz, M.G. and Adair, R.C. Jr (2008) Population ecology and phenology of Diaphorina citri (Hemiptera: Psyllidae) in two Florida citrus groves. Environmental Entomology 37, 914–924. Hall, D.G., George, J. and Lapointe, S.L. (2015) Further investigations on colonization of Poncirus trifoliata by the Asian citrus psyllid. Crop Protection 72, 112–118. He, F.M. (= Hoffmann, W.E.) and Zhou (Djou), Y.W. (1935) Notes on citrus pests. Lingnan Agricultural Journal 2(1), 165–218 [in Chinese with English summary by W.E. Hoffmann]. Hodkinson, I.D. (1974) The biology of the Psylloidea (Homoptera): a review. Bulletin of Entomological ­Research 74, 325–339. Hodkinson, I.D. and White, I.M. (1979) Homoptera Psylloidea. Handbooks for the Identification of British Insects. Volume 2, Part 5a. Royal Entomological Society, London. Hoffmann, W.E. (1936) Diaphorina citri Kuw. (Homoptera: Chermidae), a citrus pest in Kwangtung. Lingnan Science Journal 15, 127–132. Hollis, D. (1987) A new citrus-feeding psyllid from the Comoro Islands, with a review of the Diaphorina amoena species group (Homoptera). Systematic Entomology 12, 47–61. Hooker, J.D. (1875) The Flora of British India, Volume 1. L. Reeve, London, pp. 502–503. Huang, C.H., Liaw, C.F., Chang, L. and Lan, T. (1990) Incidence and spread of citrus likubin in relation to the population fluctuation of Diaphorina citri. Plant Protection Bulletin, Taiwan 32, 167–176. Hung, S.C. (2008) Ecology and vectorship of the citrus psyllid in relation to the prevalence of citrus huanglongbing. In: Ku, T.Y. and Pham, T.H.H. (eds) Proceedings of FFTC-PPRI-NIFTS Joint Workshop on Management of Citrus Greening and Virus Diseases for the Rehabilitation of Citrus Industry in the ASPAC, Plant Protection Research Institute, Hà Nội, Việt Nam, 8–12 September 2008, pp. 151–162. Hung, T.H., Wu, M.L. and Su, H.J. (2000) Identification of alternative hosts of the fastidious bacterium causing citrus greening disease. Journal of Phytopathology 148, 321–326. Husain, M.A. and Nath, D. (1927) The citrus psylla (Diaphorina citri, Kuw.) [Psyllidae: Homoptera]. Memoirs of the Department of Agriculture India, Entomology Series 10(2), 5–27; 1 plate. Johnston, N.S. (2018) Dispersal patterns of Asian citrus psyllid (Diaphorina citri Kuwayama) and secondary host interactions. Master’s thesis, University of Florida, Gainesville. Joseph, S. (2008) Curry leaf. In: Peter, K.V. (ed.) Underutilized & Underexploited Horticultural Crops, Volume 4. New India Publishing Agency, New Delhi, pp. 155–163. Joseph, S. and Peter, K.V. (1985) Curry leaf (Murraya koenigii), perennial, nutritious, leafy vegetable. Economic Botany 31, 68–73. Khan, K.M. and Borle, M.N. (1989) New host records for citrus psylla, Diaphorina citri from India. Bulletin of Entomology (New Delhi) 30(1), 118. Khan, K.M., Radke, S.G. and Borle, M.N. (1984) Seasonal activity of citrus psylla, Diaphorina citri, in the Vidarbha region of Maharashtra. Bulletin of Entomology, Loyola College 25(2), 143–146. Koizumi, M., Prommintara, M. and Ohtsu, Y. (1996) Wood apple, Limonia acidissima: a new host for the huanglongbing (greening) vector, Diaphorina citri. In: da Graça, J.V., Moreno, P. and Yokomi, R.K. (eds) Proceedings of the Thirteenth Conference of the International Organization of Citrus Virologists, Fuzhou, Fujian, China, 16–23 November 1995. International Organization of Citrus Virologists, University of California, Riverside, California, pp. 271–275. Koli, S.Z., Makar, P.V. and Choudhary, K.G. (1981) Seasonal abundance of citrus pests and their control. Indian Journal of Entomology 43(2), 183–187. Kong, Y.C., Ng, G.K.H., Wat, C.K.H. and But, P.P.H. (1986) Pharmacognostic differentiation between Murraya paniculata (L.) Jack and Murraya koenigii (L.) Spreng. International Journal of Crude Drug Research 24, 167–170. Kong, Y.C., But, P.P.H., Nguyen, K.H., Cheng, K.F., Chang, K.L., Wong, K.M., Gray, A.I. and Waterman, P.G. (1988) The biochemical systematics of Merrillia; in relationship to Murraya, the Clauseneae and the Aurantioideae. Biochemical Systematics and Ecology 16, 47–50. Kubitzki, K., Kallunki, J.A., Duretto, M. and Wilson, P.G. (2011) Rutaceae. In: Kubitzki, K. (ed.) The Families and Genera of Vascular Plants: Flowering Plants. Eudicots. Springer-Verlag, Berlin Heidelberg, pp. 276–356. Kuwayama, S. (Satoru) (1931) A revision of the Psyllidae of Taiwan. Insecta Matsumarana 5, 117–133. Kuwayama, S. (Shigeru) (1908) Die psylliden Japans. I. Transactions of the Sopporo Natural History ­Society 2, 149–189. [D. citri: p. 160–161, Plate III, Fig. 16.] Lal, M.M. (1917) Report of the Assistant Professor of Entomology. Report on the Operations of the Department of Agriculture, Punjab. In: Marshall, G.A.K. (ed.) (1920) Review of Applied Entomology, Series A: Agricultural 8, 109.



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Lal, M.M. (1918) Report of the assistant professor of entomology. Report of the Department of Agriculture Punjab for the year ended 30 June 1918: Appendix iv, p. viii. (Abstract: Review of Applied Entomology, 1920, Volume VIII, p. 109. Imperial Bureau of Entomology, London.) Lewis-Rosenblum, H., Martini, X., Tiwari, S. and Stelinski, L.L. (2015) Seasonal movement patterns and long-range dispersal of Asian citrus psyllid in Florida citrus. Journal of Economic Entomology 108, 3–10. Li, Q., Zhu, L.F., But, P.P.H., Kong, Y.C., Chang, H.T. and Waterman, P.G. (1988) Monoterpene and sesquiterpene rich oils from the leaves of Murraya species: chemotaxonomic significance. Biochemical Systematics and Ecology 16, 491–494. Li, T., Cheng, C.Z., Deng, C.J. and Chen, Z.F. (2007) Detection of the bearing rate of Liberobacter asiaticum in citrus psylla and its host plant. Acta Agriculturae Universitatis Jiangxiensis 29(5), 773–745. Lim, W.H., Shamsudin, O.M. and Ko, W.W. (1990a) Citrus greening disease and alternate hosts of the vector, Diaphorina citri Kuw., in P. Malaysia. MAPPS Newsletter (The Newsletter of the Malaysian Plant Protection Society) 13(4), 56–58. Lim, W.H., Shamsudin, O.M. and Ko, W.W. (1990b) Citrus greening disease in Malaysia: status report. In: Aubert, B., Tontyaporn, S. and Buangsuwon, D. (eds) Proceedings of the Fourth International Asia Pacific Conference on Citrus Rehabilitation, Chiang Mai, Thailand, 4–10 February 1990. FAO UNDP, Rome, pp. 100–105. Mabberley, D.J. (1997) A classification for edible citrus. Telopea 7, 167–172. Mabberley, D.J. (1998) Australian Citreae with notes on other Aurantioideae (Rutaceae). Telopea 7, 333–344. Mabberley, D.J. (2004) Citrus (Rutaceae): a review of recent advances in etymology, systematics and medical applications. Blumea 49, 481–498. Mabberley, D.J. (2016) Proposal to conserve the name Chalcas paniculata (Murraya paniculata) (Rutaceae) with a conserved type. Taxon 65, 394–395. Mabberley, D.J. (2017) Mabberley’s Plant Book: A Portable Dictionary of Plants, Their Classification and Uses, 4th Edition. Cambridge University Press, Cambridge, UK. Maki, M. (1915) Namiki oyobi Kanshôyô-Shokubutsu no Jûyô Gaichu ni kwansura Chôsa [Investigations on the principal insect pests of avenue and ornamental plants]. Ringyô Shienjô Tokubetsu Hôkoku [Special Report of the Forest Experiment Station, Government of Formosa] 1: 112 + 29 pp., 18 pls [reference to Diaphorina citri and/or Murraya paniculata on pp. 36–38, Pl. VIII]. Martini, X., Addison, T., Fleming, B., Jackson, I., Pelz-Stelinski, K. and Stelinski, L.L. (2013) Occurrence of Diaphorina citri (Hemiptera: Liviidae) in an unexpected ecosystem: the Lake Kissimmee State Park Forest, Florida. Florida Entomologist 96, 658–660. Martini, X., Pelz-Stelinski, K.S. and Stelenski, L.L. (2016) Factors affecting the overwintering abundance of the Asian citrus psyllid (Hemiptera: Liviidae) in Florida citrus (Sapindales: Rutaceae) orchards. Florida Entomologist 99, 178–186. Mathur, R.N. (1975) Psyllidae of the Indian Subcontinent. Indian Council of Agricultural Research, New Delhi. Miles, G.P., Stover, E., Ramadugu, C., Keremane, M.J. and Lee, R.F. (2017) Apparent tolerance to huanglongbing in Citrus and Citrus-related germplasm. HortScience 52, 31–39. Miyatake, Y. (1965) Notes on Psyllidae from Ryukyu Islands (Hemiptera: Homoptera). Kontyu 33, 171–189. Morton, C.M. and Telmer, C. (2014) New Subfamily Classification for the Rutaceae. Annals of the Missouri Botanical Garden 99, 620–641. Nehru, R.K., Bhagat, K.C. and Koul, V.K. (2004) Influence of citrus species on the development of Diaphorina citri. Annals of Plant Protection Science 12(2), 436–438. Nguyen, H.C. (2011) Circumscription of Murraya and Merrillia (Sapindales: Rutaceae: Aurantioideae) and susceptibility of species and forms to huanglongbing. PhD thesis, University of Western Sydney, Australia. Om, N. (2017) The roles of psyllids, host plants and environment in the aetiology of huanglongbing in Bhutan. PhD thesis, Western Sydney University, Australia. Osman, M.S. and Lim, W.H. (1992) Studies on vector distribution, etiology and transmission of greening disease of citrus in P. Malaysia. In: Setyobudi, L., Bahar, F.A., Winarno, M. and Whittle, A.M. (eds) Proceedings of Asian Citrus Rehabilitation Conference, Malang, Indonesia, 4–14 July 1989. Ministry of Agriculture, Republic of Indonesia Agency for Agricultural Research and Development. FAO UNDP INS/84/007, pp. 157–165. Osman, M.S. and Quilici, S. (1991) Trapping studies of citrus greening vector, Diaphorina citri Kuway., natural enemies and alternate hosts in Malaysia. In: Ke, C. and Osman, S.B. (eds) Proceedings of the Sixth International Asia Pacific Workshop on Integrated Citrus Health Management, Kuala ­Lumpur, Malaysia, 24–30 June 1991, pp. 118–127.

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Pande, Y.D. (1972) Seasonal fluctuations in the abundance and host preference of Diaphorina citri Kuw. in relation to certain species of Citrus. Indian Journal of Agricultural Research 6(1), 51–54. Patil, A.V., Kahare, N.P. and Raut, B.R. (1972) Screening the genetic stock of different varieties of citrus root-stocks against citrus leaf-miner and citrus psylla. Pesticides 6, 48–50. Peña, J.E., Mannion, C.M., Ulmer, B.J. and Halbert, S.E. (2006) Jackfruit, Artocarpus heterophylus, is not a host of Diaphorina citri (Homoptera: Psyllidae) in Florida. Florida Entomologist 89, 412–413. Ramadugu, C., Keremane, M.L., Halbert, S.E., Duan, Y.P., Roose, M.L., Stover, E. and Lee, R.F. (2016) Long-term field evaluation reveals huanglongbing resistance in Citrus relatives. Plant Disease 100, 1858–1869. Ramakrishna Avyar, T.V. (1924) List of Psyllidae recorded from India and Ceylon. Records of the Indian Museum 26, 621–625. Rao, K.R. and Pathak, K.A. (2001) Field evaluation of indigenous germplasm of citrus against insect pests. Indian Journal of Hill Farming 14(2), 117–119. Richardson, M.L. and Hall, D.G. (2013) Resistance of Poncirus and Citrus × Poncirus germplasm to the Asian citrus psyllid. Crop Science 53, 183–188. Roxburgh, W. (1832) Flora Indica: Descriptions of Indian Plants, Vol. II. W. Thacker and Co., Serampore, pp. 374–375, Plate 48. Sandoval, J.L. (2009) Host range study of the Asian citrus psyllid, Diaphorina citri. In: Seventh Annual Texas A&M University System Pathways Student Research Symposium, Texas A&M International University, Laredo, Texas, 13–14 November 2009. http://www.tamiu.edu/pathways/documents/ PATHWAYSONLINEPROGRAM_000.pdf. Sétamou, M., Simpson, C.R., Alabi, O.J., Nelson, S.D., Telagamsetty, S. and Jifon, J.L. (2016) Quality matters: influences of citrus flush physicochemical characteristics on population dynamics of the Asian citrus psyllid (Hemiptera: Liviidae). PLOS ONE. doi: 10.1371/journal.pone. 0168997. Shivankar, V.J., Rao, C.N. and Shyam Singh (2000) Studies on citrus psylla, Diaphorina citri Kuwayama: a review. Agricultural Research Communication Centre, Karnal, India. Agricultural Reviews 21(3), 199–204. Singh, A.B. and Nimbalkar, M.R. (1977) Murraya koenigii L. and Murraya paniculata L. – preferable hosts of Diaphorina citri Kuway. Science and Culture 43(2), 97–98. Swingle, W.T. and Reece, R.C. (1967) The botany of Citrus and its wild relatives. In: Reuther, W., Webber, H.J. and Batchelor, L.D. (eds) The Citrus Industry. Volume I: History, World Distribution Botany, and Varieties. Division of Agricultural Sciences, University of California, Berkeley, California, pp. 190–430. Thomas, D.B. (2011) Host plants of psyllids in South Texas. In: Proceedings of the 2nd International Research Conference Huanglongbing, Orlando, Florida, 10–14 January 2011, p. 64. Thomas, D.B. and De Leon, J.H. (2011) Is the Old World fig, Ficus carica L. (Moraceae), an alternative host for the Asian citrus psyllid, Diaphorina citri (Kuwayama) (Homoptera: Psyllidae)? Florida Entomologist 4, 1081–1083. Tirtawidjaja, S. (1981) Insect, dodder and seed transmissions of citrus vein phloem degeneration (CVPD). In: Matsumoto, K. (ed.) Proceedings of the Fourth International Society Citriculture Congress, Tokyo, Japan, 9–12 November 1981. International Society of Citriculture, Riverside, California, pp. 1: 469–471. Tsagkarakis, A.E. and Rogers, M.E. (2010) Suitability of ‘Cleopatra’ mandarin as a host plant for Diaphorina citri (Hemiptera: Psyllidae). Florida Entomologist 93, 451–453. Tsai, J.H. and Liu, Y.H. (2000) Biology of Diaphorina citri (Homoptera: Psyllidae) on four host plants. Journal of Economic Entomology 93, 1721–1725. Tsai, J.H., Wang, J.J. and Liu, Y.H. (2002) Seasonal abundance of the Asian citrus psyllid, Diaphorina citri (Homoptera: Psyllidae) in southern Florida. Florida Entomologist 85, 446–451. Tsai, Y.P., Hwang, M.T. and Wang, H.C. (1984) Diaphorina citri on Murraya paniculata. Plant Protection Bulletin (Taiwan) 26, 285–287. Udell, B.J., Monzo, C., Paris, T.M., Allan, S.A. and Stansly, P.A. (2017) Influence of limiting and regulating factors on populations of Asian citrus psyllid and the risk of insect and disease outbreaks. Annals of Applied Biology 171, 70–88. Viraktamath, C.A. and Bhumannavar, B.X. (2001) Biology, ecology and management of Diaphorina citri Kuwayama (Hemiptera: Psyllidae). Pest Management in Horticultural Ecosystems 7(1), 1–27. Waterhouse, D.F. (1998) Biological Control of Insect Pests: Southeast Asian Prospects. ACIAR Monograph Series No. 51. Australian Centre for International Agricultural Research, Canberra. Westbrook, C.J., Hall, D.G., Stover, E. and Duan, Y.P. (2011) Colonization of Citrus and Citrus-related germplasm by Diaphorina citri. HortScience 46, 997–1005.



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Xu, C.F., Xia, Y.H., Li, K.B. and Ke, C. (1988) Preliminary study on the bionomics of Diaphorina citri ­Kuwayama the vector of the citrus huanglungbin disease. In: Aubert, B., Ke, C. and Gonzales, C. (eds) Proceedings of the Second Asian/Pacific Regional Workshop on citrus greening, Lipa, ­Philippines, 20–26 November 1988. UNDP-FAO, Rome, pp. 29–31. Yang, Y.P., Huang, M.D., Beattie, G.A.C., Xia, Y.L., Ouyang, G.C. and Xiong, J.J. (2006) Distribution, biology, ecology and control of the psyllid Diaphorina citri Kuwayama, a major pest of citrus: a status report for China. International Journal of Pest Management 52, 343–352. Yang, Y.P., Beattie, G.A.C., Spooner-Hart, R.N., Huang, M.D., Barchia, I. and Holford, P. (2013) Influences of leaf age and type, non-host volatiles, and mineral oil deposits on the incidence, distribution, and form of stylet tracks of Diaphorina citri. Entomologia Experimentalis et Applicata 147, 33–49. Yasuda, K., Kawamura, F. and Oishi, T. (2005) Location and preference of adult Asian citrus psyllid, Diaphorina citri (Homoptera: Psyllidae) on Chinese box orange jasmine, Murraya exotica L. and flat lemon, Citrus depressa. Japanese Journal of Applied Entomology and Zoology 49, 146–149. Zhang, D.X., Hartley, T.G. and Mabberley, D.J. (2008) Rutaceae. In: Wu, Z.Y., Raven, P.H. and Hong, D.Y. (eds) Flora of China: Vol. 11 (Oxalidaceae through Aceraceae). Science Press, Beijing; Missouri Botanical Garden Press, St Louis, Missouri.

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Abiotic and Biotic Regulators of the Asian Citrus Psyllid Populations Jawwad A. Qureshi* University of Florida, Department of Entomology and Nematology, Institute of Food and Agricultural Sciences, Southwest Florida Research and Education Center, Immokalee, Florida, USA

The Asian citrus psyllid, Diaphorina citri Kuwayama, attacks citrus in several tropical and subtropical regions of the world. Its spread to these regions indicates that it has potential to survive a wide range of environmental conditions and establish in newly invaded areas. Many abiotic and biotic factors impact its colonization, development and establishment through effects on its biology, ecology and management. The abiotic factors such as temperature, humidity, light and rainfall not only have direct impact on D. citri and the biotic drivers of its populations but also play a very important role indirectly through effects on the host plants, which then affects both the pest and its natural enemies.

6.1  Abiotic Factors 6.1.1 Temperature Insects are poikilothermic, which means body temperatures are at the mercy of ambient variation. Within the range of survivable temperatures there is a narrow ideal range and a wider favorable range, beyond which certain adaptations allow them to survive. Thus, there are always higher and lower limitations to what insects can tolerate. These responses are also not static

and depend upon previous conditioning (Neven, 2000). Temperature affects all demographic parameters, including survivorship of all life stages, development rates of immatures and ­female fecundity. In regard to survivorship of D. citri adults, Hall and Hentz (2014) found 100% mortality at 41°C within 48 h although in the absence of a plant host. Similarly, Al-Riyami (2016) reported that adults held on plants at constant 43°C and 48°C were dead in about 2 h. He found that males held at 28°C, 33°C and 38°C lived for 52 ± 5, 24 ± 4 and 13 ± 2 days, respectively, and females for 40 ± 5, 31 ± 4 and 18 ± 1 days, respectively. These results were similar to those of Liu and Tsai (2000), who estimated average D.  citri female longevity as 35, 34 and 29 days at 28, 30°C and 33°C, respectively. Qureshi and Stansly (2009) reported loss of most nymphs followed during cohort studies after temperatures dipped below 0°C on 17 and 19 Feb 2007. However, Hall et  al. (2011) reported adult survival in Florida following freezing events of several hours of exposure to –8°C. Apparently, adult D. citri were cold acclimated following exposure to winter temperatures. Cool temperatures prolong survival to as long as 117 days at 15°C. In a study of different stresses on gene expression of D. citri adults, it was indicated that exposure to 42ºC for 3 h or less caused an increase in the transcriptional

*  Email: [email protected]

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­ctivity of their heat-shock gene, hsp 70 a (Marutani-­Hert et  al., 2010). That study suggested a possible response from D. citri when exposed to higher temperature extremes for short time intervals as occurs with fluctuating natural temperature which could condition them to more prolonged exposure to higher temperatures. Survivorship from egg to adult increased from 61.9% at 15°C to 83.9% at 28°C, then decreased to 73.7% at 30°C (Liu and Tsai, 2000). Greatest mortality occurred at the first instar. Al-Riyami (2016) observed average survivorship from egg to adult of 80 ± 7% and 63 ± 15% at constant 28°C and 33°C respectively. Temperatures of constant 43°C and 48°C killed nymphs in < 2 h and burned the plants. He observed nymphal survival of 21% through fourth instar at constant 38°C, although none developed to adulthood. The few that developed to adulthood at a 38°C (14 h) 33°C (10 h) cycle were deformed. Nava et  al. (2010) reported that first-­ instar nymphs failed to develop to the second instar at constant 32.5°C. At the other extreme, most nymphs survived exposure of several hours to –5°C to –6°C in the field (Hall et al., 2011). Average developmental time from egg to adult decreased from 49.3 days at 15°C to 14.1 days at 28°C, increasing to 16.3 days at 30°C (Lui and Tsai, 2000). Skelley and Hoy (2004) found that nymphs developed and emerged as adults in an average of 15 days at 27 ± 2.5°C. In Japan under laboratory conditions, it took D. citri nymphs 36.3 and 16.8 days to reach adulthood at temperatures of 15°C and 32.5°C, respectively (Nakata, 2006). Developmental time from first-instar nymph to adult at 28°C and 33°C averaged 12.6 ± 0.4 and 14.3 ± 0.3 days, respectively. An average generation time of 28.6 days was reported at a temperature of 28°C (Liu and Tsai, 2000). Female fecundity increased with increasing temperature from 15°C to 28°C and then decreased from 30°C to 33°C (Liu and Tsai, 2000). Hall et al. (2011) observed peak oviposition over 48 h at 29.6°C but none at a constant 38°C. Maximum lifetime fecundity of D. citri females at 25°C varied from 572 eggs on rough lemon to 858 eggs on grapefruit (Tsai and Liu, 2000) and a peak of 748 eggs at 28°C on seedlings of Murraya paniculata (L.) Jack (Liu and Tsai, 2000). A complete cessation of oviposition after 5 days ­exposure to 34°C was observed by Skelley and Hoy

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(2004). Oviposition was resumed in 2–3 weeks after the temperature was reduced. At the lower extreme, a threshold of 15°C was reported by Liu and Tsai (2000). By all measures, then, 28°C is the ideal temperature for D. citri. This trend is clearly seen through life-table analysis. Indeed, Liu and Tsai (2000) reported maximum jackknife estimates of the intrinsic rate of increase (per capita rate of population growth) and net reproductive rate and minimum values for mean generation time and doubling time at this temperature. More detailed discussion on effects of temperature is provided in Chapter 1. Al-Riyami (2016) observed no reproduction at a constant 38°C although fecundity and viability improved when temperature was reduced by 5°C for 10 h. This is more representative of field conditions where fluctuations in temperature are normal during different times of the day and even within tree canopies. Therefore, care must be taken when attempting to translate laboratory results to field conditions. Temperature probably plays a key role in the geographic distribution and establishment of D. citri. D. citri was not found at elevations above 1300–1500 m in Asia, possibly due to occasional frost events (Aubert, 1987). In Florida, where winters are generally mild with the possibility of occasional freeze events and summers are hot and humid, D. citri is well established. In Saudi Arabia, D. citri has been found within elevations of 1500 m in which temperatures hardly reach 34°C in summer and drop to 2.5°C in winter. Surprisingly, it has been reported that D. citri cannot survive in very hot and dry regions of Saudi Arabia such as Medina, where the temperature exceeds 45°C during the day time in summer (Aubert, 1990). Thus, contrary to laboratory studies, D. citri must be able to tolerate temperatures between the two ranges of 34°C and 45°C (Aubert, 1990), which could be due to the difference in heat tolerance extremes of different populations. This indicates that D. citri may be able to acclimate to the high temperatures exceeding 40°C. 6.1.2 Humidity Moisture requirements of an insect play a critical role in determining its establishment in a

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r­egion. McFarland and Hoy (2001) observed ­increased survival of D. citri with increasing humidity. The investigation on the combined ­ ­effects of humidity and temperature concluded that D. citri survival was low at high temperature and humidity, 34ºC and 82–92%, and high at moderate temperature and humidity, 20–30ºC and 43–75%, respectively (Yang, 1989). D. citri seems to be surviving successfully under high temperature and high humidity conditions in Florida. However, high humidity is also suitable for growth of entomopathogenic fungi that kill nymphs and adults of D. citri (Aubert, 1987).

6.1.3 Light Visual cues play an important role in host colonization behavior of D. citri and are influenced by light. D. citri are oligophagous insects utilizing some rutaceous plants for their nutritional needs and their responses to host plant orders were shown to be dependent on the presence of light (Wenninger et al., 2009). The positive phototaxis of D. citri adults was associated with short wavelengths (ultraviolet (UV), 350–405 nm, Paris et al., 2015, 2017). Psyllids are more abundant on southeastern sides of the groves and tree canopies that are more illuminated than others, suggesting strong D. citri phototaxis response to light (Sétamou et  al., 2008, 2012). Daytime capture of D. citri on yellow sticky traps was more than the capture during the night and peaked between 1200 h and 1500 h (Sétamou et al., 2012), followed by increased plant colonization and egg deposition in light. Such strong attraction to light can be exploited to enhance management of D. citri; for example: (i) planting young citrus on UV-reflective mulches which disorient psyllids from plant colonization (Croxton and Stansly, 2014); and (ii) intensifying spray applications and biological control tactics on preferred colonization sites in the borders of the groves or blocks, which could also be protected by planting windbreaks to reduce psyllids coming into groves.

r­eproduction. Aubert (1987) stated that low populations of D. citri were associated with monthly rainfall of 150 mm or more, due to nymphs being dislodged from foliage. However, Michaud (2004) did not observe this occurring in Florida in the course of cohort studies during some months when rainfall exceeded this amount. Nevertheless, populations of natural enemies such as ladybeetles and other general predators known for significant suppression of D. citri populations may also be reduced in the citrus groves after heavy rains (Qureshi and Stansly, 2009, 2010). In Florida, the typical pattern of shoot production in mature citrus trees begins with a major flush in late winter or early spring, a lesser flush in the early summer and minor flushes during late summer and fall, followed by a relatively dormant winter season in late fall and early winter with little or no new foliage growth (Hall and Albrigo, 2007; Qureshi et al., 2009). Unusual rain events during winter and excessive rains during summer promote undesired new growth, which helps psyllids to reproduce at higher rates than normal for the season and increase their populations.

6.1.5 Wind Wind speeds of greater than 0.5 m/s inhibit flight of D. citri (Aubert and Hua, 1990). Kobori et al. (2011) reported that dispersal was generally downwind, although Lewis-Rosenblum et al. (2015) found no correlation between wind direction and capture of marked D. citri. Hall and Hentz (2011) did not observe correlation between wind speed and adult captures on traps placed distant from citrus trees. Gottwald et  al. (2007) hypothesized that wind assisted dispersal of 90–145 km. However, D. citri are typically found on citrus in abundance even after defoliation by hurricanes, as indicated by rapid recolonization of the resulting induced flush (Albrigo et  al., 2005). Under laboratory conditions they were able to fly continuously up to 2.4 km without wind assistance (Martini et al., 2014).

6.1.4 Rainfall 6.1.6  Crop season There may be direct effects of rainfall on psyllid populations in addition to indirect effects related to new growth needed for development and

D. citri requires young shoots containing feather stage to recently expanded tender leaves to



Abiotic and Biotic Regulators of the Asian Citrus Psyllid Populations

­ evelop and reproduce. Adults need these tissues d to deposit eggs and nymphs to develop to adulthood. Citrus tree phenology plays a very important role in this life cycle of D. citri. During the dry winter months in Florida from November to December, mature trees produce few or no new shoots, which limits psyllid adults from reproducing, thus contributing to a reduction in psyllid populations (Hall and Albrigo, 2007; Qureshi et al., 2009). Adults must survive on mature foliage or non-reproductive hosts (Johnston, 2018) and most die before new flush is available. Additionally, this phenomenon provides an opportunity to target overwintering psyllid adults with foliar sprays of broad-spectrum insecticides such as pyrethroids and organophosphates. During this dormant season, canopies are sparse, which helps sprays to penetrate for maximum coverage. Furthermore, beneficial insects are not common, due to absence of prey such as immatures of psyllids or aphids, which develop on new growth. Thus, dormant winter sprays help reduce psyllids and conserve beneficial insects, both of which contribute to further reduction in psyllid populations during the growing season starting in spring. Generally, two dormant sprays (one each in November and January) are considered enough in Florida. However, it may be necessary to add another dormant spray during warm winters with unexpected rain events and flush. Mature trees produce major flush in the growing season in spring, followed by additional flushes during summer and fall which help psyllids to reproduce and increase their populations. Flush management to train trees to produce synchronized flushes and target sprays before the onset of the flush following the tactic of dormant sprays helps to reduce psyllids by disrupting their ability to reproduce. Biological control provides ­additional reduction through feeding by the natural enemies on the psyllid immatures developing on new growth. Thus opportunities for managing psyllids during dormant and growing seasons help reduce psyllids and the spread of the disease.

6.2  Biotic Factors 6.2.1 Parasitoids Tamarixia radiata (an ectoparasitoid) and Diaphorencyrtus aligarhensis (an endoparasitoid) are the

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only two known primary parasitoids of psyllid nymphs. These species-specific parasitoids lay eggs on (T. radiata) or inside (D. aligarhensis) the body of the psyllid nymph. The larvae consume the body contents of nymphs, thus reducing the psyllid populations before they are able to spread the disease as adults. T. radiata dominates by direct competition if it oviposits within 5 days of D.  aligarhensis (Rohrig, 2010; Vankosky and Hoddle, 2017) and is considered the more efficient parasitoid (Tang, 1990). Through combined behavior of host feeding and parasitization, one T. radiata female is capable of destroying 500 nymphs (Skelley and Hoy, 2004). These parasitoids were first reported from the northern Indian subcontinent (Waterston, 1922; Husain and Nath, 1927; Shafee et al., 1975). These and later reports suggest that these parasitoids are common in the region of origin of D. citri. T. radiata has also been successfully introduced into several countries where it reduces psyllids through behaviors of host feeding and parasitization. Examples include Réunion (Aubert and Quilici, 1984), Saudi Arabia (Aubert, 1984), Mauritius (Quilici, 1986), Indonesia (Nurhadi, 1988), Taiwan (Chien et  al., 1989), Philippines (Gavarra et al., 1990), East Java, Guadalupe (Étienne et al., 2001), Florida (Skelley and Hoy, 2004) and California (Hoddle and Pandey, 2014). In Réunion, T. radiata parasitized 60–70% nymphs, whereas, D. aligarhensis parasitism did not exceed 20% (Aubert, 1987). T. radiata was credited with reducing D. citri densities sufficiently in Réunion to mitigate the impact of huanglongbing (HLB) (Aubert and Quilici, 1984). From initial limited releases of T. radiata in Florida from Taiwan and South Vietnam populations, establishment of the parasitoid was confirmed and parasitism rates averaging less than 20% were observed during spring and summer from several citrus-­ producing regions of Florida (Qureshi et  al., 2009). Better parasitism rates were observed during fall at some locations, averaging 39% in September and 56% in November in the central and southwest regions, respectively. Hall and Rohrig (2015) reported that rates of parasitism by T. radiata in urban plantings of orange jasmine frequently exceeded 60%. Repeated releases of T. radiata in commercial groves in Florida showed that such releases provide additional control of psyllids where conventional and especially organic insecticides are used to c­ ontrol D. citri (Qureshi and Stansly, 2017). T. radiata

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was also observed contributing to psyllid mortality in other locations where no known releases were made, including Brazil (Torres et al., 2006), Argentina (Lizondo et  al., 2007), Venezuela (Cermeli et al., 2007), Mexico (De León and Sétamou, 2010), Puerto Rico (Pluke et  al., 2008) and Texas (French et al., 2001). Limited releases of D. aligarhensis adults from colonies established from Taiwan (2000– 2002) and over 11,000 from mainland China (2007–2009) were made in numerous counties throughout the citrus-growing regions of Florida (Skelley and Hoy, 2004; Rohrig et  al., 2012). However, very few were recovered and not enough to confirm establishment (Rohrig et  al., 2012). Factors including intense use of pesticides in commercial groves to control D. citri, availability of psyllid nymphs, competition with T. radiata, and predation of parasitized hosts by generalist predators may have contributed to reduced success with D. aligarhensis. However, both T. radiata and D. aligarhensis coexist in many parts of Asia and interact with the factors mentioned above. 6.2.2 Predators Generalist predators contribute significantly to psyllid mortality both in their region of origin and where psyllids invade. Husain and Nath (1927) reported coccinellids, chrysopids and syrphids as major groups of predators attacking D. citri nymphs from Punjab region of the Indian subcontinent. In Saudi Arabia, spiders comprised about 34% of the total predators that attack D.  citri (Al-Ghamdi, 2000). Species of arthropods such as a histerid beetle, Saprinus chalcites Illiger and the predaceous carabid, Egapola crenulata Dejean, were also considered important in Saudi Arabia (Al-Ghamdi, 2000). González et al. (2003) also reported on the significance of a similar predatory complex, including Coccinellidae, Chrysopidae and Syrphidae from Cuba. A study conducted soon after the invasion of D. citri in Florida did not see natural enemies as major contributors to psyllid mortality (Tsai et  al., 2002). However, within a few years of psyllid introduction in Florida, contributions of predator guilds started to become apparent. A significant increase in the native population of a ladybeetle, Olla v-nigrum Mulsant, was observed in response to psyllid populations (Michaud, 2001). Additional parallel work in Florida r­ eported a wide

range of predatory insects that fed on D. citri nymphs, including predators as diverse as lacewings (Neuroptera: Chrysopidae), spiders ­ (Aranae), and hoverflies (Diptera: Syrphidae) (Michaud, 2002). Michaud and Olsen (2003) examined the suitability of D. citri nymphs as prey for six coccinellid species: Curinus coeruleus Mulsant, Exochomus childreni Mulsant, Harmonia axyridis Pallas, O. v-nigrum Mulsant, Cycloneda sanguinea L., and Coelophora inaequalis (F.). The first five species developed successfully on the nymphal diet whereas the first four reproduced well. Qureshi and Stansly (2009) also observed significant negative impact of predators on psyllid populations employing exclusion techniques. Net reproductive rate (Ro) of D. citri was estimated to be 5–27-fold higher in the colonies protected from natural enemies compared with the unprotected colonies (Fig. 6.1). Maximum Ro ­estimated in June in caged colonies averaged 125–285 whereas all nymphs disappeared in the unprotected colonies. These findings suggested that biotic sources of mortality play a vital role in regulating the populations of D. citri. The ladybeetles O. v-nigrum, C. coeruleus, H. axyridis and C. sanguinea, the cockroach Blattella asahinai, lacewings, Ceraeochrysa sp. and Chrysoperla sp., and spiders were the most often encountered predators. Based on the evidence from different studies ladybeetles are by far the most important source of biotic mortality caused by predators to psyllid populations in Florida. However, ladybeetles were not observed as major contributors to D. citri mortality in California, but syrphids and green lacewings were (Kistner et al., 2017). 6.2.3 Entomopathogens Entomopathogenic fungi are another important source of biotic mortality to psyllid populations. The entomopathogenic fungus, Hirsutella near citriformis Speare, has been reported attacking D. citri (Rivero-Aragon and Grillo-Ravelo, 2000; Subandiyah et  al., 2000; Étienne et  al., 2001; Meyer et  al., 2007). Hall et  al. (2012) showed that naturally occurring populations of H. citriformis caused about 23% morality in the adult populations of D. citri in Florida during fall and winter seasons, with the maximum number of mummified adults sometimes exceeding 75% of the total number of adults observed. However, mummified cadavers were nearly absent during



Abiotic and Biotic Regulators of the Asian Citrus Psyllid Populations

Net reproductive rate (R0)

300

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Uncaged Caged

250 200 150 100 50

23

-J a 13 n -M a 24 r -A p 8- r M a 16 y -J un 3Ju l 31 -J u 21 l -A ug 18 -S e 16 p -O 13 ct -N o 4- v De c 5Ja 13 n -F e 13 b -M a 16 r -A pr 11 -M ay

0

Cohort initiation data (2006–2007) Fig. 6.1.  Net reproductive rate of Diaphorina citri calculated based on fecundity and survival of nymphs in colonies that were unprotected (uncaged) or protected (caged) from natural enemies to assess the impact of biotic mortality factors on psyllid populations in the citrus orchard.

spring, presumably due to low levels of relative humidity. Isaria fumosorosea (= Paecilomyces ­fumosoroseus) has also been observed infecting D.  citri at low levels in the USA (Meyer et  al., 2007, 2008; Hall et  al., 2008). Entomopathogenic fungi perform well at higher rates of relative humidity; for instance, I. fumosorosea caused more than 95% mortality of D. citri adults at 80–100% RH under laboratory conditions (Avery et al., 2009). Other species such as Lecanicillium (= Verticillium) lecanii Zimm. (Xie et  al., 1988; Rivero-Aragon and Grillo-Ravelo, 2000), Beauveria bassiana (Bals.) Vuill. (Rivero-Aragon and G ­ rillo-Ravelo, 2000), and Cladosporium sp. nr. oxysporum Berk. & Desm. (Aubert, 1987) have also been reported to infect D. citri. However, information on the phenology and impact of these fungal pathogens on D. citri populations in the field is warranted.

6.3 Management 6.3.1 Conservation Several studies have shown that natural mortality factors, particularly predators, parasitoids and pathogens, impose significant mortality on populations of D. citri. However, surviving ­populations of D. citri have proved sufficient to cause economically significant disease transmission, although the same can be said for chemical control, another tool critical for citrus protection

from D. citri and its transmission of CLas pathogens (Qureshi et al., 2014a). The intensive use of chemical control, particularly synthetic products, also comes with risks of elimination of biotic mortality, insecticide resistance and negative impact to the environment. Therefore, conservation of biotic mortality is warranted through use of selective insecticides, application methods and timing of insecticide application. Selective insecticide approaches include: (i) application of soil-applied systemic insecticides that avoid direct contact with natural enemies (Qureshi and Stansly, 2007, 2008); (ii) foliar sprays of insecticides directed mainly at adult psyllids, which are more vulnerable during periods of tree dormancy in winter when trees are not producing new growth and predators are largely absent from the groves (Stansly et al., 2009, 2010; Qureshi and Stansly, 2010); and (iii) sprays of selective insecticides such as horticultural mineral oil, lipid synthesis inhibitors and spinosyns (Stansly et al., 2002, Qureshi and Stansly, 2016, 2017). Foliar sprays of broad-spectrum insecticides applied to mature trees in winter were evaluated in a commercial citrus orchard as a tactic to reduce D. citri populations and insecticide use in spring and summer, when beneficial insects are most active (Qureshi and Stansly, 2010). A s­ ingle spray of chlorpyrifos, fenpropathrin, or oxamyl in January reduced adult psyllids by an average of 10–15-fold over 5–6 months compared with untreated trees (Fig. 6.2A (adults) and 6.2B (nymphs); results shown for chlorpyrifos).

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Adults per tap sample

(A)

4 3

Nymphs per 39 dm3

a

2

a

1 0

(B)

a Untreated Chlorpyrifos (2.8 kg/ha)

a

a ab

a b

ab

b

b

b

b

b

aa

300 a 225 150

a a

75 0

a

a aa

a

1/22

3/8

b

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ab 4/12

b 4/25

a

5/29

a

6/22

b

7/24

8/27

2007 Fig. 6.2.  Mean (±SEM) number of D. citri adults per tap sample (A) and nymphs per 39 dm3 of tree canopy (B) in Hamlin orange trees left untreated or treated with a foliar spray of chlorpyrifos on 15 January 2007 in a commercial grove in Immokalee, Florida. No other insecticides were used in the experimental blocks through August. Treated and untreated trees represented by columns with same letter for a particular date were not significantly different.

Spiders, lacewings and ladybeetles were equally abundant during the growing season in both treated and untreated trees (Fig. 6.3). Thus foliar sprays of broad-spectrum insecticides before spring growth suppressed D. citri into the growing season, with no detectable impact on key natural enemies. This tactic has been widely adopted for area-wide control of the psyllid in Florida (Stansly et  al., 2009, 2010). Additional sprays during the growing season are needed to reduce psyllids and transmission of HLB and these should be based on scouting and targeted at adult psyllids before the anticipated new flush. The tactic of limiting the use of broad-spectrum insecticides such as organophosphates and pyrethroids during the dormant winter season and reducing their use during the growing seasons also helps conserve some of the effective p ­ esticides which, if used repeatedly during the g ­ rowing season, face the risk of psyllid resistance. Potential options and supplements to conventional insecticides can be used to control psyllids and maintain citrus

production, even under conditions of high HLB incidence. For example, a program of organic insecticides sequentially mixed and alternated with horticultural mineral oil to control psyllids can provide similar or better yields than a conventional program in mature HLB-positive ‘Valencia’ oranges (Qureshi and Stansly, 2016, 2017).

6.3.2 Augmentation Inoculative releases of new strains or species of parasitoids and predators better suited to the t­ arget regions is another tactic to increase biological control through augmentation of natural populations to attack D. citri. In ­Florida, T. radiata colonies are maintained at the  ­Division of Plant ­Industry in Dundee and Gainesville for large-­scale production and maintenance of populations obtained from Taiwan, Vietnam, China and Pakistan (Qureshi et  al., 2012, 2014b). C ­ olonies from



Abiotic and Biotic Regulators of the Asian Citrus Psyllid Populations

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(A) Ladybeetles per tap sample

0.2 Untreated Chlorpyrifos (2.8 kg/ha)

0.15

0.1

0.05

0 (B)

Spiders per tap sample

0.6 0.45 * 0.3 0.15 0 (C)

Lacewings per tap sample

0.12

0.09

0.06

0.03

0.00 1/22

3/8

3/29

4/12

4/25

5/29

6/22

7/24

8/27

2007 Fig. 6.3.  Mean (± SEM) number of (A) ladybeetles, (B) spiders and (C) lacewings per tap sample in the Hamlin orange trees left untreated or treated with a foliar spray of chlorpyrifos on 15 January 2007 in a commercial grove in Immokalee, Florida. No other insecticides were used in the experimental blocks through August. Treated and untreated trees did not differ for any of the three predatory groups, except spiders on 1/22.

­ akistan populations have also been established in P California and Texas. Insectaries are producing millions of T. radiata and providing these to clientele for release in both commercial and non-commercial

environments. In southwest Florida, parasitism rates approaching 60–80% have been observed at release sites in spring and summer compared with < 20% at sites with no releases, showing that

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­ ugmentation can potentially increase parasitism a in the field (Qureshi et al., 2012). The intensive use of broad-spectrum insecticides negatively impacts the populations of the parasitoids and other natural enemies. T. radiata has been released regularly in blocks employing organic and synthetic insecticides intended for organic and conventional citrus production and in untreated blocks. Tamarixia has been recovered throughout the growing season in all programs, although more consistently in organic or untreated blocks compared with conventional blocks, and with parasitism rates exceeding 20%, with maximum parasitism in the range of 30–40% (Qureshi and Stansly, 2017). Predators observed to inflict 90% mortality to D. citri in ­Florida are now rare, due to the stepped-up use of insecticide following the introduction of HLB, and these are not available commercially. In an effort to augment the biological control of D. citri in Florida, certain citrus ­producers released several million commercially available Hippodamia convergens Guérin-­Méneville (Coleoptera: Coccinellidae) in citrus groves. This species was not common in ­Florida citrus and did not respond to the invasion of D. citri (Michaud, 2004; Qureshi and ­Stansly, 2008, 2009, 2010), but it was abundant in a study of coccinellids on citrus in Puerto Rico (Pluke et al., 2005). It is possible that this Puerto Rico population may be genetically distinct from those on the US mainland as a result of different selection pressures. The efficacy of this predator was investigated against D. citri, brown citrus aphid Toxoptera citricida Kirkaldy, and green citrus aphid Aphis spiraecola Patch, and each was found suitable for H.  convergens development and reproduction (Qureshi and Stansly, 2011). However, populations of this beetle did not increase in citrus groves ­following releases to augment its populations.

6.3.3  Commercial production and evaluation of biological control agents Predators, particularly ladybeetles that were common in Florida citrus and contributed

s­ ignificantly to the D. citri mortality (Michaud, 2004; Qureshi and Stansly, 2009, 2010), were reduced significantly with the intense use of insecticides to combat HLB (Qureshi and Stansly, 2017). These species are not available commercially and therefore there is a need for commercial production of not only these species but also others, including parasitoids. The present production sources of T. radiata in different states resulted from federal and state funding. There are some commercially available species of predators that warrant evaluation against D. citri. We evaluated Adalia bipunctata (L.) (Coleoptera: Coccinellidae) and found that it developed and reproduced successfully on a diet of D. citri (Qureshi et  al., 2013; Khan et  al., 2016). In the same study, significant reduction of D. citri nymphs averaging 54% was observed in colonies caged with adult A. bipunctata on field-­ planted citrus. Similarly, commercially available species of brown lacewing Sympherobius barberi (Neuroptera: Hemerobiidae) developed and reproduced on a diet of D. citri nymphs and provided 35% reduction in the psyllid populations when caged with the developing colonies of nymphs on field-planted citrus (Qureshi et al., 2012, 2013). A commercially available predatory mite Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae) also showed potential to impact D. citri through its ability to feed on the eggs and first-instar nymphs. Psyllid adult emergence from Murraya paniculata (L.) Jack plants infested with psyllid eggs and first-­ instar nymphs were reduced by 80% from plants caged with mites compared with the control plants without mites (Juan-Blasco et al., 2012). However, field application and evaluation of these commercially produced predators and entomopathogens such as I. fumosorosea and B. bassiana is still needed. Efforts are also warranted for commercial production of predators already shown to be significant in regulating psyllid populations in the field, particularly ladybeetles, but reduced due to increased use of ­insecticides.

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Al-Ghamdi, K.M.S. (2000) A field study on synchrony between the populations of citrus Psylla, Diaphorina citri (Kuwayama) [sic.] (Homoptera: Psyllidae) and its natural enemies in western Saudi Arabia. Bulletin of Faculty of Agriculture, Cairo University 51, 227–238. Al-Riyami, A.A. (2016) Tolerance and acclimation of Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) to high temperatures. MSc thesis. University of Florida, Gainesville, Florida. Aubert, B. (1984) The Asian and African Citrus psyllid Diaphorina citri Kuwayma, Trioza erytreae (­Del Guercio), (Homoptera Psyllidae) in the South West of Saudi Arabia. Proposals for an Integrated Control Programme. Report to the FAO (1984), p.1–28. Aubert, B. (1987) Trioza erytreae del Guercio and Diaphorina citri Kuwayama (Homoptera: Psylloidea), the two vectors of citrus greening disease: biological aspects and possible control strategies. Fruits 42, 149–162. Aubert, B. 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Hall, D.G., Wenninger, E.J. and Hentz, M.G. (2011) Temperature studies with the Asian citrus psyllid, Diaphorina citri: cold hardiness and temperature thresholds for oviposition. Journal of Insect Science 11, 83. Hall, D.G., Hentz, M.G., Meyer, J.M., Kriss, A.B., Gottwald, T.R. and Boucias, D.G. (2012) Observations on the entomopathogenic fungus Hirsutella citriformis attacking adult Diaphorina citri (Hemiptera: Psyllidae) in a managed citrus grove. BioControl. doi: 10.1007/s10526-012-9448-0 Hoddle, M.S. and Pandey, T. (2014) Host range testing of Tamarixia radiata (Hymenoptera: Eulophidae) sourced from the Punjab of Pakistan for classical biological control of Diaphorina citri (Hemiptera: Liviidae: Euphyllurinae: Diaphorinini) in California. Journal of Economic Entomology 107(1), 125–136. Husain, M.A. and Nath, D. (1927) The citrus psylla (Diaphorina citri, Kuw.) [Psyllidae: Homoptera]. Memoirs of the Department of Agriculture in India, Entomological Series 10, 1–27. Johnston, N.S. (2018) Dispersal patterns of Asian citrus psyllid (Diaphorina citri Kuwayama) and secondary host interactions. Master’s thesis, University of Florida, Gainesville, Florida. Juan-Blasco, M., Qureshi, J.A., Urbaneja, A. and Stansly, P.A. (2012) Predatory mite Amblyseius swirskii (Acari: Phytoseiidae) for biological control of Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae). Florida Entomologist 95, 543–551. Khan, A.A., Qureshi, J.A., Afzal, M. and Stansly, P.A. (2016) Two-Spotted Ladybeetle Adalia bipunctata L. (Coleoptera: Coccinellidae): a commercially available predator to control Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae). PLOS ONE 11(9), e0162843. doi: 10.1371/journal.pone.0162843. Kistner, E.J., Lewis, M., Carpenter, E., Melhem, N., Hoddle, C., Strode, V., Olivia, J., Castillo, M. and Hoddle, M.S. (2017) Digital video surveillance of natural enemy activity on Diaphorina citri (Hemiptera: Liviidae) colonies infesting citrus in the southern California urban landscape. Biological Control 115, 141–151. Kobori, Y., Nakata, T., Ohto, Y. and Takasu, F. (2011) Dispersal of adult Asian citrus psyllid, Diaphorina citri Kuwayama (Homoptera: Psyllidae), the vector of citrus greening disease, in artificial release ­experiments. Applied Entomology and Zoology 46, 27–30. Lewis-Rosenblum, H., Martini, X., Tiwari, S. and Stelinski, L.L. (2015) Seasonal movement patterns and long-range dispersal of Asian citrus psyllid in Florida citrus. Journal of Economic Entomology 108, 3–10. Liu, Y.H. and Tsai, J.H. (2000) Effects of temperature on biology and life table parameters of the Asian citrus psyllid, Diaphorina citri Kuwayama (Homoptera: Psyllidae). Annals of Applied Biology 137, 201–206. Lizondo, M.J., Gastaminza, G., Costa, V.A., Augier, L., Torres, M.L.G., Willink, E. and Parra, J.R.P. (2007) Records of Tamarixia radiata (Hymenoptera: Eulophidae) in Northwestern Argentina. Tomo 84(1), 21–22. Martini, X., Hoyte, A. and Stelinski, L.L. (2014) Abdominal color of the Asian citrus psyllid (Hemiptera: Leviidae) is associated with flight capabilities. Annals of the Entomological Society of America 107, 842–847. Marutani-Hert, M., Hunter, W. and Hall, D. (2010) Gene response to stress in the Asian citrus psyllid (Hemiptera: Psyllidae). Florida Entomologist 93, 519–525. McFarland, C.D. and Hoy, M.A. (2001) Survival of Diaphorina citri (Homoptera: Psyllidae), and its two parasitoids, Tamarixia radiata (Hymenoptera: Eulophidae) and Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae), under different relative humidities and temperature regimes. Florida ­Entomologist 84, 227–233. Meyer, J.M., Hoy, M.A. and Boucias, D.G. (2007) Morphological and molecular characterization of a Hirsutella species infecting the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), in Florida. Journal of Invertebrate Pathology 95, 101–109. Meyer, J.M., Hoy, M.A. and Boucias, D.G. (2008) Isolation and characterization of an Isaria fumosorosea isolate infecting the Asian citrus psyllid in Florida. Journal of Invertebrate Pathology 99, 96–102. Michaud, J.P. (2001) Numerical response of Olla v-nigrum (Coleoptera: Coccinellidae) to infestations of Asian citrus psyllid, (Hemiptera: Psyllidae) in Florida. Florida Entomologist 84, 608–612. Michaud, J.P. (2002) Biological control of Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae) in Florida: A preliminary report. Entomological News 113, 216–222. Michaud, J.P. (2004) Natural mortality of Asian citrus psyllid (Homoptera: Psyllidae) in central Florida. Biological Control 29(2), 260–269. Michaud, J.P. and Olsen, L.E. (2003) Suitability of Asian citrus psyllid, Diaphorina citri, as prey for ladybeetles. BioControl 49(4), 417–431. Nakata, T. (2006) Temperature-dependent development of the citrus psyllid, Diaphorina citri (Homoptera: Psylloidea), and the predicted limit of its spread based on overwintering in the nymphal stage in temperate regions of Japan. Applied Entomology and Zoology 41, 383–387.



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Nava, D., Gomez-Torres, M., Rodrigues, M., Bento, J., Haddad, M. and Parra, J. (2010) The effects of host, geographic origin, and gender on the thermal requirements of Diaphorina citri (Hemiptera: Psyllidae). Environmental Entomology 39, 678–684. Neven, L.G. (2000) Physiological responses of insects to heat. Postharvest Biology and Technology 21, 103–111. Nurhadi, F. (1988) Records of important parasites attacking Diaphorina citri in East Java, Indonesia. Regional Workshop on Citrus Greening Huanglungbing Disease, December 6–12, 1987. Paris, T.M., Croxton, S.D., Stansly, P.A. and Allan, S.A. (2015) Temporal response and attraction of Diaphorina citri to visual stimuli. Entomologia Experimentalis et Applicata 155, 137–147. Paris, T.M., Allan, S.A., Udell, B.J. and Stansly, P.A. (2017) Wavelength and polarization affect phototaxis of the Asian citrus psyllid. Insect 8, 88m. doi: 10.3390/insects8030088. Pluke, R.H., Escribano, A., Michaud, J.P. and Stansly, P.A. (2005) Potential impact of ladybeetles on Diaphorina citri (Homoptera: Psyllidae) in Puerto Rico. Florida Entomologist 88, 123–128. Pluke, R.W.H., Qureshi, J.A. and Stansly P.A. (2008) Citrus flushing patterns, Diaphorina citri (Hemiptera: Psyllidae) populations and parasitism by Tamarixia radiata (Hymenoptera: Eulophidae) in Puerto Rico. Florida Entomologist 91, 36–42. Quilici, S. (1986) Rapport de visite a` Maurice du 2 au 7 juin 1986. Doc. IRAT-Re´union, Institut de Recherches Agronomiques Tropicales, Re´union, France. Qureshi, J.A. and Stansly, P.A. (2007) Integrated approaches for managing the Asian citrus psyllid Diaphorina citri (Homoptera: Psyllidae) in Florida. Proceedings of the Florida State Horticultural Society 120, 110–115. Qureshi, J.A. and Stansly, P.A. (2008) Rate, placement, and timing of aldicarb applications to control Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae) in oranges. Pest Management Science 64, 1159– 1169. Qureshi, J.A. and Stansly, P.A. (2009) Exclusion techniques reveal significant biotic mortality suffered by Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae) populations in Florida citrus. Biological Control 50, 129–136. Qureshi, J.A. and Stansly, P.A. (2010) Dormant season foliar sprays of broad-spectrum insecticides: An effective component of integrated management for Diaphorina citri (Hemiptera: Psyllidae) in citrus orchards. Crop Protection 29(8), 860–866. Qureshi, J. A. and Stansly, P. A. (2011) Three homopteran pests of citrus as prey for the convergent ladybeetle Hippodamia convergens: Suitability and preference. Environmental Entomology 40(6), 1503–1510. Qureshi, J.A. and Stansly, P.A. (2016) Development of an Asian citrus psyllid management plan for o ­ rganic citrus. Citrograph 7(2), 52–58. Qureshi, J.A. and Stansly, P.A. (2017) Compatibility of Organic and Conventional Insecticides to Tamarixia radiata. Citrograph 8(3), 66–70. Qureshi, J.A. Rogers, M.E., Hall, D.G. and Stansly, P.A. (2009) Incidence of invasive Diaphorina citri (Hemiptera: Psyllidae) and its introduced parasitoid Tamarixia radiata (Hymenoptera: Eulophidae) in Florida citrus. Journal of Economic Entomology 102(1), 247–256. Qureshi, J.A., Rohrig, E.A. and Stansly, P.A. (2012) Introduction and augmentation of natural enemies for management of Asian citrus psyllid and HLB. Citrus Industry 93(6),14–16. Qureshi, J.A., Khan, A.A. and Stansly, P. A. (2013) Ladybeetles and lacewings for Asian citrus psyllid focused citrus pest management. Citrus Industry 94(9), 10–12. Qureshi, J.A., Kostyk, B.C. and Stansly, P.A. (2014a) Insecticidal suppression of Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae) vector of huanglongbing pathogens. PlosOne 9(12), e112331. Qureshi, J.A., Rohrig, E.A., Stuart, R.J., Hall, D.G., Leppla, N.C. and Stansly, P.A. (2014b) Imported parasitoids for biological control of Asian citrus psyllid. Citrus Industry 95(6), 10–13. Rivero-Aragon, A. and Grillo-Ravelo, H. (2000) Natural enemies of Diaphorina citri Kuwayama (Homoptera: Psyllidae) in the central region of Cuba. Centro-Agricola 27(3), 87–88. Rohrig, E.A. (2010) Biology and behavior of Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae) an endoparasitoid of Diaphorina citri (Hemiptera: Psyllidae). PhD thesis, University of Florida, Gainesville, 163 pp. Rohrig, E.A., Hall, D.G., Qureshi, J.A. and Stansly, P.A. (2012) Field release in Florida of Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae), an endoparasitoid of Diaphorina citri (Homoptera: Psyllidae), from mainland China. Florida Entomologist 95(2), 479–481. Sétamou, M., Flores, D., French, J.V. and Hall, D.G. (2008) Dispersion patterns and sampling plans for Diaphorina citri (Hemiptera: Psyllidae) in citrus. Journal of Economic Entomology 101,1478–1487.

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Sétamou, M., Sanchez, A., Patt, J.M, Nelson, S.D., Jifon, J., and Louzada, E.S. (2012) Diurnal patterns of flight activity and effects of light on host finding behavior of the Asian citrus psyllid. Journal of Insect Behavior 25, 264–276. Shafee, S.A., Alam, S.M. and Agarwal, M.M. (1975) Taxonomic survey of encyrtid parasites (Hymenoptera: Encyrtidae) in India. Aligarh Muslim University Publications (Zoological Series) of Indian Insect Types 10, 1–125. Skelley, L.H. and Hoy, M.A. (2004) A synchronous rearing method for the Asian citrus psyllid and its parasitoids in quarantine. Biological Control 29, 14–23. Stansly, P.A., Conner, J.M. and Brushwein, J.R. (2002) Control of citrus leafminer and Asian citrus psylla in sweet orange, 2001. Arthropod Management Tests 27, D10. Stansly, P.A., Arevalo, H.A., Zekri, M. and Hamel, R. (2009) Cooperative dormant spray program against Asian citrus psyllid in SW Florida. Citrus Industry 90, 14–15. Stansly, P.A., Arevalo, H.A. and Zekri, M. (2010) Area-wide psyllid sprays in Southwest Florida: An update on the cooperative program aimed at controlling the HLB vector. Citrus Industry 91, 6–8. Subandiyah, S., Nikoh, N., Sato, H., Wagiman, F., Tsuyumyu, S. and Fukatsu, T. (2000) Isolation and characterization of two entomopathogenic fungi attacking Diaphorina citri (Homoptera, Psylloidea) in Indonesia. Mycoscience 41, 509–513. Tang, Y.Q. (1990) On the parasite complex of Diaphorina citri Kuwayama (Homoptera: Psyllidae) in Asian-Pacific and other areas. Proceedings of the 4th International Conference on Citrus Rehabilitation. Chiang Mai, Thailand, 4–10 February 1990, p. 240–245. Torres, M.L., Nava, D.E., Gravena, S., Costa, V.A. and Parra, J.R.P. (2006) Registro de Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) em Diaphorina citri Kuwayama (Hemiptera: Psyllidae) em São Paulo, Brasil. Revista de Agricultura, Piracicaba - SP 81, 112–117. Tsai, J.H. and Liu, Y.H. (2000) Biology of Diaphorina citri (Homoptera: Psyllidae) on four host plants. Journal of Economic Entomology 93, 1721–25. Tsai, J.H., Wang, J.J. and Liu, Y.H. (2002) Seasonal abundance of the Asian citrus psyllid, Diaphorina citri (Homoptera: Psyllidae) in southern Florida. Florida Entomologist 85(3), 446–451. Vankosky, M.A. and Hoddle, M.S. (2017) An assessment of interspecific competition between two introduced parasitoids of Diaphorina citri (Hemiptera: Liviidae) on caged citrus plants. Insect Science doi.org/10.1111/1744-7917.12490. Waterston, J. (1922) On the Chalcidoid Parasites of Psyllids (Hemiptera, Homoptera). Bulletin of Entomological Research 13, 41–58. Wenninger, E.J., Stelinski, L.L. and Hall, D.G. (2009) Role of olfactory cues, visual cues, and mating status in orientation of Diaphorina citri Kuwayama (Hemiptera: Psyllidae) to four different host plants. Environmental Entomology 38, 225–234. Yang, Y.B. (1989) Influence of light, temperature and humidity on the development reproduction and survival of citrus psyllids. Chinese Journal of Ecology 9, 126–133. Xie, P.H., Su, C. and Lin, Z.G. (1988) A preliminary study on an entomogenous fungus [Verticillium lecanii] of Diaphorina citri Kuwayama (Hom.: Psyllidae). Chinese Journal of Biological Control 4(2), 92.

7 

Symbionts and Pathogens of the Asian Citrus Psyllid

Kirsten S. Pelz-Stelinski* Department of Entomology and Nematology, Citrus Research and Education Center, University of Florida, Lake Alfred, Florida, USA

7.1 Introduction Hemipterans are insects that have increased in agricultural importance during the past decade. These pests are a major threat to agriculture and food security due to their ability to transmit plant pathogens. Included in the numerous pathogens transmitted by this group are psyllid-­ transmitted bacteria in the genus Liberibacter. Huanglongbing (HLB), or citrus greening disease, is the most economically important disease of citrus worldwide (Halbert and Manjunath, 2004; Bové, 2006). ‘Candidatus Liberibacter asiaticus’ (CLas), one of three causal agents associated with HLB, is a phloem-limited alpha-­proteobacterium that is efficiently transmitted by its insect vector, the Asian citrus psyllid, Diaphorina citri Kuwayama. The designation ‘Candidatus’ indicates that the genus has not been cultured. D. citri acquires the pathogen while feeding on infected host plants. After ingestion, the pathogen must pass through the midgut into the hemolymph, where it replicates and disseminates throughout the insect. Once the pathogen reaches the salivary gland, it can be transmitted to susceptible host plants. The pathogen is transmitted to host plants with saliva during feeding (Roistacher, 1991; Pelz-Stelinski et  al., 2010; Ammar et  al., 2011, Chapter 8). The process of acquisition and

transmission of the pathogen requires the pathogen to overcome physical and immunological barriers in D. citri. In addition, microorganisms inhabiting the same tissues may also mediate vector–pathogen interactions. D. citri harbors a community of microorganisms that contribute to the metabolic needs, and thus the overall fitness, of its insect host. These microorganisms may also alter the transmission ­process indirectly by eliciting psyllid immune ­responses and/or directly by interacting with the pathogen. Intracellular symbionts may influence pathogen transmission by interfering with the survival and multiplication of pathogens in the psyllid. Thus, investigations of vector biology should consider reciprocal relations between the psyllid, the psyllid’s resident non-pathogenic ­microbial community, and the pathogen. Disruption of pathogen transmission through the manipulation of vector microbial communities offers an increasingly promising concept for integrated management of this pathosystem. Associations with microorganisms are common among insect species. The diversity of associations between D. citri and its community of microorganisms is more limited than those observed in many insects and consists predominantly of bacteria. Broadly speaking, the term symbiosis (‘living together’) refers to a relationship

*  Email: [email protected] © CAB International 2020. Asian Citrus Psyllid: Biology, Ecology and Management of the Huanglongbing Vector (eds. J.A. Qureshi and P.A. Stansly)

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in which organisms share a long-term, intimate association. The nature of the symbiotic relationship may range from mutualistic (both parties benefit) to commensalistic (one party benefits without affecting the other) or pathogenic (one party benefits at the cost of the other). Bacteria associated with members of the Sternorryncha, which include psyllids, aphids, mealybugs and whiteflies, are characterized as either primary or secondary symbionts. Primary, or obligate, symbionts are necessary for host survival. Insect symbionts have diverse and often profound effects on their host (Moran and Telang, 1998; Werren et  al., 2008; Engelstädter and Hurst, 2009; Feldhaar, 2011). To counter nutritionally incomplete diets, psyllids and other insects adapted by incorporation of mutualistic primary bacterial endosymbionts to supplement their nutritional needs (Douglas, 1998). In contrast, secondary symbionts are generally not required for host survival and reproduction, but instead may serve a variety of context-dependent functions. These symbionts are also referred to as facultative symbionts, as they may not occur in all individuals or necessarily contribute to host fitness. For instance, facultative symbionts may function in defense against parasitoids and pathogens (Oliver et al., 2002; Scarborough et al., 2005; Kambris et al., 2009; Moreira et al., 2009), protection from heat stress (Montllor et al., 2002), or broaden the range of suitable host plants (Feldhaar, 2011), but may also have negative effects on reproduction, longevity, or growth of their insect host (Rousset et al., 1992; Hurst et al., 1999; Werren et  al., 2008; Engelstädter and Hurst, 2009). Pathogenic or parasitic relationships occur when the symbiont benefits to the clear detriment of its host; however, the spectrum of interactions between ­organisms is such that clearly delineating a parasite from an incipient facultative association is an overly simplistic view of symbiotic associations (Pérez-Brocal et al., 2011).

7.2  Microorganisms Associated with D. citri 7.2.1  Microbial diversity Psyllids are generally phloem sap-feeding insects that rely on a diet largely deficient in amino acids

(Sandstrom and Moran, 1999). Phloem location and nutrient deficiency create an environment that supports a low diversity of bacteria (Jing et al., 2014). This contributes a correspondingly low diversity of microbiota among psyllids, including D. citri. Like humans, many insects possess diverse microbial communities throughout the digestive system and other body tissues. Establishment of these communities largely relies on acquisition of microorganisms from the environment during feeding. Thus, the sparsely populated phloem microbial community underlies the low diversity of microbiota observed in D. citri. 7.2.2  Primary and secondary ­endosymbionts Despite the lack of microbial diversity in D. citri, the phloem-feeding lifestyle contributes to uniquely strong associations with a few key microorganisms. To compensate for the dearth of nutrients in phloem sap (Sandstrom and Moran, 1999), D. citri incorporates mutualistic bacterial symbionts that supplement its nutritional needs. The gamma-proteobacteria ‘Candidatus Carsonella ruddii’ is defined as a primary endosymbiont and is housed within specialized insect cells known as bacteriocytes. Sequencing analysis of this non-culturable bacterium by ­Nakabachi et  al. (2006) demonstrated that its 0.17 Mb genome is small enough to consider the ­symbiont an ‘organelle-like’ structure with genes ­related to amino acid biosynthesis, including tryptophan and histidine, being more prominent than those considered essential. The absence of many genes essential for independent life renders Carsonella dependent on insect cells in a similar fashion to organelles. ‘Candidatus Proftella armature’ is a beta-­ proteobacterium that also resides within the D. citri bacteriocytes (Thao et al., 2000; Baumann, 2005; Nakabachi et al., 2013). Ca. P. armature is referred to as a secondary symbiont. Secondary symbionts are generally not required for host survival and reproduction, but instead may serve a variety of context-dependent functions. These symbionts are also referred to as facultative symbionts, as they may not occur in all individuals or necessarily contribute to host fitness. For instance, facultative symbionts may ­function in defense against parasitoids and pathogens,



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protection from heat stress, or broaden the range of suitable host plants, but may also have negative effects on the reproduction, longevity or growth of their insect host. Unlike many secondary, or facultative, symbionts, Ca. P. armature is universally present in D. citri, suggesting that it may be necessary for reproduction or survival. The 0.46 Mb reduced genome of Ca. P armature is comparable to the genome of Ca. C. ruddii, rendering it much smaller than the ­genomes of other facultative symbionts. The absence of essential amino acid synthesis genes indicates that any nutritional contribution by the endosymbiont would be a function of metabolic interplay between D. citri and its microbial community, rather than direct provisioning of amino acids. Around 15% of the Proftella genome contributes to the synthesis of the defensive polyketide, diaphorin, which may have antimicrobial, antiparasitic or antifungal properties (Nakabachi et  al., 2013). Diaphorin, a pederin analog extracted from D. citri, exhibits significant toxicity in pharmacological cell c­ulture assays. Genes encoding synthesis of the polyketide may have been horizontally transmitted between Proftella and a symbiont of Paederus rove beetles. To date, a biological function for diaphorin in the context of defense against predators or parasitoids has not been demonstrated. Localization of the primary and secondary endosymbionts is restricted to the U-shaped bacteriome, which consists of a unicellular bacteriocyte layer containing Ca. C. ruddii, surrounding a syncytium housing Ca. P. armature within the cytoplasm. Maintenance of endosymbionts within insect populations relies on transmission of symbionts between generations. Ca. C. ruddii and Ca. P. armature are maternally inherited through transovarial (vertical) transmission to offspring (Thao et  al., 2000). Endosymbiotic bacteria migrate to the oocytes and are transmitted through the cytoplasm of female germ cells. 7.2.3  Wolbachia D. citri also harbors a third intracellular symbiont, Wolbachia pipientis (Guidolin and Consoli, 2013; Dossi et  al., 2014; Hoffmann et  al., 2014). Wolbachia, an alpha-proteobacterium, is

characteristically transmitted through both vertical and horizontal transmission. Wolbachia, a notoriously well-known insect endosymbiont, occurs in approximately 40% of arthropod species (Zug and Hammerstein, 2012). Bacteria of the genus Wolbachia (Rikettsiales) are the most successful maternally transmitted intracellular ­bacteria (Hilgenboecker et  al., 2008) and are known to induce reproductive disorders, including sex-ratio distortion, in their hosts (Iturbe-­ Ormaetxe et al., 2011). These alterations usually ensure the maternal transmission of Wolbachia and include cytoplasmatic incompatibility (CI), induced parthenogenesis, feminization of genetic males, and male-killing (Werren et  al., 2008; van Houte et al., 2013). CI, a phenomenon where mating between same-species individuals with different Wolbachia strains or infection status fails to produce viable offspring, favors the reproduction of Wolbachia-infected females and promotes the spread of Wolbachia throughout a host population until the majority of individuals are infected. Wolbachia has been used to identify genetic differences between potato psyllids, Bacterocera erytrea, from different locations (Liu et al., 2006). Although the direct influence of Wolbachia on D. citri biology remains to be determined, the relative abundance of Wolbachia is likely associated with the abundance of CLas within hosts (Fagen et al., 2012; Hoffmann et al., 2014) and contributes to the regulation of phage lytic cycle genes in CLas (Jain et  al., 2017). A 56-amino-acid ­protein  produced by Wolbachia represses phage-­ induced lysis of CLas, putatively facilitating populations of both Wolbachia and CLas. Although the exact functions of Wolbachia on its host remain unclear, the relative abundance of Wolbachia in D. citri appears to be associated with CLas infection in the host insect (Fagan et al., 2012; Chu et al., 2016), suggesting that the abundance of Wolbachia may influence CLas acquisition or transmission, and that Wolbachia–D. citri interactions could be exploited for management of this pathosystem. In recent years, mounting evidence suggests that insect–endosymbiont interactions could be exploited for managing insect-transmitted diseases. Of particular interest are several strains of Wolbachia, which provide their host with protection against different types of infective agents and potentially reduce the transmission of insect-­borne pathogens/parasites under controlled conditions.

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Moreover, in some insects, Wolbachia-induced CI may also facilitate disease management by manipulating vector populations (Sinkins and Gould, 2006; Bourtzis, 2008; Brelsfoard et  al., 2009). High levels of Wolbachia (up to 26,000 copies of the Wolbachia outer surface protein gene wsp per copy of the D. citri wingless gene Wg) and a wide range of within-host Wolbachia densities occur in D. citri. A critical bacterial density is presumably necessary to elicit reproductive modifications, such as cytoplasmic incompatibility, in infected individuals (Noda et al., 2001). Environmental factors such as host gender (­Berticat et al., 2002), age (Unckless et al., 2009; ­Tortosa et  al., 2010), and temperature (Hurst et al., 2000; Mouton et al., 2006) are known to influence Wolbachia densities (Jaenike, 2009). Higher Wolbachia densities occur in males of D. citri compared with females, with an association between age and increased Wolbachia densities in female D. citri (Dossi et al., 2014). Several studies have investigated the genetic diversity of Wolbachia across different populations. Lashkari et  al. (2014) found that the Wolbachia wsp sequence was associated with host genetic diversity and hypothesized that Wolbachia may induce CI in its host. In general, studies are based on sequences of only one or two Wolbachia genes (wsp and ftsZ), but recent studies using multi-locus sequence typing (MLST) offer greater resolution of Wolbachia movement among D. citri populations. Geographic differences in Wolbachia densities could be influenced by environmental changes, such as temperature or insecticide exposure, or the diversity of host genotype and Wolbachia strain (Hurst et  al., 2001; Weeks et  al., 2002). Temperature-associated variation in Wolbachia densities within species may be further compounded by interactions between Wolbachia and host genotypes (Mouton et al., 2007). Diversity among Wolbachia types may occur as a result of genetic differences in host and symbiont (Jaenike, 2009). Boykin et  al. (2012) found eight haplotypes of D. citri, but only one of these was prevalent in Florida. Differences in Wolbachia diversity may be due to insertion sequences, which are recognized as major catalysts of endosymbiont genome divergence and have been found in all Wolbachia genomes to date (Duron, 2013). Characterization of the inter-population genetic

­diversity of Wolbachia among D. citri collected from different parts of the world indicates that co-infection of more than one Wolbachia strain may exist in D. citri individuals, and that the genetic diversity of the D. citri mitochondrial cytochrome oxidase I (mtCOI) sequences is associated with Wolbachia profile differences and with Wolbachia co-infection. Two dominant Wolbachia strains, ST-173 and ST-FL, occur in D. citri (Chu et  al., 2018). The distribution of each Wolbachia strain was not entirely restricted to specific geographic regions, which suggests that factors such as ­ human transportation may have played a major role in distributing D. citri (along with their Wolbachia strains) to various parts of the world. ST173 is the most prevalent strain infecting D. citri in many populations of Brazil (Guidolin and Consoli, 2013), an area located close to the ST-173-carrying Argentinean populations studied in this work. Previously, Saha et  al. (2012) showed that Wolbachia in Floridian D. citri populations belonged to a supergroup B sub-clade different from Chinese isolates and suggested that D. citri in Florida did not originate from China. Here, the data also showed clear distinction between the Florida (ST-FL) and Chinese (ST-173) Wolbachia allelic profiles, and that the ST-173 and ST-FL sequences were identical to those of the Florida and a Chinese sample from Beihai, respectively. Therefore, the data obtained from analysis of six Wolbachia genes not only supported previous findings, but also provided more robust support for the global distribution, diversity, and prevalence of major Wolbachia strains as compared with data from analysis of wsp and ftsZ alone. Co-infection of more than one dominant Wolbachia strain occurs among populations located in Thailand and the West Indies (Chu et al., 2018). Interestingly, the Wolbachia strains that the co-infected populations carry included ST173 and ST-FL, respectively. Since Thailand is located near China and Singapore (ST-173 areas), and the West Indies populations are distributed near ST-FL populations, it is plausible that D. citri in co-infected populations may have originated from their surrounding areas. Psyllids in the co-infected populations may have ­acquired additional Wolbachia strains as they moved into their current locations or have experienced changes in host–microbe interactions



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as a result of altered environments. Isopods are known to acquire different Wolbachia strains horizontally via brief blood/wound contacts with related species sharing the same ecological niches (Rigaud and Juchault, 1995) or different insect taxa may obtain exogenous strains during attack by predators and parasitoids (Werren et al., 1995). Among D. citri, populations carrying different Wolbachia strains have different m ­ tCOI, and samples with different haplotypes do not co-occur within the same population (Lashkari et  al., 2014; Chu et  al., 2018). Environmental factors, host genetics, or infection by other microbial species may also contribute to infection densities of Wolbachia strains (Goto et al., 2006; Mouton et al., 2006; Chu et al., 2016). It is possible that such interactions or factors may have occurred in some of the co-infected populations. Within populations, D. citri shares identical Wolbachia profiles. However, it is likely that other less prevalent Wolbachia profiles or mtCOI haplotypes may also exist within populations. A previous study of Brazilian D. citri populations found that, although the majority of individuals tested carried the same Wolbachia strain (ST-173), some individuals within the same populations could harbor different Wolbachia strains (Guidolin and Consoli, 2013). Previous surveys across Florida D. citri populations indicated that a very small proportion of D. citri individuals could be free of Wolbachia infection (Chu et  al., 2016), suggesting that intra-population variation in Wolbachia profiles could exist in other populations as well. Previous findings indicate that Wolbachia functions are dependent on infection levels (Breeuwer and Werren, 1993; Hurst et al., 2000; Unckless et  al., 2009); therefore, Wolbachia strains with higher within-host density may be more likely to have biologically relevant effects on the host. 7.2.4  Candidatus Liberibacter asiaticus Pathogenic or parasitic relationships occur when the symbiont benefits to the clear detriment of its host; however, the spectrum of interactions between organisms is such that clearly delineating a parasite from an incipient facultative association is an overly simplistic view of symbiotic ­associations (Pérez-Brocal et al., 2011). It is not clear where CLas and D. citri fall along this

s­pectrum of host–microbe interactions. The close relationship between these organisms is underscored by the fact that D. citri in Florida is now almost ubiquitously infected with the bacterium (Coy et al., 2014). CLas has both negative and positive effects on the life history of individual D. citri. CLas-infected psyllid adults do not survive as long as their uninfected counterparts; however, the number of eggs laid by CLas-infected females is significantly greater than by uninfected psyllids (Pelz-Stelinski and Killiny, 2016). The fecundity of D. citri infected with CLas, defined as the cumulative lifetime egg production per female, is higher than the fecundity of non-­ infected conspecifics. In addition, the mean egg production per female is significantly greater than that of non-infected females. Fertility, defined as the percentage of eggs that successfully hatch following oviposition, is not significantly affected by maternal infection status, despite the fact that it is numerically greater among eggs produced by non-infected females. The net effect of stage-specific survival indicates an overall increase in mortality when psyllids are infected with CLas (Pelz-Stelinski and Killiny, 2016). However, the net reproductive rate (R0 = Σ (lxmx), where R0 = number of female eggs per female per generation, lx = proportion of females alive at time (x), and mx = average daily number of eggs laid by females mated per treatment divided by 2 to compensate for the 1:1 sex ratio of progeny), and the finite rate of population increase (λ = R01/Σ x(lxmx)/Ro, where λ = number of females produced per female per day) indicate that the increased fecundity of CLas-infected D. citri overcomes losses due to mortality, resulting in net positive population growth. CLas is systemically distributed throughout infected D. citri, occurring in the filter chamber midgut, Malpighian tubules, hemolymph, salivary glands, ovaries, testis and other tissues (Ammar et al., 2011). CLas density is greatest in the organs and salivary glands but has a diffused distribution within the hemolymph. Horizontal acquisition from the environment (host plants) is the primary mode of CLas acquisition; however, D. citri also acquires the bacterium at low rates via transovarial and sexual transmission (Pelz-Stelinski et  al., 2010; Mann et  al., 2011). CLas present in the male genitalia can be passed to females by CLas-infested males. Horizontal transmission among members of the same sex is

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not observed, suggesting that copulation is necessary for transmission. Offspring from females exposed to CLas via infested males can inherit maternally acquired CLas. This may facilitate maintenance of the symbiotic association between the psyllid and CLas in the absence of suitable host plants. CLas also impacts the behavior of infected D. citri in several ways that contribute to the maintenance of CLas in D. citri populations. CLas infection induces release of a specific volatile chemical, methyl salicylate, which increases the attractiveness of infected plants to D. citri (Mann et  al., 2012). Behavior assays suggest that flight is initiated sooner and flight duration is greater when psyllids harbor CLas (Martini et al., 2015). Dispersal of CLas-positive D. citri to new host plants is similarly greater than the dispersal behavior of CLas-negative D. citri. ­ ­Furthermore, the attractiveness of the female to males evidently increases as her CLas titer increases. Therefore, highly infected females appear more likely to explore new hosts. Given the greater attractiveness of these females to males, there may be a greater chance not only to infect a new host, but also establish a new colony of D. citri that will become vectors of CLas following acquisition from infected plant tissue. 7.2.5  Commensal bacteria Several other bacteria have been reported in association with D. citri, including Arsenophonus from Indonesian isolates and an enteric bacterium from Florida isolates (Subandiyah et al., 2000; Saha et al., 2012). Among microbial communities in Florida D. citri populations, only the four eubacterial endosymbiont species discussed above are consistently detected among D. citri (Meyer and Hoy, 2008). In addition to the intercellular symbionts, D. citri may harbor a number of extracellular, gut-associated commensal bacteria. Unlike many insects, Sternorrhyncha such as D. citri do not possess complex gut microbial communities. When reared on citrus, they ­harbor bacteria that are characterized as citrus endophytes and insect symbionts, including Pantoea agglomerans and Alcaligenes xylosoxidans denitrificans (Kolora et al., 2015). Bacteria identified using culture and culture-dependent approaches included Bacillales species, Lysini bacillus sp.,

Paenibacillus sp., and Bacillus cereus, Staphylococcus saprophyticus, Streptomyces sp. (Gram positive), the Enterobacteriales, Enterobacter spp., Ralstonea spp., and Pantoea agglomerans, and the Pseudomonales, Pseudomonas putida, Chryseomonas luteola, Alcaligenes xylosoxidans denitrificans, and Acromobacter sp. (Gram negative). Sequences analysis of D. citri microbiota using the 454 Pyrosequencing platform indicated that the diversity of bacteria is greater in the presence of CLas than in its absence (Kolora et  al., 2015.) Although speculative, this may indicate that CLas presence alters host microbiota. This observation merits further exploration because it could be a key factor in CLas transmission success.

7.3  D. citri Immune System: ­Response to Microbial Invasion While endosymbionts are commonly found to exert metabolic influences through their associations, other functions may include effects on pathogen harboring and/or transmission. Thus, the role of psyllid endosymbionts in the ability of D. citri to transmit CLas is a fundamental question that requires further investigation. In general, the intrinsic and extrinsic factors contributing to transmission are poorly understood. A fundamental aspect of these interactions that requires investigation lies in understanding the role that the psyllid immune system and endosymbiont communities play in mediating transmission of CLas.

7.3.1  Immune system pathways in the D. citri genome Several lines of defense protect insects from invading pathogens. In addition to physical ­ ­barriers such as the outer cuticle and gut peritrophic membranes, insects may also possess obligate or facultative symbionts that assist in defense (Brownlie et al., 2009; Kaltenpoth, 2009; Oliver et al., 2010). As mentioned above, D. citri possess the defensive symbiont, Ca. Proftella armatura, whose genome encodes a polyketide toxin called diaphorin, which may have antimicrobial, antiparasitic or antifungal properties (Nakabachi et al., 2013). Endogenous insect bacterial



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microbiota can, in some cases, reduce the infection level of various insect parasitic organisms, including pathogens, by elevating the basal level of immunity in the insect host. These microbial infection barriers have been shown to inhibit transmission of disease-causing pathogens of Plasmodium falciparum and Dengue fever virus by their mosquito vectors (Hamilton and Perlman, 2013). Unlike vertebrates, insects lack an antibody-­ dependent, adaptive immune system to respond to current and future infections. Instead, they rely on an innate immune system that consists of several signaling pathways specifically triggered in response to invasion, depending on the type pathogen. Toll, Kak/Stat, and immune deficiency (IMD) pathways trigger antimicrobial peptide production, phagocytosis, melanization and encapsulation, thereby playing a role in defending against bacteria, fungi and some specific viruses (De Gregorio et  al., 2002; Agaisse et  al., 2003; Kemp et al., 2013). Genome annotation of D. citri identified most components of the Toll pathway, although recognition proteins were not identified, leaving the immune functionality of D. citri’s Toll pathway unknown (Arp et al., 2016, 2017). Similarities to Aphis pisum are apparent, including the absence of peptidoglycan recognition proteins (PGRPs) and components of the IMD pathway. The reduced immune capabilities observed in D. citri are similar to observations of immunity in A. pisum and Rhodnius prolixus. Altincicek et  al. (2008) proposed that the reduction observed in the A. pisum immune system is possibly a result of coevolution with endosymbiotic bacteria that may aid the insect by providing defense against invading microorganisms or parasites.

CLas populations and keep them from overwhelming D. citri (Ramsey et al., 2015). It is unlikely that CLas is avoiding an immune response during colonization, as it has surface components involved in immune recognition such as cell wall, membrane and flagella, with composition typical of other Gram-negative bacteria (Duan et  al., 2009). CLas is a Gram-­ negative bacterium, thus D. citri’s poor immune response and survival when exposed to other Gram-negative bacteria, such as Escherichia coli or Serratia marcescens (Arp et  al., 2017), could indicate a minimal immune response to CLas. The lower rate of D. citri survival and its inability to clear Gram-negative bacteria correlates with the absence of the IMD pathway, as this pathway is exclusively associated with recognition and ­response to Gram-negative bacteria (Arp et  al., 2016). In contrast, the D. citri immune system is evidently equipped to eliminate comparable dosages of Gram-positive bacteria, Micrococus luteus and Bacillus subtilis. These observations suggest that, in addition to the inherent colonization abilities of CLas, limitations of the D. citri innate immune system may contribute to the facilitation of this vector–pathogen interaction. D. citri likely mounts minimal defenses against CLas, improving the transmission potential of CLas facilitating the rapid progression of HLB in citrus.

7.4  Symbiont Manipulation for Pathogen and Vector Management 7.4.1 Paratransgenesis

Symbionts of D. citri may provide novel sites for the development of targeted biological control 7.3.2  Reduced function may facilitate strategies. One possible tactic would be to introsymbiont colonization duce a transmission-inhibiting transgene into the target vector populations using endosymbionts Transcriptional analysis of infected and non-­ as the vehicles for incorporating anti-pathogen infected D. citri (Vyas et al., 2015; Arp et al., 2017) or anti-vector transgenes into the vector. This corroborate the low level of D. citri ­immune re- approach, called paratransgenesis, involves gensponse to microorganisms. Very few immune-­ etic manipulation of symbiotic bacteria comassociated genes are upregulated in CLas-infected monly found in pathogen-transmitting vectors D. citri, supporting the observation that CLas in- to export anti-pathogen ‘effector’ molecules into fection does not result in high mortality (Pelz-­ the host vector. This offers several advantages Stelinski and Killiny, 2016). It is possible that over the alternative approach, i.e. modification pederin-like toxins produced by Ca. P. armature, of the vector genome, which involves insertion upregulated in CLas-infected D. citri, ­control of novel genes into the heritable genetic material

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of the vector (Riehle et al., 2003). The key to the paratransgenesis technique is to identify a symbiotic organism that has established a mutualistic or harmonious relationship with the host organism. A true symbiont that comes from the host disease cycle is most suitable; therefore, the ­selection of the proper symbiont is critical, and once selected must be malleable to genetic manipulation. The symbiont then becomes a vehicle for delivery of anti-disease gene products. The challenge is to add the anti-disease sequences to the chromosome of the symbiont, while retaining all of the properties of symbiosis and avoiding displacement by non-transgenic counterparts; in a sense, converting the symbiont into a biological control agent, but with the new wrinkle that this agent is already present in the ecosystem. Ideally, the desired outcome is to eliminate the vector transmitting the disease using more conventional integrated pest management strategies; however, given the interface of suburban, feral and unmanaged citrus with commercial citrus and the development of insecticide resistance in D. citri populations, a more practical a ­ pproach may be to eliminate D. citri’s ability to transmit the pathogen. One such strategy is to manipulate vector populations by replacing wild populations with altered populations that are unable to transmit a pathogen. This strategy has been studied most recently in several mosquito species, in which strains of Aedes aegypti were rendered unable to transmit Dengue virus following infection with the Gram-negative endosymbiotic bacterium, Wolbachia. The ability of Wolbachia to invade insect populations through a process called cytoplasmic incompatibility offers a promising, selfsustaining tool for manipulating populations. Vertically transmitted D. citri symbionts Ca. C. ruddii and Ca. P. armature possess ­reduced genomes that are consequently unlikely to be suitable for transformation. Wolbachia are present in all D. citri and are not negatively impacted by the presence of CLas. Therefore, they may be good candidates for paratransgenesis to alter the phenotype of the symbiont, such that there is a change in the fitness, behavior or vector competence of D. citri. This particular bacterium has proven recalcitrant to culturing efforts, primarily due to challenges associated with the intracellular lifestyle of this organism. The plant endophytes Alcaligenes xylosidans and Pantoea agglomerans have been cultured from D. citri reared

on citrus and may be good candidates for paratransgenesis precisely because they are culturable and occur naturally in the psyllid’s environment. 7.4.2 Antimicrobials Another symbiont-based strategy for management of the CLas–D. citri complex is the use of antimicrobial molecules to manipulate symbiont populations within the psyllid. Conventional, broad-spectrum antibiotics are one option, particularly given the recent approval of several molecules, streptomycin and oxytetracycline, for agricultural use in citrus groves, to reduce CLas in infected trees. Several challenges exist for this approach, in particular exposing D. citri to sufficient doses of molecules so as to knock down populations of the insect’s endosymbionts. While removal of the symbionts has a negative impact on psyllid survival, it requires application of antibiotics well beyond labeled rates. Synthetic nucleotide-based molecules that specifically target and eliminate CLas and D. citri symbionts are preferable to conventional, broad-­ spectrum antimicrobial molecules. Similar to RNA interference techniques for arthropod gene silencing, molecules such as morpholino oligonucleotides can be designed for antisense inhibition of endosymbiont genes, effectively silencing bacterial RNA (Weslowski et al., 2011). The specificity of antimicrobials targeting D. citri symbionts is an important feature for an agriculturally applied product to reduce D. citri and CLas transmission, particularly given concerns over antibiotic resistance. Paratransgenesis or antimicrobials could be important tools to manage natural populations of psyllids to reduce pathogen transmission in conjunction with current disease and psyllid management programs. The development of bactericidal disease management would reduce the need for reliance on pesticides for, and possibly labor costs associated with, reducing vector psyllid populations.

7.5 Conclusions Further investigations are necessary to elucidate the physiological roles of individual symbionts. Despite the limited microbiota of D. citri, the



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­ pportunity for manipulation of this commuo nity to reduce pathogen transmission remains promising. Manipulation of microbial communities to induce immune responses within D. citri populations should be investigated. Genetic manipulation of bacterial symbionts can be used to facilitate pathogen management or to regulate the dynamics of microbial populations and immune responses in D. citri. Adoption of genetic modification to disrupt CLas transmission will require assessment of the environmental, evolutionary and economic consequences of utilizing these strategies for pathogen management.

­uccessful symbiont integration into natural S D.  citri populations is a requirement for disrupting the pathogen, while selective pressure against a modified symbiont may reduce their efficacy. More work is also needed to determine the optimum method for introducing paratransgenetically modified D. citri. Fortunately, some of the groundwork for rearing and mass release has been conducted. Mass release of the D. citri parasitoid, Tamarixia radiata, is currently used for management of populations. Paratransgenesis, as a pseudo-biological control strategy, should be able to employ similar methods for releases.

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8 

Huanglongbing Pathogens: Acquisition, Transmission and Vector Interactions

El-Desouky Ammar1*, Robert G. Shatters Jr1 and Michelle Heck2,3,4 USDA-ARS, US Horticultural Research Laboratory, Fort Pierce, Florida, USA; 2 Boyce Thompson Institute, Ithaca, New York, USA; 3USDA-ARS Emerging Pests and Pathogens Research Unit, Robert W. Holley Center for Agriculture and Health, Ithaca, New York, USA; 4Plant Pathology and Plant Microbe Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, New York, USA 1

8.1 Introduction Huanglongbing (HLB), also known as citrus greening disease, is currently the most serious and devastating disease affecting citrus worldwide. The putative pathogens of HLB are the ­following three phloem-limited Gram-negative α-Proteobacteria: (i) ‘Candidatus Liberibacter ­asiaticus’ (CLas), found in Asia, North and South America, Oceania and the Arabian Peninsula (Bové, 2006; Haapalainen, 2014); (ii) ‘Ca. Liberibacter americanus’ (CLam), found in South America (Texeira et  al., 2005, 2008); and (iii) ‘Ca. Liberibacter africanus’ (CLaf) found in Africa and the Arabian Peninsula (Garnier and Bové, 1996; Pietersen et al., 2010). These three bacteria can be transmitted from plant to plant by grafting or dodder, but their natural spread is primarily by insect vectors (da Graca et  al., 2016). Two psyllid vectors of HLB-associated Liberibacter spp. have been identified: the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), natural vector of both CLas and CLam (Capoor et al., 1967; Bové, 2006), and the African citrus psyllid, Trioza erytreae del Guercio

(Hemiptera: Triozidae), natural vector of CLaf (McClean and Oberholzer, 1965, Cook et  al., 2014). Experimentally, D. citri has been shown to transmit also CLaf (Lallemand et  al., 1986), and T. erytreae has been shown to transmit CLas (Massonie et  al., 1976). Four other species of citrus-feeding psyllids have been identified in Yunnan Province, China, the most abundant of which was Cacopsylla (Psylla) citrisuga Yang & Li (Hemiptera: Psyllidae) (Yang and Li, 1984; Cen et al., 2012b). The latter species was shown to be a carrier of CLas, which has been detected by qPCR in C. citrisuga nymphs that fed on CLas-­ infected, HLB-symptomatic lemon trees (Cen et al., 2012b). However, the possible role of this species as a vector has not been proven yet. D. citri is by far the most widely spread vector of CLas, which is also the most widespread bacterium related to HLB worldwide at present. Interestingly, CLas, which is more tolerant to higher temperatures than CLam, has been reported to be gradually displacing CLam in São Paulo State, Brazil between 2004 and 2012 (Lopes et al., 2009a, b, 2013). Thus, vector relations of CLas and D. citri have been studied much

*  Email: [email protected] © CAB International 2020. Asian Citrus Psyllid: Biology, Ecology and Management of the Huanglongbing Vector (eds. J.A. Qureshi and P.A. Stansly)

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more extensively than those of the other two HLB-associated Liberibacter spp. (CLam or CLaf). While the interactions between plant-­ pathogenic viruses and their hemipteran vectors have been studied in depth for many years (Nault, 1997; Hogenhout et  al., 2008a; Gray et al., 2014), much less is known about the interactions between plant-infecting bacteria and their hemipteran vectors (Orlovskis et al., 2015; Perilla-Henao and Casteel, 2016). Recently, Perilla-Henao and Casteel (2016) used terminology from plant–virus vector relation studies to divide the hemipteran-borne bacterial plant pathogens into semi-persistent in the vector (e.g. the xylem-limited Xylella fastidiosa) and persistent (e.g. the phloem-limited Spiroplasma, Phytoplasma and Liberibacter). These three phloem-limited pathogen groups were also categorized as circulative, since they continue from the nymphal stage of the vector to the adult and are presumed to move from the vector’s gut into the hemocoel then to the salivary glands before they can be transmitted to another host plant. Additionally, these three bacterial pathogen groups were categorized as propagative, since available data support the notion that they multiply in their vectors. In this chapter, we will divide the transmission process of CLas and other HLB-associated bacteria into its main components: acquisition from infected plants; translocation from the gut into other organs and tissues of the vector; evidence for multiplication in the vector; and finally retention in the vector and inoculation of the pathogen into another host plant. We will concentrate on reviewing more recent studies on HLB-associated Liberibacter spp. Earlier studies on HLB transmission depended mainly on symptom development in infected plants rather than molecular diagnostics (such as PCR or qPCR) and have already been adequately reviewed (Capoor et  al., 1974; Grafton-­Cardwell et  al., 2013; Hall et al., 2013; da Graca et al., 2016). Finally, we will compare current knowledge of the HLB-associated bacteria pathosystem with that associated with ‘Candidatus Liberibacter solanacearum’ (CLso) and its psyllid vectors affecting potatoes, tomatoes and other host plants in Solanaceae and Apiaceae (Cooper et al., 2014; Perilla-Henao and Casteel, 2016), since the modes of transmission seem to be largely similar in both of these pathosystems.

8.2  Pathogen Acquisition from Infected Plants Acquisition of a pathogen has been defined as the ‘process by which a vector takes up a pathogen from an infected host plant during feeding’, whereas the acquisition access period (AAP) is defined as the ‘time that an insect vector is caged on (has feeding access to) infected plants during transmission studies’ (Brown, 2016). The minimum AAP necessary to be able to detect the pathogen in the vector varies with the pathogen, the vector, the host plant and environmental conditions. ‘Acquisition’ has been sometimes used to indicate that the pathogen has been ‘ingested’ from infected plant tissues into the vector’s gut (lumen). However, when the pathogen is transmitted in a persistent, circulative manner (e.g. CLas and other Liberibacter spp.) the term ‘acquisition’ usually means internalization of the pathogen into the vector’s tissues beyond the gut lumen, since pathogens that remain in the gut lumen will not be transmitted. With Liberibacter spp., acquisition by vector psyllids has been assessed more recently using PCR or qPCR of the whole body of psyllids following feeding on infected plants for various AAPs. A recent study indicated that clearing D. citri guts from CLas by feeding psyllids on healthy leaves for 24 h, following long feeding periods on infected plants, did not significantly change the rate (proportion) of CLas-infected psyllids in qPCR tests, which suggests that the effect of CLas bacteria that are present in the gut lumen is minimal in such studies (Ammar et al., 2018). Acquisition rates (sometimes referred to as infection rates) of CLas by D. citri ranging ­between 6% and 100% have been recorded in ­various studies (Table 8.1) probably reflecting ­differences in methodology such as use of nymphs versus adults for acquisition, various AAPs, source plant genotypes, infection level, CLas strains, D. citri geographic populations and/or environmental conditions. In several studies, the main factor in determining the acquisition rate of CLas by D. citri is the stage of psyllid development during the AAP. Nymphs (usually 3rd–5th instars) are much more efficient than adults in the acquisition of CLas from infected citrus plants (Inoue et  al., 2009; Pelz-Stelinski et  al., 2010; Ammar et  al., 2016; Wu et  al., 2018a).

Country/ State Brazil

China

Taiwan USA-Florida

Nymph Adult Adult Nymph+Adult Adult Nymph+Adult Adult Nymph/Adult Nymph Adult Unknown Nymph/Adult Nymph Nymph Nymph+Adult

USA-Texas S. Africa

Adult Adult Nymph Unknown

Acquisition access period

Acquisition/ infection rate (%)

Retention time

Inoculation access period

Transmission/ inoculation rate (%)

48 h 48 h 48 h – – 8h Weeks 8h 2–20 d 24 h 24 h Fieldf Weeks 1–7 d 11–15 d – Weeks Weeks 1–14 d 7–45 d 7d Fieldf

74 52 35–100 – – 6d 78 35-55d Unknowne 78 13 99 50–75 56–59 60–70 – 74–95 39–94 13–29 39–80 6–21 41–71

>31 d >33 d – – – – – – >31 d >30 d >30 d – 12 w >35 d >24 d – – – > 42 d – – –

48 h 48 h – 1–15 d 1–15 d – 24 h – 1–4 d 30 d 30 d 7d – 7d 1–24 d 30 d 7–14 d 7d 7d 7d – 7d

43 12a – 22-46a 32-76b – 18–26a – 20–50b 67b 0b 8-9a – 5–21b 4–8a 73c 5–20a 15–48b 0b 0a – 34b a

Reference Canale et al., 2017 Lopes et al., 2013 Raiol-Junior et al., 2017 Bonani et al., 2010 Wu et al., 2016 Luo et al., 2015 Xu et al., 19886 Inoue et al., 2009 Ukuda et al., 2015 Hung et al., 2004 Ammar et al., 2016 Pelz-Stelinski et al., 2010 Ammar et al., 2013b

Huanglongbing Pathogens

Japan

ACP stage during acquisition



Table 8.1.  HLB/citrus greening pathogen acquisition, retention and inoculation rates by psyllid vectors reported from several regions around the world*

Ammar et al., 2016 Pelz-Stelinski et al., 2010 Sétamuo et al., 2016 Cook et al., 2014

*All tests involved D. citri and CLas, except those in S. Africa, which involved the African psyllid Trioza erytreae and C. Liberibacter africanus (CLaf). In the transmission/inoculativity tests all psyllids were CLas or CLaf exposed prior to the test. Most transmission/inoculativity tests were done on adults, even if these have acquired the pathogen during the nymphal stage. a A single psyllid was caged per plant or excised leaf during inoculation (inoculation rates by single psyllids are in bold). b Groups of 3–10 psyllids were caged per plant or excised leaf during inoculation. c A group of 200 psyllids were caged per plant during inoculation. d Tests from electrical penetration graph (EPG) recording experiments. e Older HLB studies relied on symptom expression in test plants (PCR tests were not yet available). f In tests using field-collected psyllids the acquisition access period and stage during acquisition are not known.

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The rate of CLas acquisition by nymphs ranged between 49% and 78% following a 1–7-day AAP, whereas acquisition by adults ranged between 8% and 29% following a 1–21-day AAP (Inoue et  al., 2009; Ammar et  al., 2016). In these two studies, only CLas acquired by D. citri during the nymphal stage was successfully transmitted to healthy citrus plants. In another study, 60–100% of D. citri acquired CLas during the nymphal stage, whereas only 39% of adults acquired it during the adult stage after 35 days of AAP on infected plants (Pelz-Stelinski et  al., 2010). Possible reasons for the greater efficiency of D. citri nymphs to acquire and eventually transmit CLas from infected citrus, compared with adults, include feeding behavior, as elucidated below, as well as differences in the innate immunity between nymphs and adults, which have been suggested based on quantitative proteomic and microscopic studies of healthy and CLas-infected D. citri (Ramsey et  al., 2017; Mann et al., 2018). D. citri is capable of defending against Escherichia coli, another Gram-­ negative bacterium (as is CLas), despite lacking the immune deficiency (IMD) pathway, but the innate immunity of nymphs and adults has not been compared (Arp et al., 2017). Evidence suggests that not all D. citri populations worldwide have the same phenotype with regards to CLas acquisition and/or transmission. In Brazil, Canale et al. (2017) reported a much higher transmission rate by D. citri that acquired CLas as nymphs (43.4%) compared with those that acquired it as adults (11.8%). Interestingly, these are much higher CLas transmission/inoculation rates than those reported from Florida (Table 8.1). The greater ability of nymphs compared with adults to acquire CLas has important implications for the epidemiology and control of HLB (Gottwald, 2010; Hall et  al., 2013; Lee et  al., 2015). Differences in feeding behavior between D. citri nymphs and adults as revealed by electropenetrography (EPG) might provide an explanation for this phenomenon. EPG studies have shown that D. citri adults can acquire CLas from infected plants only during the phloem ingestion phase (E2 waveform) (Bonani et  al., 2010; Luo et al., 2015). Furthermore, George et al. (2018) found that mean duration of the phloem ingestion phase for D. citri nymphs (5.7 h) was significantly longer than that for adults (1.5 h). Also, the number of bouts and total duration of

phloem ingestion were significantly greater for nymphs compared with adults, whereas adults ingested longer from xylem tissues compared with nymphs. In this study, 58% of tested nymphs and only 6% of tested adults acquired CLas from infected plants during the 42 h test period as revealed by qPCR (George et al., 2018). Both CLas and CLam are known to be unevenly distributed in citrus tissues (Li et al., 2006; Tatineni et al., 2008; Kunta et al., 2014). Thus, increased frequency and duration of phloem ingestion could allow D. citri nymphs to tap more sieve elements, increasing the likelihood of acquiring the pathogen compared with adults. In contrast to D. citri and CLas, adults of the potato psyllid Bactericera cockerelli appear to be more efficient than 5th-­instar nymphs in acquisition of CLso, associated with zebra chip disease in potatoes and tomatoes (Cooper et  al., 2014). The distribution of CLso in potato or tomato plants has not yet been reported but could be more homogenous than that of CLas in citrus trees. Also, no EPG studies are available to compare the feeding behavior of nymphs and adults of the potato psyllid. Earlier studies on HLB suggested a minimum AAP of 15 min to 7 h of feeding by D. citri on diseased citrus (Capoor et al., 1974; Xu et al., 1988), but more recent studies using EPG and qPCR indicate shorter acquisition times. Bonani et  al. (2010), showed that 6% of adult psyllids feeding on infected plants had acquired CLas and became infected after performing E2 waveform (phloem ingestion) for at least 1 h. Another EPG study found 23 out of 24 D. citri adults that performed E2 waveform tested positive for CLas, some after only 2 min of phloem ingestion (Luo et  al., 2015). Several studies indicated that longer AAPs usually result in higher rates of CLas acquisition by the psyllids in both laboratory and field tests (Pelz-Stelinski et  al., 2010; Ammar et al., 2016). Ammar et al. (2016) found that, with D. citri nymphs, an increase in AAP between 1 and 7 days did not result in a higher proportion of CLas-infected psyllids (56.3– 59.1%), whereas with adults an increase of AAP between 1 and 14 days increased CLas-infected psyllids from 13.0 to 29.4%. However, Wu et al. (2018a) reported an increase in CLas acquisition rate by 4th-instar nymphs with increased AAPs from 30 m to 6 h, whereas no acquisition occurred by nymphs in 15 min AAP or by adults



Huanglongbing Pathogens

in AAPs shorter than 5 h. With CLso and the potato psyllid, both the acquisition rates and titer of CLso in adult psyllids increased over time following AAPs of 8–72 h on tomato or potato plants (Sengoda et  al., 2014). CLso titer in the psyllids was highest when they acquired the bacterium from tomato versus potato, apparently because CLso titer was much higher in tomato leaves, petioles and stems compared with those of potato (Sengoda et  al., 2014). Wu et  al. (2018a) also reported that both the proportion of CLas-infected D. citri and their CLas titer increased significantly with the increase of CLas titer in citrus (Shatangju) plants used for acquisition. In a field study in Florida, Hall (2018) found that the percentage of CLas-positive D. citri females was generally higher than that in males, but no differences were found among the three color morphs in adults (Hall, 2018). However, the rate of CLas acquisition by D. citri males and females was not significantly different in some laboratory tests (Ammar et al., 2018; Wu et al., 2018a). It is possible that the higher percentage of infected females in field populations may be due to the shorter life span of adult males compared with females (21–25 versus 31–32 days, respectively, at 24oC) (Nava et  al., 2007). This longevity factor may increase the importance of D. citri females as vectors, because they may have more time as compared with males to spread CLas among trees or fields. Recent work suggested that host-switching of a D. citri colony, that was highly efficient in CLas acquisition from citron plants, to Murraya plants (which are less susceptible to CLas) for 6–12 months affected CLas acquisition from infected citron negatively (Ammar et al., 2019b). Host switch effect on pathogen acquisition and transmission has been reported with other pathosystems, especially circulative aphid-­ transmitted viruses (Pinheiro et al., 2017). Also, infection-source plant genotype and age of infected leaves can affect the rate of CLas acquisition by D. citri. CLas acquisition efficiency by D. citri adults feeding on tender shoots of Citrus reticulata was significantly greater than that on Citrus sunki, while acquisition from C. sunki was significantly higher than that from C. reticulata on old leaves (Huang et al., 2015). Two studies indicated that the presence of flush shoots on infected plants significantly increases the chances of CLas acquisition by D. citri adults (Wu et al.,

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2015; Sétamou et  al., 2016). Wu et  al. (2015) suggested that D. citri adults are more attracted to infected citrus leaves because of their yellowish color, but subsequently they move to healthy citrus perhaps because of either poor nutrition or a feeding barrier in the infected hosts. Several studies indicated that D. citri can acquire CLas from asymptomatic as well as symptomatic leaves of infected plants. Coletta-Filho et  al. (2014) reported that CLas acquisition by D. citri was positively associated with plant infection level and time since inoculation, with acquisition occurring as early as 60 days after inoculation, before symptom development. In a field study in South Africa, fluctuations in the percentage infectivity of the T. erytreae psyllid populations were observed during two seasons, with infectivity peaking with or just after the citrus flush seasons (Cook et al., 2014). A new model for CLas acquisition and transmission has been proposed which stands apart from other circulative plant pathogens that typically infect the host plant systemically prior to vector acquisition from the plant. Lee et  al. (2015) proposed the idea of ‘flush transmission’, for CLas from infected D. citri adults to their young nymphs via the host plant, when these nymphs feed on the flush leaves recently infected by their parents. ‘Flush’ is any new leaf growth ranging in development from first emergence up until the leaves are fully expanded yet still tender (Hall et al., 2016). Their results suggested that young flush becomes infectious within 10–15 days after receiving CLas inoculum from infected parents that acquired CLas during the nymphal stage. As soon as the adults emerge, after acquiring CLas as young nymphs from the infected flush leaves, they repeat this process. Somewhat similar results with CLaf were obtained earlier by McClean (1974), who tested adult T. erytreae that emerged from nymphs in a prior series of transmission experiments (McClean, 1974). The second-generation of adults transmitted CLaf to several plants in three cases where there was no detectable infection in the first cycle of experiments. This newly proposed mode of CLas transmission is epidemiologically significant because the infection cycle becomes much shorter than was presumed earlier. Also, since one D. citri female lays around 572–858 eggs on citrus plants, with generation time of 17–18 days at 25oC (Tsai and Liu, 2000),

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the potential for increase in numbers of CLas-­ positive psyllids in a citrus field is substantial. ‘Flush transmission’ might explain the enormous and rapid increase in CLas infection across Florida citrus trees since this bacterium was first discovered in the state in 2005. Lee et al. (2015) proposed that psyllid flush transmission is the dominant mode of dispersal of CLas in a citrus grove. They suggested that reducing the psyllid population by 75% during the flushing periods can delay infection of a full grove, and thereby reduce the amount of insecticide used throughout the year. Early stages of citrus flush were reported to be most suitable for D. citri oviposition, nymphal survival and development, as well as adult emergence (Cifuentes-Arenas et al., 2018).

8.3  Latent Period and Pathogen Translocation in the Psyllid Vector In vector transmission of plant pathogens, the ‘latent period’ is the time that elapses between pathogen acquisition from an infected plant and the first time the insect vector becomes able to inoculate this pathogen into a new susceptible plant. Earlier studies on the latent period of Liberibacter spp., when no molecular diagnostics were available, estimated the latent period in D. citri to be 1–25 days (Xu et al., 1988; Roistacher, 1991). More recently, using qPCR on psyllids and plants, the minimum latent period of CLas in D. citri in Florida has been estimated to be less than 7 days for psyllids that acquired the pathogen as nymphs (Ammar et al., 2016). A study in Brazil reported that the minimum latent period for CLas acquired by D. citri nymphs was 7–10 days, and that the median latent period for psyllids that acquired CLas as nymphs or adults was 16.8 and 17.8 days, respectively (Canale et  al., 2017). The latent period of CLso in the potato psyllid has been reported to be around 2 weeks, and was shorter from CLso-infected tomato than from potato plants, apparently because the titer of this bacterium is higher in tomato than in potato (Sengoda et al., 2013, 2014). With the persistent, circulative mode of transmission, the latent period is presumed to be the time necessary for translocation of the pathogen from the gut lumen into gut epithelial cells, and from these to the hemolymph or other

tissues. Eventually the pathogen must reach the salivary glands before it can be injected with the saliva through the salivary canal (inside the maxillary stylets) during feeding on a susceptible plant (Gray et al., 2014). Additionally, with ‘propagative’ pathogens, the latent period may also be the time necessary for pathogen replication in gut cells, the salivary glands or other tissues of the vector (Nault, 1997; Hogenhout et  al., 2008a). Time course studies to illustrate this sequence of events in the circulative transmission process have been conducted with several hemipteran-borne plant viruses (Hogenhout et  al., 2008a) but less so with plant-infecting bacteria (Hogenhout et al., 2008b; Perilla-Henao and Casteel, 2016). Several transmission barriers have been previously proposed, and experimentally shown in some cases, for the circulative/ propagative pathogens in their vectors. These include the gut entry, gut exit/escape, salivary gland entry and salivary gland exit/escape barriers, which have been demonstrated earlier with several plant and vertebrate pathogens in their invertebrate vectors (Ammar, 1994; Hogenhout et  al., 2008a). Additionally, these pathogens may encounter other barriers in the vector, e.g. the innate immune system, which might include lack of compatibility with the hemolymph and/or salivary secretions in either the salivary glands, salivary ducts or salivary syringe (Ammar, 1994). Anatomy and ultrastructure of the alimentary canal, salivary glands and other organs and tissues in D. citri have been described earlier (Cicero et al., 2009; Ammar et al., 2017). Both the midgut and salivary glands have several membranes (e.g. microvillar and perimicrovillar membranes (Silva et al., 2004), apical and basal plasma membranes, and basal laminae) that the bacterium has to overcome to enter and/or exit these organs (Figs 8.1A, 8.1B). D. citri has a pair of accessory and principal salivary glands, and the latter have different types of acinar cells (acini), in addition to the salivary ducts and ducteoles (Fig. 8.1B) that carry the saliva, presumably with injected CLas to the salivary canal in the maxillary stylets (Garzo et al., 2012; Ammar et al., 2013a). Localization of bacteria-like spherical, oval or filamentous structures in the salivary glands, gut or other organs of infected Asian and African citrus psyllids was reported in China and South Africa, respectively, using thin-sectioning



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(A) mu bl nu

cy

mv 5 μm

lu

(B) Ac3

Ac1

dc

mv sd

ca 2 μm

Fig. 8.1.  Ultrastructure of the midgut epithelial cells (A) and the principal salivary gland (B) in D. citri; arrowheads in panel A indicate perimicrovillar membrane between microvilli and midgut lumen. Ac1, acinus 1; Ac3, acinus 3; bl, basal lamina; ca, canaliculus; cy, cytoplasm; dc, duct cell; lu, lumen; mu, muscle; mv, microvilli; nu, nucleus; sd, salivary duct. (Modified from Ammar et al., 2017).

electron microscopy (Chen et al., 1973; Moll and Martin, 1973; Xu et al., 1988). However, no immunolabeling or molecular labeling methods were available to confirm the identity of such bacteria in these studies. Since endosymbiotic bacteria of different shapes and sizes are frequently found in various tissues of hemipteran insects, and bacteria-like structures (BLS) were reported in both CLas-infected and uninfected D. citri (Ammar et al., 2017), the structures found earlier in infected psyllids should be referred to as BLS until further confirmation. Studies using fluorescent in situ hybridization (FISH) or qPCR on D. citri adults that acquired CLas as nymphs

in the laboratory, or collected from infected citrus trees in Florida fields (Ammar et al., 2011a,b; Ghanim et  al., 2016; Kruse et  al., 2017; Mann et  al., 2018) indicated the presence of CLas in most organs and tissues of infected D. citri adults, including the midgut, filter chamber, Malpighian tubules, hemolymph, salivary glands, ovaries, muscle and fat tissues as well as in the phloem of infected citrus plants (Fig. 8.2). These results were recently confirmed using immuno-gold labeling transmission electron microcopy of bacterial structures in several organs of D. citri (Achor et  al., 2019). Using qPCR, Ukuda-­ Hosokawa et  al. (2015) also reported systemic

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(A)

(B)

(C) ph ph

ph xy

5 μm

5 μm (D)

5 μm

(E)

(F)

fc n

sg

sg

mg

50 μm (G)

10 μm

10 μm (I)

n

ts ft

mu mag

10 μm (H) ft

ft

ov 40 μm

40 μm

Fig. 8.2.  Confocal laser scanning micrographs of CLas (green fluorescence) detected by FISH in the phloem of infected citrus leaves (A–C), as well as in D. citri filter chamber and midgut (D), salivary glands (E,F), muscles (G), and in the female and male reproductive systems (H, I, respectively). fc, filter chamber; ft, fat tissue; mag, male accessory gland; mg, midgut; mu, muscle; n, nucleus; ov, ovary; ph, phloem; sg, salivary gland; ts, testis; xy, xylem. (Modified from Ammar et al., 2011a).

infection of D. citri by CLas, and estimated the density of this pathogen in infected, field-collected adults (in Japan) to range between 103 to 107 bacterial cells per insect. Ammar et al. (2011a, b) reported that the proportion of D. citri with ­infected salivary glands was significantly lower than those with infected guts or other tissues.

Cooper et al. (2014) also found that fewer salivary glands of the potato psyllid were infected with CLso compared with the alimentary canals. The above results suggest a gut entry barrier in vector psyllids for both CLas and CLso (gut infection rates between 44.5% and 73.8%), and a salivary gland entry barrier (salivary gland



Huanglongbing Pathogens

infection rate significantly lower than that of the gut). Since the proportion of inoculative (infective) D. citri in Florida (when psyllids are tested individually) ranges between 4% and 20% (Table 8.1), which is much lower than the proportion of infected salivary glands (Ammar et al., 2011b), a salivary gland exit/escape barrier also likely exists in D. citri. In two recent studies, CLas was detected in D. citri tissues by FISH on the luminal membrane, in puncta within the gut epithelial cell cytoplasm, along actin filaments in the gut visceral muscles, and rarely, in association with gut cell nuclei (Kruse et  al., 2017; Mann et  al., 2018). Ghanim et  al. (2017) reported that CLas induced the formation of endoplasmic reticulum (ER)-associated bodies. They suggested that CLas may recruit those ER structures into Liberibacter-containing vacuoles (LCVs), in which bacterial cells seem to propagate. Interestingly, ER-associated LCV formations were not detected in Bactericera trigonica infected with CLso (Ghanim et al., 2017). Cicero et al. (2017) reported extensive bacterial biofilms on the outer midgut surface of CLso-­ infected adults and older nymphs. CLso cells were also observed between the basal lamina and basal epithelial membranes, on the outer basal laminar surface, in the epithelial cytosol, and filter chamber periventricular space. CLso were also found in the salivary gland pericellular spaces and in epidermal cells of the head (Cicero et al., 2017). How CLas or CLso penetrates the perimicrovillar, microvillar and basal membranes or the basal laminae to enter or exit the midgut or salivary glands of the vector is unknown. Specific mechanisms mediating Liberibacter recognition, attachment and multiplication in vector organs are not yet clear (Perilla-Henao and ­ Casteel, 2016). However, more progress has been made with the insect-borne plant pathogenic and culturable Spiroplasma spp. that have specific tip structures primarily responsible for attachment to cell membranes between microvilli (Ammar el et  al., 2004; Hogenhout et  al., 2008b). Spiroplasma kunkelii, causal agent of corn stunt disease, was found between microvilli and in endocytic vesicles of the midgut epithelium of the leafhopper vector Dalbulus maidis. Spiroplasmas with tube-like extensions accumulated between layers of the basal lamina (Ozbek et al., 2003). Spiroplasma citri, the causal agent

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of citrus stubborn disease, is thought to adhere after acquisition to receptors in the lumen of its leafhopper vector, Circulifer tenellus, midgut where endocytosis occurs (Fletcher et al., 1998; Wayadande and Fletcher, 1998; Kwon et  al., 1999). Intracellular vesicular transport mediates movement to the hemolymph and exocytosis. Once inside the hemolymph, the bacteria are transported throughout the insect body, eventually reaching the salivary glands after additional intracellular crossings (Fletcher et al., 1998). Several potential proteins required for insect attachment have been identified using S. citri mutants impaired for insect transmission and with S. citri strains that have lost insect attachment properties after multiple in vitro cultivations (Fletcher et  al., 1998; Yu et  al., 2000; Berho et  al., 2006; Mutaqin et  al., 2011). Numerous candidate attachment proteins have been identified using leafhopper monolayers, including P58, SARP, spiralin and the plasmid-borne protein P32 (Fletcher et  al., 1998; Berg et al., 2001; Gasparich, 2010), though the specific mechanisms involved remain unknown. Recently, variable Protein A of flavescence dorée phytoplasma (unculturable bacteria) has been reported to bind to the midgut perimicrovillar membrane, and to promote adhesion to gut epithelial cells, of the vector leafhopper Euscelidius variegatus (Arricau-Bouvery et al., 2018).

8.4  Pathogen Multiplication in the Vector Currently, CLas cannot be cultured and so direct evidence to support the hypothesis that CLas is propagative in the vector is difficult to obtain. Indirect evidence suggesting that CLas multiplies in D. citri includes the following: (i) at least some adult D. citri remaining inoculative with CLas through most of their lifespan after acquiring this bacterium as nymphs (Xu et  al., 1988; ­Ammar et al., 2016); (ii) almost systemic infection by CLas in the vector following CLas acquisition (Ammar et al., 2011a, b; Ukuda-Hosokawa et  al., 2015); (iii) qPCR results showing higher CLas titer in D. citri’s alimentary canal and salivary glands compared with other tissues which can be interpreted as multiplication (or alternatively, CLas accumulation) (Ammar et al., 2011b); and

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(iv) other qPCR studies showing that CLas titers in D. citri that acquired CLas during the nymphal stage are higher in adults as compared with nymphs (Ramsey et al., 2017; Mann et al., 2018). More direct evidence of CLas replication in D. citri was first reported by Inoue et al. (2009) who found that CLas titer (as estimated by qPCR) significantly increased on days 10–20 post-­ acquisition by nymphs, but not following acquisition by adults (AAP was 24 h in both cases). Later, Ammar et  al. (2016), also using qPCR analysis, tested the increase in CLas titer in D.  citri with AAPs on infected citrus plants of 1–7 days as nymphs, or 1, 7 and 14 days as adults. Following each AAP, the psyllids were transferred weekly to healthy citrus or Murraya plants throughout the rest of their adult lives and sampled weekly for qPCR, up to 35–42 days post-acquisition. CLas titer in D. citri (relative to that of the psyllid S20 ribosomal protein gene) was found to: (i) increase significantly following acquisition as nymphs or adults, but increase at a faster rate in nymphs than in adults; (ii) increase significantly with post-acquisition time peaking at 14–28 days for nymphs compared with 21–35 days for adults; and (iii) increase with longer AAP on infected plants, especially with acquisition as adults. These results strongly suggest that CLas multiplies in both nymphs and adults of D. citri but attains much higher levels in a shorter period of time post-acquisition when acquired by nymphs than when acquired by adults. Adults may require longer access to infected plants compared with nymphs for CLas to reach higher levels in the vector (Ammar et al., 2016; Ramsey et al., 2017; Mann et al., 2018). The titer of CLso in the potato psyllid vector was also reported to increase with time post-­ acquisition and to reach a plateau after an average of 15 days following AAPs of 24 and 72 h on potato and tomato plants respectively (Sengoda et  al., 2013). Wu et  al. (2018a) indicated that when CLas was acquired by D. citri nymphs, the proportion of infected hemolymph and salivary glands, as well as their CLas titer, increased ­significantly 12–18 days following a 3-day AAP. With D. citri that acquired CLas as adults, multiplication of the pathogen was poor and its titer in the salivary glands remained low (Wu et  al., 2018a). Although the data presented so far are in strong support of CLas and CLso as propagative in their vector psyllids, further studies using

insect cell lines could provide further direct evidence of bacterial replication in vector tissues. CLas titers appear to increase faster and more efficiently in nymphs as compared with adults due to differences in replication or accumulation efficiencies (Inoue et  al., 2009; ­Ammar et  al., 2016). Several factors could be responsible for this difference between adults and nymphs: •

•

• •

Younger citrus tissues upon which nymphs feed exclusively may have a higher proportion of live rather than dead CLas cells (Hu et al., 2014). CLas transmission barriers or receptor/ binding sites in the gut or salivary glands may be more permissive in psyllid nymphs than in adults. The symbiotic organisms found in the nymphs may affect CLas replication differently. There are possible differences in the immune systems of nymphs and adults (Ramsey et al., 2017; Mann et al., 2018).

Further experiments are necessary to elucidate these and other factors that may affect the efficiency of acquisition, replication and transmission of CLas and other Liberibacter spp. by D. citri and other vectors. With CLso and the potato psyllid, Cooper et  al. (2014) indicated that fewer 5th-instar nymphs were infected compared with the adults, and that 5th-instar nymphs were less likely to transmit the pathogen to non-infected host plants (Cooper et  al., 2014). However, Casteel et al. (2012) reported that adults and 5th-instar nymphs were found to carry higher titers of CLso than the younger nymphs, but young nymphs were still capable of transmitting the bacterium. With several other insect-borne plant pathogens, nymphs are much more efficient in pathogen acquisition than adults, but the role of the feeding behavior in pathogen acquisition and transmission has not been elucidated in most cases (­Ammar, 1994; Hogenhout et al., 2008b).

8.5  Pathogen Retention and Inoculation by the Vector Once CLas has been acquired during the nymphal stage, D. citri adults may remain infected and



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sometimes inoculative (infective) for most of their adult life (Xu et  al., 1988; Hung et  al., 2004). D. citri psyllids remained infected (qPCR positive) up to 35–42 days post-acquisition, and some remained inoculative up to 28 days, but not 28–35 days, post-acquisition (Ammar et al., 2016). However, several studies indicated that CLas inoculation/transmission by D. citri is intermittent, i.e. not all the plants fed on by an infected or even inoculative psyllid will be infected (Capoor et al., 1974; Xu et al., 1988; Inoue et  al., 2009; Canale et  al., 2017). Xu et  al. (1988) reported intermittent transmission of the HLB pathogen to healthy plants up to 13 transfers. Ammar et  al. (2013b) also reported intermittent CLas transmission using excised citrus leaves inoculated for two consecutive tests weekly by one, five or ten infected D. citri adults that acquired CLas during the nymphal stage. Percentage of leaves that proved CLas positive in both tests varied with the number of psyllids tested per leaf and ranged between 7.2% (1 psyllid per leaf) to 66.7% (ten psyllids per leaf). In Brazil, Canale et al. (2017), using single D. citri psyllids transferred to new healthy citrus plants every 48 h (following an AAP of 48 h), reported that among the six inoculative psyllids that acquired CLas as adults, transmission was detected on only one out of 15–18 plants consecutively exposed to each psyllid. On the other hand, with 23 inoculative psyllids that acquired CLas as nymphs, eight psyllids transmitted CLas more than once (to two to four plants out of 16–18 plants tested). Such intermittent transmission by vectors has been reported earlier for other insect-vectored plant pathogens (Hogenhout et al., 2008a). It is possible that the lower rate of CLas replication in adults, especially older ones, may be responsible for the intermittent transmission of CLas by D. citri. Alternatively, CLas replication in, or translocation to, the adult salivary glands may be slower than the rate of its release with the saliva during inoculation into healthy plants. It can also be related to a salivary gland exit barrier that may include the width of the salivary canal in the psyllid’s maxillary stylets. The diameter of this canal in D. citri adults has been reported to be 0.3–0.5 μm (Garzo et  al., 2012), whereas the diameter of presumed CLas cells in phloem tissues has been reported to be 0.33– 0.66 μm (Hartung et  al., 2010). This suggests that the salivary canal in D. citri adults, and

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probably nymphs, may not be wide enough for the larger CLas cells to go through and could thereby limit the rate of inoculation or contribute to intermittent transmission of CLas. Although the flying and more agile adults of D. citri are thought to effect most of the medium and long-range spread of CLas/HLB, Wu et al. (2013) showed that late-stage nymphs (3rd to 5th instar) of D. citri can make horizontal and vertical transfers among young citrus shoots, that nymphs transfer faster with increased density and that nymphs preferred young shoots of HLB-infected citrus. Xu et  al. (1988) reported transmission of HLB by 4th–5th-instar nymphs of D. citri but not by 1st, 2nd or 3rd instars. However, as PCR was not yet available for detection of CLas, they depended on symptom development, which takes much longer and can be less reliable. Ammar et  al. (2013a) found that the maxillary food canal in 1st-instar nymphs of D. citri is wide enough for CLas bacterial cells to go through during acquisition from infected plants, whereas the salivary canal in this instar is apparently not wide enough for this bacterium to go through with the saliva during inoculation. Thus, the size of the salivary canal may be the reason for the lack of transmission/inoculation by early-instar nymphs reported earlier (Xu et  al., 1988). A recent study suggested that D.  citri adults are more efficient than 4th–5thinstar nymphs in CLas inoculation when acquisition from infected plants started in both cases with the nymphal stage. This is apparently because, when CLas is acquired during the nymphal stage, adult psyllids become more infected with a higher titer of CLas, which also reaches the salivary glands in a higher proportion of adults compared  with  nymphs (Ammar et  al., 2019a and unpublished). This increases the epidemiological significance of D. citri adults in CLas inoculation as opposed to acquisition, which happens mainly during the nymphal stage as elucidated earlier. The piercing–sucking feeding mechanism of hemipteran vectors of phloem-limited pathogens allows these pathogens to enter the phloem directly, bypassing numerous barriers and defense mechanisms within the host plant (Bendix and Lewis, 2018). EPG studies show that inoculation of CLas by D. citri adults happens during the phloem salivation phase (waveform E1) of the psyllid feeding on a susceptible citrus plant

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(Wu et al., 2016). The minimum feeding time for successful inoculation by a single adult was 88.8 min, with a minimum E1 duration of 5.1 min, and a significantly positive correlation was found between E1 duration and transmission efficiency. In these experiments, 18% of the males and 26% of the females that performed E1 waveforms transmitted CLas to citrus plants. Another EPG study, on the carrot-infecting CLso transmitted by the psyllid B. trigonica (Antolínez et  al., 2017), also showed that Liberibacter inoculation is associated with the phloem salivation waveform (E1) where inoculation occurred in a period as short as 30 s, and that inoculation rate by single psyllids was 48%. The results also suggested that females might inoculate higher CLso titers than males, but the percentage of transmission was not affected by vector sex at a density of one or eight psyllids per plant. With CLso in potatoes, transmitted by the potato psyllid B. cockerelli, 25–30% of the psyllids reached and salivated into phloem at an inoculation access period (IAP) of 1 h, increasing to almost 80% when IAP was increased to 24 h. Probability of CLso inoculation was lower across all IAP levels than probability of phloem salivation, indicating that a percentage of infected psyllids that salivated into the phloem failed to transmit CLso. The probability of transmission increased as a function of time spent salivating into the phloem; transmission occurred as quickly as 5 min following onset of salivation. A small percentage of infected psyllids showed extremely long salivation events but nonetheless failed to transmit CLso (Mustafa et  al., 2015), which ­suggests the existence of salivary gland entry and/or exit barriers in these psyllids. Higher transmission/inoculation rates of CLas by D. citri were reported in China (Xu et al., 1988; Wu et  al., 2016), Japan (Inoue et  al., 2009) and Brazil (Raiol-Junior et  al., 2017) as compared with those in Florida (Pelz-Stelinski et al., 2010; Ammar et al., 2016) (Table 8.1). It is possible that Chinese, Japanese and/or Brazilian populations of the vector and/or the pathogen may be different than the Florida populations in vector competence (transmission efficiency). Genetic variations in D. citri populations have been reported in Brazil, Asia and other regions (Boykin et al., 2012; Guidolin et al., 2014; Lashkari et al., 2014). Also, genetic variability among D. citri populations, possibly associated with host

plants, has been reported among various locations in China (Meng et al., 2018). Additionally, differences in the experimental procedures or environmental conditions may affect the results of transmission/inoculativity tests in different laboratories or in different parts of the world. Recently, the traits of CLas acquisition and transmission competency have been demonstrated to be heritable for several generations in various field-collected populations maintained as isofemale lines from Florida (Ammar et  al., 2018). Transmission and acquisition rates were positively correlated with CLas titer in the psyllids and with each other (Fig. 8.3), as was also reported earlier (Ammar et al., 2013b). Similarly, transmission/inoculation experiments with citrus seedlings using field-collected CLas-infected D. citri in Japan suggested that CLas-transmitting insects tend to exhibit higher infection densities than do non-transmitting insects, and that a threshold level (ca.106 bacteria per insect) of CLas density in the psyllid is required for successful transmission to citrus plants (Ukuda-Hosokawa et al., 2015). Interestingly, the minimum titer for transmission appears to be lower for CLso (104 per psyllid) inoculated by the potato psyllid (Sengoda et  al., 2014). However, Gottwald and McCollum (2017) suggested that there is a ‘minimum infectious dose’ of CLas that is required in the infected psyllid if the pathogen is to proliferate in citrus and eventually lead to the development of HLB symptoms. They indicated that the probability of HLB developing in susceptible plants is strongly correlated with the proportion of D. citri populations with CLas titers that exceed ca. 105 copies per insect, and that below this level, the pathogen may be transmitted to citrus and even acquired by psyllid nymphs, but does not proliferate, move systemically, or cause HLB in citrus plants. Two distinct methods have been used to study the efficiency of CLas inoculation by D. citri into heathy citrus: whole citrus seedlings or excised citrus leaves. qPCR results of inoculated excised leaves can be known in 1–2 weeks as opposed to several months with the inoculated plants (Ammar et al., 2013b). Inoculation rates (% CLas-positive leaves or plants) using these two methods were comparable in two studies (Ammar et al., 2013b; Raiol-Junior et al., 2017), although the whole-plant method gave higher inoculation rates in a third study (Ammar et al., 2016). However, the excised leaf assay saves



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Acquisition rate

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0

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–20 28

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Mean psyllid Cycle threshold (Ct) value Fig. 8.3.  Linear regression and correlation analysis between mean Ct values of CLas in D. citri that acquired this pathogen as nymphs, and rates of CLas acquisition and transmission in 15 isofemale lines from Florida. Shaded areas indicate 95% confidence limits. Acquisition rate for each line was assessed with six qPCR tests of individual psyllids over several generations, and transmission rate for each line was evaluated in three inoculactivity tests using excised healthy citrus leaves with six to ten CLas-exposed adults per leaf per week (from Ammar et al., 2018).

considerable effort, material and especially time, which is crucial, as is the case with CLas when extensive tests are to be undertaken (Raiol-­Junior et  al., 2017; Ammar et  al., 2018). Higher proportions of CLas-positive leaves were obtained with the excised leaf method with: (i) higher densities of inoculating psyllids (five or ten adults

per leaf); (ii) longer IAPs (1 or 2 weeks); and (iii) incubation of leaves for 1 week post-inoculation prior to processing for qPCR (Ammar et al., 2013b). Efficiency was also increased by using younger citrus leaves and more sensitive CLas primers (Ammar et  al., 2013b; Raiol-Junior et al., 2017).

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CLas transmission efficiency into various citrus genotypes is usually much higher with grafting (up to 90%) than with psyllid inoculation (up to 38%) (Albrecht et al., 2014). Citrus species and genotype may affect CLas inoculation/transmission efficiency by D. citri. Citrus macrophylla and Citrus medica (citron) were 100% PCR positive for CLas in a greenhouse study using young trees of 16 citrus types exposed to free-ranging CLas-infected psyllids compared with approximately 30% infection in the complex genetic hybrids ‘US 1-4-59’ and ‘Fallglo’ (McCollum et  al., 2016). Orange jasmine, Murraya paniculata, a citrus relative that is a good host for D. citri, is much less susceptible to CLas inoculation than citrus both by graft and by psyllid inoculations (Hilf and Hall, 2014). The titer of CLas in D. citri was five times lower when the psyllids had been reared on infected M.  paniculata than when they had been reared on Citrus species, which are better hosts for CLas (Walter et al., 2012a, b). Although HLB disease symptoms rarely developed on infected Murraya plants, D. citri was nevertheless reported capable of acquiring CLas and CLam from infected Murraya and transmitting it to citrus (Damsteegt et al., 2010; Gasparoto et al., 2010). Curry leaf plant, Murraya (Bergera) koenigii, has been reported to be even less susceptible to CLas than M. paniculata, although it is also a good host for D. citri (Damsteegt et al., 2010). Differences in amino acid composition of CLas-permissive (­susceptible) and non-permissive (resistant) host plants were studied by Sétamou et  al. (2017), who reported that high proline-to-glycine ratios are characteristic of CLas-permissive hosts. Obviously, other factors may be involved in this trait. The age of the recipient plant or leaf can also affect its susceptibility to infection as well as transmission efficiency by the psyllids. In experiments using seedlings of ‘Valencia’ sweet orange as well as cv. US-942, a 1-week exposure to infected psyllids resulted in much higher infection rates (23–97%) if flush was present compared with 3–40% when no flush was present (Hall et  al., 2016). CLas variability can also affect ­vector competence and vector specificity. Pitino et al. (2014) reported that CLas can be acquired from, and inoculated into, citrus leaf discs by the striped mealybug Ferrisia virgata (Hemiptera: Pseudococcidae). Bacterial titers were positively correlated with feeding acquisition time on

CLas-infected discs, with a 2-week feeding period resulting in 1.07 × 104 to 8.24 × 107 CLas cells per mealybug. Furthermore, CLas was shown by qPCR to have moved from the mealybug gut to the salivary glands. However, no disease was observed in the host plants. Interestingly, prophage/­ phage marker iFP3 was dominant in CLas of ­infected mealybugs in contrast to FP1 and FP2 markers characteristic of infected D. citri (Pitino et  al., 2014). The authors speculated that the phage iFP3, which contains Type D prophage (Zhou et  al., 2013), may influence CLas transmission efficiency in the mealybug by expressing important genes, or by reducing CLas titer in the salivary glands by killing the bacteria in the process of phage iFP3 replication. It is not known whether CLas multiplies in F. virgata or not, but this is the first report of genetic difference among CLas populations harbored by different insect vectors with respect to whether or not they cause disease in host plants. Obviously, the question of CLas genetic diversity and regulation of gene expression in the host plant and/or in insect vectors deserves further investigation.

8.6  Vertical and Horizontal Transmission of Liberibacters Among Psyllid Individuals Transmission of pathogens from one host to another of the same species can be defined as horizontal or vertical. Vertical transmission refers to the transmission of pathogens from one generation to the next. In general, this can occur via transovarial movement of the pathogen from the mother to the egg. Horizontal transmission refers to the transmission from one organism to another of the same generation and does not require a parent–offspring relationship. This can occur among interacting individuals through casual or sexual contact or through the use of an alternative, intermediate host (in the case of Liberibacters, this would be the plant). The use of a plant-host intermediate is the predominant method of transmission of Liberibacters from one psyllid to another, since D. citri nymphs acquire CLas at a significantly higher efficiency than adults (Inoue et  al., 2009; Pelz-Stelinski et al., 2010; Ammar et al., 2016). In this mode, an infected psyllid feeds on the host plant and



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inoculates the Liberibacter that can then be acquired by another psyllid, especially nymphs, feeding on the same plant. Lee et al. (2015) demonstrated that, in citrus, the host plant does not have to be systemically infected with CLas to function as the source of inoculum (Lee et  al., 2015). A similar process may occur with CLso and the potato psyllid B. cockerelli (Torres et al., 2015). Uninfected potato psyllids acquired CLso from two non-symptomatic plants (Convolvulus and Ipomoea spp.) that harbored infected psyllids and Liberibacter DNA was detected by PCR in some of these plants. However, CLso was not acquired after infected psyllids were removed, which suggests that transmission may have occurred while the pathogen passively moved through the phloem when both infected and uninfected insects were feeding on the same plant. In the above cases, if the ‘infective’ and the ‘infected’ psyllids were of the same generation (nymphs to nymphs) this would be considered ‘horizontal’ transmission, but if the infective psyllids were adults and the infected ones were nymphs of the following generation(s), this might be considered ‘vertical’ transmission, but not of the classical sense (through the ovaries or sperms of the parents to the progeny). While plant-host-mediated transmission is widely accepted as the primary mode of transmission of Liberibacters among psyllids, the possible role of other modes of vertical transmission remains controversial. Sexual transmission from infected males to uninfected females of D. citri has been reported to occur at a fairly low rate (4%) (Mann et  al., 2011). Mating occurred in Petri dishes, with psyllids feeding on sugar solutions away from susceptible host plant material. Sexual transmission was not observed following mating of infected females and uninfected males or between adult pairs of the same sex, although CLas was detected in genitalia of both sexes. A latent period of 7 days or more was required to detect the bacterium in recipient females. However, none of the plants fed upon by these psyllids became CLas-positive by PCR (Mann et al., 2011). Transovarial transmission from an infected female to her progeny through the developing oocytes in the ovaries is known with many circulative–propagative plant pathogens, including viruses and bacteria, with varying frequencies (Ammar, 1994; Hogenhout et  al., 2008a, b;

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Haapalainen, 2014). Hansen et  al. (2008) reported that 20 of 25 eggs laid by infected females of the potato psyllid B. cockerelli were PCR positive for the zebra chip bacterium CLso (Hansen et  al., 2008). However, transovarial transmission of CLas in its psyllid vector appears to be much less efficient than that of CLso. In Taiwan, Hung et  al. (2004) reported that CLas was not detected in eggs or nymphs produced by CLas-­ infected females of D. citri, although some of these females were still infected 12 weeks after they were moved to orange jasmine (Murraya sp, which is less susceptible than citrus plants to CLas. Pelz-Stelinski et  al. (2010) reported that CLas from infected D. citri female progeny reared on healthy plants was detected by qPCR in 2% of pooled egg samples, 6.3% of pooled 3rd–5th-­ instar nymphs and 2.4% of newly eclosed adults. Eggs laid on PCR-negative sweet orange were transferred to B. koenigii, considered less susceptible to CLas, to minimize likelihood of nymphs being infected from host plants possibly inoculated by their infected mothers (Pelz-Stelinski et al., 2010). Furthermore, CLas has been found by FISH in the ovaries of D. citri adults (Ammar et  al., 2011a), and CLas-like structures were found in the D. citri ovaries by TEM (Mann et al., 2011). It is important to note that none of the above-mentioned studies have demonstrated that transovarial or sexual transmission of Liberibacter results in progeny that are actually capable of infecting susceptible plants. Thus, the epidemiological significance of transovarial and sexual transmission of CLas and/or CLso is still in question. Grafton-Cardwell et al. (2013) suggested that occurrence of high populations in the field as well as possible persistence of the pathogen on low CLas-susceptible host plants like orange jasmine could make these important supplementary mechanisms of transmission. On the other hand, CLas titers were typically very low in the few psyllids from orange jasmine testing positive for CLas in Florida (Walter et al., 2012a, b).

8.7  Effects of Liberibacter on the Vector Biology and Fitness Many pathogens manipulate their vectors to promote their own spread, potentially at some

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cost to the vector (Ingwell et  al., 2012). Vector characteristics subject to manipulation may include movement, probing, feeding, flight, mating, fitness, immunity and host selection. For ­example, D. citri is known to be preferentially ­attracted to the volatile compound methyl salicylate produced by HLB-infected trees (Mann et  al., 2012). After feeding on infected plants, psyllids then prefer to settle on healthy plants (Mann et al., 2012). Signaling to D. citri via methyl salicylate may be key to the HLB pathogen dynamics because it increases the proportion of infected psyllids and host plants (Martini et  al., 2016). However, methyl salicylate is also attractive to the parasitoid wasp, Tamarixia radiata, resulting in higher incidence of parasitsim (Martini et  al., 2014). Disruption of host preference and increased parasitism suggested possible use for methyl salicylate in HLB management (Martini et al., 2016). CLas may also cause changes in feeding behavior of adult D. citri reared on CLas-infected plants, reduction in durations of non-probing period before first phloem contact, and phloem salivation event before phloem ingestion (Killiny et  al., 2017a). Feeding on CLas-infected plants increased the frequency (number of bouts) of xylem ingestion by non-infected D. citri (George et  al., 2018). Symptom expression in the plant also influenced feeding characteristics. In plants with moderate HLB symptoms, adult psyllids spent more time salivating during phloem feeding, and the percentage of time spent feeding on the phloem decreased with increasing symptom severity (Cen et al., 2012a). The impacts of Liberibacter infection in the plant or insect on psyllid biology and fitness are complex and likely dependent on vector-­pathogen, genotype-by-genotype interactions. Both positive and negative impacts have been reported in the literature. CLas-infected D. citri exhibited a higher finite rate of population increase and net reproductive rate as compared with their uninfected conspecifics (Pelz-Stelinski and Killiny, 2016; Ren et al., 2016; Wu et al., 2018b). CLas-­ positive nymphs developed faster on CLas-infected plants as compared with healthy plants (Ren et al., 2016). The impacts on fecundity and development come at a cost to the insect. CLas negatively impacts the survival of both D. citri adults (Pelz-Stelinski and Killiny, 2016; Killiny et  al., 2017b; Wu et  al., 2018b) and nymphs

(Ren et al., 2016). The costs of carrying CLas to the psyllid’s metabolism are also shared by the psyllid’s immune system. CLas-infected D. citri are more susceptible to infection by three different species of entomopathogenic fungi as ­compared with CLas-uninfected populations (Orduño‐Cruz et al., 2015). With the zebrachip Liberibacter, CLso infection in B. cockerelli negatively impacted fecundity and nymphal survival (Nachappa et  al., 2012b), but had no effect on hatching percentage, incubation time, nymphal development time or total development time (Nachappa et al., 2012b). The impacts of Liberibacters on the psyllid are mediated, in part, by the psyllid immune system (Nachappa et al., 2012a; Orduño‐Cruz et al., 2015; Ramsey et  al., 2015, 2017; Kruse et  al., 2018). D. citri resembles its close Sternorrhynchan hemimetabolous aphid relative, Acyrthosiphon pisum, in that they both have a reduced suite of immune-system genes against Gram-­ negative bacteria compared with holometabolous insects (International Aphid Genomics Consortium, 2010; Arp et al., 2016; Saha et al., 2017). This is especially true of the IMD pathway which coordinates the immune response of Drosophila melanogaster and other holometabolous insects in response to infection with Gram-negative bacteria. The majority of transcripts in the IMD pathway were also lacking in the B. cockerelli transcriptome (Nachappa et al., 2012a). The reduced immune systems of D. citri, other psyllids and perhaps all insects in the hemipteran suborder Sternorrhyncha were likely favored by ­selection due to a close relationship between the insect and Gram-negative ­ b acterial endosymbionts. D. citri has a complex microbiome involving a variety of microbial endosymbionts, including ‘Candidatus Carsonella rudii’, ‘Candidatus Profftella armatura’ and Wolbachia pipientis. The roles of ‘Ca. C. ruddii’ and ‘Ca. P. armatura’ can be inferred from genome sequencing (Nakabachi et al., 2013b) and are described in more detail in Chapter 7, this volume. ‘Ca. C. ruddii’ is the most ubiquitous symbiont among different psyllids (Tamborindeguy et al., 2017). Genome sequencing revealed that it is a nutritional symbiont and that metabolic interdependence between ‘Ca. C. ruddii’ and its insect host is a hallmark feature of the symbiosis, where genes absent from the endosymbiont genome can be found in the



Huanglongbing Pathogens

host genome and vice versa for the biosynthesis of amino acids. All three species of bacteria are maternally inherited through the reproductive tract, although their distribution in the insect varies. The Wolbachia strain found in D. citri was phylogenetically described by Saha et al. (2012) and is genetically distinct from previously characterized Wolbachia strains in other insects. Whereas ‘Ca. C. ruddii’ and ‘Ca. P. armatura’ are enclosed in a specialized organ (Nakabachi et al., 2013b), the bacteriome Wolbachia is known to reside in several tissues of psyllids where CLas has been found, such as the fat body, salivary glands, ovaries, tracheal cells and midgut (Ammar et  al., 2011a, b; Kruse et al., 2017; Mann et al., 2018; Achor et al., 2019). CLas infection may affect the interplay among the D. citri bacterial endosymbionts. Wolbachia co-localizes in the same cells of the gut as CLas, though in distinct subcellular locations involving little overlap (Kruse et al., 2017; Mann et  al., 2018). Wolbachia titers were found to be more variable in the guts of CLas-infected adult insects (Kruse et  al., 2017) and are positively correlated with the titer of CLas (Fagen et  al., 2012; Mann et  al., 2018). The strain of Wolbachia from D. citri was recently shown to encode a small, secreted protein that suppresses expression of the holin promoter in the bacteriophage of CLas, suggesting that Wolbachia may directly suppress the phage’s lytic cycle in D. citri (Jain et  al., 2017). A prediction from this finding is that the titers of Liberibacter in the insect may be regulated by Wolbachia via controlling the bacteriophage. The density of Wolbachia in the psyllid is likely controlled by the genotype of Wolbachia and/or the psyllid (Chu et al., 2016). In the mosquito Aedes aegypti, Wolbachia infection leads to a reduction in the density of four different pathogens via a proposed innate immune system priming method whereby the presence of Wolbachia activates the insect’s basal immune response to better defend against an invading pathogen (Ye et  al., 2013), but it is not known whether this occurs in D. citri. ‘Ca. P. armatura’ has been reported so far only from D. citri and has been found in all D. citri populations analyzed worldwide (Nakabachi et  al., 2013b). ‘Ca. P. armatura’ has a reduced genome at 0.54 Mb, and approximately 15% of its genes are predicted to be involved in polyketide

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metabolism (Nakabachi et  al., 2013b). A novel polyketide, diaphorin, was discovered in abundant quantities (up to 3 μg per 500 μg insect) using HPLC mass spectrometry and was determined to be structurally related to the rove beetle (family: Staphylinidae) metabolite pederin using NMR (Nakabachi et  al., 2013b, Szebenyi et al., 2018). Diaphorin was found to have mild cytotoxicity to mammalian cells in culture at low doses (Nakabachi et al., 2013b), but its toxicity to bacteria has not been investigated. Despite ‘Ca. P. armatura’ being sequestered in the bacteriome, Liberibacter and ‘Ca. P. armatura’ have a history of horizontal gene transfer. Nakabachi and colleagues discovered phylogenetic evidence that the Liberibacter bacteria (CLas, Clam and CLso) share an amino acid transporter gene (LysE) with ‘Ca. P. armatura’ acquired by horizontal gene transfer (Nakabachi et  al., 2013a). Given this close relationship, it is not surprising that the two bacteria interact, specifically by CLas infection inducing ‘Ca. P. armatura’ to alter the production of diaphorin and a structurally related diaphorin molecule, along with the ‘Ca. P. armatura’ proteins involved in the polyketide biosynthesis (Ramsey et al., 2015). They further proposed a metabolic interdependence among D. citri and ‘Ca. C. ruddii’ and ‘Ca. P.  armatura’ in the production of diaphorin through valine metabolism.

8.8  Molecular and Proteomic Interactions between Liberibacter and the Vector A draft assembly of the D. citri genome is available, and community efforts in the manual annotation of more than 500 gene models have resulted in an improved genome assembly for bioinformatics analysis (Saha et al., 2017). The psyllid genome has paved the way for transcriptomic and proteomic studies to examine the impact of Liberibacter on the psyllid vector at the molecular level (Nachappa et al., 2012a; Reese et al., 2014; Ramsey et al., 2015; Vyas et al., 2015; Kruse et  al., 2017, 2018; Ramsey et  al., 2017). Breakthrough mass spectrometry technologies for protein interaction analysis has enabled proteome-wide studies of D. citri and CLas protein interactions (Ramsey et al., 2017).

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At the proteome level, CLas infection has an impact on adult D. citri defense and immunity proteins (Kruse et  al., 2018) as well as on proteins involved in energy storage and utilization, endocytosis, cellular adhesion and cytoskeletal remodeling (Ramsey et  al., 2015, 2017). Proteins in the tricarboxylic acid (TCA) cycle are upregulated in CLas-infected insects (Ramsey et al., 2015) and CLas induces the production of TCA cycle metabolites by the psyllid (Killiny et  al., 2017b). The titer levels of CLas, as well as the host plant variety, have major impacts on the insect’s proteome response. Proteomic signatures vary if the psyllids are reared on CLas-infected sweet orange or citron (Ramsey et  al., 2015, 2017). One of the most highly expressed proteins in CLas-exposed nymph and adult D. citri is a hemocyanin, an oxygen transport protein with functions in immunity and defense reported in other arthropods (Lee et al., 2003). The C-terminus of D. citri hemocyanin-1 was shown to interact physically with the CLas coenzyme A (CoA) ­biosynthesis enzyme phosphopantothenoylcysteine synthetase/decarboxylase (Ramsey et al., 2017). Hemocyanins have conserved histidine residues forming a coordination complex with copper ions to bind oxygen for transport in the hemolymph (Markl, 2013). The expression of hemocyanin is correlated to color morphology in D. citri. There are at least three D. citri color morphs: blue, gray and yellow. Hemocyanin transcript expression in blue morphs is > threefold greater than in gray and yellow D. citri (Ramsey et  al., 2017). Blue D. citri have enhanced flight capabilities, and coupled to hemocyanin expression data, it is possible that the greater levels of hemocyanin in the blue color morphs provide the insect with enhanced metabolic capacity that could benefit vector performance and transmission. However, hemocyanin may also play a role in D. citri immunity against CLas. The role of hemocyanin in arthropod innate immunity has been previously documented, including the conversion of hemocyanin into phenoloxidase, which is a critical component of the insect’s immune response (Decker and Jaenicke, 2004). An antimicrobial peptide derived from the C-terminus of crayfish hemocyanin was shown to inhibit the growth of both Gram-negative and Gram-positive bacteria (Lee et  al., 2003). Because not all insects within a population of D. citri acquire or transmit CLas (Ammar et al., 2018), and the proteomic analysis

was conducted on pools of CLas-exposed insects collected from CLas-positive trees, the role of hemocyanin in acquisition and transmission ­remains to be investigated. Extensive interactions between CLas and D. citri first occur in the alimentary canal. Some anatomical features of the psyllid alimentary canal are specific to psyllids and some are broadly shared with other species in the Sternorrhyncha (Cicero et al., 2009). At the molecular level, the changes induced by CLas in the psyllid gut are distinct from the whole insect response, suggesting that the gut interface sustains and protects the psyllid from most of the damage caused by defensive compounds produced by an HLB-infected tree. In the gut, CLas infection reduces the expression of D. citri proteins in the TCA cycle, iron metabolism, insecticide resistance and immunity genes (Kruse et  al., 2017). There is a concerted downregulation of all detectable D. citri enzymes in the TCA cycle in D. citri reared on CLas-infected trees (Kruse et al., 2017). CLas-induced oxidative stress in the gut is more significant in males as compared with females (Mann et  al., 2018). CLas also induces changes in the nuclear morphology of the midgut epithelial cells consistent with programmed cell death (Ghanim et  al., 2016; Mann et  al., 2018). It is possible that programmed cell death in the psyllid midgut is induced by the expression of CLas effector proteins. Like other bacterial pathogens, CLas encodes effector proteins that induce cell death in plant cells (Pitino et al., 2016). In contrast, nymph midguts are nearly totally resistant to changes in midgut nuclear morphology induced by CLas (Mann et  al., 2018). One hypothesis to explain the difference between the adult and nymph midgut response, which is also supported by proteomics analysis (Ramsey et al., 2017), is that nymphs have an attenuated immune response at the molecular level as compared with adults. At the wholebody level, proteome analysis comparing CLas-­ exposed D. citri to those reared on healthy citrus plants shows 356 versus 82 proteins differentially expressed in adults and nymphs, respectively. Furthermore, the magnitudes of the changes in the nymph proteome in response to CLas are far smaller as compared with the changes induced by CLas in adult insects (Ramsey et  al., 2017). A dampened immune response may provide a window for CLas establishment in close coordination with the psyllid’s bacterial



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endosymbionts during nymphal development. This may be another reason why D. citri nymphs have a greater ability to acquire CLas and become infected by it, as compared with adults, in addition to the longer phloem ingestion by nymphs (George et al., 2018) as explained earlier.

8.9  Conclusions and Future Directions Long-term management of HLB and other bacterial diseases caused by Liberibacter species using insecticides against the psyllid vectors is not environmentally sustainable or efficient, because insect transmission of these bacteria may occur even under low psyllid vector populations (Lee et  al., 2015). Blocking bacterial spread by disrupting transmission of the bacteria by the insect vector is a new approach in the fight against vector-borne diseases. The severity of HLB and its devastating effects on citrus production in Florida and other places are fueling scientific discoveries in this pathosystem at an increasingly accelerating pace. However, many unanswered questions remain, especially regarding the mechanisms of CLas acquisition and translocation in the vector, why some psyllid populations are better CLas acquirers and/or transmitters than others, as well as the possible role of CLas receptors in the gut, salivary glands or other tissues of the psyllid vectors. Also, the question of CLas diversity in the host plant and/or in insect vectors deserves further investigation to deepen our understanding of Liberibacter pathogenesis and transmission. In reviewing the research on modes of psyllid to psyllid transmission of Liberibacters, it is clear that more definitive experiments need to be conducted to verify specifics of the ‘flush transmission’ mode (Lee et al., 2015), CLas replication in psyllid tissues and organs, and to determine both the occurrence and importance of transovarial and sexual transmission in the epidemiology of this devastating vector pathogen complex. The interactions among the

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psyllid endosymbionts, Liberibacter and the psyllid remain an underexplored research frontier. Developing a better understanding of diaphorin in the biology of D. citri may lead to the development of new strategies to control the psyllid due to the unique and highly specific relationship ­between D. citri and ‘Ca. P. armatura’. Investigating the interaction between Wolbachia and CLas may lead to the development of new tools using Wolbachia for controlling CLas transmission by D. citri. A high-resolution transcriptomic proteomic analysis of specific insect tissues, as was done for the D. citri gut, will provide a more comprehensive understanding of vector–pathogen interactions and address the questions of cooperation and conflict between the vector and the pathogen at the molecular level. Tissues of interest include the hemolymph, reproductive organs, bacteriome, salivary glands and fat body. A repository for these data exists within the Psyllid Expression Network, an online tool available on www.citrusgreening.org, for researchers to mine quantitative omics data for protein pathway discovery in response to CLas in the psyllid. Further knowledge about CLam, CLaf and CLso and their psyllid vectors may also shed light on Liberibacter–vector relations in general. These research efforts will be greatly aided by interdisciplinary teams of researchers and including industry partners bringing new technologies and creative ideas to bear on studying these emerging and economically important crop diseases.

Acknowledgements The authors gratefully acknowledge the California Citrus Research Board, USDA Agricultural Research Service (ARS), the Florida Citrus Research and Development Foundation, and USDA National Institute of Food and Agriculture (NIFA) for funding many studies that were cited in this chapter. We thank Dr David Hall (USDA ARS at Fort Pierce, Florida) for a critical review of a previous version of this chapter.

References Achor, D., Ammar, E.-D. and Levy, A. (2019) Localization of Candidatus Liberibacter asiaticus in Diaphorina citri at the ultrastructural level supports a circulative-propagative transmission mode. In:

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Orduño-Cruz, N., Guzmán-Franco, A.W. and Rodríguez-Leyva, E. (2015) Diaphorina citri populations carrying the bacterial plant pathogen ‘Candidatus Liberibacter asiaticus’ are more susceptible to infection by entomopathogenic fungi than bacteria-free populations. Agricultural and Forest Entomology 18(1), 95–98, doi: 10.1111/afe.12138. Orlovskis, Z., Canale, M.C., Thole, V., Pecher, P., Lopes, J.R. and Hogenhout, S.A. (2015) Insect-borne plant pathogenic bacteria: getting a ride goes beyond physical contact. Current Opinion in Insect Science 9, 16–23. Ozbek, E., Miller, S.A., Meulia, T. and Hogenhout, S.A. (2003) Infection and replication sites of Spiroplasma kunkelii (Class: Mollicutes) in midgut and Malpighian tubules of the leafhopper Dalbulus maidis. Journal of Invertebrate Pathology 82, 167–175. Pelz-Stelinski, K.S. and Killiny, N. (2016) Better together: association with ‘Candidatus Liberibacter asiaticus’ increases the reproductive fitness of its insect vector, Diaphorina citri (Hemiptera: Liviidae). Annals of the Entomological Society of America 109, 371–376. Pelz-Stelinski, K.S., Brlansky, R.H., Ebert, T.A. and Rogers, M.E. (2010) Transmission parameters for ‘Candidatus Liberibacter asiaticus’ by Asian citrus psyllid (Hemiptera: Psyllidae). Journal of Economic Entomology 103, 1531–1541. Perilla-Henao, L.M. and Casteel, C.L. (2016) Vector-borne bacterial plant pathogens: interactions with hemipteran insects and plants. Frontiers in Plant Science 7, 1163. Pietersen, G., Arrebola, E., Breytenbach, J., Korsten, L., Le Roux, H.F., La Grange, H., Lopes, S., Meyer, J.B., Pretorius, M. and Schwerdtfeger, M. (2010) A survey for ‘Candidatus Liberibacter’species in South Africa confirms the presence of only ‘Ca. L. africanus’ in commercial citrus. Plant Disease 94, 244–249. Pinheiro, P.V., Ghanim, M., Alexander, M., Rebelo, A.R., Santos, R.S., Orsburn, B.C., Gray, S. and Cilia, M. (2017) Host plants indirectly influence plant virus transmission by altering gut cysteine protease activity of aphid vectors. Molecular & Cellular Proteomics 16, S230–S243. Pitino, M., Hoffman, M.T., Zhou, L., Hall, D.G., Stocks, I.C. and Duan, Y. (2014) The phloem-sap feeding mealybug (Ferrisia virgata) carries ‘Candidatus Liberibacter asiaticus’ populations that do not cause disease in host plants. PLOS ONE 9, e85503. Pitino, M., Armstrong, C.M., Cano, L.M. and Duan, Y. (2016) Transient expression of ‘Candidatus Liberibacter asiaticus’ effector induces cell death in Nicotiana benthamiana. Frontiers in Plant Science 7, 982. Raiol-Junior, L.L., Baia, A.D.B., Luiz, F.Q.B.F., Fassini, C.G., Marques, V.V. and Lopes, S.A. (2017) Improvement in the excised leaf assay to investigate inoculation of ‘Candidatus Liberibacter asiaticus’ by the Asian citrus psyllid Diaphorina citri. Plant Disease 101, 409–413. Ramsey, J.S., Johnson, R.S., Hoki, J.S., Kruse, A., Mahoney, J., Hilf, M.E., Hunter, W.B., Hall, D.G., Schroeder, F.C., Maccoss, M.J. and Cilia, M. (2015) Metabolic interplay between the Asian citrus psyllid and its profftella symbiont: an Achilles’ heel of the citrus greening insect vector. PLOS ONE 10, e0140826. Ramsey, J.S., Chavez, J.D., Johnson, R., Hosseinzadeh, S., Mahoney, J.E., Mohr, J.P., Robison, F., Zhong, X., Hall, D.G., Maccoss, M. et al. (2017) Protein interaction networks at the host-microbe interface in Diaphorina citri, the insect vector of the citrus greening pathogen. Royal Society Open Science 4, 160545. Reese, J., Christenson, M.K., Leng, N., Saha, S., Cantarel, B., Lindeberg, M., Tamborindeguy, C., Maccarthy, J., Weaver, D., Trease, A.J. et al. (2014) Characterization of the Asian citrus psyllid transcriptome. Journal of Genomics 2, 54–58. Ren, S.L., Li, Y.H., Zhou, Y.T., Xu, W.M., Cuthbertson, A.G., Guo, Y.J. and Qiu, B.L. (2016) Effects of ‘Candidatus Liberibacter asiaticus’ on the fitness of the vector Diaphorina citri. Journal of Applied Microbiology 121, 1718–1726. Roistacher, C.N. (1991) Graft-transmissible Diseases of Citrus: Handbook for Detection and Diagnosis. UN Food & Agriculture Organization, Rome. Saha, S., Hunter, W.B., Reese, J., Morgan, J.K., Marutani-Hert, M., Huang, H. et al. (2012) Survey of endosymbionts in the Diaphorina citri metagenome and assembly of a Wolbachia wDi draft genome. PLoS ONE 7(11), e50067. doi: 10.1371/journal.pone.0050067. Saha, S., Hosmani, P.S., Villalobos-Ayala, K., Miller, S., Shippy, T., Flores, M., Rosendale, A., Cordola, C., Bell, T., Mann, H. et al. (2017) Improved annotation of the insect vector of citrus greening disease: ­biocuration by a diverse genomics community. Database 2017, bax032-bax032. Sengoda, V.G., Buchman, J.L., Henne, D.C., Pappu, H.R. and Munyaneza, J.E. (2013) ‘Candidatus ­Liberibacter solanacearum’ titer over time in Bactericera cockerelli (Hemiptera: Triozidae) after acquisition from infected potato and tomato plants. Journal of Economic Entomology 106, 1964–1972.

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Sengoda, V.G., Cooper, W.R., Swisher, K.D., Henne, D.C. and Munyaneza, J.E. (2014) Latent period and transmission of ‘Candidatus Liberibacter solanacearum’ by the potato psyllid Bactericera cockerelli (Hemiptera: Triozidae). PLOS ONE 9, e93475. Sétamou, M., Alabi, O.J., Kunta, M., Jifon, J.L. and Da Graca, J.V. (2016) Enhanced acquisition rates of ‘Candidatus Liberibacter asiaticus’ by the Asian citrus psyllid (Hemiptera: Liviidae) in the presence of vegetative flush growth in citrus. Journal of Economic Entomology 109, 1973–1978. Sétamou, M., Alabi, O.J., Simpson, C.R. and Jifon, J.L. (2017) Contrasting amino acid profiles among permissive and non-permissive hosts of ‘Candidatus Liberibacter asiaticus,’ putative causal agent of Huanglongbing. PLOS ONE 12, e0187921. Silva, C.P., Silva, J.R., Vasconcelos, F.F., Petretski, M.D., Damatta, R.A., Ribeiro, A.F. and Terra, W.R. (2004) Occurrence of midgut perimicrovillar membranes in paraneopteran insect orders with comments on their function and evolutionary significance. Arthropod Structure & Development 33, 139–148. Szebenyi, D.M., Kriksunov, I., Howe, K.J., Ramsey, J.S., Hall, D.G., Heck, M. and Krasnoff, S. (2018) Crystal structure of diaphorin methanol monosolvate isolated from Diaphorina citri Kuwayama, the insect vector of citrus greening disease. Acta Crystallographica Section E: Crystallographic Communications 74, 445–449. Tamborindeguy, C., Huot, O.B., Ibanez, F. and Levy, J. (2017) The influence of bacteria on multitrophic interactions among plants, psyllids, and pathogen. Insect Science 24, 961–974. Tatineni, S., Sagaram, U.S., Gowda, S., Robertson, C.J., Dawson, W.O., Iwanami, T. and Wang, N. (2008) In planta distribution of ‘Candidatus Liberibacter asiaticus’ as revealed by polymerase chain reaction (PCR) and real-time PCR. Phytopathology 98, 592–599. Texeira, D.D.C., Ayres, J., Kitajima, E., Danet, L., Jagoueix-Eveillard, S., Saillard, C. and Bové, J. (2005) First report of a huanglongbing-like disease of citrus in São Paulo State, Brazil and association of a new Liberibacter species, ‘Candidatus Liberibacter americanus,’ with the disease. Plant Disease 89, 107–107. Teixeira, D., Eveillard, S., Sirand-Pugnet, P., Wulff, A., Saillard, C., Ayres, A. and Bové, J. (2008) The tufB–secE–nusG–rplKAJL–rpoB gene cluster of the liberibacters: sequence comparisons, phylogeny and speciation. International Journal of Systematic and Evolutionary Microbiology 58, 1414–1421. Torres, G.L., Cooper, W.R., Horton, D.R., Swisher, K.D., Garczynski, S.F., Munyaneza, J.E. and Barcenas, N.M. (2015) Horizontal transmission of ‘Candidatus Liberibacter solanacearum’ by Bactericera cockerelli (Hemiptera: Triozidae) on convolvulus and ipomoea (Solanales: Convolvulaceae). PLOS ONE 10, e0142734. Tsai, J.H. and Liu, Y.H. (2000) Biology of Diaphorina citri (Homoptera: Psyllidae) on four host plants. Journal of Economic Entomology 93, 1721–1725. Ukuda-Hosokawa, R., Sadoyama, Y., Kishaba, M., Kuriwada, T., Anbutsu, H. and Fukatsu, T. (2015) Infection density dynamics of the citrus greening bacterium ‘Candidatus Liberibacter asiaticus’ in field populations of the psyllid Diaphorina citri and its relevance to the efficiency of pathogen transmission to citrus plants. Applied and Environmental Microbiology 81, 3728–3736. Vyas, M., Fisher, T.W., He, R., Nelson, W., Yin, G., Cicero, J.M., Willer, M., Kim, R., Kramer, R., May, G.A. et  al. (2015) Asian citrus psyllid expression profiles suggest ‘Candidatus Liberibacter asiaticus’-­ mediated alteration of adult nutrition and metabolism, and of nymphal development and immunity. PLOS ONE 10, e0130328. Walter, A.J., Duan, Y. and Hall, D.G. (2012a) Titers of ‘Candidatus Liberibacter asiaticus’ in Murraya paniculata and Murraya-reared Diaphorina citri are much lower than in citrus and citrus-reared psyllids. HortScience 47, 1–4. Walter, A.J., Hall, D.G. and Duan, Y. (2012b) Low incidence of ‘Candidatus Liberibacter asiaticus’ in Diaphorina citri and its host plant Murraya paniculata. Plant Disease 96, 827–832. Wayadande, A.C. and Fletcher, J. (1998) Development and use of an established cell line of the leafhopper Circulifer tenellus to characterize Spiroplasma citri–vector interactions. Journal of Invertebrate Pathology 72, 126–131. Wu, F., Liang, G., Chen, J., Huang, J. and Cen, Y. (2013) The movement and spread of nymphs of Diaphorina citri Kuwayama on host plants. Journal of Environmental Entomology 35, 578–584. Wu, F., Cen, Y., Deng, X., Chen, J., Xia, Y. and Liang, G. (2015) Movement of Diaphorina citri (Hemiptera: Liviidae) adults between huanglongbing-infected and healthy citrus. Florida Entomologist 98, 410–416. Wu, F., Huang, J., Xu, M., Fox, E.G., Beattie, G.A.C., Holford, P., Cen, Y. and Deng, X. (2018a) Host and environmental factors influencing ‘Candidatus Liberibacter asiaticus’ acquisition in Diaphorina citri. Pest Management Science 74, 12.



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Wu, F., Qureshi, J.A., Huang, J., Fox, E.G.P., Deng, X., Wan, F., Liang, G. and Cen, Y. (2018b) Host plant-mediated interactions between ‘Candidatus Liberibacter asiaticus’ and its vector Diaphorina citri Kuwayama (Hemiptera: Liviidae). Journal of Economic Entomology 111, 2038–2035. Wu, T., Luo, X., Xu, C., Wu, F., Qureshi, J.A. and Cen, Y. (2016) Feeding behavior of Diaphorina citri and its transmission of ‘Candidatus Liberibacter asiaticus’ to citrus. Entomologia Experimentalis et Applicata 161, 104–111. Xu, C., Xia, Y., Li, K. and Ke, C. (1988) Further study of the transmission of citrus huanglungbin by a psyllid, Diaphorina citri Kuwayama. International Organization of Citrus Virologists Conference Proceedings (1957–2010), 1988. Yang, C. and Li, F. (1984) Nine new species and a new genus of psyllids from Yunnan. K'un ch'ung fen lei hsueh pao= Entomotaxonomia 6, 251–266 Ye, Y., Woolfit, M., Rances, E., O'Neill, S. and McGraw, E. (2013) Wolbachia-associated bacterial protection in the mosquito Aedes aegypti. PLOS Neglected Tropical Diseases 7, e2362. Yu, J., Wayadande, A. and Fletcher, J. (2000) Spiroplasma citri surface protein P89 implicated in adhesion to cells of the vector Circulifer tenellus. Phytopathology 90, 716–722. Zhou, L., Powell, C.A., Li, W., Irey, M. and Duan, Y. (2013) Prophage-mediated dynamics of ‘Candidatus Liberibacter asiaticus’ populations, the destructive bacterial pathogens of citrus Huanglongbing. PLOS ONE 8, e82248.

9 Epidemiology of Huanglongbing: Implications of Infective Colonization Events

Susan Halbert1* and Burton Singer2 Florida Department of Agriculture and Consumer Services, Division of Plant Industry, Gainesville, Florida, USA; 2University of Florida, Emerging Pathogens Institute, Gainesville, Florida, USA 1

This chapter discusses the epidemiology of huan­ glongbing (HLB, citrus greening) in light of a new transmission mechanism presented by Lee et  al. (2015). Our examples are from Florida, where incursion of HLB has been observed in detail over the past 14 years. We conclude with implications for future management of the dis­ ease. Most vectored plant pathogens can be ­acquired by their vectors at or just prior to the onset of symptoms. Until then, the plant might be infected, but no transmission occurs to other plants. The interval between infection and the time when vectors can acquire the pathogens is the latent period. The time between infection and the onset of symptoms in the plant is the incubation period (Chiyaka et  al., 2012). Incu­ bation period and latent period have been used interchangeably because, in many cases, they are the same. In the case of HLB, the latent period and the incubation period are not the same. The latent period is equal to one generation of psyllid vec­ tors (Lee et al., 2015), and the incubation period is long and variable, up to at least 6 years (Shen et al., 2013). This situation is due to the recently identified transmission mechanism, discovered by Dr William O. Dawson (UF, IFAS, CREC),

detailed in Lee et  al. (2015). This chapter will discuss the implications of the new mechanism on spread of HLB at various spatial scales. A final section suggests how the variability of the incu­ bation period could be used to advantage in management of disease.

9.1  The New Transmission Mechanism: Infective Colonization Events Lee et al. (2015) presented the experiments and results demonstrating the brief duration of the latent period and the phenomenon of infective colonization events. Briefly, several healthy citrus seedlings were placed in a cage with Diaphorina citri Kuwayama (Hemiptera: Psylloidea) from an infected culture (both plants and insect vectors exposed). Plants with new growth were used, be­ cause D. citri requires new citrus sprouts (flush) for reproduction. After about 2 weeks, all adults were removed, leaving only their progeny. The nymphs were allowed to mature on new growth of the plants, and the resulting adults were tested for ‘Candidatus Liberibacter asiaticus’ (CLas), the

*  Email: [email protected]

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presumed pathogen that causes HLB. In most cases, many of the adults were positive for CLas, indicating that the pathogen was being passed to the progeny of the insects. We refer to this phe­ nomenon as an infective colonization event. Such events can happen in at least two ways. Either there is transovarial transmission, or the nymphs acquire CLas from their feeding site infected by their parents. Transovarial trans­ mission has been demonstrated by Pelz-Stelinski et  al. (2010), but it occurs at a very low rate. Dr  Dawson tested the psyllid colonized shoots and they also were positive for CLas, indicating that the nymphs are acquiring CLas from the feeding site (Lee et al., 2015). Thus, the latent period for HLB equals one generation of psyllid vectors. Depending on tem­ perature and availability of flush, this can occur in 2–3 weeks. A female psyllid can lay up to an average of 748 eggs under laboratory conditions (Tsai and Liu, 2000). The increase in numbers of positive psyllids can be enormous, especially if abundant new colonization sites are available for emerging infected adults. Some of the plants in the cages were not col­ onized by psyllids. Most of the colonized plants in Dawson’s experiments subsequently developed symptoms, but the non-colonized plants rarely developed HLB (Lee et  al., 2015). This indicates that colonization likely plays a significant part in determining whether potential infection by CLas-positive psyllids results in disease develop­ ment. It also suggests that there is a minimum concentration of CLas in young flush required for eventual development of HLB symptoms. Many questions remain. For example, acqui­ sition of CLas in infective colonization events can be variable (0–83% in Dawson’s experiments) (Lee et al., 2015). The parameters that influence the efficiency of infective colonization events are largely unknown. Similarly, the relative import­ ance of infective colonization events compared with acquisition from chronic systemically infected trees in HLB epidemiology is unknown.

9.2  Significance of Positive Psyllids and Psyllid Testing A direct practical result of the new transmis­ sion mechanism described above is that testing

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psyllids can provide early warning for presence of CLas, prior to symptom development. The po­ tentially profuse proliferation of CLas-positive psyllids through infective colonization events can result in detectable CLas-positive psyllids long before symptoms of HLB develop (Manju­ nath et  al., 2008; Halbert et  al., 2010, 2012; Shen et  al., 2013) (Fig. 9.1). In several cases, positive psyllids were found a year or more before symptoms developed, even though the plants were inspected regularly. In the case of the Flor­ ida Department of Agriculture and Consumer Services, Division of Plant Industry (DPI) Citrus Arboretum, the first positive psyllids were found in 2005, and the first positive tree was found about 2 years later in 2007. All the plants were examined and tested by molecular analysis every year (Manjunath et  al., 2008). Testing psyllids has been adopted as a means of early detection and has resulted in discovery of positive plants in Texas and California (e.g. Wesson, 2017). Psyllid testing also can be used to learn about disease dynamics. The appearance of posi­ tive psyllids indicates bacterial presence, even though there are no visible symptoms. Much can be learned about the movement of HLB by testing psyllids (Manjunath et al., 2008). Unfor­ tunately, the percentage of positive psyllids still is annoyingly unpredictable. A high percentage of psyllids may be positive, even though plants are asymptomatic. This is explained at least in part by Dawson’s transmission mechanism, but the story appears to be more complicated. It re­ mains difficult to maintain a laboratory colony with 100% positive insects, even if all the plants are positive. Similarly, it is sometimes difficult to find a majority of psyllids positive in a com­ pletely infected citrus grove. In the Bactericera cockerelli (Šulc)/Candidatus Liberibacter solan­ acearum (CLso) system, there was a dramatic decline in proportions of positive psyllids after several generations in field-cage trials (Workneh and Paetzold, 2016). This result could indicate that the infective colonization event mechanism does not occur in the CLso system, such that infectivity in succeeding generations would ­depend on transovarial transmission. Alterna­ tively, there could be another mechanism at work that, if elucidated, could help explain the variability seen in positive D. citri. Similarly, presence of CLas in a psyllid does not automatically mean that it can transmit the

12/2005

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Orlando

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Tampa

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Tampa

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+ Plants + Psyllids 2008

Miami

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Miami

Miami

+ Psyllids 2007 + Psyllids 2006 + Psyllids 2005

Fig. 9.1.  Maps of the Florida peninsula showing the distribution of plants (2005–2012) and psyllids (2005–2008) positive for Candidatus Liberibacter asiaticus. Huanglongbing (HLB) was found in Florida in August 2005. By 2010, HLB was widespread, especially along major road corridors. By 2012, most commercial groves were affected. Positive psyllids sometimes were found in places where the disease had not been detected yet. Maps are courtesy of Alicia B. Lawrence, Florida Department of Agriculture and Consumer Services, Division of Plant Industry.



Epidemiology of Huanglongbing

pathogen. Ammar et al. (2016) and Inoue et al. (2009) found that D. citri that acquired CLas as adults did not transmit the pathogens. Pelz-­ Stelinski et  al. (2010) found rare transmission events by psyllids that acquired CLas as adults. The percentage of positive psyllids varies seasonally, with a build-up of positive psyllids during the rainy season that allows back-to-back colonization (Hall, 2017). Ukuda-Hosokawa et al. (2015) showed an increase in titer (bacteria per insect) during spring, coinciding with spring flush. In Pakistan, Razi et  al. (2014) showed a marked decrease in numbers of positive psyllids in the hot summer months. The insects survived, but they did not test positive for the pathogens. The authors attributed this decrease to natural thermotherapy. Although ‘citrus dieback’ has been a problem for a century or more (Husain and Nath, 1927), production is somewhat easier in Pakistan than in humid subtropical climates such as southern Florida, where flushing can be nearly continuous, with no hiatus in infective colonization events. Much remains to be learned about the parameters that drive acquisition of CLas and subsequent transmission efficiency, especially in field settings. Particularly vexing is the lack of empirical information on psyllid invasions at the grove and multi-grove level. We know from mi­ crosimulation studies (Lee et al., 2015) that the rate and pattern of spread of asymptomatic in­ fection is sensitive to the frequency and pattern of arrival of infected psyllids to a grove. Lee et al. (2015) considered some scenarios where psyl­ lids invaded a previously uninfected grove from an adjacent infected grove along an edge and/or in a corner, or blown in by the wind and dropped into the uninfected grove in a random pattern. The distinct entrance patterns led to totally dif­ ferent subsequent patterns of HLB spread. Enter­ ing an uninfected grove in present-day Florida is not a likely situation. However, invasions of al­ ready infected groves by either of the previously mentioned patterns serve to enhance the spread of infection, but branching out from places cor­ responding to the new invasion site(s). Rather than considering only a single new invasion, there can be edge/corner invasion followed a few days later by psyllids blown in by the wind and dropped into the uninfected, or previously infected, grove in a random pattern. Here, the relative sizes of psyllid populations in successive

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invasions and the percentage of infected psyllids among them (infectivity) can greatly influence the subsequent pattern of CLas spread. Meas­ urements of psyllid invasion and concurrent in­ fectivity are needed to better understand disease ­dynamics and to validate, or adaptively adjust, what has been seen in simulated invasions.

9.3  Single Trees One of the salient qualities of Candidatus Liberibac­ ter spp. is their ability to hide (Wang et al., 2017). CLas can be graft-transmitted from both asymp­ tomatic and symptomatic bud sources (Lin and Lin, 1990). A single shoot is infected after an infective colonization event, but the tree can re­ main asymptomatic for years. The long incuba­ tion period may be due in part to slow movement through phloem, although it is not clear what possible barriers are responsible. There also is evidence that the bacteria move vertically much more readily than they move laterally (Wang et al., 2017). However, CLso moves very quickly to potato tubers, even if the plant is infested with infective psyllids a few days prior to harvest (Rashed et  al., 2013). The rate of movement within the tree is one of the most important parameters necessary for a mathematical model to determine the relative roles of vector infection and systemic tree infection in acquisition of CLas by psyllids (Chiyaka et al., 2012). Equally important, if not more so, is the need to understand the processes that lead to onset of symptoms. A much deeper understand­ ing of the within-tree dynamics of CLas and phloem will be necessary before mechanistically based prediction of symptom onset can be made. Recent progress in this direction is the detailed proposal of a process of dynamic switching by bacteria to facilitate their dispersal throughout a flow network and, thereby, maximize colon­ ization (Kannan et  al., 2018). This is only one piece of a much larger puzzle that needs to be filled in for HLB. One significant discovery that emerged from the intense research effort in Florida was the extent of root damage due to CLas infection (Dung et al., 2009; Johnson et al., 2014). It had been assumed that root damage was a second­ ary result of phloem plugging in the canopy.

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In fact, roots often are heavily colonized by CLas before symptoms appear in the canopy. Johnson et al. (2014) demonstrated 26% root loss prior to foliar symptom development. Detection of CLas in roots also is more consistent than detection in the canopy; and in the majority of trees, root in­ fection was detected prior to canopy infection, even though twice as many foliar samples were taken per tree (Johnson et  al., 2014). It is not known how much of the observed canopy dam­ age is the result of prior damage to the roots. It is also possible that invisible phloem damage in the canopy contributes to root damage. Direct tissue blot immunoassay confirms root infection (Ding et al., 2015). The method is a highly useful one for direct observation of location of the pathogen. These experiments should be done over time to see where and when the pathogens move in the tree. The incubation period for HLB can be long and variable in the field (Shen et al., 2013). Even in the laboratory, the incubation period is vari­ able. Albrecht et  al. (2014) reported 50% graft transmission 6–11 weeks after inoculation, and 80–90% 16–26 weeks after inoculation. In the field, also with young trees, detectable CLas inci­ dence remained below 50% for at least a year after planting. The long incubation period pre­ sumably means that plants resist disease; the variability suggests that some plants fight better than others. Environmental (or even microenvi­ ronmental) conditions could enable some plants to resist disease development. The incubation period also is likely to be dependent on the dose of CLas in a given tree, which is determined largely by psyllid invasion patterns over time. At least two mechanisms provide inoculum for psyllids. Below, we discuss root-sourced inocu­ lum from chronically infected trees, and inocu­ lum from infective colonization events. Johnson et  al. (2014) demonstrated that infected roots provide a reservoir of inoculum for the canopy. When the tree flushes, CLas flows from the in­ fected roots to the new shoots. Coletta-Filho et al. (2014) tested psyllid acquisition from graft-­ inoculated trees at varying times after inocula­ tion. Acquisition, measured by CLas-positive psyllids, increased with bacterial titer and time after infection, but acquisition occurred as early as the first measurement, taken at 60 days post-­ inoculation. They conclude that this shows psyl­ lids can acquire CLas during the asymptomatic

phase of infection. Since the experiment was done only with adult psyllids, inoculation of test plants by positive psyllids was very low – only three of a total of 419 plants. Thus, this experi­ ment did not test the contribution of chronically infected trees (root-sourced infection) adequately. Infective colonization events also can produce colonies of infective psyllids. Stansly et al. (2014) demonstrated that insecticide alone could in­ crease yield of citrus in a commercial grove. Their results show that reinfection, presumably by infective colonization events, matters. Indeed, this is where fresh invasion – especially multiple invasions during a flushing season – can nullify the effect of infrequent insecticidal spraying. The interplay between these two mechan­ isms has not been elucidated. Hilf and Luo (2018) measured CLas in shoots that grew from heavily pruned small trees in order to determine timing of bacterial colonization of new growth. They took measurements 30, 60 and 90 days after pruning. The first measurement (30 days) was too late to determine whether shoots be­ come positive from root-sourced inoculum prior to becoming positive from an infective coloniza­ tion event, because psyllid colonies mature in much less than 30 days. Does colonization of more shoots increase disease because the bac­ teria do not move horizontally in vascular tis­ sue? Does root infection distribute bacteria to the whole tree? Are psyllids more likely to acquire CLas from root-sourced inoculum, or from a colony infected by its founding female? Does bac­ terial titer in a tree decline over time without re­ infection? These questions have a bearing on both epidemiology and management of the disease.

9.4  Grove-scale Movement Spread within a citrus grove has been studied extensively (Gottwald et  al., 1989, 1991a, b; Aubert, 1990; Gottwald, 2010), but surprisingly these authors do not consider the effect of differ­ ent psyllid invasion scenarios or even measure psyllid pressure or infectivity. Moreover, all these studies assumed a constant incubation period. Symptoms are observed over time, and plants that develop symptoms first are assumed to have been infected first. Temporal and spatial patterns are developed based on appearance of symptomatic



Epidemiology of Huanglongbing

plants. Since the parameters that control symptom development are understood poorly, it is possible that this underlying assumption of a constant incubation period should be ques­ tioned. If after just a few life cycles, enormous numbers of CLas-positive psyllids can material­ ize via infective colonization events, it is possible that, in some cases, whole blocks are infected nearly simultaneously, and symptom develop­ ment depends on dosage of CLas, or even under­ lying physiological parameters determined by the locations of the trees. Albrecht et al. (2014) planted trees in June 2005 and again in August 2007. All the trees had standard psyllid control and fertilizer. In ap­ proximately 3 years, nearly all the plants were positive for CLas. Symptoms eventually appeared in all the PCR-positive plants, but often PCR de­ tection preceded symptom expression. Approxi­ mately 2 years after planting, the number of trees detected positive by PCR was double or triple the number that expressed the disease. In the second experiment (planted in 2007), all the plants were positive by PCR almost 2 years be­ fore all had symptoms. Only 50–60% of the plants showed symptoms by the time all were positive by PCR (see Figure 1 in the article). The average length of time between positive PCR and symptoms can be estimated at about 1 year from experiments by Albrecht et al. (2014) and Shen et al. (2013); however, symptoms may take consid­ erably longer to develop on some trees (Albrecht et al., 2014). Probably symptom expression also is dependent on the age of the trees. The actual incubation period also includes the time be­ tween psyllid inoculation and PCR detection. Given that positive psyllids, including nymphs, have been discovered years prior to PCR detec­ tion (Shen et al., 2013), it is possible that the in­ cubation period is even longer and more variable than studies on the time lag between PCR detec­ tion and symptoms might indicate. The plant physiological processes involved in disease expression seem to be important. In the CLso system, Rashed et  al. (2016) showed that severity of disease, measured by biochem­ ical analysis, usually was correlated with vector density, but there was also a significant cultivar effect. Stansly et  al. (2014) found that insecti­ cides alone can mitigate symptoms, indicating that reinfection could be important. Thus, it might be that plants developing early symptoms

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were infected by more psyllids rather than earl­ ier psyllids. These findings would support the possibility that more infective colonization events could result in earlier symptom expres­ sion. Similarly, microenvironment might play a role in early disease development. Research is needed to match disease spread information with some reproducible measure of vector abun­ dance and infectivity over time. This is precisely where the lack of documentation of psyllid inva­ sion patterns creates severe difficulties. It is not enough to observe symptom emergence patterns over time and then ask what psyllid invasion pat­ terns might be consistent with them. There are simply too many possible invasion scenarios that could give rise to a given symptom emergence pattern. We find it curious that the HLB litera­ ture to date is almost devoid of consideration of the large literature on invasions and spatial spread in the extant ecology and plant pathology literature (e.g. Hastings et al., 2005; Gilligan and van den Bosch, 2008; Melbourne and Hastings, 2009). Having said this, doing field measure­ ment and modeling on insect dispersal for peren­ nial plants is quite difficult. Nevertheless, there has been recent progress in the study of the aphid-borne plum pox virus in a multi-orchard setting (Pleydell et al., 2018). Udell et  al. (2017) provided useful psyllid and flush information in commercial citrus in southwest Florida. An outbreak of D. citri ­occurred in one of the study groves (Felda) in 2013. The grove in Felda flushed continuously for about 6 months, beginning in January 2013. Consequently, there was opportunity for back-to back-colonization by D. citri for the entire period. The percentage of plants positive for CLas in­ creased much more rapidly after the outbreak. The model developed by Lee et  al. (2015) pre­ dicted that back-to-back colonization in the presence of the CLas pathogen would result in a high rate of increase in infected trees. It appears that this may have occurred in Felda in 2013. Several authors have noted edge effects and directional bias in appearance of HLB symptoms in a grove (Albrecht et al., 2014; Sétamou and Bartels, 2015). More D. citri were trapped at the grove edges than in the center, and especially on the southeastern edge. Our observations in ­Florida (S.E. Halbert and M.W. Brodie, 2005– 2007, unpublished results) indicated that HLB symptoms often appeared first in the southeastern

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edges of groves. In field studies by Albrecht et al. (2014), trees in the southern part of each row became positive first. Pelz-Stelinski et al. (2016) found that groves planted with rows oriented east–west (southern sun exposure along the row) had more psyllids than those oriented north–south. Sétamou and Bartels (2015) speculated that the reason for higher numbers of psyllids on edges could be due to psyllid ­behavior, or it could be due to increased citrus flush, which promotes psyllid reproduction (and infective colonization events). Edges re­ ceive more light than inner trees. Higher avail­ ability of light could influence both the insects, which have shown highly complex reactions to various light regimes (Paris et  al., 2017), and the plants. In summary, HLB usually appears on the edges of groves (and grove blocks) before it appears in the centers (Albrecht et  al., 2014; ­Sétamou and Bartels, 2015). Edges remain more ­severely damaged, even as the disease claims the remainder of the planting. Sétamou and Bartels (2015) showed that more psyllids are found on the edges of groves and blocks. It is not known whether trees on the edges manifest symptoms earlier because they were infected first, because more insects inoculated them, or because of mi­ croenvironmental factors such as more light. Trees are infected, as shown by PCR assays, be­ fore they manifest symptoms (Shen et al., 2013; Albrecht et  al., 2014). The actual extent and progression of an epidemic in a grove is difficult to measure, because trees might be infected long before they manifest symptoms or even become PCR positive. This point leads naturally to an important open empirical and theoretical problem: namely, characterization of the dynamics of both the new infection front and the symptom front at the grove and multi-grove level. There is a large vol­ ume of theoretical literature dating back to R.A. Fisher’s classic 1937 paper on wave of advance of advantageous genes (Fisher, 1937; Hastings, 1996), extensions of which would be relevant for immediate use in characterizing the wave of advance of new CLas infection fronts. The larger the separation in time between this front and the onset of symptoms front, the longer there will be productive citrus trees. However, specifying the process and mechanics of onset of symptoms in comparison with the infection front is a critical

missing link in our ability to develop a rigorous understanding of how grove damage develops.

9.5  Scale of the Grove Neighborhood Both landscape and management factors influ­ ence the activity of D. citri and movement of CLas in a grove neighborhood. Individual grove management plays a defining role, because area-wide management of the insects is needed to reduce populations by avoiding unmanaged ref­ uges. Landscape factors that influence the extent of HLB include proximity to urban areas (Pelz-­ Stelinski et al., 2016) and presence or absence of windbreaks (Martini et  al., 2015). Curiously, elevation also was significant (Pelz-Stelinski et al., 2016), even though the variation in elevation in peninsular Florida is less than 100 m. Grove management on a regional basis de­ termines the abundance of D. citri. In Brazil, the fate of new plantings depended on area-wide management, with new citrus blocks planted close to ‘bad neighbors’ faring poorly (Bassanezi et al., 2013). Similarly, Boina et al. (2009) marked D. citri in managed and unmanaged plots using a novel immuno-marking technology. They recaptured more D. citri that had moved from unmanaged citrus to managed citrus than D. citri that moved from managed to unmanaged citrus. Lewis-Rosenblum et  al. (2015) used similar marking technology to demonstrate movement from adjacent unmanaged to managed groves. Additionally, they showed that long-range move­ ment was sufficient to put managed groves at risk if abandoned or marginally managed groves were present in the vicinity. Marginally man­ aged groves had more D. citri than abandoned ones in a survey by Pelz-Stelinski et  al. (2016). Grove neighborhood management practices had a major effect on the abundance of D. citri (Pelz-Stelinski et al., 2016). Proximity to urban areas had a significant effect on the abundance of D. citri (Pelz-Stelinski et al., 2016). Reasons for this might include un­ managed infected citrus in residential areas, presence of untreated plantings of ornamental citrus relatives like Murraya paniculata (L.) Jack (orange jasmine), and D. citri hosts grown in protected environments near buildings, where



Epidemiology of Huanglongbing

D. citri can overwinter easily. Proximity to urban development also might restrict spray options for commercial growers. Bishop and Guthrie (1964) demonstrated a similar phenomenon for po­ tato leafroll virus and its aphid vector Myzus (Nectarosiphon) persicae (Sulzer). Aphid-infested bedding plants (flowers and vegetables) were available in stores in the spring, and saved potato seed pieces perpetuated the virus from year to year. The closer the potato seed production farm was to a village, the more likely it was that the crop would have unacceptable levels of virus-­ infected seed pieces. The problem was solved by inspecting bedding plants and providing free clean seed pieces to home gardeners (Bishop, 1967). Unfortunately, the solutions are not so simple for citrus, because it is a perennial crop. However, the findings of Pelz-Stelinski et  al. (2016) underscore the importance of getting urban gardeners on board with area-wide man­ agement strategies. Sétamou and Bartels (2015) found the same edge factor in a grove neighbor­ hood as in a single block. Groves at the outer edges of the study area had significantly more D. citri. Windbreaks reduced the numbers of D. citri (Martini et al., 2015). Apparently, there was no difference in numbers of natural enemies, which ruled out one of the known causes of reduction in pest numbers near barriers. Another possible explanation for reduced numbers of D. citri near windbreaks is shade. This also could explain their finding that trees in newly planted entire blocks had more D. citri than resets in mature groves (Martini et al., 2015). A microsimulation study analogous to that of Lee et  al. (2015), but focused on dynamics across multiple neighboring groves, has not been carried out to date, but such a study could be valuable for providing insight about a diver­ sity of management strategies. An important as­ pect of a study that builds on the Lee et al. (2015) analyses would be to include more flexible dis­ persal kernels in the model, and particularly to allow for occasional long-distance flights (both between groves and within larger groves) by migrating psyllids. Such flights can initiate infective colonization events in previously clean parts of a grove and provide a basis for irregu­ lar patterns of later symptom development. Alternative management strategies and their consequences could be explored in such a mod­ eling study.

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In summary, numbers of D. citri and conse­ quent spread of HLB in a grove neighborhood is variable, but overall citrus health can be im­ proved by area-wide insect management. Other contributing factors include proximity to urban areas and landscape architecture.

9.6  Statewide Movement Statewide movement of HLB in Florida was ­facilitated greatly by human activities (Shimwela et al., 2018), both in movement of fruit (Halbert et  al., 2010) and movement of nursery stock, especially M. paniculata, a popular ornamental citrus relative that is a favored host for D. citri (Halbert et al., 2012). Gottwald et al. (2007) esti­ mated that HLB moves regionally at a rate of about 20 km per year. HLB colonized the Florida peninsula much faster than that. This section will attempt to explain why that occurred. It appears that D. citri sometimes can fly about 70 km on its own, if there are no barriers (Halbert et al., 2008). The eastern edges of com­ mercial groves just west of the Florida Everglades began to develop symptoms of HLB in 2005 (Fig. 9.1). The urban areas along the Florida east coast, about 70 km away, were heavily infected at the time. There is little if any citrus in the Everglades, and no roads for transport-assisted psyllid movement. Even accounting for this oc­ casional long-distance dispersal capability, the Florida peninsula became contaminated with HLB far faster than would be expected without human-assisted movement of insects and patho­ gens (Shimwela et al., 2018). Delimiting surveys for D. citri after it was discovered in 1998 indicated that it had spread coastally about 40 km north and south of an ap­ parent point of introduction in east central Palm Beach County. Based on rate of spread indicated by subsequent survey data, we believe that we found D. citri within 6 months to 1 year of its ori­ ginal introduction. Thus, we have a fairly firm date for the beginning of our experience with D. citri, which spread to southern Miami-Dade County on its own in about a year after its detec­ tion in central Palm Beach County. Southern Miami-Dade County has large numbers of ornamental nurseries. One of the most popular plants produced in the early 2000s

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was M. paniculata. It is a beautiful ornamental plant that takes little care and produces fragrant flowers and bright red fruits. It was used widely for hedges around homes, apartment complexes and parking lots. During the real-estate boom in the early 2000s, many thousands of these plants were transported to projects around the state. M.  paniculata also is a favored host of D. citri. Soon after D. citri became established in the ­nursery production area, it spread almost imme­ diately throughout the state on M. paniculata ­nursery stock (Halbert et al., 2003). Initial sur­ veys for HLB, which began immediately after the vector was discovered and continued into 2001, indicated that the initial D. citri population prob­ ably was not contaminated with HLB. Tested DPI inspector samples (n = 627) from 1998 to 2001 were all negative for CLas (S.E. Halbert and M.L. Keremane, unpublished results). Since we know now that positive D. citri are the most reliable early warning for the presence of CLas, we are fairly confident that our initial population of D. citri was clean. By the time we found HLB in south Miami-Dade County, it had spread exten­ sively and could not be eradicated. We know now that the incubation period for HLB is vari­ able and potentially a year or more. Thus, the pathogens probably were spreading asympto­ matically for several years before symptoms developed. We suspect that the psyllids encoun­ tered the pathogens in about 2000 or 2001, probably in southern Miami-Dade County. Although it is difficult to find a M. paniculata plant that tests positive for CLas, it can support infective colonization events (Lee et  al., 2015). Thus, CLas probably spread in the same way as D. citri did. This asymptomatic ornamental plant transported CLas-positive psyllids from southern Miami-Dade County to retail nurseries and de­ velopment projects statewide (Manjunath et al., 2008; Halbert et  al., 2012). Retail nurseries often carried both M. paniculata and citrus, so ongoing epidemics developed in the venues themselves. The continual supply of new flushing healthy citrus plants and the resident CLas-­ positive psyllid populations in these locations provided an ideal environment for production of nearly unlimited numbers of CLas-positive D. citri through infective colonization events (resident positive psyllids; unlimited amounts of colonizable new flush). Trees were purchased, and the disease spread to neighborhoods.

In 2005, our (DPI and USDA/APHIS/PPQ) extensive delimiting survey indicated that we had symptomatic plants only in the southern portion of the Florida peninsula, as far north as Ft Pierce on the east coast and in isolated small pockets in southwestern Florida commercial groves; however, there were positive psyllids found in 2005 in many places in ­Florida where there was no known HLB. One positive psyllid sample came from Nassau County, just south of the Georgia border from M. paniculata in  a discount garden center (Manjunath et al., 2008) (Fig. 9.1). The plant material was traced back to Miami-Dade County. Many early samples of positive psyllids came from retail venues (Manjunath et al., 2008; Halbert et al., 2012). Positive psyllids also moved on trailers of bulk fruit being moved to juice factories. Halbert et al. (2010) sampled D. citri from trailers of un­ processed juice oranges. The insects were distrib­ uted throughout the loads, and some of them proved to be positive for CLas. As the Florida map of HLB-positive square mile sections filled in, major fruit transportation highways were out­ lined in positive finds (Fig. 9.1). Positive psyllids were found in the envir­ onment in Polk County in 2005, in the center of our commercial citrus production area, but the first symptomatic plant in Polk County was not found until 2007. In one grove neighbor­ hood in Polk County, positive psyllids, including nymphs, were found in several places in 2005 (Manjunath et  al., 2008). The first positive plants at that location were not found until 6  years later, in spite of yearly surveys in the area and multiple molecular tests on various plants throughout the intervening years (S.E. Halbert, October 2011, unpublished results). Probable sources of infected psyllids in 2005 would have been M. paniculata plants at the dis­ count garden center, approximately 0.5 km away, or fruit trailers from southern Florida. The in­ fective colonization event mechanism explains how positive psyllids multiplied in numbers that made them easy to find, years before symp­ toms became evident. In 2008, DPI instated new regulations that required all citrus and citrus relatives (including M. paniculata) to be produced in enclosed structures that are inspected every 30 days for breaches and any sign of infestation. Prior to



Epidemiology of Huanglongbing

those regulations, up to about 25% of D. citri infestations discovered by DPI inspectors on ­ plants for sale were in commercial propagating nurseries. After the regulations became manda­ tory, the percentage of infestations in propagating nurseries (as opposed to retail venues) became negligible (Halbert et al., 2012). The Florida nur­ sery industry now is among the cleanest in the world. Another consequence of the regulations was that it became unprofitable to produce M. paniculata plants. Unfortunately, in hindsight, the regulations came too late to delay the distri­ bution of D. citri and the HLB pathogens. This occurred because there was no way to under­ stand the significance of M. paniculata until Dawson’s experiments indicated that it could support infective colonization events. In summary, although our population of D. citri originally was not contaminated with CLas, the pathogens spread very quickly once they encountered D. citri, probably in southern Miami-Dade County. Much of the long-distance spread occurred via plants for sale (mostly M.  paniculata out of Miami-Dade County) and transport of bulk fruit for juice production. The infective colonization event mechanism explains how CLas-positive psyllids could be relatively easy to find and easily transported years before symptoms became evident in the environment. Movement of both vectors and pathogens was facilitated by movement of M. paniculata plants, especially during the real estate boom in the early 2000s. The nursery regulations of 2008 have ended this pathway and ensured that ­Florida citrus nursery stock production is clean and free of HLB.

9.7  Management Implications The infective colonization event mechanism (Lee et al., 2015) has profound implications for management. Su et al. (1986) were the first to define the three-part management strategy that is now recommended around the world. Their program included production of clean plants, excellent psyllid control and elimination of known inoculum. The new transmission mechanism has complicated production and regulation of clean nursery stock and grove management, especially inoculum removal in places like ­

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­­ Florida where citrus flush can be available most of the year.

9.7.1  Nursery management It is essential to eliminate psyllids completely from elite nursery stock production. If even a single gravid CLas-positive female psyllid finds her way into a nursery setting, it can spell ser­ ious trouble. She can initiate an infective colon­ ization event, and her progeny also can be CLas-positive, colonizing and infecting other plants. Any colonized plant is potentially in­ fected. Testing the psyllids is the best way to de­ termine whether the incursion included the pathogens. However, when an incursion occurs, the first thing the grower usually does is to spray the plants to kill the psyllids. Dawson’s experi­ ments (Lee et al., 2015) indicated that colonized shoots are positive for CLas for a while after an infective colonization event. Thus, testing shoots with psyllid damage (even shortly after the in­ sects are gone) should provide evidence for whether CLas is present. A rigorous experiment should be done to check this. Plants should continue to be protected in retail environments. A continual influx of un­ protected plants and a resident CLas-positive insect population provide ideal conditions for infective colonization events, and infected plants for sale (Halbert et al., 2012). It is a chal­ lenge to regulate sale of nursery stock under these conditions, because of the long and vari­ able incubation period for the disease. One can quarantine for the insects alone. In that case, if the insects are gone, either because of treat­ ment or because the flush where they grew has matured, the plants are released from quaran­ tine. The CLas pathogens nevertheless might be present, but without HLB symptoms. Inspec­ tors are not able to quarantine for the disease unless there are symptoms of HLB. On average, DPI inspectors find CLas-positive psyllids in a retail environment about 9 months before they find symptomatic plants in the same venue (Halbert et  al., 2012). Since the life cycle of the insects is a few weeks (Tsai et  al., 2000), there is opportunity for many infective cycles between the appearance of CLas-positive psyl­ lids and the discovery of CLas-positive plants.

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Asymptomatically infected plants inevitably will be sold in the interim. It is not practical to test all the trees for sale or to hold all the nur­ sery stock for at least 9 months to determine if pathogens are present. Another issue in retail citrus regulation is the longevity and efficacy of psyllid treatments. In California, the efficacy of the pesticide treat­ ment for regulatory purposes expires in 90 days (Byrne et  al., 2018). A survey of retail sites in southern California showed that one-third of the plants had been treated more than 1 year prior to inspection, and the majority had been treated more than 90 days prior to inspection. A frequency distribution graph (Byrne et al., 2018) of infested samples showed that there was an in­ crease in numbers of infested plants (and by def­ inition, failed treatments) found by inspectors over the first 3 months after treatment. We have seen very similar results in Florida over the past decade (Halbert, 2017). DPI inspectors collected 1872 regulatory samples of D. citri from plants for sale between January 2007 and September 2016. On average 7.3% of the infested citrus trees had been treated within the past 30 days, and 17.3% of the infested trees had been treated between 31 and 60 days prior to sampling, sug­ gesting that the treatment could be failing by the second month after treatment. The retail envir­ onment is a difficult place to maintain efficacy of the pesticide. Re-entry requirements and cost prevent retreatment that is required to maintain a lethal dose in plant tissues. Watering and care regimes can eliminate soil-applied materials. Additionally, pesticide resistance could be a fac­ tor (Langdon et  al., 2018). The ability of these plants with failing pesticide residues to support colonization of D. citri promotes infective colon­ ization events, and consequently, infected plants for sale. All these factors make it very challen­ ging to keep plants for sale free of CLas, espe­ cially in a retail setting. 9.7.2  Grove management Grove management is no less challenging, espe­ cially inoculum removal. Most tree removal in commercial groves is based on scouting for symptoms, but it is possible to have 100% infec­ tion before symptoms appear (Lee et al., 2015). Even if early detection were to advance to the

point where an infected tree could be identified at the instant of infection, it would be impossible in most cases to mobilize removal fast enough to prevent late-instar infected D. citri from matur­ ing and migrating to another tree. It was a nearly universal experience in ­Florida that rogueing symptomatic trees could not stay ahead of the epidemic. We returned sev­ eral months later to blocks where a few positive trees had been found, only to discover that much of the block was symptomatic, even if the original positive trees had been removed (S.E. Halbert and M.W. Brodie, 2006–2007, unpublished observa­ tions). In the early years after the disease was found, many (especially large-scale) growers were eager to follow the advice to scout several times each year and remove symptomatic trees. By 2010, most growers had quit removing trees (Muraro, 2012). Udell et  al. (2017) showed that continual flushing promoted D. citri and an increase in HLB. It is possible that in dry climates, or in citricul­ ture regimes that lack irrigation, flush is inter­ mittent enough to restrict infective colonization events. In those situations, it might be possible to achieve benefit by rogueing symptomatic plants (Razi et al., 2014).

9.7.3  Psyllid control It might be impossible to control psyllids well enough to prevent infection. In Dawson’s experi­ ments (Lee et  al., 2015), most colonized plants later developed symptoms of HLB. They used tiny plants, and it might take more than one in­ fective colonization event to infect a large tree permanently; however, it likely is not possible to prevent infective colonization events over the lifespan of the tree. That said, Stansly et  al. (2014) and Tansy et  al. (2017) showed that insecticide alone increased yield of oranges. Thus, psyllid control still is an important and cost-effective component of management (Tansy et al., 2017).

9.7.4 Replanting If the industry as a whole is to continue, it is ne­ cessary to replant in cases where trees become



Epidemiology of Huanglongbing

irredeemably unproductive due to HLB or other causes. Martini et  al. (2015) found that resets have fewer psyllids than trees in newly planted solid blocks. Solid set blocks are particularly challenging to manage prior to productive ma­ turity. Hall et al. (2013) found that nearly all the trees were infected in less than 4 years, despite a rigorous insecticide program. Probably it is im­ portant to practice excellent area-wide psyllid control if new blocks are to survive to bearing age. It is easier to lower overall psyllid popula­ tions than to prevent transmission to young trees by direct pesticide treatment. Nevertheless, protecting solid set blocks remains one of the most challenging aspects of HLB management. These issues can be analyzed quantitatively via modeling studies analogous to, but much more detailed than, those in Lee et  al. (2015) with the aim of providing additional guidance for HLB management beyond the information currently available. In summary, there are no easy answers for how to manage HLB. Growing clean nursery stock is the most straightforward strategy, and even that has problems, especially in retail set­ tings. As soon as the trees are planted in the field, they are available for psyllid colonization and infective colonization events. It probably is not possible to prevent infection. Current man­ agement in Florida primarily relies on pesticide and nutritional treatments, which may or may not be sustainable in the long run. Until resist­ ant plants are available, management must focus instead on mitigation of disease, a topic that has received little attention.

9.8  Can the Long and Variable Incubation Period Help to Mitigate Disease? This section will come full circle. The preceding sections of this chapter have emphasized the challenges presented by the infective coloniza­ tion event mechanism. It makes it difficult to regulate nursery stock, difficult to detect infec­ tion and remove inoculum in a timely manner, and even difficult to do research about disease progress. These challenges are due to the long and variable incubation period. In this section, we will suggest that this very feature of the

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HLB pathosystem can be exploited for disease management – with some additional research. The best pest management strategies ex­ ploit natural or observed variation in pest or disease impact. For example, if there is observ­ able variation in severity of a new pest among cultivars of a crop, it is likely that plant breeders will be able to develop a resistant plant. Similarly, if significant geographic variation in pest popu­ lations is observed, and it is determined that nat­ ural enemies are present where the impact is minimal and not present where the pest causes economic damage, classical biological control is a good option for successful management. Fi­ nally, if there is variation from one season to the next in pest populations on an annual crop, ad­ justment of planting date to avoid the pest could be an option. Unfortunately, in the HLB pathosystem, we do not have much variation among cultivars of citrus, and the problem is severe in the Indian subcontinent, where the vector, D. citri, and its parasites are native (Wang et  al., 2018). Thus, the opportunities to exploit genetic and geo­ graphic variation are limited. Seasonal variation does not apply, because citrus is a perennial crop. For best management, we need another source of variation to exploit. As we have discussed at length, there is sig­ nificant variation in the time it takes for disease to develop. The long and variable incubation period might thus be able to be used in our favor, if we managed disease rather than infection. Given the important role played by infective col­ onization events, it might not be possible to elim­ inate infection. Although we would prefer that citrus trees never became infected, we can live with infection if it does not produce disease. The long incubation period indicates that the plants are fighting disease development; the variable incubation period indicates that some plants fight more successfully than others. Compared with genetic and geographic variability in the HLB pathosystem, the variability in the incu­ bation period is consistent and enormous. This makes it a prime candidate for exploitation for a successful management strategy. The source of and reasons for this variation are not understood. Exploitation of the highly variable incubation period could offer the best chance to develop a successful and sustainable management strat­ egy for HLB.

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Acknowledgments We thank Alicia B. Lawrence, DPI, for creating our maps, Philip Stansly, University of Florida, Pamela Roberts, University of Florida, and Paul

Skelley, DPI, for reviewing the manuscript. We also thank the many DPI and USDA/APHIS/PPQ inspectors, without whose samples many of the concepts presented here would not have been discovered.

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(2013) Evaluation of management programs for protecting young citrus plantings from huanglongbing. HortScience 48, 330–337. Hastings, A. (1996) Models of spatial spread: a synthesis. Biological Conservation 78, 143–148. Hastings, A., Cuddington, K., Davies, K.F., Dugaw, C.J., Elmendorf, S. et al. (2005) The spatial spread of invasions: new developments in theory and evidence. Ecology Letters 8, 91–101. Hilf, M. and Luo, W.-Q. (2018) Dynamics of ‘Candidatus Liberibacter asiaticus’ colonization of new growth of citrus. Phytopathology 108, 1165-1171. doi: 10.1094/PHYTO-12-17-0408-R. Husain, M.A. and Nath, D. (1927) The citrus psylla (Diaphorina citri, Kuw.) [Psyllidae: Homoptera]. Memoirs of the Department of Agriculture of India. Entomology Series 10, 5–27. Inoue, H., Ohnishi, J., Ito, T., Tomimura, K., Miyata, S., Iwanami, T. and Ashihara, W. (2009) Enhanced proliferation and efficient transmission of Candidatus Liberibacter asiaticus by adult Diaphorina citri after acquisition feeding in the nymphal stage. Annals of Applied Biology 155, 29–36. Johnson, E.G., Wu, J., Bright, D.B. and Graham, J.H. (2014) Association of ‘Candidatus Liberibacter asiaticus’ root infection, but not phloem plugging with root loss on huanglongbing-affected trees prior to appearance of foliar symptoms. Plant Pathology 63, 290–298. Kannan, A., Yang, Z., Kim, M.K., Stone, H.A. and Siryapon, A. (2018) Dynamic switching enables efficient bacterial colonization in flow. Proceedings of the National Academy of Sciences 115(21), 5438–5443. Langdon, K.W., Schuman, R., Stelinski, L.L. and Rogers, M.E. (2018) Spatial and temporal distribution of soil-applied neonicotinoids in citrus tree foliage. Journal of Economic Entomology 111, 1788–1798. doi: 10.1093/jee/toy114. Lee, J.A., Halbert, S.E., Dawson, W.O., Robertson, C.J., Keesling, J.E. and Singer, B.H. 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Lin, K.-H. and Lin, K.-H. (1990) The citrus huang lung bin (greening) disease in China. In: Aubert, B., Tontyaporn, S. and Buangsuwon, D. (eds) Rehabilitation of Citrus Industry in the Asia Pacific Region. Proceedings Asia Pacific International Conference on Citriculture, Chiang Mai, Thailand, 4–10 ­February 1990. FAO-UNDP, Rome, pp. 1–26. Manjunath, K.L., Halbert, S.E., Ramadugu, C., Webb, S.E. and Lee, R.F. (2008) Detection of ‘Candidatus Liberibacter asiaticus’ in Diaphorina citri and its importance in the management of citrus huanglongbing in Florida. Phytopathology 98, 387–396. Martini, X., Pelz-Stelinski, K.S. and Stelinski, L.L. (2015) Absence of windbreaks and replanting citrus in solid sets increase density of Asian citrus psyllid populations. Agriculture, Ecosystems and Environment 212, 168–174. Melbourne, B.A. and Hastings, A. (2009) Highly variable spread rates in replicated biological invasions: fundamental limits to predictability. Science 325(5947), 1536–1539. Muraro, R.P. (2012) Evolution of citrus disease management programs and their economic implications: the case of Florida’s citrus industry. Proceedings of the Florida State Horticultural Society 125, 126–129. Paris, T.M., Allan, S.A., Udell, B.J. and Stansly, P.A. (2017) Evidence of behavior-based utilization by the Asian citrus psyllid of a combination of UV and green or yellow wavelengths. PLOS ONE 12(12), e0189228. doi: 10.1371/journal.pone.0189228. Pelz-Stelinski, K.S., Brlansky, R.H., Ebert, T.A. and Rogers, M.E. (2010) Transmission parameters for Candidatus Liberibacter asiaticus by Asian citrus psyllid (Hemiptera: Psyllidae). Journal of Economic Entomology 103, 1531–1541. Pelz-Stelinski, K.S., Martini, X., Kingdom-Gibbard, H. and Stelinski, L.L. (2016) Patterns of habitat use by the Asian citrus psyllid, Diaphorina citri, as influenced by abiotic and biotic growing conditions. Agricultural and Forest Entomology 19, 171–180. doi: 10.1111/afe.12197. Pleydell, D.R.J., Soubeyrand, S., Dallot, S., Labbonen, G., Chadoeuf, J., Jacquot, E. and Thebaud, G. (2018) Estimation of the dispersal distances of an aphid-borne virus in a patchy landscape. PLOS Computational Biology 14(4), e1006085. doi: 10.1371/journal.pcbi.1006085. Rashed, A., Wallis, C.M., Paetzold, L., Workneh, F. and Rush, C.M. (2013) Zebra chip disease and potato biochemistry: tuber physiological changes in response to ‘Candidatus Liberibacter solanacearum’ infection over time. Phytopathology 103, 419–426. Rashed, A., Wallis, C.M., Workneh, F., Paetzold, L. and Rush, C.M. (2016) Variations in zebra chip disease expression and tuber biochemistry in response to vector density. Phytopathology 106, 854–860. Razi, M.F., Keremane, M.L., Ramadugu, C., Roose, M., Khan, I.A. and Lee, R.F. (2014) Detection of citrus huanglongbing-associated ‘Candidatus Liberibacter asiaticus’ in citrus and Diaphorina citri in Pakistan, seasonal variability, and implications for disease management. Phytopathology 104, 257–268. Sétamou, M. and Bartels, D.W. (2015) Living on the edges: spatial niche occupation of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), in citrus groves. PLOS ONE 10, e0131917. doi: 10.1371/journal.pone.0131917. Shen, W., Halbert, S.E., Dickstein, E., Manjunath K.L., Shimwela, M.M. and van Bruggen, A.H.C. (2013) Occurrence and in-grove distribution of citrus huanglongbing in North Central Florida. Journal of Plant Pathology 95, 361–371. Shimwela, M.M., Schubert, T.S., Albritton, M., Halbert, S.E., Jones, D.J., Sun, X.-A., Roberts, P.D., Singer, B.H., Lee, W.S., Jones, J.B., Ploetz, R.C. and van Bruggen, A.H.C. (2018) Regional spatial-temporal spread of citrus huanglongbing is affected by rain in Florida. Phytopathology 108, 1420–1428. doi: 10/1094/PHYTO-03-18-0088-R. Stansly, P.A., Arevalo, H.A., Qureshi, J.A., Jones, M.M., Hendricks, K., Roberts, P.D. and Roka, F.M. (2014) Vector control and foliar nutrition to maintain economic sustainability of bearing citrus in Florida groves affected by huanglongbing. Pest Management Science 70, 415–426. Su, H.-J., Cheon, J.-U., and Tsai, M.-J. (1986) Citrus greening (Likubin) and some viruses and their control trials. In: Plant Virus Diseases of Horticultural Crops in the Tropics and Subtropics. Proceedings of the Food and Fertilizer Technology Centre for the Asian and Pacific region, September, Taipei, Taiwan. FFTC Book Series 33, pp. 143–147. Tansey, J.A., Vanaclocha, P., Monzo, C., Jones, M. and Stansly, P.A. (2017) Costs and benefits of insecticide and foliar nutrient applications to HLB-infected citrus trees. Pest Management Science 73(5), 904–916. doi: 10.1002/ps.4362. Tsai, J.H. and Liu, Y.H. (2000) Biology of Diaphorina citri (Homoptera: Psyllidae) on four host plants. Journal of Economic Entomology 93, 1721–1725.



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10 

Sampling and Economic Thresholds for Asian Citrus Psyllid

Cesar Monzo1* and Philip A. Stansly2 Instituto Valenciano de Investigaciones Agrarias, Centro de Protección Vegetal y Biotecnología, Unidad de Entomología, Valencia, Spain; 2University of Florida, Department of Entomology and Nematology, Institute of Food and Agricultural Sciences, Southwest Florida Research and Education Center, Immokalee, Florida, USA

1

Integrated pest management (IPM) seeks to combine, in a sustainable way, all available management techniques to prevent or reduce losses attributable to pests in cropping systems (Kogan, 1998). Economic constraints, human health and environmental requirements have motivated transformation of crop production into more complex and efficient systems that favor non-­ aggressive management tools such as biological control over reductionist and impacting approaches like insecticidal control. Under this paradigm, information about all the processes involved in agrosystems is utilized to understand complex interactions and predict whether and which management action may be required. Knowledge of the biology, ecology and damage potential of the target organism are needed for efficient pest management where damage ­potential is usually linked to pest demography. Therefore, the decision to intervene should be based on pest and natural enemy density data. In some cases, the bare detection of the target organism will be sufficient to take action. In other cases, intervention will be determined by pre-­ established pest and natural enemy densities through the concepts of economic injury levels and economic thresholds (Stern et  al., 1959; Higley and Pedigo, 1996).

Systematic and consistent demographic information derived from representative samples of the whole population is a critical component of IPM. The quality of this information is linked to the effort invested in obtaining it. We have therefore to strike a balance between effort invested and data quality. Material costs and time spent to gather and interpret collected data are variables commonly employed to measure sampling effort. Accuracy and data precision have, on the other hand, frequently been used as quality indicators of the information obtained (Pedigo et al., 1972). Objectives of a monitoring plan will define the equilibrium point. Sampling aimed at research or pest detection at very low densities requires high-quality data. However, some sensitivity and precision can be sacrificed in favor of less costly sampling protocols needed for routine management purposes (Southwood and Henderson, 2009).

10.1  Asian Citrus Psyllid Monitoring 10.1.1  Goals of ACP monitoring plans Risks associated with the presence of huanglongbing (HLB) and/or its vectors require implementation of vigorous management programs to

*  Email: [email protected]

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maintain the viability of citrus production. Vector control has become the basis of these programs in lieu of curative remedies against HLB. Management strategies and thus goals of vector monitoring will vary by region, depending on epidemiological status (Chapter 9). In areas where Asian citrus psyllid (ACP) is still absent or scattered, prevention policies to impede the establishment of the vector are called for. The success of HLB management in these cases will mostly depend on a rapid response to any potential threat thus identified. Early ACP detection, especially at very low densities, therefore requires an exhaustive monitoring program to detect presence of ACP. These requirements favor high sensitivity and low data-processing time. Where ACP is ubiquitous and HLB is already widespread, monitoring goals are different. Recent studies have demonstrated that vector control has a positive effect on yield of mature trees, even when most are infected with HLB (Stansly et  al., 2014; Monzo and Stansly, 2017; Tansey et al., 2017). This effect alone is sufficient to economically justify continued efforts to suppress ACP under these conditions. The likely mechanism related to the as yet unproven hypothesis is that continuous bacteria re-inoculation by HLB-vectoring ACP in already infected trees plays a central role in their decline. Therefore, the positive effect of ACP suppression is due to reduced rates of pathogen re-­ inoculation (Monzo and Stansly, 2017). Additional benefits gained by thus protecting young trees from HLB are intuitive but as yet not quantified. Spatial and temporal information regarding ACP density changes enables estimation of potential risks and therefore planning of management actions. Coordinated monitoring at the regional scale has proven highly efficient in creating a dynamic picture of ACP demography throughout the season and across extensive ­citrus-growing areas, providing valuable information for regional vector management (www.crec.ifas.ufl.edu/extension/ chmas/chma_websites.shtml). In this second epidemic scenario, monitoring plans usually prioritize implementation even though this entails some loss of quality data, namely accuracy and ­precision. 10.1.2  ACP sampling methods A diverse set of sampling methods have been proposed, tested and implemented to detect or monitor ACP field populations. In most cases,

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psyllid adults are the chosen target stage. Adults are in general relatively easy to monitor and ultimately responsible for disease transmission, ­although nymphs also play a fundamental role in acquisition (Lee et  al., 2015; Ammar et  al., 2016) (Chapter 8). Thus, data on nymphal populations can be of great utility to better understand and predict HLB epidemiology (Chapter 9). Although active sampling methods are ­recommended for pest management purposes (Yi et al., 2012), many current ACP monitoring protocols use passive techniques to randomly sample the overall population. Nevertheless, ACP is known to move positively in response to different kinds of external stimuli (Chapter 4). Chemotaxis to female sexual lures has been ­described (Wenninger et al., 2008). Pheromones or other lure compounds have proven of great value for monitoring other pests, owing to high specificity and sensitivity. However, no specific compounds have been isolated and identified to implement this approach, although recent studies have shown how acetic acid baits enhance attraction of male ACP to sticky traps (Zanardi et  al., 2018). Phonotaxis has also widely been studied in Hemiptera. ACP adult males and females use vibrational communication during courtship. Preliminary trap prototypes able to reproduce female vibrational signals and to effectively trap adult males have been developed (Mankin et  al., 2015) (Chapter 3). Phototaxis has been extensively studied on ACP (Hall et al., 2010; Paris et al., 2015, 2017a), demonstrating positive response of ACP to yellow and ultraviolet (Chapter 4). Furthermore, addition of ultraviolet light to green and yellow wavelengths seems to importantly enhance ACP attraction (Paris et al., 2017b). In the following subsections some of the most used and evaluated passive sampling methods for ACP monitoring and detection are described. 10.1.2.1  Stem-tap sampling Stem-tap or limb-tap sampling has long been used to sample arthropods in fruit tree crops (Thistlewood, 1986) and recently adapted to monitor ACP in citrus (Hall et al., 2007; Qureshi and Stansly, 2007). Stem-tap sampling consists of striking a randomly selected branch of a tree with a length of PVC pipe or similar blunt

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i­nstrument to dislodge ACP adults resting or feeding on foliage. To standardize this procedure, it is advisable to fix the number of strikes (e.g. three strikes per sample) and the number of samples per tree (e.g. two samples per tree on two opposite sides of the canopy). A laminated white sheet or a white tray is held horizontally under the branch to collect the dislodged adult ACP, which are rapidly counted in the field. Other pest or beneficial insects and mites can also be evaluated. Material costs for stem-tap sampling are very low, as is implementation time. Data can be processed immediately, in contrast to some other sampling techniques. These qualities and reproducibility among practitioners make tap sampling convenient for generating large data sets quickly and at low cost. It has been adopted by the Citrus Health Response Program (CHRP) (www.fdacs. gov/Divisions-Offices/Plant-Industry/AgricultureIndustry/Citrus-Health-Response-Program) to monitor over 5000 citrus blocks in ­Florida every 3 weeks (www.flchma.com). However, exhaustive evaluations of this methodology have shown that it lacks sensitivity compared with other techniques and precision may be compromised at very low ACP densities (Monzo et al., 2015). Since counts are done in the field, scout training is required and no further data validation can be made. At the other extreme, inaccuracies may occur at high densities due to mobility of ACP adults. Also, tap sampling is not practical in very young trees, due to limited canopy volume. 10.1.2.2  Sticky traps Sticky traps are widely used to monitor ACP populations (Aubert and Quilici, 1988; Hall et al., 2007; Hall and Hentz, 2010; Monzo et al., 2015). Several studies have evaluated this methodology and compared it to stem-tap sampling (Hall and Hentz, 2010; Hall et al., 2010; Monzo et al., 2015; Miranda et al., 2018). Owing to the demonstrated attraction of ACP adults to yellow (Hall et al., 2010; Paris et al., 2015), yellow traps are used to maximize capture efficacy, thus making sticky trapping an active capturing sampling method. Sticky traps are generally deployed at approximately 1.5 m height on the periphery of the tree canopy. No orientation effects on the number of captures have been found. Nevertheless, to minimize any trap position bias, deploying

them alternately at one and other side of the canopy is recommended. In blocks where tree rows run north and south, traps would be installed alternately at the east and west sides of the tree canopy, whereas in blocks with rows running east to west they would be deployed at the north and south sides. Traps are usually replaced weekly or bi-weekly. Collected traps are examined in the laboratory to count the number of ACP captured. Since collected material can be stored and further revised, scouting error may be controlled better than with stem taps. Sticky trap sampling has also been proved to be more sensitive than stem taps (Hall and Hentz, 2010; Monzo et  al., 2015). Field evaluations of the two methodologies showed that sticky traps are able to capture up to 14 times more ACP adults than stem taps and they were twice as likely to detect the presence of ACP. However, the higher material costs and especially implementation time render this methodology less efficient except at very low ACP densities (Monzo et al., 2015). Sticky traps would therefore be advisable for early ACP detection and to monitor young plantations where stem tapping is not efficient. 10.1.2.3  Visual sampling Visual sampling consists of directly observing and counting ACP in the field during a predetermined time frame and/or in a sampling area or unit. This methodology has the lowest implementation costs and, as with stem taps, information can be rapidly processed. Again, training is required and data consistency may be compromised by scouting ability and expertise. Visual sampling protocols are used to monitor adults and nymphs (Sétamou et al., 2008; Monzo et al., 2015; Tansey et al., 2017). Sampling for nymphs is done upon new growth shoots (flushes), mostly during the plant growing season. Flushing intensity is estimated by counting total number of shoots in a predetermined canopy area and nymphs are sampled from a prefixed number of flushes. Both enumerative and binomial nymph sampling have been used. For the enumerative approach, the number of nymphs per flush is counted with a magnifying hand-held lens or counted inside under a stereoscopic microscope. For binomial sampling, nymph/egg presence or absence per flush is noted and the proportion of



Sampling and Economic Thresholds

infested flushes estimated (Sétamou et al., 2008; Tansey et al., 2017; Udell et al., 2017). For visual adult sampling, a protocol was developed to regularly monitor close to 4000 ha of mature citrus trees in Florida (Monzo et al., 2015). Samples were taken throughout the season and observations were made during a short period of time (e.g. 40 s) on pre-selected trees, in either of the two sides of the tree canopy. Adult visual sampling was more sensitive than stem taps and precise at low ACP densities, although stem taps would give better results at increasing ACP densities (Monzo et al., 2015). 10.1.2.4  Suction sampling Suction sampling is widely used for all kinds of arthropod studies in agriculture and ecology. This method uses powered devices able to intake and to capture target organisms. Converted hand-held leaf blowers, powered by two-cycle engines, have proved highly efficient for capturing ACP adults (Thomas, 2012; Monzo et al., 2015; Miranda et al., 2018). One study showed that a 5 min suction sample upon 2 × 2 m highly ACP-infested tree canopies captured more than 60% of all existing adults (Thomas, 2012). Time per tree canopy is fixed to standardize sampling protocols. Monzo et al. (2015) proposed a protocol consisting of ten-tree composite samples at pre-fixed stops in the grove. Each tree canopy was struck and then vacuumed for about 1.5 s to dislodge and intake the ACP adults. This was repeated ten times for each tree. Collected material was taken to the laboratory for examination. Captured psyllids can be kept alive for laboratory studies such as bioassay to evaluate insecticide susceptibility (Tiwari et al., 2011). Material costs of suction sampling are greater than for other methodologies. Time used to take the samples, process them and obtain the data is greater than other sampling methods except for sticky traps. Great sensitivity offsets these disadvantages if estimation or detection of a low-density population is the goal. Suction samples collected an average 28 times more ACP adults than stem taps, which was fourfold more ACP per unit of sampling time (Monzo et  al., 2015). Weight, noise and vibration make handling the device arduous and so other sampling methods are advisable for routine ACP monitoring, except for early ACP detection and research purposes.

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10.1.2.5  Sweep nets Sweep nets have also long been used for sampling, including of ACP (Weinert et  al., 2004). The net is swept in an arc of approximately 180° so that half the rim strikes the tree canopy. Adults trapped in the net are visually counted immediately. Protocols proposed using this ­methodology are similar to those defined for stem taps (Monzo et al., 2015). Sensitivity was similar to stem taps but it had a higher implementation time and yielded less precise results for any pest density (Monzo et al., 2015). Composite-sampling such as for suction sampling would probably improve efficiency.

10.1.3  Criteria for selection of sample method Differences between ACP sampling methods in terms of time invested to obtain data (sampling effort), ability to detect the target organism (sensitivity), precision (standard error, mean ratio) information generated, the time spent between taking the sample and extracting the data (data processing time) and adaptability to young or mature trees, permit us to rank these sampling methods differentially, depending on the requirements of a pre-planned scouting program (Fig. 10.1). Suction sampling gets the highest sensitivity rating per unit sampling effort (Table 10.1). This trait would make it the most appropriate for ACP detection. It also requires less sampling effort to reach similar precision levels than stem taps at low ACP density. These two traits make suction the most reliable and efficient for regular ACP monitoring, were it not for lag time ­between sampling and data collection as well as burdensome handling. As a result, its use is ­generally relegated for detection and specialized ­research. Stem-tap, sticky trap and sweep-net sampling attain similar sensitivity per sampling effort. Stem taps have the advantage of rapid data processing time and less sampling effort required to attain similar precision levels at all but the lowest ACP densities and are thus recommended for large-scale ACP monitoring plans. Adult visual sampling has a low data-­ processing time, is easy to implement and slightly

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Goals of monitoring

Research Young trees

Sticky traps Suction Visual (nymphs)

Early detection

Mature trees

Sticky traps

Young trees

Stem taps Visual (nymphs)

Mature trees

Sticky traps

Suction

ACP management Young trees

Mature trees

Sticky traps

Sticky traps

Stem taps

Suction

Visual (nymphs)

Visual (adults)

Suction Visual (nymphs)

Fig. 10.1.  Diagram for selection of ACP sampling method according to the goals of a monitoring plan. Table 10.1.  Comparison of ACP sampling methods to the stem tap (Monzo et al., 2015). Sampling effort to stem taps measured as time invested to implement the sample and process data, sensitivity to stem taps, relative sensitivity/relative sampling effort ratio, invested sampling effort relative to stem taps to attain similar precision levels, time needed to process and obtain the data once the sample was taken and adaptability to sample young trees are the proposed criteria to further select a method according to the needs of a sampling plan.

Relative sensitivity

Relative sensitivity/ Relative sampling effort

Sampling effort for similar precision

1 16

1 14

1 1

Very low High

Low High

7

28

4

*** Less at < 0.017 ACP adults per stem tap Less at < 0.14 ACP adults per stem tap

High

Medium

Less at < 0.007 ACP adults per stem tap More at any ACP density

Very low

High

Low

Low

Relative sampling effort

Stem taps Sticky traps

Suction

Sampling method

Visual

2

Sweep net

2

> 1 at < 0.013 ACP adults per stem tap < 1 at > 0.013 ACP adults per stem tap 1

> 0.5 at < 0.013 ACP adults per stem tap < 0.5 at > 0.013 ACP adults per stem tap 1

improves sensitivity with respect to stem taps at very low ACP densities. It also needs less sampling effort to attain similar levels of precision than stem taps at low ACP densities. These traits make visual sampling a cheap alternative to stem tapping in cases of low ACP infestation levels.

Data Adaptability processing to young time trees

Stem tapping and sweep netting do not adapt well to ACP sampling in young trees. Under those conditions, suction or sticky traps are good for detection and visual sampling is a good alternative for regular ACP monitoring.



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10.2  Economic Thresholds for ACP Management

10.2.2  Economic thresholds for ACP control at low HLB incidence

10.2.1  Economic thresholds for disease vectors

For reasons mentioned above, aggressive vector control is one management tactic almost universally recommended to impede disease establishment and propagation (Belasque et  al., 2010; Bassanezi et  al., 2013a) (Chapters 13, 14, 15 and 16). Rogueing symptomatic trees, although widely recommended, has not been clearly demonstrated to be effective (Bassanezi et al., 2013b), due to the long latent period between infection and symptom expression (Chapters 8 and 9). With the exception of exclusion methods (Chapter 11), the lack of alternative strategies has left frequent calendar insecticide applications as the only accepted means to accomplish the goal of impeding HLB propagation. The ultimate sustainability of this practice is still open to question.

The concept of economic injury levels (EIL) was initially proposed by Stern et al. (1959) as a way of rationalizing pesticide use and thereby facilitating integration with biological control. EIL has been defined as ‘the lowest population density that will cause economic damage’ (Stern et al., 1959). EIL is a theoretical concept applied in the field by way of an economic threshold (ET), variously defined as the pest population density at which control must be initiated to avoid surpassing the EIL. ET is therefore based on the EIL but includes estimated population increase during time lags between decision making and execution. EIL and ET are thus curative rather than preventive measures and therefore require a quantitative relationship between pest injury and crop damage. Consequently, ET has its main application against phytophagous pests that damage crop yield or quality. Economic loss from systemic infection is generally considered as a function of disease incidence (Jones, 2004). A vector like D. citri is a pest in its own right whose damage potential is increased in proportion to the number capable of transmitting disease. This complication has resulted in few studies of EIL or ET applied to disease vectors. The difficulty was illustrated by a study of Brust and Foster (1999), who sought an ET on cantaloupe for the cucumber beetle Acalymma vittatum vector of bacterial wilt caused by Erwinia tracheiphila. Regressions of beetle populations on symptomatic plants in different experiments yielded slopes as divergent as 0.16 and 0.94, indicating differences in incidence and/or titer of the pathogen in the vector population. The authors then settled on an arbitrary ‘action’ threshold based on the result that bacterial wilt was not spread by one beetle per plant. Perennial crops present an additional challenge for vector management in that disease incidence is cumulative rather than reset every cycle, as in annual crops. Thus, a preventive approach would seem to make more sense in a perennial crop, especially early on, as the biological and economic impact of disease is greatest on young plants.

10.2.3  Economic injury levels for ACP control at moderate-to-high HLB incidence Scenarios where both HLB and ACP are well established and the disease is widely spread are common in Asia and the Americas (Hall et  al., 2013). Under these circumstances, neither eradication nor preventive measures for either vector or disease are feasible. Nevertheless, recent research demonstrates that productivity in citrus groves with high HLB incidence can be maintained (Stansly et  al., 2014; Tansey et  al., 2017). These studies also give evidence that the success of this approach is conditioned, in part, by low ACP densities. A logical explanation for this result is that tree decline is mediated by bacterial re-inoculation into already infected trees (Plotto et al., 2017; Tansey et al., 2017). These findings changed the conception of HLB management in areas with high disease incidence and triggered the following questions: (i) is plant damage (ultimately yield loss) and pathogen injury a function of ACP density; and (ii) if so, how much vector suppression can we afford? Given that a certain level of ACP is tolerable, the concept of economic threshold become feasible. For its efficiency and ease of application, the stem tap was chosen as the sampling method to implement this strategy. Preliminary research

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to establish the utility of ACP control to improve yield began by implementing arbitrary ‘action’ thresholds, first of five ACP per ten taps, later ­reduced to two and finally to one per ten taps (Stansly et  al., 2014; Tansey et  al., 2017) as area-­wide management reduced ACP populations (Bassanezi et  al., 2013a). Economic analysis of the results demonstrated the utility of the approach (Monzo and Stansly, 2015). Nevertheless, these thresholds did not consider the ­dynamic nature of most of the variables used to calculate EILs and estimate ETs. Rather than a fixed ACP density boundary above which it would be necessary to spray, specific treatment levels were required based on particular economic circumstances to include treatment efficacy, material and application costs, ACP sampling costs, market juice prices and juice quality. 10.2.3.1  Relationship between cumulative tap results and yield Two replicated experiments were conducted over 4 years in two commercial groves of mature orange trees under high HLB incidence (Monzo and Stansly, 2017). Two arbitrary threshold levels (two and seven ACP per ten taps) were adopted as triggers for ACP-directed sprays and compared with monthly applications and an untreated control. ACP populations were monitored by tap samples conducted every 2 weeks. The total number of ACP adults per season, expressed as cumulative number per tree and season, was later related to yields in blocks under different vector densities. The novel approach consisted of expressing EILs as a function of vector cumulative numbers instead of vector densities at a particular time. This opened the door for estimates of treatment thresholds based on previously fixed variables such as costs of dormant sprays and monitoring, as well as juice quality and market juice price. A post-dormant season insecticide application would be triggered only when a predicted ACP cumulative boundary was reached. The process would then be repeated to calculate a new threshold. The number of insecticide applications to optimize HLB management in a given growing season and particular citrus block would vary depending on economic variables, insecticide efficacy and even neighboring pest pressure. A spreadsheet was proposed to do the calculations based on adult

ACP cumulative numbers, estimated ACP management costs, juice prices and amount of expected solids in juice. A hypothetical practical case is presented next to exemplify the implementation of these thresholds. 10.2.3.2  Practical use of cumulative stem-tap results for making spray decisions The management strategy proposed to implement treatment thresholds based on the EIL calculations for 10–15-year-old sweet orange trees under moderate to high incidence of HLB, with production destined for the processed industry, is as follows: 1. Irrespective to ACP densities, two insecticide applications during tree dormancy (December and January in Florida) with broad-spectrum ­insecticides (organophosphates or pyrethroids) aimed at reducing ACP adults prior to the subsequent flush and first generation in the growing season (Qureshi and Stansly, 2010). The availability of hyper-abundant new-growth flushes following tree dormancy results in an almost unlimited resource for oviposition (Udell et  al., 2017). In addition, the role of natural enemies as ACP demographic regulating forces is marginal in the absence of flush (Monzo et al., 2014). 2. ACP adult density monitored bi-weekly throughout the season using stem-tap sampling protocols (Monzo et al., 2015). 3. A first treatment threshold for the growing season (February to November) is calculated­ based on management costs: ACP monitoring, dormant season sprays and the first growing-­ season spray with a more selective insecticide. Total management costs are then balanced against predicted yield losses associated with different adult ACP densities, expressed as ­cumulative number (k), using an empirical mathematical relationship (Equation 10.1). The amount of ACP (k) equaling management costs to yield losses would be set as the estimated treatment threshold. For the allocated management costs in this example, the first growing season treatment would be made once k exceeds 4.5 ACP cumulative adults per tree (Fig. 10.2). 4. If ACP adult cumulative numbers do not reach that value, no insecticide applications would be made until the next dormant-season spray. Otherwise, a new treatment threshold would be set by adding costs of a second growing-season



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Growing season

ACP cumulative (k)

Beginning of the season

End of the season 4th treatment threshold (4)

9.5 7.7 6.1 1st treatment threshold

4.5

3rd treatment threshold (3) 2nd treatment threshold (2)

(1) (1)

Winter

Winter

Fig. 10.2.  Implementation of the treatment threshold strategy during the growing season based on ACP adult cumulative data. The season would begin with two winter insecticide applications (1). Once growing season begins first treatment threshold is calculated (4.5 ACP adults per tree) based on allocated management costs: ACP monitoring = $141/ha; dormant season sprays = $75/ha and application; first growing season spray = $130/ha. Insecticide treatment would be made whenever this threshold is reached (2). A second threshold would then be calculated (6.1 ACP adults per tree) and second growing season spray made once the threshold is reached (3). The procedure would be repeated (4) until the end of the growing season.

spray to all previously considered variables, considering that expected price could be modified based on economic outlook. Assuming similar application costs (k = 6.1), cumulative adults per tree would balance ACP management costs with associated yield losses. 5. The same procedure will be repeated until the growing season is over. As can be observed, the number of applications throughout the growing season will be mainly conditioned by the ability to suppress ACP. To optimize ACP management, and reduce insecticide application frequency, ­effectiveness of insecticide treatments must be maximized through judicious choice of active ­ingredients. The demonstrated ability of natural enemies to suppress ACP, particularly in summer (Qureshi and Stansly 2009; Monzo et al., 2014) provides impetus to use of selective mode of

­ctions and treatment threshold to further a ­conservation biological control. Grove-to-grove ­migrations can also be an important factor influencing ACP growth rates in a particular block. In this sense, coordinated management strategies will greatly help optimize ACP control. Equation 10.1. Empirical relationship ­obtained by Monzo and Stansly (2017) in which ACP management costs (C) are balanced to the economic yield losses associated to changing ACP densities, expressed as ACP adult cumulative numbers per tree (k). æ ö ç 0.97 × k ÷ -1 C = P × 1988.3 × ç ÷ × 100 0.97 k × çç 1 + ÷÷ 50.91 ø è

References Ammar E.-D., Ramos, J.E., Hall, D.G., Dawson, W.O. and Shatters, R.G. Jr (2016) Acquisition, replication and inoculation of Candidatus Liberibacter asiaticus following various acquisition periods on huanglongbing-infected citrus by nymphs and adults of the Asian citrus psyllid. PLOS ONE 11, e0159594. Aubert, B. and Quilici, S. (1988) Monitoring adult psyllas on yellow traps in Reunion Island. In: Timmer, L.W., Garnsey, S.M. and Navarro, L. (eds) Proceedings of the 10th Conference of the International Organization of Citrus Virologists, 17–21 November 1986, Valencia, Spain. International Organization of Citrus Virologists, University of California, Riverside, California, pp. 249–254.

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Bassanezi, R.B., Montesino, L.H., Gimenes-Fernandes, N., Yamamoto, P.T., Gottwald, T.R. et al. (2013a) Efficacy of area-wide inoculum reduction and vector control on temporal progress of huanglongbing in young sweet orange plantings. Plant Disease 97, 789–796. doi: 10.1094/PDIS-03-12-0314-RE Bassanezi, R.B., Belasque, J. Jr and Montesino, L.H. (2013b) Frequency of symptomatic trees removal in small citrus blocks on citrus huanglongbing epidemics. Crop Protection 52, 72–77. Belasque, J. Jr, Bassanezi, R.B., Yamamoto, P.T., Ayres, A.J., Tachibana, A., Violante, A.R. and Dragone, J. (2010) Lessons from huanglongbing management in São Paulo state, Brazil. Journal of Plant Pathology 285–302. Brust, G.E. and Foster, R.E. (1999) New economic threshold for striped cucumber beetle (Coleoptera: Chrysomelidae) in cantaloupe in the Midwest. Journal of Economic Entomology 92, 936–940. doi: 10.1093/jee/92.4.936. Hall, D.G. and Hentz, M.G. (2010) Sticky trap and stem-tap sampling protocols for the Asian citrus psyllid (Hemiptera: Psyllidae). Journal of Economic Entomology 103(2), 541–549. Hall, D.G., Hentz, M.G. and Ciomperlik, M.A. (2007) A comparison of traps and stem tap sampling for monitoring adult Asian citrus psyllid (Hemiptera: Psyllidae) in citrus. Florida Entomologist 90(2), 327–334. Hall, D.G., Richardson, M.L., Ammar, E.D. and Halbert, S.E. (2013) Asian citrus psyllid, Diaphorina citri, vector of citrus huanglongbing disease. Entomologia Experimentalis et Applicata 146(2), 207–223. Hall, D.G., Sétamou, M. and Mizell III, R.F. (2010) A comparison of sticky traps for monitoring Asian citrus psyllid (Diaphorina citri Kuwayama). Crop Protection 29(11), 1341–1346. Higley, L.G. and Pedigo, L.P. (eds) (1996) Economic Thresholds for Integrated Pest Management, Vol. 9. University of Nebraska Press, Lincoln, Nebraska. Jones, A.C. (2004) Using epidemiological information to develop effective integrated virus disease management strategies. Virus Research 100, 5–30. Kogan, M. (1998) Integrated pest management: historical perspectives and contemporary developments. Annual Review of Entomology 43(1), 243–270. Lee, J.A., Halbert, S.E., Dawson, W.O., Robertson, C.J., Keesling, J.E. and Singer, B.H. (2015) Asymptomatic spread of huanglongbing and implications for disease control. Proceedings of the National Academy of Sciences 112(24), 7605–7610. Mankin, R.W., Rohde, B. and McNeill, S. (2015) Vibrational duetting mimics to trap and disrupt mating of the devastating Asian citrus psyllid insect pest. Journal of the Acoustical Society of America 138(3), 1790. Miranda, M.P., dos Santos, F.L., Bassanezi, R.B., Montesino, L.H., Barbosa, J.C. and Sétamou, M. (2018) Monitoring methods for Diaphorina citri Kuwayama (Hemiptera: Liviidae) on citrus groves with different insecticide application programmes. Journal of Applied Entomology 142(1–2), 89–96. Monzó, C. and Stansly, P.A. (2015) Thresholds for vector control and compatibility with beneficial fauna in citrus with high incidence of huanglongbing. Acta Horticulturae 1065, 1137–1144. Monzo, C. and Stansly, P.A. (2017) Economic injury levels for Asian citrus psyllid control in process oranges from mature trees with high incidence of huanglongbing. PLOS ONE 12(4), e0175333. Monzo, C., Qureshi, J.A. and Stansly, P.A. (2014) Insecticide sprays, natural enemy assemblages and predation on Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae). Bulletin of Entomological Research 104(5), 576–585. Monzo, C., Arevalo, H.A., Jones, M.M., Vanaclocha, P., Croxton, S.D., Qureshi, J.A. and Stansly, P.A. (2015) Sampling methods for detection and monitoring of the Asian citrus psyllid (Hemiptera: Psyllidae). Environmental Entomology 44(3), 780–788. Paris, T.M., Croxton, S.D., Stansly, P.A. and Allan, S.A. (2015) Temporal response and attraction of Diaphorina citri to visual stimuli. Entomologia Experimentalis et Applicata 155, 137–147. doi: 10.1111/eea.12294 Paris T.M., Allan, S.A., Udell, B.J. and Stansly, P.A. (2017a) Wavelength and polarization effect phototaxis of the Asian citrus psyllid. Insect 8, 88m. doi: 10.3390/insects8030088. Paris, T.M., Allan, S.A., Udell, B.J. and Stansly, P.A. (2017b) Evidence of behavior-based utilization by the Asian citrus psyllid of a combination of UV and green or yellow wavelengths. PLOS ONE 12(12), e0189228. Pedigo, L.P., Lentz, G.L., Stone, J.D. and Cox, D.F. (1972) Green cloverworm populations in Iowa soybean with special reference to sampling procedure. Journal of Economic Entomology 65(2), 414–421. Plotto, A., Baldwin, E., Bai, J., Manthey, J., Raithore, S., Deterre, S. and Zhao, W. (2017) Effect of vector control and foliar nutrition on the quality of orange juice affected by Huanglongbing: sensory evaluation. HortScience 52(8), 1092–1099. Qureshi, J.A. and Stansly, P.A. (2007) Integrated approaches for managing the Asian citrus psyllid Diaphorina citri (Homoptera: Psyllidae) in Florida. Proceedings of Florida State Horticultural Society 120, 110–115.



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Qureshi, J.A. and Stansly, P.A. (2009) Exclusion techniques reveal significant biotic mortality suffered by Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae) populations in Florida citrus. Biological Control 50(2), 129–136. Qureshi, J.A. and Stansly, P.A. (2010) Dormant season foliar sprays of broad-spectrum insecticides: An effective component of integrated management for Diaphorina citri (Hemiptera: Psyllidae) in citrus orchards. Crop Protection 29(8), 860–866. Sétamou, M., Flores, D., French, J.V. and Hall, D.G. (2008) Dispersion patterns and sampling plans for Diaphorina citri (Hemiptera: Psyllidae) in citrus. Journal of Economic Entomology 101(4), 1478–1487. Southwood, T.R.E. and Henderson, P.A. (2009) Ecological Methods. John Wiley & Sons, Chichester, UK. Stansly, P.A., Arevalo, H.A., Qureshi, J.A., Jones, M.M., Hendricks, K., Roberts, P.D. and Roka, F.M. (2014) Vector control and foliar nutrition to maintain economic sustainability of bearing citrus in Florida groves affected by huanglongbing. Pest Management Science 70(3), 415–426. Stern, V.M.R.F., Smith, R., Van den Bosch, R. and Hagen, K. (1959) The integration of chemical and biological control of the spotted alfalfa aphid: the integrated control concept. Hilgardia 29(2), 81–101. Tansey, J.A., Vanaclocha, P., Monzo, C., Jones, M. and Stansly, P.A. (2017) Costs and benefits of insecticide and foliar nutrient applications to huanglongbing-infected citrus trees. Pest Management Science 73(5), 904–916. Thistlewood, H.M.A. (1986) The bionomics and monitoring of Campylomma verbasci (Meyer) on apple in the Okanagan Valley, British Columbia. Doctoral dissertation, Theses, Dept of Biological Science, Simon Fraser University, Burnaby, British Columbia, Canada. Thomas, D.B. (2012) Comparison of insect vacuums for sampling Asian citrus psyllid (Homoptera: Psyllidae) on citrus trees. Southwestern Entomologist 37(1), 55–60. Tiwari, S., Mann, R.S., Rogers, M.E. and Stelinski, L.L. (2011) Insecticide resistance in field populations of Asian citrus psyllid in Florida. Pest Management Science 67(10), 1258–1268. Udell, B.J., Monzo, C., Paris, T.M., Allan, S.A. and Stansly, P.A. (2017) Influence of limiting and regulating factors on populations of Asian citrus psyllid and the risk of insect and disease outbreaks. Annals of Applied Biology 171(1), 70–88. Weinert, M.P., Jacobson, S.C., Grimshaw, J.F., Bellis, G., Stephens, P.M., Gunua, T.G., Kame, M.F. and Davis, R.I. (2004) Detection of huanglongbing (citrus greening disease) in Timor-Leste (East Timor) and in Papua New Guinea. Australasian Plant Pathology 33(1), 135–136. Wenninger, E.J., Stelinski, L.L. and Hall, D.G. (2008) Behavioral evidence for a female-produced sex ­attractant in Diaphorina citri. Entomologia Experimentalis et Applicata 128(3), 450–459. Yi, Z., Jinchao, F., Dayuan, X., Weiguo, S. and Axmacher, J.C. (2012) A comparison of terrestrial arthropod sampling methods. Journal of Resources and Ecology 3(2), 174–182. Zanardi, O.Z., Volpe, H.X.L., Favaris, A.P., Silva, W.D., Luvizotto, R.A.G., Magnani, R.F., Esperança, V., Delfino, J.Y., de Freitas, R. et al. (2018) Putative sex pheromone of the Asian citrus psyllid, Diaphorina citri, breaks down into an attractant. Scientific Reports 8(1), 455.

11 

Management Objectives and Integration of Strategies for the Asian Citrus Psyllid

Philip A. Stansly and Jawwad A. Qureshi* University of Florida, Department of Entomology and Nematology, Institute of Food and Agricultural Sciences, Southwest Florida Research and Education Center, Immokalee, Florida, USA

11.1  Objectives of Asian Citrus Psyllid Management Asian citrus psyllid (ACP) poses a major threat to citrus everywhere and is the prime focus of citrus pest management wherever it is present. The level of preoccupation is largely a function of where each growing region is presently on a continuum from threatening to invade, to early invasion, to endemic. As of this writing, ACP has not yet been detected in the Mediterranean, ­Australia or South Africa, and the objective where ACP is not present is to forestall an invasion, primarily by educational and regulatory actions. South Africa is a special case in that the African citrus psyllid (Trioza erytreae Del Guercio) and greening are both endemic. However, the ­African vector and disease are largely confined to the cooler parts of the African citrus growing area. ACP and Asian huanglongbing (HLB) are better adapted to hot climates and would, therefore, pose additional problems for the African citrus industries (Bove, 2006). Diaphorina citri was recently found in Tanzania and the presence of the causal agent ‘Candidatus ­Liberibacter asiaticus’ (CLas) could not be ruled out (Shimwela et al., 2016).

The next stop on the continuum is the early stages of invasion, as exemplified by California and described in detail in Chapter 16. Here the objective of psyllid management is to slow the establishment of the psyllid and disease by regulatory actions and also by regional ‘eradication’ efforts in response to local detection of ACP or HLB. The next stage is illustrated by the situation in Texas and southern California where ACP is endemic, but HLB has not yet been detected throughout the zone, as described in Chapters 15 and 16. Finally, there are the situations in Brazil, Florida and southern China where both vector and disease are endemic. However, objectives still differ somewhat, due to different histories and economics in these regions. The many large producers of the huge Brazilian processed fruit industry are using aggressive vector control to hold off the worst ravages of HLB (Chapter 13). In contrast, costs and regulations restrict use of insecticides in the smaller and more diverse Florida industry compared with Brazil, and ­ most bearing trees are now HLB positive. The industry in Florida is struggling to maintain ­ productivity by reducing ACP populations to ­ ­decrease re-inoculation rates as well as ‘spoon-­ feeding’ with water and nutrients to optimize

*  Email: [email protected]

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Management Objectives and Integration of Strategies

tree health and vigor (Chapter 14). The Chinese industry has the longest history of HLB, primarily in the southern tier of provinces. Citrus production there consists largely of small producers supplying local markets with high-priced produce that covers the cost of rapid turnover of trees. Chemical control of ACP has been largely inefficient or neglected until recently, resulting in conditions conducive to biological control.

11.2  Pre-ACP Strategies Given the proven ability of ACP to be transported on both host plants and fruit (Chapter 9), the arrival of ACP in these areas is probably just a matter of time. Indeed, T. erytreae is already present in the Iberian Peninsula (Cocuzza et al., 2017). This recent invasion has engendered concern and a call to action as outlined by the Citrus Health Response Program (CHRP) adopted in Florida (https://www.freshfromflorida.com/DivisionsOffices/Plant-Industry/Agriculture-Industry/ Citrus-Health-Response-Program). These are largely regulatory measures aimed at preventing entry into the country or assuring a quick ­response in the event of detection. 1. Raise awareness within the citrus production community and public at large regarding dangers and risks associated with ACP and HLB. 2. Strengthen the letter and enforcement of phytosanitary regulations on movement of plants and fruit. 3. Regulate and implement protection of nursery stock in ACP-proof structures. 4. Plan and implement inspection of commercial and dooryard citrus and alternate hosts of ACP and HLB. 5. Prepare an eradication plan to set in motion upon first detection of ACP or HLB.

11.3  Early-stage Invasion Strategies This stage commences with the first detection of ACP in small regions. In California, the first four regulatory actions were implemented as well as an eradication plan with the overarching goal of reducing the incidence and spread of ACP to prevent the establishment of HLB and buy time for the research community to develop better disease

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management methods, or even a cure for the disease. This required, among other actions, an extensive ACP monitoring system using yellow sticky traps and pre-emptive sprays of commercial and residential citrus at the invasion front (Chapter 16). Initially, these eradicative treatments were applied within 800 m of a psyllid find and 5-mile quarantines were set up around each find. As populations expanded and were found repeatedly, the quarantines shifted to full county and eventually multi-county regions (southern California). At present, most of the main commercial citrus area (central San Joaquin Valley) is still in a quarantine comprised of counties that are partially infested with ACP but HLB not yet detected, with a geographic barrier between it and adjacent contiguous citrus growing regions (https://www.cdfa.ca.gov/plant/acp/ regulation.html). Treatment and movement of nursery stock are carefully regulated and bulk fruit moving between these regions is treated before harvest and trucks are tarped. Compared with the rapid spread of HLB in Brazil and Florida, these measures have been effective in slowing the epidemic, although different climatic conditions probably also come into play (see Section 11.6).

11.4  Mid-stage Invasion Strategies This situation is exemplified by Brazil, Texas and southern California where ACP has spread throughout commercial and residential citrus but detectable HLB is still confined to certain areas. In Brazil, HLB was detected in 2004 but is still at an estimated 17% in the main citrus growing area in the state of São Paulo and adjacent areas of Minas Gerais. The relatively slow spread of HLB compared with Florida can be attributed to initially lower populations of ACP in Brazil, plus an aggressive spray program that for many large farms includes surrounding groves and residential citrus (Chapter 13). Surprisingly, no insecticide resistance has been detected in tested ACP populations, possibly because numbers are maintained at such low levels that selection cannot operate efficiently or that the few psyllids collected come from unsprayed areas. In southern California, growers have organized into psyllid management areas or pest control districts and apply three to four psyllid effective treatments per year in a coordinated area-wide

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fashion to suppress psyllids. Tamarixia radiata has been released in a grid pattern in residences throughout southern California. In addition, treatments are applied to residential trees neighboring citrus groves participating in the coordinated treatments. Where HLB has been found in residences, insecticide treatments are applied to the residential citrus and their neighboring trees. Actions taken in Texas are similar to those outlined below (Section 11.5) for Florida, but with greater emphasis on dooryard citrus, which comprises a large portion of citrus trees in the region. Given the difficulty of using insecticides under these conditions, an intense campaign of biological control was implemented focused on mass rearing and release of T. radiata (Chapter 15) (Qureshi et al., 2014a). In commercial citrus, an area-wide spray program was instituted with sprays early and late in the ‘dormant’ season and another pair of sprays at 2-week intervals in late summer. Between times, grower-­ initiated whole block and border sprays are based on scouting to target multiple pests (Chapter 15). Estimated production of grapefruit and oranges in Texas has been maintained or increased since the 2014/15 season (4.25 and 1.45 million boxes, respectively) to an estimated 4.1 and 1.83 million boxes, respectively, in 2017/18 (NASS, 2018).

11.5  Late-stage Invasion This stage characterizes Florida, parts of Mexico, the Caribbean and presumably all or most of Asia. The situation in Florida is described in Chapter 14. Given an incidence of HLB in bearing trees of close to 100%, the justification for controlling D. citri is the yield response, presumably to reduce ‘re-inoculation’ of CLas (Stansly et  al., 2014; Tansey et al., 2015; Monzo and Stansly, 2017; Qureshi and Stansly, 2019). However, legal, social and economic factors prevent growers in Florida from intensifying spray programs to the extent of Brazil. Instead, improved horticultural practices, in particular plant nutrition, plus the demise of the most susceptible scion varieties appear to be increasingly important factors in maintaining the productivity of the better citrus orchards in Florida (Chapter 14). In contrast to Florida, citrus has all but disappeared in much of the Caribbean, with the possible e­ xception of the Dominican Republic.

11.6  Climate, Psyllids and HLB While time and technology are largely responsible for contrasts between regions mentioned above, climate also plays a role that may become more evident over time. Temperature affects psyllid development, pathogen titer and tree growth. Favorable temperatures for D. citri lie in the range of 20–30°C and are ideal at 28°C (Liu and Tsai, 2000) (Chapter 1). The favorable range for CLas is similar (Gasparoto et al., 2012). Psyllid reproduction will only occur in the presence of young shoots (flush). Therefore, flush cycles are the ultimate determinants of ACP population growth and dispersal. Shoot and root growth alternate with root growth following shoot growth in the presence of adequate soil moisture (Bevington and Castle, 1985) and both are optimal within the range of 27–32°C, again depending on soil moisture (Davies and Jackson, 2009). However, shoot growth was found to depend more on soil temperature than air temperature (Khairi and Hall, 1976). Mature trees typically have a major flush in spring which averaged 76% and 78% of the annual total in Clermont (Florida) and Weslaco (Texas) compared with 93% in Riverside, California (Cooper et al., 1963). Thus, secondary flushes in summer and early autumn depend on moisture and tend to be suppressed during hot dry summers characteristic of Mediterranean climates such as California (Cooper et al., 1963; Garcia Mari et al., 2002). Long flush-free periods thus limit opportunities for ACP reproduction and persistence. A comparison of climate data among the principal New World citrus growing regions reveals important contrasts that are likely to affect the severity of ACP infestation and thus of HLB (Table 11.1). California has a dry Mediterranean-­ type climate characterized by cool winters and dry hot summers with little or no rainfall in summer (Fig. 11.1). Texas and Florida have higher average temperatures, though cooler in winter, and more even annual distribution of rainfall (Figs 11.2 and 11.3). São Paulo, situated closer to the equator, has lower but less variable temperatures and a relatively dry cool season during which most citrus is still not irrigated. Therefore, one would predict the greatest amount of off-­season flush and higher ACP populations in Florida, ­followed by Texas, Brazil and finally California, which is consistent with observed patterns.



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Table 11.1.  Annual average high, average low, average temperature and average rainfall for Visalia (California), McAllen (Texas), Immokalee (Florida) (www.usclimatedata.com) and Araraquara (São Paulo, Brazil). Location

Latitude

Visalia CA McAllen TX Immokalee FL Araraquara SP

36.3 N 26.2 N 26.4 N 21.8 S

Av. high°C

Av. low°C

23.8 29.9 29.7 26.2

Av. temp°C

10.9 18.5 16.6 14.6

17.4 24.2 23.2 20.4

Av. rain (mm) 278 565 1,265 1,352

40°C

60mm 50mm

30°C

40mm 20°C 30mm 10°C 20mm 0°C

10mm 0mm

Low

High

Dec

Nov

Oct

Sep

Aug

Jul

Jun

May

Apr

Mar

Feb

Jan

–10°C

Precipitation

Fig. 11.1.  Temperature and precipitation in Visalia, California.

11.7  Insecticidal Control Insecticides have been and still are the first line of defense against ACP for many reasons: ­efficacy, cost, availability of products and application equipment, and familiarity with use. Practical drawbacks include toxicity to users, export issues with maximum residue levels (MRLs), suppression of natural enemies and insecticide resistance. Criteria for choice of product, timing and application method aim at maximizing benefits while minimizing drawbacks.

11.7.1  Criteria for choice Efficacy is probably the most important criterion in the choice of insecticide to target a particular

pest. Qureshi et al. (2014b) summarized the results of multiple field tests evaluating 44 separate active ingredients against ACP. More than 90% reduction of adults for at least 3 weeks was observed following sprays of 23 insecticides representing Insecticide Resistance Action Committee (IRAC) groups 1B, 3A, 4A, 4C, 4D, 5, 21, 23 and 28. The ratio of labeled rate to the sensitivity of ACP to the active ingredient also impacts efficacy in the field. For instance, the highest labeled rates for the organophosphates dimethoate and malathion were more than 30 times higher than their respective residual LC90s (lethal concentration 90%) in contrast to abamectin, zeta-cypermethrin and fenpyroximate which were four- to sevenfold lower (Vanaclocha et al., 2018). Additionally, group 4A products thiamethoxam and especially imidacloprid are many times more active against nymphs than against adults (Vanaclocha et al., 2018).

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40°C 120mm 30°C

100mm 80mm

20°C

60mm 40mm

10°C

20mm 0°C

Low

High

Dec

Nov

Oct

Sep

Aug

Jul

Jun

May

Apr

Mar

Feb

Jan

0mm

Precipitation

Fig. 11.2.  Temperature and precipitation in McAllen, Texas.

40°C 200mm 180mm 30°C

160mm 140mm 120mm

20°C

100mm 80mm 60mm

10°C

40mm 20mm 0mm

Low

High

Dec

Nov

Oct

Sep

Aug

Jul

Jun

May

Apr

Mar

Feb

Jan

0°C

Precipitation

Fig. 11.3.  Temperature and precipitation in Immokalee, Florida.

Spirotetramat has no adult activity (Vanaclocha et al., 2018) but is also effective against scales and mites and forgiving of beneficial insects (Brück et al., 2009). Selectivity is a quality that can be seen in two contrasting lights. Compatibility with natural

enemies is a favorable quality but so is activity on a range of pests. Thus, conservation of natural enemies and activity against more than one pest that may require control can be opposing criteria for choice. As broad-spectrum products in IRAC group 1 (organophosphates and carbamates) have



Management Objectives and Integration of Strategies

been phased out through regulation, premixes of more than one active ingredient that broaden the target range become ever more popular. Furthermore, certain products may be synergistic against ACP, such as mixtures of thiamethoxam with abamectin or chlorantraniliprole (Qureshi et al., 2013; Vanaclocha et al., 2018). Cost is necessarily a factor in any input decision and insecticides are no exception. In general, new products still on patent are expensive and older products available as generics are cheap. Thus, cost considerations favor older products, like pyrethroids, which tend to be broad spectrum and labeled for most crops, including citrus. Timing may also affect the choice of insecticide based on predominant target stage or secondary pest presence, factors in turn related to tree growth and climate. Trees are generally not flushing during cool or dry periods, so the predominant ACP stage will necessarily be adults. Insecticides applied at this time can significantly reduce founding populations in subsequent flush and thus the potential to increase and eventually disperse to spread disease (Qureshi and Stansly, 2010). Another advantage to applying prior to flush is the relative absence of vagile predators, such as ladybeetles, that feed on insects like psyllids, aphids and whiteflies that develop in flush. Just prior to a major flush is a good time to apply spirotetramat, which is systemic and can move into developing shoots (Brück et al., 2009). Nymphs would be the principal target during flush periods. However, much of the major spring flush corresponds with bloom, which limits options, due to label restrictions aimed at protecting bees (Davies and Jackson, 2009). Canopy penetration can be ­ ­improved by increasing spray volume or droplet size, but these measures also increase run-off, especially from the outer canopy. 11.7.2  Population detection and monitoring This is an essential component IPM, for application of economic thresholds as well as assessing the effectiveness of control actions. Several methods of monitoring ACP have been used, including visual sampling, yellow sticky cards, sweep nets, tap sampling and suction devices. Each has its advantages and disadvantages. Yellow sticky

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traps are sensitive and assess ACP in flight, so results may not always correlate with what is in the canopy. Using them is time consuming and labor intensive, requiring deployment, collecting and reading, and expensive, about US$1 per trap (Hall and Hentz, 2010). Visual sampling is rapid but tends to vary between practitioners. Suction sampling is most sensitive of all and thus best for detection but also labor intensive. Tap sampling was the most efficient of all methods tested for estimating population density above one ACP pre 100 taps (Monzo et al., 2015). Tap sampling requires striking a randomly selected branch with a stick or length of PVC pipe and counting ACP adults falling on to a laminated sheet held below (Qureshi and Stansly 2007, 2010; Qureshi et al., 2009). Because of its ease and reliability, the tap sample has been adopted by many growers as well as APHIS-PPQ (Animal and Plant Health Inspection Service: Plant Protection and Quarantine) and FDACS-DPI (­ Florida Department of Agriculture and Consumer Services: Division of Plant Industries) as an integral part of the CHRP in Florida. In California, yellow sticky traps are used in areas for detecting new infestations of ACP and visual sampling of flush is used by the pest control advisors to trigger ­area-wide treatments in southern California. 11.7.3  Economic injury levels Economic injury levels for ACP in mature trees with the high incidence of HLB have been estimated for two commercial orchards of process oranges in Florida (Monzo and Stansly, 2015, 2017). Two nominal thresholds were chosen (two and seven ACP adults per ten tap samples) based on preliminary data. ACP populations, HLB incidence, yield and fruit quality in response to these two treatments were estimated for 4 years and compared with a monthly calendar spray on the one hand and an untreated check on the other in a randomized block design with four replications. A rectangular hyperbolic model was used to correlate cumulative ACP counts at 2-week intervals during the growing season with yield loss. These results were then used to estimate economic injury levels given different prices that are paid in the juice market based on yield and solids concentration. This research demonstrated the feasibility of an economic threshold approach for managing ACP

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with insecticides. Thresholds based on nymphs and efficiently throughout the canopy, especially are being developed in California (E. Grafton-­ where pests may be concentrated. Engine or PTO-driven polar air-blast sprayers have been Cardwell, personal communication). the standard in the industry for over 50 years (Fox et al., 2008). A powerful fan pushes a large 11.7.4  Where to spray: the ‘edge effect’ volume of air forward against a bulkhead which directs it 90 degrees out perpendicular Several studies have noted and confirmed the pro- to the direction of travel through openings, pensity of ACP to accumulate along the edges of creating air jets into which the spray mixture citrus blocks (Gottwald et al., 2008; Boina et al., is introduced generally through disk and core 2009; Anco and Gottwald, 2015; Sétamou and hydraulic nozzles (Fig. 11.4). Design factors Bartlets, 2015), which is consistent with higher include air velocity and volume, number, disincidence of HLB along block borders (Aubert, position and type of nozzles; operational fac1990; Gottwald et al., 2008). Colonization clearly tors include travel speed and pump pressure; begins on the borders but the permanence of this tree factors include tree spacing and canopy distribution and its prevalence adjacent to open size and density; and meteorological factors areas as opposed to contiguous blocks of trees in- include wind speed and direction, temperature dicates more complex relationships. Sétamou and and humidity (Farook and Salyani, 2006; Fox Bartlets (2015) hypothesized that preference for et al., 2008). border trees is a characteristic innate behavior of Coverage generally decreases toward the inD. citri. These authors noted that trees on open terior of the canopy and toward the top of large borders get more light than interior trees and trees. (Stansly et al., 1996). However, most flush would likely flush earlier. It may also be that short- and therefore psyllids are found in the first range movement stimulated by lack of flush would 30  inches (76 cm) of the canopy (Davies and eventually result in arrival to a border where the Jackson, 2009). Nevertheless, deposition in the insect would at least temporarily arrest flight. Re- inner canopy can be improved by increasing gardless of the explanation, the edge effect has ob- spray ­volume or droplet size, but these measures vious implications for ACP management. For one also increase run-off. Droplet size can be reduced thing, ACP monitoring can focus on block bor- by increasing pump pressure or using smaller oriders. An example is the protocol adopted by the fice nozzles, but will increase drift. Vertical distriCHRP in Florida of ten tap samples on each of four bution of spray material can be improved by tower corners and one in the center of each sampled arrangements that emit spray at different heights block. Arevalo et al. (2011) recommended five ten- and therefore closer to the target in the upper tap samples along the borders and the same num- canopy (Stansly et al., 1996; Farook and Salyani, ber in the interior of each block, possibly leading 2004; Fox et al., 2008). This would be beneficial, to a decision to spray only borders. California is since ACP tends to infest the upper canopy, predeveloping a similar decision for border-only treat- sumably in response to light (Chapter 3). Coverment based on examining ten flushes in the inter- age is better on smaller trees, which also open ior of the orchard. Border versus whole-block the door to over-the-top applications that insprays provide savings in spray materials and clude tunnel sprayers to minimize drift (Fox spraying times. Furthermore, less ACP exposure et al., 2008). to often-used insecticides reduces selection for reTiming sprays to flush cycles requires rapid sistance while at the same time conserving benefi- application afforded by low-volume sprays by cial arthropods needed for biological control of air or ground. Aerial sprays of pyrethroids or this and other pests. ­organo-phosphates applied at 47–140 l/ha have proven effective as ‘dormant sprays’ during the cool dry season in Florida (Stansly et al., 2009; 11.7.5  Application method Qureshi et al., 2010). Frequent applications of horticultural mineral oil (HMO), either neat at The application method is also subject to choice. 19 l/ha or suspended in an equal volume of The objective is to deposit spray material evenly water, were effective in controlling ACP and



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Fig. 11.4.  Air-blast sprayer working in Florida citrus (photo J. Qureshi).

maintaining yields in Florida (Tansey et al., 2015). Applications were made with a sprayer equipped with a fan-assisted rotary atomizer fed by a peristaltic pump. While most insecticides are applied to the foliage as sprays, some systemic products can be applied to the soil where they are taken up by the roots and translocated through the xylem up to the foliage, particularly young flush. Such products include the 4A neonicotinoids imidacloprid, thiamethoxam and clothianidin, the group 28 cyantraniliprole, and the carbamate aldicarb. The neonicotinoids and cyantraniliprole are used only on young trees, due to label restrictions on rate, whereas aldicarb was used also on mature trees, though it is no longer permitted on citrus in most countries (Qureshi and Stansly, 2008). The remaining products are applied to the soil as drenches or through drip irrigation. Drip irrigation is more efficient once root systems have established 6 months to a year after planting. Cyantraniliprole has been too expensive for widespread use, leaving only neonicotinoids. Unfortunately, this has resulted in extensive resistance to this mode of action in Florida (see next section).

11.7.6  Resistance management Minimizing selection for resistance is largely a question of minimizing exposure of the pest population to particular insecticide modes of action. In practice, this boils down to eliminating unnecessary sprays and rotating modes of action. Greater reliance on biological and cultural controls would relieve pressure on insecticides. Additionally, the first three actions outlined above to conserve natural enemies also serve to eliminate unnecessary sprays as well as lay a groundwork for rotation by limiting the use of broad-spectrum insecticides. The rotation objective can then be completed by making use of the full range of selective insecticides available for any additional sprays. These could include rotations of products such as spinetoram, spirtotetramat, tolfenpyrad, cyantraniliprole and flupyradifuron. Optimal timing for these will depend on label restrictions and effects on different life stages, secondary pests and beneficial arthropods. Application of multiple modes of action in premixes or tank mixes compromises this strategy by reducing the number of potential rotation partners (Qureshi et al., 2014b).

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11.7.7  Area-wide management Insecticidal control of ACP is seen as the principal recourse for forestalling the spread of HLB and managing it once established. There can be little debate as to the short-term benefits of this approach, although sustainability over the long term is open to question. The effects of immigration from untreated or insufficiently treated citrus are a major shortcoming, engendering an area-wide approach that is credited with the relative success of chemical control. However, after 10 years, the voluntary program in Florida and California has met with only partial success (Chapter 14). Problems include lack of full ­compliance and growing disillusionment with insecticidal control, in part because of costs and insecticide resistance, especially with neonicotinoids, as well as influx of psyllids from residential areas and abandoned orchards. In comparison, larger citrus growers in Brazil administer and pay for ACP control in their immediate neighborhoods with apparent success (Chapter 13). The more recently instituted program in the relatively small Texas growing region also appears to be working, bolstered by a strong program of biological control in residential neighborhoods. The program in California of treating groves with area-wide treatments, applying insecticides to neighboring residential areas in some regions, and releasing T. radiata in residential areas has thus far held HLB to residential citrus and slowed the spread of ACP into the main citrus growing areas except for the south (Chapter 16). Thus, area-wide management based on ­insecticidal control has provided at least shortterm suppression of HLB but is subject to problems of insecticide resistance and disaccord among clientele.

11.8  Integration with Biological Control A typical pattern for many invasive pests is an initial explosive phase followed by gradual attenuation, presumably in response to recruitment by natural enemies. In the case of ACP, this process may have been truncated by the ­necessity of intensive insecticide use to slow the ­epidemic of HLB. Given the efficiency of the

acquisition/transmission process, it is not surprising that HLB has yet to be halted by biological or insecticidal control. Nevertheless, biological control has a lot to offer to an integrated management system, including levels of mortality comparable or superior to those conferred by insecticides at certain times of the year (Qureshi and Stansly, 2009) (Chapter 6). The challenge is how to integrate two apparently incompatible control methods. A successful plan might include actions to conserve or increase natural enemies including (i) limiting use of broad-spectrum insecticides to when most natural enemies are scarce or absent; (ii) refuges from broad-spectrum insecticides where natural enemies can build up; (iii) targeted sprays of selective products only when economic thresholds are exceeded; and (iv) reinforcement by early-season inoculative releases of key natural enemies. The elements of a program incorporating these principles are outlined below. Broad-spectrum insecticides (pyrethroids and organophospates) have been shown to be most effective and with least collateral damage to natural enemies during dry and/or cool seasons when trees normally do not flush. This practice takes advantage of the weak link in the ACP life cycle with the objective of further reducing populations before a major flush and when most natural enemies are scarce (Qureshi and Stansly, 2010). A related practice is to restrict further use of pyrethroids and organophosphates to block borders. This takes advantage of the so-called ‘edge effect’ based on the observation that ACPs are often denser on the perimeters of citrus blocks, especially those adjacent to open areas (Luo et al., 2012; Sétamou and Bartles, 2015) (Chapter 15). Border sprays thus control psyllids where they are most numerous with inexpensive products, reducing the need for spraying the entire orchard and at the same time providing refuge for natural enemies in block interiors. Economic thresholds based on economic injury levels are fundamental to integrated pest management systems. However, they are typically difficult to employ when the pest is a disease vector, because of uncertainty as to the proportion of potential vectors capable of transmitting the pathogen. Furthermore, the damage potential of the disease is to some extent a function of the age of the plant when infected. These factors



Management Objectives and Integration of Strategies

become inconsequential once most plants become infected with HLB and the damage potential becomes a function of the psyllid population. At that point, the principle of economic injury level can be applied to eliminate unnecessary sprays and, consequently, negative impacts on natural enemies (Chapter 10) (Monzo and Stansly, 2015). The parasitoid T. radiata is being mass-reared and released in Florida, Texas, California, Brazil, Costa Rica and perhaps elsewhere. The objective is generally to suppress ACP in residential and abandoned citrus which is difficult or costly to spray. Augmentative releases of T. radiata during spring flush could be beneficial in commercial citrus when parasitism is normally low, especially following dormant sprays that are detrimental to overwintering parasitoids. However, the expense is currently too high to release them at the level needed.

11.9  Exclusion Methods The experience in Florida has shown that a certain level of productivity of HLB-infected bearing trees can be maintained through a combination of vector control and horticultural practices aimed at maintaining tree health. Economic sustainability is thus a function of costs, yield and commodity price. Notwithstanding, the ability to bring new plantings into profitable production remains a crucial question for the future of the industry. Resistance to insecticides in ACP in Florida has rendered efficacy of soil applications of neonicotinoid insecticides transitory in frequently flushing young trees. Whether frequent spraying can compensate for this shortcoming is an open question. Deficiencies and doubts regarding the present insecticide-­ based program for young trees have engendered experimentation with exclusion methods. These include screen coverings for entire blocks and individual trees as well as repellency afforded by

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metalized polyethylene ground covers (Croxton and Stansly, 2013; Schumann et al., 2017). All these methods imply additional costs that must be compensated for by savings in inputs and/or increased production.

11.10  The Way Forward ACP suppression is not an end in itself but merely one component of an HLB management system. The other two components focus on the pathogen and host susceptibility. The need for vector control will be lessened commensurate with successful implementation of methods to reduce the titer of CLas in the plant or vector, or with increased tolerance to HLB through genetics and improved horticultural practices. Traditional methods of vector control, in particular those based on insecticides, are clearly stopgap measures needed to hold off the worst consequences of HLB until other management options are in place. Nevertheless, it is unlikely that new developments will ever completely obviate the need for ACP suppression, although biological and cultural controls could to some degree reduce the present dependence on insecticides. Other novel methods of ACP suppression, such as suppression using RNAi or genetic modification to reduce vector capacity of D. citri, may also become functional in the future (Chapter 17). Meanwhile, insecticides will likely continue to play an important role in integrated strategies both to slow the spread of HLB and to reduce the impact of the disease on infected trees.

Acknowledgement Thanks to E. Grafton-Cardwell for a helpful review.

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Monzó, C. and Stansly, P.A. (2015) Thresholds for vector control and compatibility with beneficial fauna in citrus with high incidence of Huanglongbing. Acta Horticulturae 1065, 1137–1144. Monzo, C. and Stansly, P.A. (2017) Economic injury levels for Asian citrus psyllid control in process oranges from mature trees with high incidence of huanglongbing. PLOS ONE 12(4), e0175333. Monzo, C., Arevalo, H.A., Jones, M.M., Vanaclocha, P., Croxton, S.D., Qureshi, J.A. and Stansly, P.A. (2015) Sampling methods for detection and monitoring of the Asian citrus psyllid (Hemiptera: Psyllidae). Environmental Entomology 44(3), 780–788. NASS (2018) Citrus Fruits 2018 Summary. USDA National Agricultural Statistics Service, 35. Available at: http://usda.mannlib.cornell.edu/usda/current/CitrFrui/CitrFrui-08-28-2018.pdf. Qureshi, J.A. and Stansly, P.A. (2007) Integrated approaches for managing the Asian citrus psyllid Diaphorina citri (Homoptera: Psyllidae) in Florida. Proceedings of Florida State Horticultural Society 120, 110–115. Qureshi, J.A. and Stansly, P.A. (2008) Rate, placement, and timing of aldicarb applications to control Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae) in oranges. Pest Management Science 64, 1159–1169. Qureshi, J.A. and Stansly, P.A. (2009) Exclusion techniques reveal significant biotic mortality suffered by Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae) populations in Florida citrus. Biological Control 50, 129–136. Qureshi, J.A. and Stansly, P.A. (2010) Dormant season foliar sprays of broad-spectrum insecticides: an effective component of integrated management for Diaphorina citri (Hemiptera: Psyllidae) in citrus orchards. Crop Protection 29, 860–866. Qureshi, J.A. and Stansly, P.A. (2019) Organic and conventional ACP management. Citrograph 10(3), 66–69. Qureshi, J.A., Rogers, M.E., Hall, D.G. and Stansly, P.A. (2009) Incidence of invasive Diaphorina citri (Hemiptera: Psyllidae) and its introduced parasitoid Tamarixia radiata (Hymenoptera: Eulophidae) in Florida citrus. Journal of Economic Entomology 102, 247–256. Qureshi, J.A., Kostyk, B.C. and Stansly, P.A. (2010) Ground application of foliar insecticides to ‘Valencia’ oranges for control of Diaphorina citri Kuwayama (Hemiptera: Psyllidae). Proceedings of the Florida State Horticultural Society 123, 109–112. Qureshi, J.A., Kostyk, B. and Stansly, P.A. (2013) Control of Asian citrus psyllid and citrus leafminer in oranges with foliar sprays of horticultural mineral oil, microbial and synthetic insecticides. Proceedings of Florida State Horticultural Society 126, 62–67. Qureshi, J.A., Rohrig, A.A., Stuart, R.J., Gall, D.G., Leppla, N.C. and Stansly, P.A. (2014a) Imported parasitoids for biological control of Asian citrus psyllid. Citrus Industry June 2014. Qureshi, J.A., Kostyk, B. and Stansly, P.A. (2014b) Insecticidal suppression of Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae) vector of huanglongbing pathogens. PLOS ONE 9(12), e112331. doi: 10.1371/journal.pone.0112331. Salyani, M. (2000) Optimization of deposition efficiency for airblast sprayers. Transactions of the ASAE 43(2), 247–253. doi: 10.13031/2013.2699. Schumann, A.W., Singerman, A., Wright, A.L. and Ferrarezi, R.S. (2017) 2019–2020 Florida Citrus Production Guide: Citrus Under Protective Screen (CUPS) Production Systems. Available at: http://edis.ifas. ufl.edu/hs1304 (accessed 30 November 2019). Sétamou, M. and Bartels, D.W. (2015) Living on the edges: spatial niche occupation of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), in citrus groves. PLOS ONE 10(7), e0131917. doi: 10.1371/journal.pone.0131917. Shimwela, M.M., Narouei-khandan, H., Halbert, S.E., Keremane, M.L., Minsavage, G.V. and Timilsina, S.C. (2016) First occurrence of Diaphorina citri in East Africa, characterization of the Ca. liberibacter species causing huanglongbing (HLB) in Tanzania, and potential further spread of D. citri and HLB in Africa and Europe. European Journal of Plant Pathology 146(2), 349–368. doi: http://dx.doi.org. lp.hscl.ufl.edu/10.1007/s10658-016-0921-y Stansly, P.A., Rouse, R.E. and Cromwell, R.P. (1996) Deposition of spray material on citrus fruit and ­foliage by air and ground application. Proceedings of the Florida State Horticultural Society 109, 34–40. Stansly, P.A., Arevalo, H.A., Zekri, M. and Hamel, R. (2009) Cooperative dormant spray program against Asian citrus psyllid in SW Florida. Citrus Industry 90, 14–15. Stansly, P.A., Arevalo, H., Qureshi, J.A., Jones, M.M., Hendricks, K., Roberts, P.D. and Roka, F.M. (2013) Vector control and foliar nutrition to maintain economic sustainability of bearing citrus in Florida groves affected by huanglongbing. Pest Management Science 70(3), 415–426. doi: 10.1002/ps.3577.

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Stansly, P.A., Arevalo, H.A., Qureshi, J.A., Jones, M.M., Hendricks, K., Roberts, P.D. and Roka, F.M. (2014) Vector control and foliar nutrition to maintain economic sustainability of bearing citrus in Florida groves affected by huanglongbing. Pest Management Science 70(3), 415–426. Tansey, J.A., Jones, M.M., Vanaclocha, P., Robertson, J. and Stansly, P.A. (2015) Costs and benefits of frequent low-volume applications of horticultural mineral oil for management of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae). Crop Protection 76, 59–67. Vanaclocha, P., Jones, M.M., Tansey, J.A., Monzó, C., Chen, X. and Stansly, P.A. (2018) Residual toxicity of insecticides used against the Asian citrus psyllid and resistance management strategies with ­thiamethoxam and abamectin. Journal of Pest Science 92, 871–883.

12 

Management of the Asian Citrus Psyllid in Asia

George A.C. Beattie* School of Science and Health, Western Sydney University, Penrith, NSW, Australia

12.1  Origin and Spread Diaphorina citri Kuwayama (Hemiptera: Sternorrhyncha: Liviidae) is assumed to have evolved in South Asia (Hollis, 1987; Halbert and Manjunath, 2004; Beattie and Barkley, 2009). It was described by Kuwayama (1908), based on specimens collected from mandarin trees in ‘Shinchiku Prefecture’ in northeastern Taiwan in 1907. It was then erroneously described as Euphalerus citri (Kuwayama) by Crawford (1912). Crawford’s description was based on a single female in the Indian Museum, Calcutta in Bengal (now Kolkata in West Bengal) that was collected by James Jenkins in Adra, now in West Bengal, in 1909, and 17 specimens (both sexes) collected by George Compere on citrus trees in the Philippines. Crawford (1912) noted that Compere had observed the psyllid in considerable numbers in India, and that seven specimens collected from orange trees were also present in the insect collection at the Imperial Agricultural Research Institute in Pusa, Bihar. Records in the US National Museum of Natural History (NMNH) and in formal publications, newspapers and magazines (Harwood, 1905–1906a, b; Anon., 1907; Crafts, 1907; Back and Pemberton, 1917; Essig, 1931; Flanders, 1949; Compere, 1961; Wilson, 1960) indicate

that Compere either observed or collected D. citri on the Indonesian islands of Java and Timor in 1900 and 1905, respectively, and in Sri Lanka (Ceylon) and India between 1900 and 1907, as well as in the Philippines in 1907–1908 and 1909. He visited India once in 1903, once in 1906, and twice in 1907, and collected parasitoids of red scale (Aonidiella aurantii (Maskell) (Hemiptera: Coccomorpha: Diaspididae)) and/or purple scale (Lepidosaphes beckii (Newman) (­ Diaspididae)) in southern India at or near Bangalore in 1906, Chennai (Madras) and Thoothukudi (Tuticorin) in Tamil Nadu, Bengalura (Bangalore) in Kanataka, and Goa, and in central India in Mumbai (Bombay), Nagpur, Pune (Poona) and Karad in Maharashtra in 1907 (Compere, 1912). These records suggest that he observed D. citri in India in 1906 and/or 1907, and possibly at more than one location. Chandra Shekar ­ Misra and Dwarka Prasad Singh collected specimens on orange at Pusa in 1908 and 1909 (­Mathur, 1975). The psyllid was recorded in Macao in 1906, Amboina (Moluccas) in 1907 (Crawford, 1919), and Hong Kong in July 1908 (Martin and Lau, 2011). However, there are no records to suggest that George Compere observed it during nine trips he made to southeast China to collect parasitoids of armoured and soft scale pests

*  Email: [email protected] © CAB International 2020. Asian Citrus Psyllid: Biology, Ecology and Management of the Huanglongbing Vector (eds. J.A. Qureshi and P.A. Stansly)

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of citrus between 1899 to 1909. Nor was it mentioned as a pest of citrus in southern China in a 1918 report of surveys of citriculture in Guangdong undertaken by George Groff and Kwok Wah Shau (WS Kwok: Guo Huaxiu) (see Cooper 1989), or by Reinking (1919, 1921), Groff and Howard (1924), Hoffmann (1931) and Hu (1931). Reinking (1921) listed pests of citrus in Thailand, southern Vietnam, southern China, and the Philippines. Groff and Howard (1924) reported their observations on predation on insects by the weaver ant Oecophylla smaragdina (Fabricius) (Hymenoptera: Formicidae) in citrus orchards in Guangdong. Hoffmann (1931) listed Hemiptera and Homoptera in Guangzhou, and Hu (1931) noted pests recorded during surveys related to citriculture in Guangdong, Fujian and Zhejiang during 1928 and 1929. D. citri was first recorded in mainland China, in Guangdong Province, in 1934 (He and Zhou, 1935; Jiang et al., 1935; Hoffmann, 1936). Phylogenetic studies reported by Wu et al. (2018) suggest that it was introduced to the mainland near Chaoshan on psyllid-infested host plants from Taiwan in the late 1920s or early 1930s. Movement of infested plants from Chaoshan, as documented by Groff (1934), appear to have led to the establishment of populations in Guangzhou in south-central Guangdong. From the mid-1930s, the psyllid spread to citrus-producing regions in the north and west of Guangdong, and southwards to the island of Hainan; this occurred over several decades (Wu et al., 2018). Although it was recorded in Macao in 1906 and in Hong Kong in 1908, it does not appear have spread northwards into mainland China through villages and orchards on the Pearl River (Zhujiang) Delta before 1934. The distribution of orchards in the delta, as depicted in US Army topographic map NF 49–8, Series L500 (Army Map Service, Corps of Engineering, Washington, DC, 1954, available at: http:// w w w. lib.utexas.edu/maps/ams/china/txuoclc-10552568-nf49-8.jpg), suggests that such spread could have been hindered by the presence of few, if any, orchards within 80 km north of Hong Kong on the eastern side of the river, and 50 km north of Macao on the western side, before 1950. Spread westward from Guangdong to Yunnan in southwest China appears to have involved adaptation of the psyllid to elevations above 800 m (Wang et  al., 2018). It may also

have involved incursions from Myanmar or nearby northwestern Indochina. Reports by Clausen (1933) and Clausen et  al. (1933) suggest that D. citri was first recorded in Myanmar between 1924 and 1928. It was first recorded in Thailand in 1965 (Catling, 1970), Vietnam in 1967–1968 (Anonymous, 1978), Cambodia in 1995, and Laos in 1997 (Garnier and Bové, 2000). It has occurred widely in Bangladesh (formerly East Bengal) since at least the mid-1970s (Catling et  al., 1978) and was first recorded in Nepal and ­Bhutan in the 1960s (Catling, 1968; Catling et  al., 1978; Lama and Amatya, 1991, 1993; Ohtsu et al., 1998). Aubert (1990b) recorded it in the Indonesian province of Papua on the ­island of New Guinea; and Davis (2003) and Weinert et  al. (2004) reported its detection at Vanimo in northwestern Papua New Guinea in 2002. Twenty specimens were collected by ­Gerald Hill in 1915 at Stapleton, Northern Territory, ­Australia (Bellis et  al., 2005); however, it was eradicated from the ­Northern Territory by chance between 1916 and 1922 during a successful campaign to eradicate citrus canker (Xanthomonas citri subsp. citri (ex Hasse 1915) Gabriel et  al. 1989 (Pseudomonadales: Pseudomonadaceae)) (Hill, 1918; Bellis et al., 2005). Recent molecular studies (Wang et  al., 2017, 2018; Wu et al., 2018) suggest that genetic variation in D. citri and its primary endosymbiont, ‘Candidatus Carsonella ruddii’, in South Asia and Southeast Asia reflect adaptation of the psyllid to humid tropical and subtropical environments as it spread eastward from hot-arid regions of South Asia where it evolved. The above records suggest that spread of D. citri eastward from South Asia to Southeast Asia was related to movement of infested host plants from Dutch and Portuguese colonies in southern India (e.g. Goa, Kerala and Tamil Nadu) and Sri Lanka to Java, Ambon, Timor, Luzon, Macao and Formosa (Taiwan). If so, the host plants may have been lemon and/or lime seedlings taken by sea in the 16th and 17th centuries, perhaps through to 1800, for planting in or near ports for year-round production of fresh fruit required to prevent scurvy (Bonavia, 1886, 1888–1890; Bowman, 1955; Webber, 1967; Moore, 2001; De Villiers, 2006; Baron, 2009; Hoogervorst, 2013). These plants may have harboured ‘Candidatus Liberibacter asiaticus’ (CLas),



Management of the Asian Citrus Psyllid in Asia 181

the putative causal agent of huanglongbing (HLB). However, if some trees were infected, records related to spread of the disease in Southeast Asia would suggest that it may not have persisted, due to death of infected trees and limited spread by D. citri in the absence of monocultures. There is no indication that high-density populations occurred at these localities until after 1950. This suggests that populations may have occurred at low densities due to influences of abiotic factors, particularly rainfall, and natural enemies, including the weaver.

12.2  Pest Status The presence of D. citri in considerable numbers on citrus was first observed by Compere in India, then including Pakistan (Crawford, 1912). The records of Compere’s travels in Asia cited above suggest that his observations were made in 1903, 1905 and/or 1909. Fletcher (1917, 1919) regarded it as minor pest in India but noted that it sometimes occurred in large numbers and caused considerable damage. Lal (1918) mentioned that it caused great damage in the Punjab in northwestern India. Husain and Nath (1927) noted that, in extreme cases, trees in the Punjab suffered complete defoliation and that apart from defoliation and loss of sap, the nymphs also injected some toxic substance into plant tissues during feeding. Undersized fruit, poor and insipid juice were attributed to this ‘toxin’ and branches not directly attacked became prematurely dry. The income of one grower in the Punjab was reduced by 90% after a severe attack by the psylla (Husain and Nath, 1927). Pruthi and Mani (1945) noted that the psyllid was so destructive in several parts of India that the citrus industry was often completely ruined. They commented that it was not so destructive in other countries. The severe impacts observed by Husain and Nath (1927) were most probably caused by ‘CLas’. Ahmad (1961) estimated that > 4.5 million adults and nymphs were present in March (spring) 1959 in a 1 acre (0.4 ha) irrigated orchard comprising 110 mature sour lime (Citrus × aurantiifolia (Christm.) Swingle) trees in the hot desert of the Multan (30.1981°N, 71.4685°E, 129 m above sea level

(asl)) region of the Punjab. This capacity to increase is related to high fecundity and the relatively short generation times when flush growth suitable for development of nymphs is available (Husain and Nath, 1927). D. citri was not regarded as a serious pest of citrus in India and China before the 1960s and 1970s after the psyllid was recognised as the vector of CLas (­Capoor et  al., 1967; Zhao, 1981). ‘Shinchiku Prefecture’, where Shigeru Kuwayama collected the psyllid specimens he described as Diaphorina citri (Kuwayama 1908), was a northern administrative division of Taiwan during Japanese rule of Taiwan from 1895 to 1945. The region now comprises Hsinchu and Miaoli counties, and Taoyuan. From 1642 to 1662 parts of the region were colonized by the Dutch.

12.3  Industries and Orchards Citrus industries in Asia in 1960 were relatively small compared with today, and orchards were small and sparse. Large, irrigated monocultures that favour D. citri (and spread of HLB) were uncommon. In the 1990s, citrus in Southeast Asia was grown as backyard trees and in small groves < 1–2 ha. Citriculture in the Philippines consisted of many small groves and a few large corporate estates, while citrus in Vietnam was mainly grown in large state farms. In Thailand, farm sizes of > 10 ha were larger than in many other countries. Orchards in southern China ranged from small growers to large state farms (Catling, 1996). FAO statistics (FAOSTAT: http:// www.fao.org/faostat/en/), despite apparent inaccuracies, indicate that 54,000, 23,300, 16,000, 372, 6000, 20,000, 2900 and 8500 ha of oranges (presumably including mandarins) were harvested in India, Pakistan, mainland China, Taiwan, Vietnam, Indonesia, the Philippines and Thailand, respectively, in 1961. In 2015, there were 574,000, 135,590, 498,600, 5277, 45,410, 49,914, 1417 and 23,161 ha, respectively. There were 36,000, 14,000, 1600, 87, 100 and 11,500 ha of lemons and limes harvested in India, Pakistan, mainland China, Taiwan, the Philippines and Thailand, respectively. In 2015, there were 268,000, 7713, 106,000, 2432, 505 and 13,324 ha, respectively. Despite large increases in areas harvested

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in most of these countries, there were noticeable periods of decline due to HLB. Most orchards and blocks of trees within them are still small, with high border (edge) to area ratios that favour rapid ingress of D. citri and CLas (Gottwald et al., 2008; Bassanezi et al., 2013; Anco and Gottwald, 2015) and limit area-wide management options. These orchards are on land ranging from flat (some interspersed with canals) to gently undulating to steep contoured hills.

12.4  Current Distribution and Influence of Abiotic Factors The current distribution of D. citri in Asia spans seven Köppen-Geiger climate zones (Peel et  al., 2007) (Table 12.1) ranging from hot desert ­regions of Saudi Arabia, Yemen, Iraq and Iran in Western Asia, and Afghanistan, Pakistan and India in South Asia, through tropical and subtropical regions in India, Nepal, Bhutan and Bangladesh in South Asia, and Myanmar, ­Thailand, Indonesia, Malaysia, Laos, Cambodia, Vietnam and the Philippines in Southeast Asia, and south and east China and southern Japan in East Asia. Several authors have reported that the ­incidence of D. citri on citrus declines with increasing altitude above sea level. Aubert (1987) ­reported that it was common from sea level to 1500 m in southwestern Arabia but absent between 1700 m and 1800 m. Maximum summer temperatures ranged from 32–34°C at 1500 m, and minimum temperatures in winter were as low as 2.5°C. Occasional frosts occurred in ­orchards at 1700–1800 m asl (Aubert, 1987). Bové and Garnier (1984) recorded D. citri and CLas near Ta’if (~1450 m asl) in Saudi Arabia. In Pakistan, it occurs as far north as 35° at 1300 m near Charbagh in the Swat Valley of Kyber Pakhtunkhwa (North-West Frontier Province) ­ (Zeb et al., 2011). In Nepal, it has been recorded at 1350 m in the Kathmandu Valley (Lama et al., 1988; Regmi et  al., 2010) and at 1300 m at Bhakimli in the west (Manandhar et al., 2001). In Bhutan it has been recorded at elevations up to 1380 m (Om, 2017) but incidence above 1200 m appears to be sporadic and possibly related to the movement of infested seedling mandarin trees from nurseries at elevations < 1200 m.

In China, heaviest infestations of the psyllid and the greatest incidence of HLB occur in the southeast in Guangxi, Guangdong, Fujian and Taiwan. The boundary separating high from low incidence runs from 29°N in Zhejiang to 25°N in Yunnan in a sea-facing arc tracking slightly inland from the mountain ranges that separate inland Sichuan, Guizhou, Hunan and Jiangxi from coastal Guangxi, Guangdong, ­Fujian and Zhejiang (Yang et  al., 2006; Wang et  al., 2018). Elevations of localities between 26.00°N and 29.00°N where D. citri occurs in these provinces range from ~500 m to 1200 m (Chao et al., 1979; Yang et al., 2006). Wang et al. (2018) reported the northernmost boundaries of populations from west to east in China as 26.63°N in Yunnan, 28.40°N in Sichuan, 25.87°N in Guizhou, 26.32°N in Hunan, 27.50°N in Jiangxi and 29.48°N in Zhejiang, with the highest elevation occurring at 1284 m (23.5892°N, 103.3168°E) in Yunnan. In peninsular Malaysia the altitudinal limit for D. citri is about 1200 m where minimum temperatures of 14°C occur (Aubert, 1990b). Medium infestations were reported in villages and small farms at 1000 m in East Java in Indonesia by Aubert et  al. (1985). Beattie et  al. (2010) ­reported that the psyllid was common at 60 m, uncommon at 640 m, and never observed at 1300 m asl, in Central Java, Indonesia, over 2–3 years. Average temperatures and annual rainfall at the three elevations during the study were ~26.5°C and 2447 mm, 22.1°C and 3068 mm, and 17.4°C and 2908 mm, respectively, with peak monthly rainfall in December and January of approximately 470 mm at 60 m, 580 mm at 640 m, and 630 mm at 1300 m (Siti Subandiyah and ­Rachmad Gunadi, Universitas Gadjah Mada). Abiotic factors that affect survival of D. citri and influence where citrus can be grown with negligible risk of HLB (provided that farmers plant pathogen-free trees) include rainfall, ambient temperature, and saturation deficits (relative humidity) (Aubert, 1987, 1990b; Yang et  al., 2006). They may also include extreme UV. Such factors can reduce or preclude the need to use insecticides to suppress psyllid populations. Prolific flushing during seasonally warm dry months, generally in spring and autumn depending on the location, favours rapid increases in populations. Aubert (1988, 1990b) regarded D. citri as a surprisingly enduring insect capable



Management of the Asian Citrus Psyllid in Asia 183

Table 12.1.  Representative features of Köppen-Geiger climate zones where Diaphorina citri occurs in Asia. The zones are arranged in order of increasing annual precipitation. Records rounded to the nearest whole number. Temperatures (°C)

Köppen-Geiger Zone

Rainfall (mm)

Average Average Average monthly monthly annual Record minimum maximum peak maximum

Desert BWh: Khanpur, Punjab 16–19 Province, Pakistan; Ta’if in Saudi Arabia. Hot semi-arid BSh: Regions of the 16–19 Kyber Pakhtunkhwa (North-West Frontier Province) and Punjab provinces of Pakistan; Punjab State, and the Deccan Plateau states of Rajasthan, Gujarat, Madhya Pradesh, Telangana, Karnataka and Andhra Pradesh in India. Humid subtropical Cfa: Regions of 13.7–19 southern Zhejiang and Jiangxi, coastal Fujian, Guangdong and Guangxi in China Tropical wet monsoon with pronounced 21–25 dry season Aw: Mumbai in Maharashtra and Kolkata in West Bengal, India; Dhaka in Bangladesh; Yangon in lower Myanmar; Bangkok on the central plains and Chiang Mai in mountainous northern region of Thailand; Phnom Penh in southcentral Cambodia; Saigon in southern Vietnam; Kaohsiung and Tainan on the island of Taiwan; Manila on Luzon Island in the Philippines; Kupang and Dili on the island of Timor in Indonesia Tropical monsoon climate with driest 21–23 month at or soon after winter solstice Am: Jakarta and Yogyakarta, Java, in Indonesia; Chittagong in Bangladesh; Hue in central Vietnam Monsoon influenced humid subtropical 12–21 Cwa: In India, Dharamshala in Himachal Pradesh, Srinagar in Uttarakhand, Jalpaiguri in West Bengal and Bongaigaon in Assam; Kathmandu in Nepal, Gelephu, Phuentsholing and Damphu in Bhutan; Ruili, Longchuan and Xiaopingtian (Lujiangba) in Yunnan, China; Hanoi in northern Vietnam. Tropical rainforest climate Af: Kuching, 23–24 Sarawak, in Borneo, and Kuala Lumpur in Peninsular Malaysia

Annual

Monthly range

30–33

36–43

41–48

97–180

1–37

29–32

37–40

48–49

400–760

78–230

22–26

32–34

39–42

1350–1900 210–350

29–33

33–37

40–44

1300–2680 170–602

30–31

31–35

35–41

1855–2794

25–29

29–39

35–47

1142–3732 290–818

32–32

33

37–39

2628–4117 131–648

6–743

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of surviving a wide range of temperature extremes, from 45°C in Saudi Arabia to –7°C or –8°C in subtropical China. Environments in which daily maximum temperatures of 38°C and low saturation deficits (high relative humidity) prevail, such as in Guangdong in China at elevations below 300 m can be more limiting than where environments with daily maximum temperatures of 45–50°C and high saturation deficits (low relative humidity) prevail, such as in Pakistan at 160 to > 600 m asl. The insect thrives under the latter conditions when leaves of irrigated host plants are turgid. This suggests that survival of the psyllid is related to evaporative cooling of leaves and evaporative cooling of psyllid adults and nymphs, the former enhanced by the 35° angle of the adult body to the leaf surface when feeding, and the latter by dorsoventrally flat nymphs with relatively large wings pads. Under conditions where high temperatures and high saturation deficits prevail, evaporative cooling leads to leaf temperatures several degrees below those that would occur at lower ambient temperatures and higher relative humidity (Hoffmann et al., 1975; Gates, 2003; Beattie et al., 2012). Low saturation deficits at high ambient temperatures lead to leaf temperatures above ambient, reduced natural cooling of leaves, and increased mortality of psyllid nymphs and adults above the levels found at the same ambient temperatures at higher saturation deficits. Overhead irrigation and misting of water into canopies could have similar effects at ambient temperature above 35°C. Hoffmann et al. (1975) attributed higher survival of Acizzia russellae Webb & Moran (Hemiptera: Psyllidae) on Acacia karoo Hayne (Fabales: Fabaceae) at critically high temperatures (43°C and 46°C) and moderate saturation deficits (approximately 3.3–5.3 kPa) and lower survival at lower and higher saturation deficits to evaporative cooling by the insect or the plant, or both. Hoffmann et al. (1975) concluded that at low saturation deficits, the insects suffered heat-related death, as they could not lower their body temperatures below their upper thermal death point, and that at very high saturation deficits desiccation caused death. Low saturation deficits also favour entomopathogens. Heavy rainfall, particularly monsoon rains over several months in Köppen–Geiger Aw, Am, Cwa zones, and monthly falls in Af regions, washes eggs and young nymphs from immature flush

growth on host plants. Aubert (1987) mentioned monthly rainfall above 150 mm being associated with low populations due to eggs and young nymphs being washed off plant surfaces. Atwal et al. (1970) attributed a decline in psyllid populations in late summer in the hot semi-arid BSh Punjab region of India, to harmful effects (mechanical injury or fungal epizootics, or both) of rainy weather. Two severe typhoons in October 1970 in Albay Province on the island of Mindanao in the Philippines, where an Aw tropical wet monsoon climate with a pronounced dry season prevails, led to almost complete disappearance of psyllid populations for almost 5 months due to extensive damage to crops (Bigornia and Obana, 1974). Regmi and Lama (1988) reported declines in populations during the rainy season (July to October) in the Pokhara Valley (935 m asl) in Nepal, noting that heavy rain eliminated eggs and nymphs, whereas adults hid on the abaxial leaf surfaces and on twigs in inner-lower portions of canopies. They recommended that any measures for control of the psyllid should be undertaken mainly at the end of the rainy season. The region falls within the CWa humid subtropical monsoon zone with dry winters and wet summers. Lakra et  al. (1983) mentioned heavy mid-summer rainfall (> 300 mm) adversely affecting populations in the Punjab region of India. Chavan and Summanwar (1993) recorded the lowest population densities near Pune in Maharashtra, India, after tropical wet-monsoon rains from June to October. Average rainfall (World Weather Online, available at: https:// www.worldweatheronline.com.) in Pune during this interval is 165 mm, ranging from 126 mm to 207 mm per month. Leong et al. (2011) noted that a decline in populations of eggs and adults from October 1999 to January 2000 in their studies in Sarawak (tropical rainforest Af zone) was probably due to harmful effects of rain. They considered it possible that monthly rainfall > 150 mm and high relative humidity are major limiting factors to the abundance and distribution of the psyllid in Malaysia. However, populations of nymphs remained high during December 1999 and ­January–February 2000. This was ­attributed to possible lower susceptibility of older instar nymphs being dislodged from immature flush growth, and less susceptible to impacts of high relative humidity. Razi et al. (2014) reported low populations of the psyllid in BSh regions of Punjab



Management of the Asian Citrus Psyllid in Asia 185

Province of Pakistan during hot-summer months being associated with ~68, 96 and 149 mm of rain in June, July and August, respectively. Such impacts of torrential rainfall suggest that heavy overhead irrigation would dislodge eggs and nymphs. Recent studies in Bhutan (Om, 2017) suggest that UV radiation affects the survival of D. citri and, directly or indirectly, the presence of CLas, at elevations > 1200 m asl, through impacts on insect and/or plant tissues. Impacts on the psyllid, if they occur, could be related to morphology (Pérez-Valencia and Moya-Raygoza, 2015), physiology and/or primary and secondary endosymbionts. Molecular differences in the primary endosymbiont ‘Ca. C. ruddii’ in psyllids sampled at 200–450 m and 1040 m in Bhutan were reported by Wang et al. (2018). Impacts on plants could be related to anatomy and physiology, including phytochemistry. The psyllid and HLB are common below 1100 m, and few of possibly 2–2.5 million trees planted below 1200 m in 1990s were still alive in 2016, whereas most of some 0.5–1 million trees planted from 1200 m to 1700 m had not been killed by HLB. The abrupt change in both the incidence of the psyllid and the disease is not related to ambient temperatures, saturation d ­ eficits or rainfall. Other arthropods present in the orchards above, but infrequently below, 1200 m appear to have evolved to minimize ­impacts of UV. In contrast to eggs and nymphs of D. citri, which are exposed directly to light, nymphs of Cacopsylla heterogena Li (Psyllidae) develop within pouch galls formed immediately after eggs are laid on the adaxial surfaces of immature flush leaves some 10 mm long. Populations of C. heterogena occur at elevations up to 2440 m but decline abruptly below 1200 m, probably because increasing ambient temperatures and falling relative humidity increase the risks of egg and nymph mortality, despite protection afforded to these immature stages by the pouch galls in which they develop. An unidentified midge (­Diptera: Cecidomyiidae) that occurs in Bhutan at elevations ranging from 800 m to 1400 m d ­evelops within wart-like, tubular leaf galls formed by adaxial leaf surfaces rolled inwards along midribs. Unidentified spider mites (Acari: Tetranychidae) were also observed on abaxial leaf surfaces of mandarin trees at 650 m and leaflet surfaces of Zanthoxylum sp. at 1600 m. Some spider mites are known to occur on ­abaxial leaf surfaces in

order to avoid UV (­ Ben-Yakir and Fereres, 2016). Intensity of UV radiation increases with increasing elevation and declines with increasing latitudes north and south of the equator. At sea level it is greatest within ± 10° of the Equator. Levels are also influenced by environmental factors such as cloud cover. Bhutan, between 26.7 and 28.3°N, lies within a region of the earth’s surface where high UV radiation occurs (Liley and McKenzie, 2006). Intensity is higher than in Arabia (Liley and McKenzie, 2006) where D. citri occurs, as noted above, at Ta’if at ~1450 m asl (Bové and Garnier, 1984). Blumthaler et  al. (1997) noted that total irradiance and UVA radiation under clear-sky conditions in summer in Switzerland increased by 8% and 9%, respectively, per 1000 m between 580 and 3580 m asl. UV radiation (UVA and UVB) causes high mortality of immature stages of the chrysomelid, Cassida rubiginosa Müller (Bacher and Luder, 2005), and reduces oviposition by two-spotted mite (Tetranychus urticae Koch) (Acari: Tetranychidae) (Kuhlmann and Müller, 2011). Indirect effects on herbivores are associated with changes in plant morphology, physiology and photochemistry (Kuhlmann and Müller, 2011). Under enhanced UVB radiation, probing behaviour as well as the nymphal development period, reproductive and post-reproductive period, and difference in weight after moulting of the grain aphid (Sitobion avenae) (Hemiptera: Aphididae) are negatively affected (Hu et al., 2013a, b). Hu et al. (2013b) noted differences in response to UV radiation among the brown and green morphs of the grain aphid. Further, UV radiation causes changes in plant secondary metabolites, which are known to contribute to protection against pathogens and insects (Katerova et  al., 2012). Salt et  al. (1998) observed progressively lower populations of the psyllid, Strophingia ericae (Curtis) (Hemiptera: Liviidae), and reduced level of the amino acid, isoleucine, in heather plants (Calluna vulgaris (L.) Hull) (Ericales: Ericaceae) compared with the controls over a 27-month period.

12.5  Use of Insecticides and Spread of HLB Equipment used by farmers in Asia to apply sprays ranges from simple bucket pumps, through

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knapsacks (hand-pump, battery-powered and motorized) and stationary motorized units with long hoses, to drive-past and boat-mounted air-blast (with and without towers) and boom sprayers in larger orchards. The widespread practice of adding more than one product to a spray tank sometimes leads to physically and chemically incompatible mixtures of active i­ngredients and emulsifiers that may lead to precipitation of chemicals in the spray tanks, inactivation of active ingredients, reduced spray deposition and increased risk of phytotoxicity. Poor water quality and poor agitation of tank mixes also influence tank mix stabilities, spray deposition and efficacy. Dose–response relationships have not been derived for impacts of chemicals on D. citri versus volume of spray per hectare for any of these sprayers for any given concentration of active ingredient. Spray coverage from hand-held wands ranges from haphazard (sometimes sparse) to thorough. Even when effective coverage can be achieved with younger trees, farmers may be reluctant to use the volumes of water required to effectively cover trees. Coverage for all types of equipment declines with increasing tree heights, which can exceed 6 m. Insecticide residues on sprayed surfaces decline as chemicals break down and volatilize, and as plant tissues grow. Concentrations of systemics decline as they break down, volatilize from, or dissipate within, plants, or cease to be present at effective doses as plant tissues grow. Tree height also influences the efficacy of systemic pesticides ­ ­applied to soil and trunks. For example, 100% mortality of citrus leafminer (Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae)) in rough lemon seedlings 150 mm high following application of soil-applied imidacloprid (18.0 mg/plant), thiamethoxam (22.5 mg/plant) and acetamiprid (18.0 mg/plant) occurred 2.3, 1.8 and 2.7 days after application, respectively. As the height of seedlings increased to 660 mm, maximum larval mortality caused by the insecticides occurred after 47.6 and 58.6 days, and 19.9% after 6.6, 5.9 and 8.7 days, respectively (Sharma and Chadda, 2010). Initial recommendations for use of chemicals to suppress populations of D. citri in citrus orchards were made in India. Spraying with 0.78% crude oil in water was considered effective in the Punjab (Lal, 1917), as were tobacco

decoction sprays (Lal, 1918). Fish oil soap and fish oil–resin soap were recommended as contact sprays (Fletcher, 1917, 1919). Husain and Nath (1927) recommended sprays containing crude oil, tobacco decoction or resin compound. Pruthi and Mani (1945) made similar recommendations. Initial use of synthetic insecticides to control D. citri in China was linked to management of citrus leafminer (Chen, 1985) which, like the psyllid, was not reported in mainland China until the 1930s (Clausen, 1931; Tan and Huang, 1996). The 1950s heralded the use of synthetic organochlorines, organophosphates and carbamates (Ahmad, 1961). Pyrethroids dominated in the mid and late 1980s. In the 1990s, benzoylurea, pyrazolecarbamides and neonicotinoids gained favour. Currently used pesticides include avermectin and spinosyn insecticides. Insecticides currently registered for use in China include bifenthrin, betacyfluthrin, tolfenpyrad, abamectin/tolfenpyrad, pyriproxyfen, thiomethoxam, acetamiprid/chlorpyrifos, cypermethrin/ chlorpyrifos and beta-cypermethrin/chlorpyrifos (Tao Lei, South China Agricultural University, personal communication, 6 June 2017). Catling (1968) was alarmed by the intensive pest control programs used in many large citrus estates in the Mindanao region of the Philippines in 1968. Two large estates he visited applied combinations of insecticides including the organochlorines, endrin and lindane, and the organophosphates, parathion and EPN, every 14 days throughout the year. He subsequently reported many instances of heavy spraying and indiscriminate pesticide use in China, Thailand, Malaysia, Indonesia and the Philippines in a survey of 54 citrus farms in Asia in 1985 (Catling, 1996). Of more than 50 materials recorded, the most frequently used insecticides were organophosphates, including monocrotophos and parathion. Data from 12 farms in Thailand in 1985 revealed an average of 21.8 sprays annually, with a maximum of 35. ‘Good growers’ in Indonesia and the Philippines applied averages of 31.2 (17–44) and 15.6 (9–26) sprays annually, respectively, and lowland and highland growers in Malaysia applied averages of 2.8 (2–5) and 24.2 (20–28) sprays annually. In China, 5–12 sprays were applied annually (Catling, 1996). At one farm in Thailand, sprays were applied every 5 days in the early 1990s; the



Management of the Asian Citrus Psyllid in Asia 187

frequency dropped to every 10 days when no fruit were present. One grower applied ‘an incredible’ 73 sprays annually (Catling, 1996). Two or three materials were often mixed together as ‘cocktails’ in Thailand, Indonesia and the Philippines (Catling, 1996). In China and Southeast Asia, the use of more than 70 insecticides, miticides and fungicides with a plethora of names were recorded during a similar survey in the above countries and Vietnam and Laos in 1991. Seven leading state farms in northern Vietnam applied an average of 8.4 sprays a year. Averages in China of 9.8 in 1985 and 12 in 1991 were attributed, in part, to the then common practice of removing major midsummer flush by hand (Catling, 1996). Catling (1996) regarded the typical heavy spray schedules in Thailand and parts of the Philippines and Indonesia as costly, hazardous (particularly ­ azinphos methyl, methomyl, monocrotophos, ­ parathion and phorate), and frequently counter-­ productive. Extension services were considered poor with very few, if any, subject matter specialists trained in citriculture, let alone citrus IPM. Extension workers were invariably unqualified, underpaid, poorly trained generalists moulded along traditional lines, and often overburdened with other official duties and ‘the delivering of edicts’. These factors led to particularly negative consequences in Thailand, where growers were virtually at the mercy of unscrupulous pesticide salesmen (Catling, 1996). Quach et al. (2008) evaluated the impact of three rates of imidacloprid applied as a soil drench to caged potted sweet orange trees in a screenhouse on adult D. citri mortality. The aim was to determine the longevity of adults alighting on treated plants. The insecticide was applied as 0.2%, 0.4% and 0.6% w/v. Twenty adults were released into each cage 2, 4, 6, 8 and 10 weeks after application of the insecticide. Mortality was assessed 1, 2, 4 and 6 h after release of the adults, and then daily for 4 days. All adults released on to plants 2 weeks after application of 0.6% w/v imidacloprid died within 2 days. All adults released on to plants 6, 8 and 10 weeks after application of imidacloprid at this concentration died within 3 days. In each instance ‘CLas’-harboring adults would have been able to transmit the pathogen. Leong et al. (2002) assessed the efficacy of an nC24 horticultural mineral oil (HMO) for

control of D. citri in three commercial orchards in southern Sarawak, Malaysia. The HMO was applied at 0.33%–0.5% (v/v) (mostly 0.5%) at all locations, weekly in one orchard and fortnightly in the other orchards. The efficacy of these sprays was compared with two alternative control programs based on synthetic pesticides applied at the manufacturers’ recommended rates. One of these programs was based on imidacloprid alternated with chlorpyrifos or deltamethrin, the other on triazophos alternated with a product containing chlorpyrifos and cypermethrin. All control programs were similarly effective against the psyllid and several other pests. There is no instance in which use of chemicals to suppress populations of D. citri in Asia has prevented spread of HLB. In many instances, where control of the psyllid has been reported as significant, or very effective, the impacts on spread of the disease are most probably minimal given the size of populations when treated and generally short-term suppression of psyllid populations. Field studies that have determined impacts of suppression of psyllid populations on spread of the disease include those reported by Ke and Xu (1990), Gatineau et  al. (2010) and Leong et al. (2012). Ke and Xu (1990) suggested that effective management of HLB in the presence of D. citri required annual rates of spread (new positive detections) of the disease in orchards by psyllid to be < 0.2% (1 in 500 trees). They reported successful management programs based on planting of pathogen-free trees, removal of diseased trees and judicious application of pesticides in several orchards in Guangdong and Fujian. In one instance at the Yangcun Overseas Chinese Citrus farm in Guangdong (where numbers of trees rose from 558,453 in 1978 to 1,804,588 in 1989), annual incidence of HLB declined from 6.9% to 0.8% of trees. The spray programs in this instance involved one to two applications before the spring budbreak, and single applications to flush growth in spring, summer and autumn. Nevertheless, the 2000 ha ­orchard succumbed to the disease in the late 1990s when centrally directed area-wide management ended and responsibility for managing trees became the individual responsibility of some 3000 farmers or families (Zhuang and Cai, 2003; Cen Yijing, South China Agricultural University, personal communication, 8 June 2018).

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Gatineau et  al. (2010) compared the incidence of D. citri and HLB in three 0.5 ha King orange (Citrus × aurantium) orchards in the Mekong Delta in Vietnam for two consecutive years after planting initially pathogen-free trees. All trees in one of the orchards were not treated with insecticides. All trees in the other orchards were treated with either fenobucarb or imidacloprid. Fenobucarb was applied fortnightly in sprays at 250 g a.i./ha; imidacloprid was applied monthly. On each occasion, 75 ml of 200EC product was applied 300 mm above ground level to the trunks. High numbers of adults and nymphs were observed in the untreated orchard. Numbers in the two insecticide treatments were more than 90% lower than the untreated control. Very few adults and no 5th-instar nymphs were observed in the imidacloprid orchard. After 2 years, CLas was detected by PCR in approximately 96% of trees in the untreated orchard, and in 75% and 24% of trees in the fenobucarb and imidacloprid treated orchards, respectively. Modeling in Brazil has indicated that 100% of trees in an orchard are likely to be infected (CLas-positive by PCR) when 28%, or less, of trees in the orchard are symptomatic (Lee et  al., 2015; Craig et  al., 2018). Thus, trees in the untreated and fenobucarb-­ treated orchards were possibly infected within 6–12 months of planting, and 100% infection in the imidacloprid orchard would probably have occurred within 26 months. Leong et  al. (2012) compared the impacts of four treatments over 3 years (January 1999 to December 2001) on the incidence of HLB in a honey tangerine orchard: (i) unsprayed; (ii) 0.35% v/v aqueous nC24 92% unsulfonatable residues (UR) HMO emulsions; (iii) triazophos (0.75 ml 40EC product per litre) alternated with chlorpyrifos/cypermethrin (2.25 ml 500EC/ 50EC product per litre); and (iv) imidacloprid (0.5 ml 200EC product per litre). The HMO was applied weekly and the insecticides fortnightly. Levels of HLB, as determined by PCR, rose over the 3 years to reach 42.2, 9.4, 11.4 and 22.7% in the unsprayed, imidacloprid, nC24 HMO and triazophos/cypermethrin/chlorpyrifos treatments, respectively. Use of mineral oils and insecticides for suppressing the incidence of D. citri and HLB were also compared in a 1 ha Siem mandarin orchard at Purworejo (7.70°S, 109.93°E 60 m asl) in

Java, Indonesia (Poerwanto, 2010). Trees were planted in October 2005 but the ingress of D. citri into the orchard was slow and eggs, nymphs or adults were not observed until July 2007. There were six treatments: two untreated, one with initially pathogen-free trees (PFT), the other with trees of uncertain pathogen status (non-PFT); two oil treatments, one with an nC21 92% UR HMO, the other with a broader boiling point range agricultural mineral oil (AMO) nC24 99.8% UR; and two insecticide treatments, one based on farmer practice, the other on imidacloprid. All trees in these four treatments were initially pathogen-free. The farmer’s practice entailed monthly applications of 0.025% lambda cyhalothrin and 0.05% profenofos. Applications of imidacloprid (200SL) commenced 2 weeks after planting. In the first year, 50 ml of 0.6% solution of the product was applied every 3 months to soil around the drip line under each tree. It was then applied monthly and undiluted to the trunks of each tree at the rate of 1 ml per tree (0.2 ml imidacloprid per tree). Average numbers of adult psyllids observed per tree from July 2007 to April 2009 were 0.9, 1, 3, 1, 11.6 and 4.8% in the nC21 HMO, nC24 AMO, farmer practice, imidacloprid, control PFT and control non-PFT treatments, respectively. The percentage of CLas PCR-positive trees in each of the treatments was 6.3, 6.3, 0, 12.5, 25 and 6.3%, respectively. No oil-induced phytotoxicity was observed, but some transient and inconsequential oil soaking was observed. Trees receiving the imidacloprid treatment did not appear to be any better than trees in other treatments. A similar study at Cai Be (10.3521°N, 105.8993°E, 4 m asl) in Tien Giang Province southwest of Ho Chi Minh City in the Mekong Delta of southern Vietnam commenced in a 0.65 ha King orange orchard in June 2004 (Beattie et al., 2010). Flushing was almost continuous with three to four major cycles per year. The synthetic pesticide treatments ceased in March 2006 due to impacts of guava (Psidium guajava L. (Myrtales: Myrtaceae)) interplants on ingress of D. citri and HLB, and rapid growth of trees that hampered sampling and application of sprays. Two adult psyllids were observed in June 2004 in one treatment, none thereafter. Psyllids were present in surrounding groves in the absence of guava interplants and, in 2006, levels of PCR-positive HLB infections in orchards to the



Management of the Asian Citrus Psyllid in Asia 189

north, east, south and west of the site were 37.5, 72.7, 34.4 and 57.1%, respectively. From March 2006 until July 2009, mineral oils (0.5% v/v) were applied to all treatments about 12 times a year. No phytotoxicity was observed. In 2008, 5.2% of trees were dead and 23.3% of live trees were PCR-positive. In 2009, 20.6% of trees were dead and 28.6% of live trees were infected: 34.3% of the dead trees were PCR-positive for HLB in 2008. Most deaths were attributed to phytophthora root and collar rots caused by Phytophthora spp. (Peronosporales: Peronosporaceae), gummosis caused by Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (syn. Diplodia natalensis Pole-Evans) (Botryosphaeriales: Botrysphaeriaceae), and/or pink disease caused by Erythricium salmonicolor (Berk. & Broome) Burds. (syn. Corticium salmonicolor Berk. & Broome) (Polyporales: Phanerochaetaceae). Observations indicated that very few deaths, perhaps none, were caused by HLB alone. More than 25 t of fruit valued at AU$2/kg were harvested from the orchard in 2009 despite the serious impacts of the diseases.

12.6  Resistance to Insecticides Resistance to currently used pesticides has been reported in Pakistan and China. Naeem et  al. (2016) reported that psyllids sampled from orchards in Punjab Province were highly resistant to imidacloprid as compared with the susceptible population. The resistance ratios were in range of 237–760, 56–213, 13.1–46, 31–217 and 9–89-fold for imidacloprid, acetamiprid, chlorfenapyr, nitenpyram and thiamethoxam, respectively, and 40–107 and 33–125-fold in the case of conventional insecticides, i.e. bifenthrin and chlorpyrifos, respectively. Nitenpyram and thiamethoxam, with no or very low resistance, were recommended for use in combination or in rotation with other pest management tactics for managing resistance in D. citri (Naeem et al., 2016). In China, Tao et al. (2017) reported that psyllids from Jiangxi Province have become less susceptible to beta-cypermethrin, imidacloprid, chlorpyrifos, acetamiprid and thiamethoxam. The decline in sensitivity to the insecticides was greatest for beta-cypermethrin, and then imidacloprid.

12.7  Health and Environmental Impacts of Pesticide Use Long-term and short-term health effects in farmers exposed to pesticides have been recorded in China (Snyder and Ni, 2017). Parveen et  al. (2004) reported that the pyrethroids bifenthrin, cypermethrin, fenpropathrin and fenvalerate, the organophosphates chlorpyrifos and profenofos, the organochlorines endosulfan and dicofol, the carbamates carbosulfan and carbofuran, and the fungicides benomyl and thiabendazole were frequently found in citrus fruits. Residues exceeded maximum residue levels (MRLs) in 15–36 of samples. Days after harvest were not cited. Pesticides banned for use in agriculture in China include the organochlorines, DDT, HCH and endosulfan, the organophosphates, chlorpyrifos, diazinon, dimethoate, methidathion, omethoate, phosmet and phosphamidon, and the pyrethroid, bifenthrin (Snyder and Ni, 2017). These authors noted that many banned pesticides in China are available in the countryside, especially in small and remote stores. Once farmers show an interest in banned pesticides such as methamidophos and monocrotophos, the stores will contact farmers and send pesticides to their home. The stores may also work with illegal manufacturers and sometimes sell ‘bundled’ pesticides. Farmers buy one kind of pesticide which is allowed, and the store will send another free of charge. This practice is strictly illegal, but farmers may not be aware that a pesticide is banned (Snyder and Ni, 2017). Faheem et  al. (2015) noted a huge gap between environmental legislation and implementation. Although organochlorines (e.g. DDT, BHC and endosulfan) and organophosphates (e.g. methyl parathion, methamidophos and monocrotophos) were banned in Pakistan when their research was undertaken, some, including organochlorines, were still available. D. citri is now a self-created problem in Pakistan where indiscriminate use of synthetic chemicals for the psyllid and other citrus pests has resulted in residual toxicity endangering human and animal health as well as causing environmental degradation (Mahmood et  al., 2014). They reported that estimates of as much as 17% of pesticides used in Pakistan are applied to fruits and vegetables. Studies conducted there

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during 1980s and 1990s showed that 422 out of 1059 samples of fruits and vegetables were found to be contaminated with pesticide residues and 71 of these had residue levels exceeding the limits set by the FAO/WHO Codex A ­ limentarius Commission. Most of this usage was related to management of D. citri. Most farmers in Asia are illiterate or have limited education. They may not be able to read labels or read with limited comprehension (Chalermphol and Shivakoti, 2009; Faheem et al., 2015; Snyder and Ni, 2017). Thai farmers do not follow recommended precautions when using pesticides, even if they are aware of the risks (Chalermphol and Shivakoti, 2009). Moreover, they regularly mixed two or more pesticides in spray tanks and commonly apply pesticides at higher than recommended label rates (Chalermphol and Shivakoti, 2009). Like other farmers in Asia, they depend on advice from pesticide companies, neighbors and information heard on radios or viewed on television (Chalermphol and Shivakoti, 2009; Faheem et  al., 2015; Snyder and Ni, 2017). Chalermphol and Shivakoti (2009) noted that learning from academic documents and programs on television did not interest most Thai farmers. Farmer field schools are regarded as more effective than top-down learning programs (Nicetic et  al., 2010), but such schools are limited by the resources, sheer numbers of farmers and the excessive promotion of pesticides.

12.8  Natural Enemies Natural enemies of D. citri in Asia include two solitary primary parasitoids, numerous predators (see Table 12.2), O. smaragdina, and six entomopathogens: Beauveria bassiana (Bals.) Vuill. (Hypocreales: Cordycipitaceae) (Xie et al., 1988; Ye et al., 1994; Yang et al., 2006), an unidentified Beauveria sp. (Gavarra and Mercado, 1988), Hirsutella citriformis Speare (Hypocreales: Ophiocordycipitaceae) (Subandiyah et al., 2000), Isaria fumosorosea Wize (Hypocreales: Cordycipitaceae) (Subandiyah et  al., 2000), Isaria javanica (Friederichs & Bally) Samson & Hywel-Jones (Xie et  al., 1988; Ye et  al., 1994  ; Yang et  al., 2006) and Lecanicillium lecanii (Zimm.) Zare & W. Gams (Hypocreales: Cordycipitaceae) (Xie

et al., 1988; Ye et al., 1994; Yang et al., 2006). Aubert (1987) associated fungal epizootics in artificially reared colonies of D. citri with the sooty mold fungus, Capnodium citri Berk. & Desm. (Capnodiales: Capnodiaceae), in Réunion (21.1144°S, 55.5334°E) in the Indian Ocean Mascarene Islands, noting that the effects were density dependent and sometimes severe. Such impacts have not been reported in Asia, and the epizootics may have been caused by other factors, as C. citri is a saprophyte (Reynolds, 1999; Chomnunti et al., 2014) and there are no other reports of the fungus or other sooty molds being entomopathogens. The two primary parasitoids, Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) and Diaphorencyrtus aligarhensis (Shafee, Alam & Agarwal) (Hymenoptera: Encyrtidae), are both solitary, arrhenotokous, koinobionts of D.  citri nymphs. T. radiata is an ectoparasitoid and D. aligarhensis is an endoparasitoid. The former was described from specimens reared from D. citri collected in Lyallpur, now Faisalabad (31.4195°N, 73.0800°E, 189 m asl) in the Punjab region that is now part of Pakistan, then India, in January 1921 (Waterston, 1922). The latter was described from specimens collected in Aligarh (27.8992°N, 78.0797°E, 191 m asl), Uttar Pradesh in 1968, and near Jaipur (26.9141°N, 75.7873°E, 433 m asl), Rajasthan, in India (Shafee et al., 1975; Hayat, 1999). These records, and those for D. citri, suggest that both parasitoids are native to South Asia. T. radiata is host-specific (Aubert and Quilici, 1984). Its life history was first studied in the Punjab by Husain and Nath (1924). It was introduced into Taiwan from Réunion between 1983 and 1986 and confirmed as present in 1987 (Chien and Chu, 1996). Identification of parasitoids collected in Guangxi in 1976 and in Fujian between 1982 and 1985 indicated that it was present in both of these mainland provinces before it was introduced into China from Réunion for mass-rearing and release (Tang, 1988; Xia, 1988). Tang (1988) considered it possibly indigenous to mainland China and present in Taiwan before it was introduced to the island from Réunion. However, no parasitoids were associated with D. citri populations when the psyllid was first recorded in Guangzhou (Hoffmann, 1936). A report by Catling (1968) suggested that it was not present in the Philippines before it



Management of the Asian Citrus Psyllid in Asia 191

was introduced from Réunion in 1988 by Gavarra and Mercado (1988). The parasitoid was first recorded in Indonesia in East Java in 1984 (Nurhadi and Whittle, 1989). At this point, no parasitoids had been recorded in West Java (Nurhadi and Whittle, 1989). Om et  al. (2017) reported another species of Tamarixia, Tamarixia drukyulensis Yefremova & Yegorenkova, parasitizing Diaphorina communis Mathur on Bergera koenigii L. (Rutaceae: Aurantioideae: Clauseneae) in Bhutan, where both D. citri and D. communis are present in citrus orchards (Om, 2017). Tang and Aubert (1990) mentioned Diaphorina aegyptiaca Puton (syn. Diaphorina cardiae (Crawford)), Diaphorina auberti Hollis, and Psylla sp. as hosts of D. aligarhensis. These records relate to Shafee et  al. (1975) for Psylla sp., Hayat (1981) and Prinsloo (1985) for D. aegyptiaca, Hayat (1981) for Diaphorina sp., and Aubert (1987) for D. auberti. The parasitoid was recorded in 1972 in Taiwan as Psyllaephagus diaphorinae Lin & Tao and considered endemic (Lin and Tao, 1979). Kohno et al. (2002) reported an unidentified encyrtid associated with D. citri on Ishigaki Island, in southern Japan. They thought the parasitoid may have been Diaphorencyrtus diaphorinae (= D. aligarhensis). Catling (1968) reported a parasitoid, later identified as D. aligarhensis, as widespread in the Philippines in 1968 and the most abundant parasitoid reared from D. citri nymphs collected in the provinces of Batangas, Laguna, Sorsogon and Albay on the northern ­island of Luzon (12.6–18.7 °N), and Davao and Cotabato provinces on the southern island of Mindanao (5.6–9.9 °N). The parasitoid was described as Aphidencyrtus diaphorinae by Myartseva and ­Trjapitzin (1978) based on specimens ­collected from a psyllid-infested orange tree in Hanoi, Vietnam, in April 1972. It was present in East Java in Indonesia, where it was initially referred to as Psyllaephagus sp. (Kalshoven, 1981) and Psyllaephagus pulvinatus Waterston (Nurhadi and Whittle, 1989), before 1981. Prior to 1989, no primary parasitoids of D. citri had been recorded in Malaysia, but both T. radiata and D. aligarhensis were recorded during surveys undertaken in 1989 and 1990 (Osman and Quilici, 1991). Collectively, these records suggest that both parasitoids were widely dispersed in East and Southeast Asia by the 1980s as a consequence of the movement of parasitized nymphs on psyllid-­infested plants.

Rates of parasitism of D. citri by T. radiata were first recorded in 1921 and 1922 for psyllid nymphs collected in citrus gardens at Faisalabad, Sargodha (32.0754°N, 72.6849°E, 188 m asl) and Gujranwala (32.1618°N, 74.1929°E, 228 m asl) in the Punjab (Husain and Nath, 1924). Parasitism ranged from zero to 95% on 25 occasions (generally monthly) and averaged 61.5%. The authors did not mention primary endoparasitoids. Ninety years later, Khan et al. (2014) reported average parasitism of 26% on ACP-infested Kinnow mandarin (Citrus reticulata Blanco) and sweet orange (Citrus × aurantium L.) trees. Husain and Nath (1927) reported that parasitism of the psyllid by T. radiata in the Punjab was often high and that as many as 95% of nymphs may be attacked. Nurhadi and Whittle (1989) noted that levels of parasitism by T. radiata and D. aligarhensis in Java varied, ranging, when present, from 62–70% and 7–67%, respectively, and higher in unsprayed backyards than in orchards where pesticides were used regularly or irregularly. Liu (1991) reported average parasitism by T. radiata (cited as ‘Tetrastichus sp.’) of 36% on summer flush and 46% on autumn flush in a mature orange orchard in Guangzhou. Lama and Amatya (1991) reported 90% parasitism of D. citri by T. radiata and D. aligarhensis in the Phuentsholing district of Bhutan. In Nepal, Regmi (1992) reported that T. radiata reduced D. citri populations by 90% under screenhouse conditions and by up to 50% in the field. Huang et al. (1999) recorded 30–50% parasitism of nymphs by T. radiata and D. aligarhensis (cited as Psyllaephagus sp.) in citrus orchards near Fuzhou. Levels of parasitism by the encyrtid reported recorded by Kohno et  al. (2002) on Ishigaki Island was on average one-eighth that recorded for T. radiata. In Sarawak, East Malaysia, parasitism by T. radiata and D. aligarhensis recorded by Leong et al. (2011) ranged from 4.4 to 22.3%, and 2.6 to 35.3%, respectively, with three seasonal peaks annually. Dawane et  al. (2016) reported seasonal parasitism by T. radiata on Ambia Bahar sweet orange in Maharashtra ranging from zero to 15.4%. Host feeding by both T. radiata and D. aligarhensis also leads to death of psyllid nymphs. Ratios of parasitized nymphs to host-fed nymphs recorded by Tang and Huang (1991) were 2:1 for T. radiata and 2.6:1 for D. aligarhensis. Host

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feeding led to death, and nymphs that were fed on were not parasitized (Tang and Huang, 1991). In laboratory studies at 25°C, Chien (1995) showed that D. aligarhensis and T. radiata can kill, by host-feeding and parasitism, 177 and 245 nymphs, respectively, when provided with 20 hosts per day, and 288 and 513 nymphs, respectively, when provided with 40 hosts per day. The effectiveness of both primary parasitoids is affected by more than 15 hyperparasitoids, the identities of which vary between regions (Tang, 1990; Tang and Aubert, 1990; Gavarra et al., 1992; Waterhouse, 1998; ­Halbert and Manjunath, 2004; Yang et al., 2006). Some attack both primary parasitoids and others only one (Waterhouse, 1998; Yang et  al., 2006). Most attack Diaphorencyrtus aligarhensis (Tang, 1990; Chien et  al., 1991; Waterhouse, 1998). Levels of hyperparasitism can be high, thus impeding the effectiveness of the two primary parasitoids. Predators recorded in Asia in association with D. citri are listed in Table 12.2. Scant information is known about the impact of these natural enemies in orchards and on the psyllid on host plants in natural environments. Oecophylla smaragdina Fabricius (Hymenoptera: Formicidae), commonly known as the weaver ant, golden weaver ant, green tree ant and yellow tree ant, was the first insect used by humans to control, through predation, large insects, ­caterpillars and stinkbug pests, on citrus some 1700 years ago near Hanoi, which was then part of the Chinese Han Dynasty province of ­Jiāozhı̌ (‘Chiāo-chı̌h’: 交趾, 交阯) (Needham, 1986). It has also been used to suppress insect pests of litchi (Litchi sinensis Sonnerat (Sapindales: Sapindaceae)), including Tessaratoma ­papillosa (Drury) (Hemiptera: Heteroptera: ­ Tessaratomidae) in southern China (Needham, 1986) and, recently, red-band thrips (Selenothrips rubrocinctus (Thysanoptera: Thripidae)) on mango in the Northern Territory, Australia (Peng and Christian, 2004), and thrips and citrus blossom midge (Contarinia citri Barnes (Diptera: Cecidomyiidae)) damage in citrus orchards in Yunnan, China (Zhang et al., 2010). It is native to tropical and subtropical regions of Asia and Australasia. It belongs to an Old World genus that has persisted through most of the Tertiary with little speciation (Wilson and Taylor, 1964) and now comprises two recognized species, O. smaragdina and, in trop-

ical Africa, Oecophylla longinoda Latreille. However, recent molecular studies suggest geographic diversity among populations sampled from regions in Asia and Australasia (Azuma et al., 2002, 2006). For centuries O. smaragdina has been reared in orchards in China and Indochina. Nests have been sold by farmers, and bamboo bridges have used been to facilitate movement of workers between trees (McCook, 1882; Groff and Howard, 1924; Chevalier, 1934; Needham, 1986; Huang and Yang, 1987; Van Mele, 2008). In Java, larvae and pupae, called ‘kroto’, are collected in the wild from trees and commercialized as food for songbirds and as fishing bait. This brings substantial income to numerous rural households (Césard, 2004). In addition to ‘kroto’, the ant is used in Asia for Chinese and Indian traditional medicines, and as a prized human delicacy (Sribandit et al., 2008). A growing interest in the eating of ants has led to higher demand throughout Thailand. Collection of ants is profitable, and the harvest pressure on local O. smaragdina populations may potentially lead to unsustainable overexploitation in natural habitats (Sribandit et al., 2008). Ants collected in northern Australia are sold for up to AU$650/kg and used to make boutique gin (Adelaide Hill Distillery, Nairne, South Australia) and cheese (Woodside Cheese Wrights, Woodside, South Australia) in South Australia (Staight, 2017). Groff and Howard (1924) questioned the value of O. smaragdina in the management of citrus pests, noting that its usefulness appeared be related to destruction of caterpillars and other large insects, as it did not destroy scale insects and ‘not always plant lice’, presumably aphids, that ‘are also serious enemies of citrus trees’. Nests were usually full of soft-scale insects that adult O. smaragdina tended for honeydew. However, Groff and Howard (1924) noted that the adult ants never seemed to tend aphids, and that when aphids were placed near a nest the ants initially were aggressive towards the moving bodies of the aphids. The ants then seized the aphids with their jaws and dropped them off trees. These observations, on the campus of Lingnan University (now Zhongshan/Sun Yat-sen University), appear to have been made shortly before D. citri was first recorded in Guangzhou in 1934 (Hoffmann, 1936) and, therefore, do not relate to the psyllid.

Order

Family

Species

Coleoptera

Coccinellidae

Brumoides suturalis (Fabricius) Cheilomenes lunata (Fabricius) Cheilomenes (= Menochilus) sexmaculata (Fabricius)

Cited as

Cydonia lunata (Fabricius) Cheilomenes quadriplagiata Swartz



Table 12.2.  Predators recorded in association with Diaphorina citri in Asia. Country

References

India

Husain and Nath (1927); Pruthi and Mani (1945) Irshad (2001) Al-Ghambi (2000)

Pakistan Saudi Arabia China

Nepal China

Regmi (1992) Wei et al. (1995); Yang et al. (2006)

India

Ramya and Thangjam (2016)

India Iran Nepal Pakistan India

Chilocorus nigritus (Fabricius)

Nepal India

Coccinella septempunctata (L.)

Coccinella transversalis Fabricius

Coccinella repanda Thunberg

Coelophora biplagiata (Mulsant) Coelophora circumusta (Mulsant) Cryptolaemus montrouzieri (Mulsant) Parexochomus nigripennis Erichson

Lemnia (Artemis) circumustus (Mulsant)

Exochomus nigripennis (Erichson)

Iran Nepal Pakistan India

Rakhshani amd Saeedifar (2013)

Continued

Management of the Asian Citrus Psyllid in Asia 193

China

Chien amd Chu (1996); Yang et al. (2006) Husain and Nath (1927); Pruthi and Mani (1945); Sharga (1948) Rakhshani and Saeedifar (2012, 2013) Regmi (1992) Irshad (2001) Husain and Nath (1927); Pruthi and Mani (1945) Regmi (1992) Husain and Nath (1927); Pruthi and Mani (1945) Rakhshani and Saeedifar (2013) Regmi (1992) Irshad (2001) Husain and Nath (1927); Pruthi and Mani (1945); Ramya and Thangjam (2016) Yang et al. (2006)

Order

Family

194

Table 12.2.  Continued. Species

Cited as

Harmonia axyridis (Pallas) Harmonia dimidiata (Fabricius)

Neuroptera

Staphylinidae Chrysopidae

Harmonia conglabata (L.) Saprinus chalcites

Chrysopa ­septempunctata Wesmael.

Chrysoperla carnea (Stephens) Chrysopa vulgaris Schneider

Thysanoptera

Phlaeothripidae

Diptera

Syrphidae

Mallada (Chrysopa) boninensis Okamoto Pseudomallada eurydera (Navás) Aleurodothrips fasciapennis (Franklin) Allobaccha sapphirina (Weideman) Allographa asp.

Apertochrysa crassinervis (Esben Pettersen

References

China India Nepal China

Yang et al. (2006) Ramya and Thangjam (2016) Regmi (1992) Yang et al. (2006)

Nepal China China

Regmi (1992) Yang et al. (2006) Yang et al. (2006)

Iran China India

Rakhshani and Saeedifar (2013) Chien and Chu (1996) Shivankar et al. (2000)

India Saudi Arabia Saudi Arabia

Ramya and Thangjam (2016) Al-Ghambi (2000) Al-Ghambi (2000)

Saudi Arabia China

Al-Ghambi (2000) Yang et al. (2006)

Iran

Rakhshani and Saeedifar (2013)

Saudi Arabia

Al-Ghambi (2000)

China India

Chien and Chu (1996); Yang et al. (2006) Shivankar et al. (2000)

China

Chen et al. (2002)

Iran

Rakhshani and Saeedifar (2013)

Nepal

Regmi (1992)

G.A.C. Beattie

Carabidae Histeridae

Harmonia octomaculata (Fabricius) Jauravia quadrinotata Kapur Propylea japonica (Thunberg) Phrynocaria congener (Billberg) Scymnus levaillanti Mulsant Serangium sp. Serangium parcesetosum Sicard Oenopia conglobata (L.) Egapola crenulata Dejean Saprinus (Saprinus) chalcites (Illiger) Paederus alfierii Koch Chrysopa pallens (Rambur)

Country



Hemiptera

Geocoridae

Mantodea Acari

Mantidae Anystidae Phytoseiidae

Araneae

Clubionidae Gnaphosidae Salticidae

China Saudi Arabia Saudi Arabia China China China

Chien and Chu (1996) Al-Ghambi (2000) Al-Ghambi (2000) Wu (1994) Fang et al. (2013) Fang et al. (2013)

Iran Iran India

Rakhshani and Saeedifar (2013) Rakhshani and Saeedifar (2013) Sadana (1991); Sadana and Kaur (1980) Al-Ghambi (2000)

Saudi Arabia

Management of the Asian Citrus Psyllid in Asia 195

Spider complex

Geocoris sp. Coranus aegyptius (Fabricius) Sphodromantis viridis Forsskäl Anystis baccarum (Linnaeus) Neoseilus barkeri (Hughes) Neoseiulus cucumeris (Oudemans) Cheiracanthium sp. Zelotes sp. Marpissa tigrina Tikader

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Before 1960, in the apparent absence of HLB, citrus farmers at Wangtong near Guangzhou in the Pearl River Delta in Guangdong, China, applied one to two sprays annually to control citrus flea beetle, Clitea metallica Chen (Coleoptera: Chrysomelidae) (Chen, 1985). They relied on O. smaragdina to control other pests. Under such circumstances, D. citri was not regarded as a serious pest. Increased use of synthetic pesticides after 1960 led to sprays being required annually for control of D. citri, citrus red mite (Panonychus citri (McGregor) (Acari: Tetranychidae)), citrus rust mite (Phyllocoptruta oleivora (Ashmead) (Acari: Eriophyidae)), aphids, and citrus stink bug (Rhynchocoris humeralis Thunb. (Hemiptera: Pentatomomorpha: Pentatomidae)). In many orchards, scale insects, whiteflies and leafrollers also became serious pests. Chen et  al. (1980) attributed ‘rampancy’ of citrus red mite after the 1960s to the increased use of synthetic pesticides. It is now a major pest of citrus in China. In a field experiment conducted in the region during 1979– 1981 (Chen, 1985), less than 1% of eggs that hatched on immature flush growth on trees in an orchard with low pesticide use completed nymphal development. The incidence of pests other than the psyllid was low and HLB was not detected. In another orchard, where chemical insecticides were used 12–18 times annually, survival of nymphs over 18 generations was 2.2–17.5%. The incidence of pests other than the psyllid was high and the disease was present. Whittle (1992) noted, during pest and disease surveys in 1990 and 1991, that backyard citrus trees in the Mekong Delta in southern Vietnam were practically free of foliar pests, largely because of O. smaragdina. Nguyen (Cuc) (1995) reported that O. smaragdina can reduce D. citri populations, and the incidence of HLB, to low levels in the Delta. The ant preys on eggs of the psyllid (Nguyen (Cuc), 1995). According to Van Mele and Cuc (Nguyen) (2000), pesticide use can be halved when the ant is present in citrus orchards. Despite these reports and that of Chen (1985), it is not fully evident how O. smaragdina suppresses populations of D. citri. Vu et  al. (2012) observed it removing eggs and nymphs of Trioza hopeae Burckhardt & Vu (Triozidae), and either no galls, or very few, were formed on the new leaves of the host plant, Hopea odorata Roxb. (Malvales: Dipterocarpaceae) in southern

Vietnam. They observed the ant feeding on T. hopeae. However, there are no reports of it removing or preying on D. citri nymphs and adults. O. smaragdina workers move rapidly, even in the absence of D. citri eggs and nymphs, over surfaces within citrus canopies, including immature flush growth suitable for oviposition by D. citri females and subsequent nymphal development (personal observation). As alluded to by Nguyen (Cuc) (1995), this suggests that abdominal Dufour’s gland secretions (Bradshaw et al., 1979; Keegans et  al., 1991) deposited as ant trails by O. smaragdina workers on plant surfaces may repel D. citri adults. The secretions, which are produced by both O. longinoda and O. smaragdina, persist on plant surfaces for extended periods. O. longinoda deposits can be detected by workers 11 months after they have been deposited, in spite of rain (Beugnon and Dejean, 1992). Offenberg (2007) proposed that the persistence of deposits and coverage of entire ant territories may present reliable cues of ant presence and predation risk and therefore warn potential prey. Van Mele et  al. (2009) reported that female Bactrocera dorsalis (Hendel) (Bactrocera invadens Drew, Tsuruta & White) are reluctant to land on fruits exposed to abdominal Dufour gland secretions of O. longinoda, and that after landing female flies often take off quickly and fail to oviposit. Secretions of O. smaragdina are known to also repel pollinators (Tsuji et al., 2004; Rodríguez-Gironés et al., 2013). The natural distribution of the ant (Wetterer, 2017) is similar to the distribution of D. citri in Asia and Australasia, with the exception of northern Australia, where the psyllid is not present, and where the psyllid occurs in coastal regions in East China north of Shajian (24.7521°N), Hu’aan, to the west of Xiamen in Fujian (Yang et al., 1983), or in Hunan, Jiangxi, Taiwan and the southern islands of Japan in the absence of the ant. Wetterer (2017) mapped > 2500 sites where the ant occurs in 21 countries: Australia (northern parts of Western A ­ ustralia, the Northern Territory and Queensland), ­Bangladesh, Bhutan, Brunei, Burma (Myanmar), Cambodia, China, India, Indonesia, Laos, Malaysia, Nepal, Palau, Papua New Guinea, ­ Philippines, Singapore, Solomon Islands, Sri Lanka, Thailand, Timor Leste and Vietnam. The vast majority of records were from areas with tropical Köppen-Geiger rainforest (Af), monsoon



Management of the Asian Citrus Psyllid in Asia 197

(Am), and savanna (Aw) climates. However, > 250 records were from other areas, most with a dry winter subtropical climate (Cwa), in the Himalayan foothills of India and Nepal, southern China, northern Vietnam and the southern coast of Queensland, Australia. A few records were from sites within warm semi-arid (BSh) areas (see Table 12.1). O. smaragdina does bite humans and will attack if teased or disturbed (Groff and Howard, 1924). Risk of being bitten in orchards can be readily addressed by enticing ants out of orchards when access to trees is required and by application of wood ash to exposed limbs (Van Mele and Nguyen (Cuc), 2007). Its use in biological control is compatible with other natural enemies and of minor concern with respect to attendance of honeydew-producing soft-scales and mealybugs (Huang and Yang, 1987). Use with mineral oils is also compatible (Huynh et al., 2002; Peng and Christian, 2004). Most recent research on the use of Oecophylla species in biological control of pests of tree crops has been in Africa and in southern Vietnam (Van Mele, 2008). There is a clear need for O. smaragdina to be used in conjunction with other natural enemies to suppress populations of D. citri and other citrus pests, and levels of HLB. It could be used in environments where the ant occurs naturally, and where it can be cultivated, nurtured, and managed within orchards. Van Itterbeeck (2014) discussed prospects for semi-cultivating the ant. Research is required to empirically demonstrate that O. smaragdina secretions repel D. citri and, if so, which of the known constituent molecules in the secretions affect psyllid behavior, alone or in specific proportions, and whether the compounds can be incorporated in sprays applied in orchards to suppress psyllid populations. Repellent effects of mineral oils on D. citri (Rae et al., 1997; Poerwanto et al., 2012; Yang et  al., 2013) may be related to mimicry of the O. smaragdina secretions, as has been postulated for responses of Bactrocera tryoni (Froggatt) to deposits of horticultural and agricultural mineral oils (Nguyen et al., 2017). The cost-effectiveness of mass-rearing O. smaragdina for biological control of insect pests in orchards, including D.  citri on citrus trees, and for medicinal uses, food (animals and humans) and beverages should be determined. Wetterer (2017), citing

molecular studies by Azuma et al. (2002, 2006), considered it possible that cold-tolerant Oecophylla from subtropical climates in northern Asia may represent a separate species. Studies are required to determine genetic variation between populations of O. smaragdina and, if populations do differ, how differences may be related to cold tolerance, for example southern Hunan, Jiangxi and coastal regions in Fujian and Zhejiang.

12.9  Plant Volatiles and Repellency Cen et al. (2005) found that volatiles of Mikania micrantha Kunth., Praxelis clematidea (Griseb.) R.M. King & H.Rob., Wedelia chinensis (Osbeck) Merr. (Asterales: Asteraceae) and Lantana camara L. (Lamiales: Verbenaceae) repelled adult psyllids placed in a four-armed olfactometer. Three of these species, L. camara, M. micrantha and P. clematidea, are invasive weeds and are, therefore, not suitable for interplanting in orchards. Regmi and Lama (1988) reported potential impacts of interplanted Tropaeoleum majus L. ­ (Brassicales: Tropaeolaceae) (cited as ‘Nasturtium tropaelum’) on D. citri in Nepal. Yang et al. (2013) showed in laboratory experiments that leaf volatiles of billygoat weed (Ageratum conyzoides L. (­Asterales: Compositae)) and greenleaf desmodium (Desmodium intortum (Mill.) Urb. (Fabales: Leguminosae)) had significant impacts on the behavior of adult psyllids. If such impacts can be ­demonstrated in orchards, cultivation of these weeds may be more suitable for limiting D. citri populations than interplanting with guava, particularly in situations where cultivation of guava may not be profitable. Billygoat weed and greenleaf desmodium are grown as understorey green-manure crops in orchards in southern China in order to enhance the incidence, biodiversity and effectiveness of natural enemies of arthropod pests (Liang and Huang, 1994; Liu et  al., 1999a, b, 2001; Ouyang et  al., 2006). Greenleaf desmodium is also grown as a forage crop (Liang and Huang, 1994; Liu et al., 2001). Both plants are known to have behavioral impacts on arthropods (Khan et  al., 2000, 2010; Kong et al., 2005). Field studies in Vietnam (Beattie et al., 2006; Ichinose et  al., 2012) and Indonesia (Beattie et  al., 2010) have shown that interplanting

198

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c­ itrus with guava slows rates of ingress of D. citri and HLB into orchards. Laboratory studies (Cen et  al., 2008; Zaka et  al., 2010, 2015) indicate that the effects are related to guava volatiles. The studies in Indonesia suggested differences between the guava varieties, red-seeded ‘Jakarta’ and white-seeded ‘Sukan’ (Beattie et al., 2010). However, impacts of interplanting appear to be limited to < 2 years (Beattie et al., 2010; Ichinose et al., 2012). Both studies were undertaken on flat land, and the density of trees within and surrounding orchards in Vietnam may have enhanced concentrations of volatiles within orchards by limiting movement of air.

personal observation). More recently, farmers in Sihui near Guangzhou have used 0.0008% 1-naphthaleneacetic acid (NAA) for the same purpose, but care is required as over-application of the plant growth regulator can be phytotoxic. The benefits of high-density plantings in the face of an HLB epidemic (Aubert, 1990) are debatable. Dense overcrowded canopies are incompatible with thorough coverage of insecticide sprays, reduce the yields on the sides of canopies and increase risks of plant diseases other than HLB within canopies. However, such plantings can lead to more rapid returns on investment in the first few years after planting than from less dense plantings.

12.10  Physical Controls and General Orchard Practices

12.11  Concluding Remarks

Physical controls limiting the spread of HLB include removal of infected trees (Ke and Xu, 1990), use of windbreaks to reduce natural dispersal of psyllids within and among orchards (Ke and Xu, 1990), hand removal of summer flush growth (Barkley et  al., 1980; Aubert, 1990a; Catling, 1996) particularly on small farms (Chen et  al., 2009) and overhead irrigation to reduce survival of eggs and nymphs (G.A.C. Beattie, personal observation). Ke and Xu (1990) mentioned use of horsetail beefwood (Casuarina equisetifolia subsp. incana (Benth.) L.A.S. Johnson (Fagales: Casuarinaceae)) windbreaks to separate every 10 ha of trees. Use of such physical barriers warrants further research to determine the most suitable plants, heights, planting distances and patterns in relation to topography, and wind directions and speeds. Hand removal of summer flush (Aubert, 1990a) to reduce the impacts of citrus leafminer and D. citri now appears to be less common in China than several decades ago (G.A.C. Beattie,

As a vector of CLas, D. citri has provided a perfect opportunity for pesticide manufacturers, distributors and retailers to sell insecticides to citrus farmers in Asia. These manufacturers, distributors and retailers exploit a general lack of knowledge among farmers about pests and diseases, and how to control them. As companies reap profits, the plight of farmers remains unresolved due to a thus far incurable disease. The folly of excessive use of synthetic pesticides for suppressing populations of D. citri and incidence of HLB needs to be addressed through government policies and legislation, education of farmers and curtailment of exploitative marketing leading to misuse and overuse use of synthetic pesticides. It is unlikely that rates of spread of HLB would be any worse than if soft options such as effectively-implemented biological control and mineral oils were used in lieu of synthetic pesticides. Orchards would regain biodiversity and the need to use pesticides to manage induced secondary pests such as mites, thrips and scales would decline.

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13 Asian Citrus Psyllid Management in São Paulo, Brazil

Marcelo Pedreira Miranda* and Antonio Juliano Ayres Fundo de Defesa da Citricultura (Fundecitrus), Araraquara, São Paulo, Brazil

13.1 Introduction Brazil is the world’s largest producer of oranges with an average of 500 million boxes produced over the last season (2016/17, USA marketing year; 2017/18, Brazil marketing year) of which 79% (395 million boxes) comes from the state of Sao Paulo (USDA, 2017). The Asian citrus psyllid (ACP) Diaphorina citri was reported in Brazil in the 1940s (Costa Lima, 1942) but considered as a secondary pest until the first report of huanglongbing (HLB) in Brazil in 2004 (Coletta-Filho et  al., 2004; Teixeira et  al., 2005). HLB quickly spread throughout São Paulo State (SPS), requiring the citrus industry to adapt to a new reality, with many growers opting to replace citrus with another crop. Current citrus orchard area in the state is 445,232 ha, of which oranges represent 93%, with the remaining 7% divided among acid lime, lemons and tangerines. Meanwhile, planting density has increased from a mean 476 trees per acre (1176 trees per hectare) before HLB to 678 (1675) currently (Fundecitrus, 2017a). Incidence of visually symptomatic orange trees was recently estimated for SPS and west-southwestern Minas Gerais state at ≈17% (Fundecitrus, 2017b). This compares favorably with other countries from Central and North America where disease incidence is much higher

(López-Hernández et  al., 2014; Singerman and Useche, 2016). In part, this difference could be explained by the previous experience that the Brazilian citrus growers had with citrus variegated chlorosis (CVC), a disease caused by the xylem-limited bacterium Xylella fastidiosa. CVC is transmitted by a xylem-feeding group of leafhoppers (Cicadellidae) known as ‘sharpshooters’ (Redak et al., 2004). Measures used to manage CVC, and later adapted for HLB, include nursery production under greenhouse conditions, removal of symptomatic trees in the field and insect vector control. This last measure has been greatly studied and improved in recent years in regard to ACP. In this chapter, the main management strategies used by citrus growers from SPS for ACP control will be presented.

13.2  Monitoring and Phytosanitary Alert System In SPS as elsewhere, greatest ACP populations occur mainly during spring and summer, coinciding with the vegetative flushing of citrus trees (Yamamoto et al., 2001; Hall et al., 2008). Due to the greater frequency of insecticide application, psyllid population density in SPS commercial

*  Email: [email protected]

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citrus groves is very low (0.09 psyllid per yellow sticky card); however, in areas where there is poor or no insecticide application, the ACP population is 24 times higher (Fig. 13.1). Monitoring is considered an important component of ACP management in SPS as elsewhere. Therefore, a comparative study of psyllid monitoring methods (visual inspection, tap sampling, sweep net, motorized suction device and yellow sticky cards) was undertaken in blocks with and without insecticide application. Results demonstrated that, although the sampling methods that involve a direct observation (e.g. stem tap and visual inspection) are faster and less expensive than sticky cards (Sétamou et al., 2008; Monzo et al., 2015), they were less effective in detecting the presence of D. citri, especially in areas where chemical control was applied (Miranda et al., 2018a). In these areas, the frequency of D. citri detection by yellow sticky traps (YSTs) was up to 32-fold greater than with visual inspection, and in some cases no ACP was detected by stem tap or visual inspection. These two methods register ACP presence only at the moment of sampling, whereas psyllids in contact with treated plants may die before being observed by the scouts. In contrast, the sticky card is a passive method, collecting ACP during the

entire time it is in the field. Consequently, in areas where HLB is present and ACP chemical control must be applied, the use of sticky cards is necessary for effective psyllid monitoring (Miranda et al., 2018a). Yellow sticky card traps are the main method used for ACP monitoring in SPS. The recommendation is to install the traps in the upper third and external part of the tree canopy. Traps are preferably set on orchard borders, especially along perimeters of the property. Traps should be assessed weekly and replaced biweekly, or before if covered with dirt or insects. Even if visual inspection has low efficacy in areas where intense insecticidal control is applied, this monitoring method can still be used to assess the efficacy of insecticide spray application. The presence of any ACP stage, mainly nymphs of 4th and 5th instar, may indicate failure of the chemical control program (e.g. low application frequency, inadequate insecticide rate or low spray coverage). The recommendation is to perform visual inspection on 1% of the trees along block borders, especially along grove perimeters. Inspectors should assess three to five branches per tree, focusing on young shoots to look for nymphs and adults. Periodic training of inspectors at least once a year is recommended to

7

Number of psyllids/yellow sticky trap

ACP control

Poor or No ACP control

6 5 4 3 2 1 0

J FMAM J J A SOND J FMAM J J A SOND J FMAM J J A SOND 2015

2016

2017

Fig 13.1.  Number of Diaphorina citri adults per yellow sticky trap in citrus orchards in São Paulo State, Brazil. ACP control: citrus orchards receiving ≥ 12 spray applications per year. Poor or No ACP control: citrus orchards receiving ≤ 6 spray applications per year or without chemical control.

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maintain and improve the accuracy of ACP detection. Inspectors are oriented to look for the ACP size, shape and forewing that are key features for identification of this insect. The establishment of coordinated regional management (removal of symptomatic trees and frequent ACP control) has proved essential for successful HLB containment. According to ­Bassanezi et  al. (2013), incidence of HLB was 91% lower in areas with regional management compared with areas with only local management. Fundo de Defesa da Citricultura (Fundecitrus) developed the Phytosanitary Alert System (PAS) in 2011 with the objective of helping citrus growers to better understand the ACP population in the region where their farms are located and ­coordinate applications of insecticides on a regional scale. PAS consists of an online system that organizes information about the ACP population and citrus vegetative stage and can be accessed by computer, smartphones or tablets. PAS is free for the growers, as is all information and training needed to participate provided by the Fundecitrus extension service. Data on ACP population is obtained from sticky traps distributed over the state. These traps are georeferenced and installed along the property perimeter at a spacing that can range from 100 m to 500 m. Costs of traps, assessment and replacement are responsibility of the citrus grower. The grower is also expected to assess the development stage of the shoots at the same time the traps are read. The Fundecitrus team follows these same procedures in areas with higher psyllid pressure such as small orchards with poor management, abandoned or non-commercial orchards. Currently, PAS is available for 11 regions of SPS including the west-southwest of Minas Gerais state, employing 27,000 sticky traps (92% from growers and 8% from Fundecitrus), distributed in 1462 orchards, with a total monitored area of 270,955 ha (44.5% of the citrus area). The regions are divided into quadrants (2 ×2 km), each of which houses an average of nine traps. All data are input fortnightly by the citrus growers and Fundecitrus team (15th and the last day of each month), and automatically processed and presented as maps (GIS format using Google Maps), tables and interactive graphics. The PAS provides two levels of information: regional and available to the public (students,

researchers, growers, etc.) and at orchard level available only to the grower. The PAS webpage displays regional information divided into four sections: (1) map with the quadrants of different colors indicating ACP incidence; (2 and 3) graphics with indices regarding ACP and vegetative stage; and (4) coordinated spray alerts by region. For maps and graphics, the user can select a specific period of the year. About 85% of the growers regularly send data to the system. An additional incentive for registered growers is availability of 70 tools to help them to visualize and analyze the data related to the psyllid incidence and tree vegetative state on their own property. The main indexes used by the growers are: (1) mean number of ACP per trap; (2) traps capturing high numbers; (3) traps with higher frequency of psyllid capture; and (4) psyllid population around the orchard (radius 0–10 km). All this information is able to be selected in specific periods of the year. Besides online visualization, PAS can generate a report as an Excel file. All this information informs the grower about the time and location of ACP entry into the orchard, and defines specific control strategies for each sector of the property. In addition, the PAS sends a message by phone or email to schedule a regional coordinated spray application whenever the ACP population increases.

13.3  Chemical Control Chemical control is considered the most used and proven method for management of D. citri (Boina and Bloomquist, 2015). Also, significantly reduced spread of HLB has been seen when control is applied on a regional scale (Bassanezi et  al., 2013). Growers in Brazil must only use ­insecticides for ACP control found in the Prote­ Citrus (Citrus Protection Products) list to comply with regulations of the importing countries. Considering that a single psyllid is able to inoculate Candidatus Liberibacter asiaticus in several citrus plants (Canale et al., 2017), and in areas where HLB is endemic there is a greater likelihood of bacteriliferous psyllids (Sassi et al., 2017), the presence of a single psyllid is sufficient to indicate the need for control. Chemical control strategies for ACP are contingent on tree



Asian Citrus Psyllid Management in São Paulo, Brazil 213

age: (i) nursery trees; (ii) young orchard (≤ 3 years old); and (iii) mature orchard (> 3 years old).

13.3.1  Nursery trees The usual practice by citrus growers in SPS is to apply neonicotinoid insecticides (imidacloprid or thiamethoxam) by drench (volume application 50 ml per tree) on nursery trees 1–5 days before planting. This application provides effective control (ACP mortality ≥ 80%) for up to 90 days after planting. Moreover, drench applications of thiamethoxam and imidacloprid on sweet orange nursery trees disrupt the probing behavior of D. citri, mainly during the phloem ingestion phase (overall reduction of 90%) (­Miranda et al., 2016). Therefore, this treatment may reduce the probability of Ca. Liberibacter spp. transmission in citrus plants by D. citri.

13.3.2  Young orchard (≤ 3 years old) The recommendation at this growth stage is to apply a systemic (neonicotinoid) insecticide as a soil drench or trunk treatment because of the frequency of vegetative flushes, along with foliar sprays of different mode of actions. Studies conducted under SPS conditions showed a reduction of 60% in HLB incidence in blocks that received sprays plus drench applications compared with blocks that just received spray applications, reinforcing the recommendation mentioned ­ above (M.P. Miranda, São Paulo, 2018, personal ­communication). Three to four systemic applications are recommended per year, mainly in anticipation of vegetative flushes, i.e. between the end of winter and early spring (first application), in early summer (second application) and late summer (third application). An additional (fourth) application can be performed in the early fall, preferably by trunk (due to low soil moisture during this time of year). Rates of systemic insecticides are calculated based on the plant size. For insecticides applied by drench, an amount of product (milliliters (ml) or grams (g)) per meter of tree height is used. In the case of trunk application, the rate is calculated according to diameter of the trunk. These types of application provide an

effective ACP control up to 50–70 days after application. The most commonly used insecticides for ACP control by foliar application belong to the pyrethroid, organophosphate and neonicotinoid modes of action. ACP control is typically greater than 80% over a range of 5–14 days. This effect will depend on the rate used, weather conditions (occurrence of rain) and presence of shoots. The recommendation is to apply at intervals of 7–14 days. Actual frequency of application is determined by block according to incidence of HLB, ACP monitoring and the location. Thus, blocks located on the perimeter of the property are usually sprayed more frequently than in the interior of the farm. In order to avoid ACP resistance to insecticides, most citrus companies are rotating insecticides with different modes of action. ACP resistance has not yet been observed for the insecticides commonly used in citrus production in SPS, even with frequent chemical control (Poltronieri, 2013). This may be related to the fact that the psyllids captured in commercial citrus orchards are from areas with little or no insecticide selection pressure (i.e. abandoned orchards and backyards).

13.3.3  Bearing orchard (> 3 years old) Several experiments were performed in SPC to establish a recommendation for application of systemic insecticides (drench and/or trunk) on mature orchards. In most cases, ACP control was not effective at the 80% mortality level. This result was related to the low concentrations of systemic insecticides in foliage of large citrus tree (M.P. Miranda, São Paulo, 2018, personal communication). Therefore, the recommendation for trees > 3 years old is foliar application at intervals of 14 or 28 days. Again, application frequency is based on a per block basis with the same criteria as for young blocks. Due to the need for frequent foliar application to control ACP, several experiments were conducted to assess the possibility of reducing the spray volumes, based on the tree-row volume (TRV) concept (Byers et al., 1971). TRV per hectare is calculated by dividing the area of 1 ha (in  square meters) by the inter-row spacing (in  meters) and multiplying by the tree canopy

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height and width (in meters). With this concept, it is possible to standardize the spray volume per cubic meter of canopy regardless of the citrus tree size. It was demonstrated that reduction of the standard volume used by citrus growers (80 ml spray mixture/m³ of tree canopy) to a volume of 25–40 ml of spray mixture/m³ of tree canopy, maintaining the same insecticide concentration (grams of active ingredient per liter), resulted in the same control efficacy against D. citri (M.P. Miranda, São Paulo, 2018, personal communication). These reduced volumes also represented an environmental and economic gain because of a reduction of up to 69% in insecticide and water consumption and 32% in diesel per hectare, totaling a reduction of up to 52% in spray cost. Another advantage is that a gain in the operational efficiency is also obtained, by spraying a greater area in a shorter period of time. This is an important consideration in the management of HLB, as some sites of the farm require a greater frequency of spraying, in particular orchard borders. Moreover, spraying large areas in a short period of time is fundamental to avoiding ACP dispersal among farms participating in regional management programs. Based on these results, the current volume application recommendation for ACP control in SPS is 25–40 ml of spray mixture/m³ of tree canopy, regardless of orchard age. In general, these volumes are 50% less than the volumes used in other citrus-growing countries in Central and North America. In addition, insecticide rates (ml or g/l water) used for ACP control can be reduced by 66%. Consequently, even with the frequent insecticide application in citrus orchards in SPS, the amount of insecticide applied per area per year is equal to or less than that applied in these other countries. Fundecitrus has also created software (SPIF – Sistema de Pulverização Integrado do Fundecitrus) to help growers calculate the appropriate spray volume, insecticide rates and sprayer set-up according to orchard age. Growers can thus reduce spray volume and consequently chemical drift and/or run-off. In addition, alternative compounds to control ACP, with promising results, are being studied such as botanic and biological insecticides, and kaolin (Volpe et al., 2016; Ausique et al., 2017; Miranda et al., 2018b). The idea is to replace some chemical applications with these selective products.

13.4  Biological Control Natural enemies (predators, parasitoids and entomopathogenic fungi) have an important role in the regulation of ACP populations in the field (Qureshi and Stansly, 2009). Through a partnership among Esalq/USP, Koppert and Fundecitrus, several studies have been conducted in Brazil in recent years to develop biopesticides with high efficacy against ACP adults. Field studies have demonstrated that application of two virulent fungal strains, Isaria fumosorosea ESALQ-1296 and Beauveria bassiana ESALQPL63, resulted in mortality of adult ACP ranging from 96.1% (rainy season) to 57.8% (dry season) (Ausique et al., 2017). Based on these results, a commercial biopesticide was released on the market in 2018 as a new tool to manage ACP. Additionally, the parasitoid Tamarixia radiata Waterston, 1922 (Hymenoptera: Eulophidae) is already available to citrus growers. T. radiata adults can feed on nymphs, mainly of first instars, and females parasitize nymphs from 3rd to 5th instar of ACP (Chu and Chien, 1991). In Brazil, the first report of T. radiata was in 2005 in the central region of SPS (Gómez-Torres et  al., 2006). Subsequent studies were carried out in the department of entomology of Esalq/USP in Piracicaba-SP to better understand the biology and develop a rearing system for this parasitoid under Brazilian conditions. Later surveys showed that the parasitoid was widely distributed throughout SPS, although at low rates of natural parasitism (average 12.4%) (Paiva and Parra, 2012). However, a mean increase of 4.3 times in incidence of parasitism and a mean reduction of 69% in the nymph population was observed ­following sequential release of T. radiata (Diniz, 2013), demonstrating good potential for this parasitoid under SPS environmental conditions. Due to the high susceptibility of T. radiata to insecticides, Parra et al. (2010) proposed to release only in areas without chemical control, such as abandoned orchards, organic orchards and dooryards, to reduce the ACP population and consequent dispersal into commercial ­orchards. In 2015, a second facility for rearing T. radiata was built in partnership between Fundecitrus and Bayer CropScience in ­Araraquara,



Asian Citrus Psyllid Management in São Paulo, Brazil 215

SP with the objectives of: (i) producing ­parasitoids for release in unmanaged areas indicated by PAS; and (ii) training citrus growers to install their own rearing program. The T. radiata rearing system used by Fundecitrus is an adaptation of the methodology described by Gomez-Torres (2009). ACP rearing is carried out in two conditions: a climate-controlled room (28 ± 2°C, 14 h photophase and 70 ± 10% RH) and in a greenhouse (Fig. 13.2A, B). The greenhouse system has the advantage of lower cost (construction and maintenance) than growth chambers, but is subject to environmental conditions. Seedling Murraya paniculata are first pruned to induce flushing. When shoots reach 0.3–0.5 mm length, 12 seedlings with 10–12 young shoots each are placed inside cages (55 × 84 × 55 cm) into which are released 900 adult psyllids (10–15 days old) for 7 days of oviposition. The adult psyllids are then removed using an electric aspirator and transferred to new cages for a new cycle of oviposition. In this system, 80% of the resulting

nymphs are used for the T. radiata rearing and 20% are kept to maintain the ACP colony. Cages containing ACP 3rd- and 4th-instar nymphs are then transferred to a climate-­ controlled room (25 ± 2°C, 14 h photophase, and 70 ± 10% RH). T. radiata adults (1–2 days old) are released at a ratio of one parasitoid to an estimated 25 nymphs. The first adults begin to emerge 12 days later, and are collected daily ­directly from the walls or ceiling of the cage for a period of 5 days using an electric aspirator (Fig. 13.2C). They are then stored, 200 wasps per 50 ml plastic tube, containing some drops of honey, and packed in polystyrene boxes for transport to the field for releasing the same afternoon or next morning (Fig. 13.2D). For abandoned or organic orchards, 400 parasitoids per hectare are released, and in dooryards, 30 parasitoids per tree (citrus or orange jasmine). Over the past 3 years, 2,171,409 parasitoids were produced at Fundecitrus, of which 1,811,200 (83.4%) were released in areas determined by PAS (Fig. 13.3).

(A)

(B)

(C)

(D)

Fig. 13.2.  (A, B) Diaphorina citri rearing under greenhouse and laboratory conditions (28 ± 2°C, 14 h photophase and 70 ± 10% RH) for Tamarixia radiata mass rearing, respectively. (C) T. radiata harvested directly from the rearing cage using an electric aspirator. (D) T. radiata stored in plastic tubes (detail) and released in field.

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M.P. Miranda and A.J. Ayres

Period: 2015–2018

GO

Number of Tamarixia radiata 5,000 10,000 15,000 20,000 25,000 Phytosanitary alert regions

MS

MG

RJ

N

PR 0

100

200

300 km

Source: Fundecitrus

Fig. 13.3.  Number of Tamarixia radiata released in areas determined by the Phytosanitary Alert System (PAS).

In this rearing system in the laboratory, the mean parasitism is around 68%, where 20% of the parasitoids produced are used to maintain the colony and 80% released in the field. The production cost (labor and maintenance) is around US$0.07 per T. radiata, of which 67% is labor. New studies are being developed to reduce the cost, especially of labor. In addition to the facilities at Esalq/USP and Fundecitrus, there are six more facilities producing T. radiata in SPS. These are maintained by the citrus companies with a rearing capacity about 100,000 parasitoids per month from each laboratory.

13.5  External Actions In orchards with a rigorous program of HLB-symptomatic tree removal and ACP control, it is thought that little or no secondary spread occurs; however, disease incidence can continue to increase due to the primary spread (Bergamin Filho et al., 2016). This is caused by constant short- and long-range dispersal of

ACP from unmanaged areas to commercial citrus orchards (Boina et  al., 2009; Lewis-­ Rosenblum et al., 2015; Tomaseto et al., 2015). Insecticides used for ACP control can disrupt probing behavior and consequently may reduce Ca. ­Liberibacter spp. inoculation (Miranda et al., 2011, 2016; Ammar et  al., 2015). However, most insecticides have a short residual period when sprayed on citrus, which can be further reduced by young shoot growth and weather conditions (De Carli et al., 2016; M.P. Miranda, São Paulo, 2018, personal communication). Therefore, growers must extend the disease management measures to areas around their orchards to achieve successful and sustainable HLB control. The highest priority external action is elimination of ACP host plants (citrus and/or orange jasmine) requiring an agreement with the land owner where plants will be removed. In most cases, the plants are eliminated in exchange for some benefit, mainly replacement with other fruit trees. It is recommended to spray insecticide immediately before the elimination wherever



Asian Citrus Psyllid Management in São Paulo, Brazil 217

possible, to prevent ACP dispersal to managed orchards. If plant removal is not possible, the second priority is the application of contact insecticides to citrus and/or orange jasmine trees located in dooryards and orchards whenever ACP is detected visually or by sticky cards. Residents should always be agreeable and informed. If none of the forgoing actions are possible, the release of T. radiata is recommended. External actions should be conducted in a 5 km radius from the orchard, starting with those locations closest to the commercial orchard. Michigami et al. (2015) reported that for each R$1.00 spent in external actions, approximately R$8.00 are saved in losses. Fundecitrus teams facilitate first contact between citrus growers and property owners of the areas where the external actions are desired. The team also puts on events to inform citrus growers and the general public about the importance of eliminating ACP host plants (sources of HLB inoculum). Televized campaigns are used to inform the community regarding this issue where HLB incidence is high. Currently, some citrus companies have a specific team to conduct external actions. Around 233,963 citrus and/or orange jasmine trees have been eradicated in rural and urban areas of different regions of SPS through collaboration between these companies and Fundecitrus over the past 2 years.

13.6  Successful Cases of HLB Management Farm size and the presence or absence of neighbor areas without ACP control are important factors determining the success or failure of HLB management (Belasque et  al., 2010). While these factors are not under direct control of the grower, implementation of a rigorous ACP control program inside and outside the farm can stabilize or reduce the annual HLB ­infection rate (percentage of new HLB-­ symptomatic trees found per year). External actions include voluntary removal of dooryard or abandoned trees, release of T. radiata or treatment of remaining trees (Table 13.1). The following case of four farms located in regions of SPS with medium or high HLB incidence

i­llustrates the point. Farms 1 and 3 were located in the Bebedouro region where HLB incidence increased from an average of 2.43% in 2012 to 7.7% in 2017 (Fundecitrus, 2017b). In response, growers intensified chemical control and initiated external actions (Table 13.1). Farm 1 was a small orchard whose owner was able to remove a neighboring abandoned citrus orchard 60% the size of his own. With these measures, Farms 1 and 3 were able to reduce the annual HLB infection rate in their orchards in spite of the disease increase in the ­region (Fig. 13.4). Farm 2 was actually a composite of 11 small bearing groves located in ­Pirassununga with the second highest HLB incidence (25.43%) in SPS. As consequence, this farm has the highest HLB incidence among the farms presented here. In 2015, the grower increased the frequency of foliar insecticide and began a campaign to remove trees outside the farm in July 2016 in anticipation of replanting. These actions were likely responsible for the reduction of the annual HLB infection rate ­ ­observed in the past 2 years (Fig. 13.4). Farm 4 was one of the largest farms of SPS and located in Matão region, where average HLB incidence was estimated at 21.63% (Fundecitrus, 2017b). This farm was one of the pioneers in removing HLB-symptomatic trees and performing a rigorous ACP management since HLB detection in SPS. Due to its size (6700 ha of citrus), Farm 4 functions as an area-wide management unit. Nevertheless, there was a strong influence from surrounding areas with little or no ACP control (around 100 neighbors). In an effort to reduce the primary infection, internal ACP control was intensified and a program of external actions initiated. These measures were likely responsible for the decrease of the annual HLB infection rate documented in Fig. 13.4.

13.7 Acknowledgements We would like to thank the agronomists of Fundecitrus extension service (Ivaldo Sala, Luis Scandelai, Bruno Daniel and Guilherme Rodriguez) and citrus growers for providing the data on HLB management; and Dr. Philip A. Stansly for reading and giving his useful comments on drafts of this chapter.

218

Table 13.1.  Internal and external Diaphorina citri management in farms from São Paulo State, Brazil. ACP management inside the farm Chemical control Orchard age (years)

1

25,000

1–15

2

85,000

3

4

Young orchard (≤ 3 years)

Bearing orchard (≥ 3 years)

5–44

Drench: four applications/year Foliar application: 7-day intervals –

180,000

6–12

–

Foliar application: 10–14-day intervals

3,037,012

0–21

Drench: two applications/year Foliar application: 7–10-day intervals

Foliar application: 7–10–14-day intervals

*ACP visual inspection is performed along with other pests (mites and scales)

ACP monitoring*

External actions (radius up to 5 km)

Foliar application: 14-day intervals

YST 14-day interval

Foliar application: 10–14-day intervals

YST and visual inspection: 7- and 14-day intervals, respectively YST and visual inspection: 7- and 14-day intervals, respectively YST and visual inspection: 7- and 14-day intervals, respectively

Eradication of 120 citrus trees from dooryards and 15,000 citrus trees from abandoned orchard. Release of 2000 T. radiata Eradication of 642 citrus trees from dooryards and 8000 citrus trees from abandoned orchard. Release of 2200 T. radiata Eradication of 20 citrus trees from dooryards and 500 citrus trees from abandoned orchard. Release of 8000 T. radiata Eradication of around 119,000 citrus trees from dooryards and mainly from abandoned orchards. Monthly foliar insecticide application on 97,000 citrus trees. Release of 62,970 T. radiata

M.P. Miranda and A.J. Ayres

Total number of citrus trees

Farm



Asian Citrus Psyllid Management in São Paulo, Brazil 219

5.0

Farm 1

Farm 2

4.3

Farm 3

4.0

HLB annual incidence (%)

3.0 2.0

2.5

5.0

2.6 2.0

2015

0.5

0.3

2016

2017

2015

2016

2017

2015

2016

3.0 2.2

2.0

2017 Farm 4

3.9

4.0

1.0

2.8

1.4

1.0 0.0

2.9

1.8

2.0

2.4 1.7

1.6

1.0

1.3 0.8

0.5

0.0 2007

2008

2009

2010

2011

2012

2013

2014

2015

2016

2017

Fig. 13.4.  Annual huanglongbing (HLB) incidence in citrus orchards in São Paulo State that perform internal and external actions against Diaphorina citri.

References Ammar, E., Hall, D.G. and Alvarez, J.M. (2015) Effect of cyantraniliprole, a novel insecticide, on the inoculation of Candidatus Liberibacter asiaticus associated with citrus huanglongbing by the Asian Citrus Psyllid (Hemiptera: Liviidae). Journal of Economic Entomology 108, 399–404. doi: 10.1093/jee/tov016 Ausique, J.J.S., D’Alessandro, C.P., Conceschi, M.R., Mascarin, G.M. and Delalibera Júnior, I. (2017) Efficacy of entomopathogenic fungi against adult Diaphorina citri from laboratory to field applications. Journal of Pest Science 90, 947–960. doi: 10.1007/s10340-017-0846-z Bassanezi, R.B., Montesino, L.H., Gimenes-Fernandes, N., Yamamoto, P.T., Gottwald, T.R. et  al. (2013) Efficacy of area-wide inoculum reduction and vector control on temporal progress of Huanglongbing in young sweet orange plantings. Plant Disease 97, 789–796. doi: 10.1094/PDIS-03-12-0314-RE Belasque, J. Jr, Bassanezi, R.B., Yamamoto, P.T., Ayres, A.J., Tachibana, A., Violante, A.R., Tank, A. Jr, Di Giorgi, F., Tersi, F.E.A., Menezes, G.M. et al. (2010) Lessons from huanglongbing management in São Paulo state, Brazil. European Journal of Plant Pathology 92, 285–302. Bergamin Filho, A., Inoue-Nagata, A.K., Bassanezi, R.B., Belasque, J. Jr, Amorim, L. et al (2016) The importance of primary inoculum and area-wide disease management to crop health and food security. Food Security 8, 221–238. doi: 10.1007/s12571-015-0544-8 Boina, D.R. and Bloomquist, J.R. (2015) Chemical control of the Asian citrus psyllid and of Huanglongbing disease in citrus. Pest Management Science 71, 808–823. doi: 10.1002/ps.3957 Boina, D.R., Meyer, W.L., Onagbola, E.O. and Stelinski, L.L. (2009) Quantifying dispersal of Diaphorina citri (Hemiptera: Psyllidae) by immunomarking and potential impact of unmanaged groves on commercial citrus management. Environmental Entomology 38, 1250–1258. doi: 10.1603/022.038.0436 Byers, R.E., Hickey, K.D. and Hill, C.H. (1971) Base gallonage per acre. Virginia Fruit 60, 19−23. Canale, M.C., Tomaseto, A.F., Haddad, M.L., Coletta-Filho, H.D. and Lopes, J.R.S. (2017) Latency and persistence of ‘Candidatus Liberibacter asiaticus’ in its psyllid vector, Diaphorina citri (Hemiptera: Liviidae). Phytopathology 107, 264–272. doi: 10.1094/PHYTO-02-16-0088-R Chu, Y.I. and Chien, C.C. (1991) Utilization of natural enemies to control of psyllid vectors transmitting citrus greening. In: Kiritani, K., Su, H.J. and Chu, Y.I. (eds) Integrated Control of Plants Virus Disease. Food and Fertilizer Technology Center Asian and Pacific Region, Taipei, pp. 135–145.

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Coletta-Filho, H.D., Targon, M.L.P.N., Takita, M.A., De Negri, J.D., Pompeu, J. Jr et al. (2004) First report of causal agent of huanglongbing (‘Candidatus Liberibacter asiaticus’) in Brazil. Plant Disease 88, 1382. doi: 10.1094/PDIS.2004.88.12.1382C Costa Lima, A.M. da (1942) Insetos do Brasil. Vol. 3: Homópteros. Série Didática No. 4, Escola Nacional de Agronomia, Rio de Janeiro, Brazil. De Carli, L.F., Miranda, M.P., Volpe, H.X.L., Zanardi, O.Z., Vizoni, M.C. et al. (2016) Young shoots affecting the efficacy of insecticide spray applications against Diaphorina citri. Proceedings of the International Citrus Congress. Foz do Iguaçu, Paraná/Brazil, p. 238. Diniz, A.J.F. (2013) Otimização da criação de Diaphorina citri Kuwayama, 1908 (Hemiptera: Liviidae) e de Tamarixia radiata (Waterston, 1922) (Hymenoptera: Eulophidae), visando a produção em larga escala do parasitoide e avaliação do seu estabelecimento em campo. Doctoral thesis. Escola Superior de Agricultura ‘Luiz de Queiroz’, University of São Paulo, Brazil. López-Hernández, D., Luis-Pantoja, M., Llauger-Riverón, R., González-Fernández, C., Casín-Fernández, J.C. et al. (2014) Situación de huanglongbing de los cítricos en Cuba siete años después de su detección. CitriFrut 31, 3–9. Fundecitrus (2017a) Tree inventory of the São Paulo and west-southwest of Minas Gerais citrus belt – snapshot of groves in March 2017. Fundecitrus, Araraquara, São Paulo, Brazil. Fundecitrus (2017b) Greening/HLB. Available at: http://www.fundecitrus.com.br/levantamentos/greening (accessed September 2017). Gomez-Torres, M.L. (2009) Estudos bioecologicos de Tamarixia radiata (Waterston, 1922) (Hymenoptera: Eulophidae) para o controle de Diaphorina citri Kuwayama, 1908 (Hemiptera: Psyllidae). Doctoral thesis. Escola Superior de Agricultura ‘Luiz de Queiroz’, University of São Paulo, Brazil. Gomez-Torres, M.L., Nava, D.E., Gravena, S., Costa, V.A. and Parra, J.R.P. (2006) Registro de Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) em Diaphorina citri Kuwayama Hemiptera: Psyllidae) em São Paulo. Revista de Agricultura 81, 112–117. Hall, D.G., Hentz, M.G. and Adair, R.C. Jr (2008) Population ecology and phenology of Diaphorina citri (Hemiptera: Psyllidae) in two Florida citrus groves. Environmental Entomology 37, 914–924. doi: 10.1603/0046-225X Lewis-Rosenblum, H., Martini, X., Tiwari, S. and Stelinski, L.L. (2015) Seasonal movement patterns and long-range dispersal of Asian citrus psyllid in Florida citrus. Journal of Economic Entomology 108, 3–10. doi: 10.1093/jee/tou008 Michigami, F.A.B., Girotto, L.F. and Bassanezi, R.B. (2015) Effect of internal and external inoculum control practices on HLB epidemic progress in a commercial citrus grove. In: 4th International Research Conference on Huanglongbing, Orlando, Florida. Miranda, M.P., Felippe, M.R., Garcia, R.B., Yamamoto, P.T. and Lopes, J.R.S. (2011) Effect of insecticides and mineral oil on probing behavior of Diaphorina citri Kuwayama (Hemiptera: Psyllidae) in citrus. In: 2nd International Research Conference on Huanglongbing, Orlando, Florida. Miranda, M.P., Santos, F.L., Bassanezi, R.B., Montesino, L.H., Barbosa, J.C. et al. (2018a) Monitoring methods for Diaphorina citri Kuwayama (Hemiptera: Liviidae) on citrus groves with different insecticide application programmes. Journal of Applied Entomology 42, 89–96. Miranda, M.P., Zanardi, O.Z., Tomaseto, A.F., Volpe, H.X.L., Garcia, R.B. et al. (2018b) Processed kaolin affecting the probing and settling behavior of Diaphorina citri (Hemiptera: Lividae). Pest Management Science 74(8), 1964–1972. doi: 10.1002/ps.4901 Miranda, M.P., Yamamoto, P.T., Garcia, R.B., Lopes, J.P.A. and Lopes, J.R.S. (2016) Thiamethoxam and imidacloprid drench applications on sweet orange nursery trees disrupt the feeding and settling behavior of Diaphorina citri (Hemiptera: Liviidae). Pest Management Science 72, 1785-1793. doi: 0.1002/ps.4213 Monzo, C., Arevalo, H.A., Jones, M.M., Vanaclocha, P., Croxton, S.D. et al. (2015) Sampling methods for detection and monitoring of the Asian citrus psyllid (Hemiptera: Psyllidae). Environmental Entomology 44, 780–788. doi: 10.1093/ee/nvv032 Paiva, P.E.B. and Parra, J.R.P. (2012) Natural parasitism of Diaphorina citri Kuwayama (Hemiptera, Psyllidae) nymphs by Tamarixia radiata Waterston (Hymenoptera, Eulophidae) in São Paulo orange groves. Revista Brasileira de Entomologia 56, 499–503. doi: 10.1590/S0085-56262012000400016 Parra, J.R.P., Lopes, J.R.S., Gomez-Torres, M.L., Nava, D.E. and Paiva, P.E.B. (2010) Bioecologia do vetor Diaphorina citri e transmissão de bactérias associadas ao Huanglongbing. Citrus Research & ­Technology 31, 37–51.



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Poltronieri, A.S. (2013) Bases para o manejo da resistência de Diaphorina citri (Hemiptera: Liviidae) ao inseticida neonicotinoide imidacloprid em pomares de citros. Doctoral thesis. Escola Superior de ­Agricultura ‘Luiz de Queiroz’, University of São Paulo, Brazil. Qureshi, J.A. and Stansly, P.A. (2009) Exclusion techniques reveal significant biotic mortality suffered by Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae) populations in Florida citrus. Biological Control 50, 129–136. doi: 10.1016/j.biocontrol.2009.04.001 Redak, R.A., Purcell, A.H., Lopes, J.R.S., Blua, M.J., Mizell, R.F. et al. (2004) The biology of xylem fluid-feeding insect vectors of Xylella fastidiosa and their relation to disease epidemiology. Annual Review of Entomology 49, 243–270. Sassi, R.S., Bassanezi, R.B., Sala, I., Coletti, D.A.B., Rodrigues, J.C. et al. (2017) Incidence and distribution of Diaphorina citri carrying ‘Candidatus Liberibacter asiaticus’. In: 5th International Research Conference on Huanglongbing. Orlando, Florida. [Abstracts: Journal of Citrul Pathology, iocv_journalcitrulpathology_34714.] Sétamou, M., Flores, D., French, J. and Hall, D. (2008) Dispersion patterns and sampling plans for Diaphorina citri (Hemiptera: Psyllidae) in citrus. Journal of Economic Entomology 101, 1478–1487. doi: 10.1603/0022-0493 Singerman, A. and Useche, P. (2016) Impact of citrus greening on citrus operations in Florida. Available at: http://edis.ifas.ufl.edu/fe983 (accessed 6 January 2018). USDA (2017) Citrus Annual Report Brazil. USDA Global Agricultural Information Network (GAIN) Report BR 17012. USDA Foreign Agricultural Service, Washington, DC. Available at: usdabrazil.org.br/pt-br/ reports/citrus-annual-4.pdf (accessed December 2017). Teixeira, D.C., Danet, J.L., Eveillard, S., Martins, E.C., Jesus, W.C. Jr et al. (2005) Citrus huanglongbing in São Paulo State, Brazil: PCR detection of the ‘Candidatus Liberibacter’ species associated with the disease. Molecular and Cellular Probes 19, 173–179. doi: 10.1016/j.mcp.2004.11.002 Tomaseto, A.F., Krugner, R. and Lopes, J.R.S. (2015) Effect of plant barriers and citrus leaf age on dispersal of Diaphorina citri (Hemiptera: Liviidae). Journal of Applied Entomology 140, 91–102. doi: 10.1111/ jen.12249 Volpe, H.X.L., Fazolin, M., Garcia, R.B., Magnani, R.F., Barbosa, J.C. et al. (2016) Efficacy of essential oil of Piper aduncum against nymphs and adults of Diaphorina citri. Pest Management Science 72, 1242–1249. doi: 10.1002/ps.4143 Yamamoto, P.T., Paiva, P.E.B. and Gravena, S. (2001) Flutuação populacional de Diaphorina citri Kuwayama (Hemiptera: Psyllidae) em pomares de citros na região norte do estado de São Paulo. Neotropical Entomology 30, 165–170. doi: 10.1590/S1519-566X2001000100025.

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Integrated Management of Asian Citrus Psyllid and Huanglongbing in Florida: Past, Present and Future Philip A. Stansly and Jawwad A. Qureshi* University of Florida, Department of Entomology and Nematology, Institute of Food and Agricultural Sciences, Southwest Florida Research and Education Center, Immokalee, Florida, USA

First detected in 1998, the Asian citrus psyllid (ACP) Diaphorina citri swept through Florida in record time, comparable in spread only to the citrus leafminer Phyllocnistis citrella detected 5 years earlier. Movement of both pests was aided by commercial transport of plants throughout the state. Huanglongbing (HLB), or citrus greening disease, was detected 7 years later in pommelos in the extreme southeast of the peninsula. Subsequent delimiting surveys discovered positive citrus trees up the east coast and across the state. Roguing proved ineffective. The genie was out of the bottle and the Florida citrus industry had to learn to live with HLB. Thus began a protracted battle, with vector control and tree health management as the basic tools, gradually aided by a reactivated breeding program. More definitive solutions have proved elusive and remain on the horizon. Nevertheless, it is likely that help will come from more directed ACP management strategies and new plant improvement techniques.

14.1  Early Detection and Spread ACP was detected in June 1998 on the east coast of Florida, first Palm Beach County (2 June),

soon followed by finds of ACP in neighboring Broward (4 June) and Martin counties (8 June) on citrus and the citrus relative, orange jasmine Murraya paniculata (Rutaceae). The initial infestation was estimated to have occurred within 6 months or a year before these first detections. A heavy ACP infestation was found on citrus of mixed varieties in a discount store in Broward county on 12 June 1998 (Halbert, 1998). The psyllid spread rapidly and by 2001 was reported from 31 Florida counties. Many subsequent early detections were reported from retail stores selling M. paniculata, even in the northern part of the state where neither citrus nor orange jasmine can grow without judicious freeze protection (Halbert et al., 2002). Spread of HLB in Florida was estimated at 50–60 km p.a., aided greatly by retail distribution of citrus and orange jasmine and movement of D. citri in fruit trucks from orchard to packing or processing facilities (Halbert et al., 2010, 2012; Hall and McCollum, 2011). Early studies (1999–2000) showed that populations of adult ACP occurred on potted orange jasmine throughout the year and depended on new leaf flushes (Tsai et al., 2002). The authors suggested that continuous flushing by orange jasmine could serve to maintain ACP populations

*  Email: [email protected]

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l­ ocally. Populations built up rapidly in commercial citrus, reaching an average of 13.3 and 39.9 on adults and nymphs, respectively, per flush shoot at four locations in the heart of the citrus district in Polk County (Michaud, 2001). Similar population levels were documented in a later study conducted in 2006/07 that included the three principal citrus-growing areas of the state (Qureshi et al., 2009). Surveys conducted to detect HLB using PCR were negative until the first detection in backyard pomelo trees in extreme southern Miami-Dade County in August 2005 (Halbert et al., 2012). By January 2006 the disease was confirmed in the five southeastern counties of the state and in Hendry County in the southwest. In six of 13 counties, positive psyllids were found prior to finding HLB-positive plants in surveys conducted in 2005 and 2006 (Manjunath et al., 2008). HLB was confirmed in most citrus-producing counties in Florida by the end of 2007 (https://crec.ifas.ufl. edu/extension/greening/history.shtml).

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and first released in Florida in 1999 (Hoy, 2005). Rapid spread was observed and later confirmed in all areas of the state (Qureshi et al., 2009). Early efforts to control ACP were largely limited to young plantings where direct damage from ACP feeding was intense enough to kill young flush (Stansly et al., 2002). These efforts were largely limited to soil applications of imidacloprid also used in young trees for citrus leafminer control (P.A. Stansly, unpublished data). It was not until after the detection of HLB in 2005 that large-scale applications on more mature trees commenced, especially in the southern part of the growing regions. Insecticides identified early as effective against ACP included fenpropathrin, chlorpyrifos, imidacloprid, thiamethoxam and aldicarb (Childers and Rogers, 2005).

14.2  Critical Elements of ACP Management in Florida 14.2.1  Insecticidal control

14.1.1  Regulatory actions Federal law restricts the movement of live citrus plants, plant parts, budwood or cuttings outside Florida. Availability of disease-free nursery stock was an early and key recommendation for HLB management (Bove, 2006), requiring major changes in production practices as mandated by a Citrus Health Response Plan (CHRP) developed by the USDA Animal and Plant Health Inspection Service (APHIS) and Florida Department of Agriculture and Consumer Services/Division of Plant Industries (FDACS/DPI) (FDACS/DPI, 2007). Citrus nursery stock and budwood sources must now be grown in insect-resistant structures constructed at a minimum with poly/ polycarbonate covering or screened with a maximum screen size of 266 × 818 μm designed to exclude psyllids, aphids, leaf miners or other pests. In addition, nurseries had to be set back at least 1 mile and budwood production 10 miles from concentrations of citrus trees. 14.1.2  Early control efforts Tamarixia radiata, the principal parasitoid of D. citri, was brought into quarantine in 1998

14.2.1.1  Transmission and acquisition A detailed description of the acquisition and transmission of the causative pathogen, Candidatus Liberibacter asiaticus (CLas) is provided in ­Chapter 8. For this discussion, it is sufficient to state that both transmission and acquisition of the CLas bacteria can occur on a single flush shoot within a single psyllid generation. This is the case because developing nymphs can acquire and presumably amplify bacteria injected by adults on the same shoot (Lee et al., 2015). The result can be that both emerging adults and the young shoot become infected with CLas. In contrast, HLB symptoms may not appear for months or even longer when compromised phloem function results in root degeneration. For this reason, CLas is often detected in the vector population before it can be found in trees (Manjunath et al., 2008). 14.2.1.2  Time and space considerations D. citri depends on emerging leaf shoots (‘flush’) for maturing eggs, oviposition and nymphal growth and survival (Tsai et al., 2002). Flushing patterns depend on climate, mitigated somewhat by irrigation and to a lesser extent, by fertilization.

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The subtropical weather pattern in Florida is characterized by wet warm summers and cool dry winters. Approximately 75% of new flush is put on by mature trees in concert with warming patterns in late winter and early spring (Cooper et al., 1963). Additional flushes occur in early summer and early fall, with sporadic flush in summer. Young trees may flush almost continuously. Principal movement of ACP occurs after flushes, particularly in spring (Hall and Hentz, 2011). Highest incidence of ACP testing positive for CLas occurs in psyllids flying after the spring flush and fall flush (Manjunath et al., 2008). However, adults are much more numerous in the spring flight, which initiates a transmission cycle that repeats throughout the growing season. Therefore, curtailing the spring flight is a principal objective in ACP management. 14.2.1.3  Dormant sprays In the absence of flush, adult ACP survives by feeding on mature host plant foliage and even non-host plants (Tsai and Lui, 2000; Martini et al., 2013; Johnston, 2018). The normally cool dry winter relatively devoid of flush imposes a constraint on ACP reproduction, resulting in ­declining populations in anticipation of the spring flush. It was shown early on that the most effective way to suppress spring flight and consequent disease spread was to pre-empt colonization of the spring flush by controlling the overwintering primarily adult population with the so-called ‘dormant sprays’ (Qureshi and Stansly, 2010). Two commercial-scale trials documented in this study demonstrated that one or two applications of broad-spectrum insecticide in winter resulted in significant ACP suppression for as long as 6 months. Another advantage was the absence of any negative impacts on generalist predators such as ladybeetles and lacewings, which migrated into the groves during spring flush and were thus unaffected by sprays applied weeks or months earlier. This strategy became a key component of ACP management in Florida, including area-wide control. 14.2.1.4  Edge effect Another commonly observed characteristic of ACP distribution is the so-called edge effect: higher populations observed on perimeter trees

(Sétamou and Bartels, 2015) (Chapter 11). The edge effect is especially notable where the perimeter faces an open area versus adjoining blocks of citrus. Whether the effect is due to psyllids from the outside resting on the edges before moving into the interior or accumulating on open perimeters rather than flying into a void is still not clear. The long-term and seemingly universal existence of the edge effect would seem to favor the latter explanation. Regardless, the management response has been to concentrate some sprays on block perimeters to reduce the number of applications made to the entire grove (Sétamou and Bartels, 2015; Stansly, 2016). 14.2.2  Area-wide management The creation of ‘Citrus Health Management Areas’ (CHMAs) including area-wide vector control was the top priority recommendation made by a strategic planning committee addressing HLB in Florida (National Research Council, 2010). The first area-wide sprays against ACP in Florida were applied in late fall and winter 2008–2010 in southwest Florida (Stansly et al., 2009, 2010). Gulf Coast Citrus Growers and Hendry County Cooperative Extension promoted the effort, the University of Florida Institute of Food and Agricultural Sciences (UF IFAS) provided technical advice, and the Immokalee office of FDACS/DPI monitored ACP populations using the ‘tap sample’ method (Qureshi et al., 2009). More than 70,000 acres (28,000 ha) were sprayed by air and 30,000 acres (12,000 ha) by ground. An organophosphate insecticide was ­recommended for the first (late fall) spray and a pyrethroid for the winter spray. Results indicated a more than 15-fold decrease in ACP numbers the following spring compared with untreated blocks. The CHMA program per se was initiated in 2011 with 68 management areas designated by 2017 (https://crec.ifas.ufl.edu/extension/chmas/ index.shtml). In the more successful of these, individual growers stepped forward to take leadership in organizing cooperative sprays throughout the year. The CHRP program undertook the task of monitoring ACP in 6000 blocks throughout the citrus growing area using 50 tap samples evenly divided between the four corners and center of each block. ACP numbers steadily declined through



Integrated Management – Past, Present and Future

2014; additionally, the CHMA program was credited with success toward a major goal of reversing early trends t­oward insecticide resistance, although no clear explanation was provided (Coy et al., 2016) (Figs 14.1, 14.2). 14.2.2.1  Demise of CHMA ACP tap-sampled numbers rebounded from 2015 through 2017 in a total reversal of early positive trends (Fig. 14.1). Reasons for this rebound were variously attributed to warm winters with consequent tree flushing, a fall-off in spray frequency for economic reasons and/or insecticide resistance. Additionally, while the strategic planning committee recommended government support and CHMA managers were empowered to enforce best management practices (National Research Council, 2010), grower organizers were pretty much left on their own. Non-cooperating growers (‘bad neighbors’) were often blamed for the inability to control ACP (Boina et al., 2009) but there was no way to force compliance. Furthermore, there was a growing skepticism among growers regarding yield and economic benefits of ACP control once trees were infected with HLB, in spite of ample evidence to the contrary (Stansly et al., 2014; Morzo and Stansly, 2017; Tansey et al., 2017). These studies demonstrated a direct relationship between

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ACP control and yield gain. While it is not clear how ACP control can improve the yield from infected trees, indications are that reducing the re-inoculation rate allows the tree to produce clean flush and thus re-establish movement of photosynthate from foliage to roots. Nevertheless, many grower organizers became frustrated with the lack of cooperation and ceased efforts to coordinate sprays.

14.2.3  Effect of hurricanes Given the profound impacts on the Florida citrus industry in recent years (Albrigo et al., 2005), it is surprising how little has been published about the effects of hurricanes on ACP. Studies made on the effects of wind direction on flight direction have largely been inconclusive (Martini et al., 2014; Lewis-Rosenblum et al., 2015). Albrigo et al. (2005) did mention the likelihood that psyllids would rapidly inhabit new flush stimulated by hurricane-induced defoliation. In fact, that is exactly what happens. Hurricane Irma devastated Florida citrus on 10 September 2017, causing an estimated $760 million in damage, including 65% crop loss and hitting especially hard in the southwest growing region. Defoliation was widespread and severe, followed by a massive flush and explosion of ACP. Tap samples

Statewide average ACP population

Average ACP population per block

35 30 25 2012 2013 2014 2015 2016 2017

20 15 10 5 0 Jan

Feb March April May June July

Aug Sept Oct

Nov

Dec

Fig. 14.1.  Average number of adult ACP per 50 tap samples over some 5000 sample sites throughout Florida, prepared by Brandon Page (https://crec.ifas.ufl.edu/extension/chmas/index.shtml).

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25

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15

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Arbitrary threshold used for EIL Studies

5

0

11

1–

1 20

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–0

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–0

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Fig. 14.2.  Average number of psyllid adults per 50 tap samples in the CHMAs and arbitrary threshold used for economic injury level (EIL) studies.

conducted by the CHRP program on some 850 blocks in the southwest region averaged 17.7 ACP per 50 taps in November compared with an average of 2.01 that month for the previous 6 years. 14.2.4  Economic injury levels Chapter 10 provides a full explanation of current progress establishing economic injury levels (EILs) for ACP control in mature citrus with high incidence of HLB. Here we only repeat the main points and limitations. The initial impetus for the study came from previous research in a ­commercial citrus grove that showed significant yield responses from bearing orange trees with high incidence of HLB in response to insecticidal control targeting ACP. Treatments included dormant sprays and additional applications based on nominal thresholds that decreased from 0.5 to 0.1 per tap sample in ACP populations globally from 2008 through 2016 (Stansly et al., 2014; Tansey et al., 2017).

A follow-up study established EILs based on annual cumulative numbers of ACP in two other commercial orange groves from 2010 through 2013 (Monzo and Stansley, 2017). This study included four treatments: sprays at two different threshold levels (two and seven per ten tap samples), a more typical frequency of 10–11 sprays a year and an untreated check. While the threshold system has yet to be widely employed in Florida citrus, it has the potential of optimizing spray frequency in mature groves with high incidence of HLB. On the other hand, the EILs for ACP in young citrus during the first 3–5 years after planting has not been addressed. The economic value of protecting young trees from HLB is presumably quite high. The corresponding EIL would be low and would require evaluation of pathogen incidence and titer in low populations of ACP population and several years of production data. Nevertheless, it is clear that, due to their small size and constant flushing patterns, early infection spells trouble for the young tree and for the grower hoping to make a profit before HLB has too great an impact on tree health.



Integrated Management – Past, Present and Future

14.2.5  Product choices and efficacy Early studies indicated the efficacy of pyrethroids (fenpropathrin), organophosphates (chlorpyrifos), neonicotinoids (imidacloprid) and aldicarb (Stansly et al., 2002; Childers and Rogers, 2005). The list of effective products was lengthened in subsequent years to include other chemical classes, including: spinetoram (model of action (MoA) 5); fenpropathrin and tolfenpyrad (MoA 21A); spirotetramat (MoA 23); and cyantraniliprole (MoA 28) (Qureshi et al., 2014a). The efficacy and persistence against ACP of these and other foliar and soil-applied insecticides, as indicated by numerous replicated field studies conducted in Florida between 2005 and 2013, were summarized in Qureshi et al. (2014a). Reduction of adult and/or nymphal populations of 90% or more was documented with 23 insecticides in nine MoAs. 14.2.6  Resistance management The likelihood of resistance to key MoAs was always a concern, given the high degree of dependency on insecticides to manage ACP and thus mitigate the effects of HLB. As mentioned above, monthly foliar applications in Florida directed mostly against ACP were common at the time of the study (Monzo and Stansley, 2017) (Chapter 10). Soil applications of systemic neonicotinoid insecticides may be applied every 30 days, interspersed with foliar sprays of different active ingredients (http://edis.ifas.ufl.edu/in686). Early studies at only five sites around the state indicated moderate levels of resistance to the most used insecticides: imidacloprid (resistance ratio (RR) 35) and to a lesser extent chlorpyrifos (RR 18) and fenpropathrin (RR 5). There was also an RR of 15 to thiamethoxam (even though it had not yet been labeled for citrus), indicating likely crossresistance with imidacloprid. Increased levels of detoxifying enzymes such as general esterase, glutathione S-transferase and monooxygenase levels in field populations compared with the susceptible laboratory colony were documented. Increased activity of cytochrome P450 enzymes in field populations of ACP was also documented in a later paper (Tiwari et al., 2011, 2013). Complacency, particularly with regard to neonicotinoid resistance, was shattered in the spring of 2017 when control failures were

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documented in several new plantings in northern, eastern and central citrus growing regions (L.L. Stelinski, unpublished data). Blame was placed on the until-then recommended practice of applying neonicotinoids to young trees as soil drenches or by fertigation (mortality by ingestion), although foliar applications of different modes of action were also urged, to delay selection for resistance (Stansly et al., 2016a). Subsequent research indicated that ingestion required greater concentrations of active ingredient to attain levels of mortality in adult ACP comparable with direct contact (Langdon and Rogers, 2017). However, nymphs were found to be ten and 28 times more sensitive to thiamethoxam and imidacloprid, respectively, than adults. Nymphs are primarily, if not wholly, responsible for the acquisition of CLas (Chapter 8), and therefore are the principal target of systemic insecticides applied to young trees. Continuous soil applications would presumably result in long periods of sublethal exposure and thus rapid selection for resistance by the ACP population. Therefore, conserving the effectiveness of these chemicals necessitates rotation with other modes of action. 14.2.7  Secondary pest resurgence Given the dependence on insecticidal control for ACP management and the relatively low cost of broad-spectrum compared with selective materials, the decimation of natural enemies and consequent secondary pest resurgence were to be ­expected (Qureshi and Stansly, 2007). Indeed, increased incidence and persistence of citrus rust mite (Phyllocoptruta oleivora) and citrus leafminer (P. citrella) is a general trend, to a lesser extent with aphids (Aphis spiraecola and A. gossypii), various scale insects, and spider mites (Panonychus citri, Eutetranychus banksi), with sporadic flare-ups of mealybugs (Planococcus citri) and whiteflies (Singhiella citrifolii) (Stansly, ­unpublished data).

14.3  Biological Control 14.3.1  Natural control Chapter 6 deals with biotic sources of mortality, so here we merely provide some highlights as well

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as implications for integrated pest management. Repeated observations of cohorts on young flush as well as exclusion techniques conducted prior to widespread insecticidal control indicated high levels of mortality to immature ACP (Michaud, 2004; Qureshi and Stansly, 2009). Mortality averaged 80.2 ± 4.5% in replicated cohorts observed over 17 months (Qureshi and Stansly, 2009). Mortality was highest in summer and fall, averaging over 90% and reached 100% in June. Estimated net reproductive rate averaged 27.0 ± 5.0 on uncaged shoots compared with 180.0 ± 16.2 on caged shoots. Ladybeetles appeared to be responsible for the largest proportion of this mortality, with certain species, especially Olla v-nigrum, enjoying a numerical response to the huge availability of an especially suitable prey species (Michaud, 2001; Michaud and Olsen, 2004). Additional contributions to ACP mortality came from lacewings, spiders and apparently even Asian cockroaches (Blattella asahinai). In contrast, apparent parasitism by T. radiata was low, in part at least due to intraguild predation (Michaud, 2004).

14.3.2 Augmentation The ectoparasitoid T. radiata (Waterston) (Hymenoptera: Eulophidae) is the more abundant and widespread of the two known parasitoids of D. citri. It occurs naturally or has successfully established wherever ACP is found, including ­Florida, where it was first released in 1999 from wasps collected in Taiwan and Vietnam (Hoy, 2005; Chen and Stansly, 2014). In contrast, the endoparasitoid Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae) did not establish in Florida, despite considerable effort (Rohrig et al., 2012). D. citri is the only known host of T. radiata, although Bactericera cockerelli Sulc was found parasitized by it at low levels (5%) (Hoddle and Pandey, 2014). A program of augmentative release was initiated in 2007 with the original ‘Florida Strain’ of T. radiata using M. paniculata as a plant host for ACP and augmented in 2009 by wasps from Guangdong Province (China), Vietnam and Punjab, Pakistan (Qureshi et al., 2009). Early results indicated the significantly increased incidence of parasitism in

blocks receiving releases (Qureshi et al., 2012, 2014b). FDACS-DPI opened the new Dundee FL rearing facility in 2014 to augment production from the Gainesville Biological Laboratory and a year later produced 2.3 million wasps for release throughout the state, with a focus on urban and abandoned citrus. Little information is available on the impact of these releases. Effects on key natural enemies of different-­ intensity spray programs were documented by Monzo et al. (2014), using exclusion techniques and direct observation. Monthly sprays of recommended insecticides for control of ACP significantly reduced predation on immature ­ stages, especially during the critical late winter/ early spring flush. Significantly reduced populations of ladybeetles, spiders and pseudomyrmicine ants associated with insecticide use were seen over seasons and in most seasons. Reversing the effects of over a decade of intensive insecticide use to re-establish the former natural enemy complex will take a concerted effort of reducing insecticide use, especially of broadspectrum insecticides. Nevertheless, the potential for biotic mortality is equal to or better than most insecticide treatments, especially in summer. Given the present trends of increasing ACP populations, decreasing confidence in insecticidal control, and the success of horticultural practices to mitigate effects of HLB, a more integrated approach to psyllid control is warranted and would likely be welcome.

14.4  Cultural Control Cultural controls are by definition practices directed at improving crop performance but also may help manage pests, or at least mitigate damage. By this broad definition, enhanced ­nutrition, including foliar sprays, slow-release or ­frequent liquid fertilizer application, soil pH adjustments, and soil amendments such as compost, are cultural practices that mitigate HLB symptoms by improving tree growth and yield but may actually increase ACP populations by enhanced flushing. Practices such as flush management, UV-reflective mulch or citrus under cover are directed specifically at ACP control but also have major effects on tree performance.



Integrated Management – Past, Present and Future

14.4.1  Foliar nutrition, soil pH and amendments Foliar application of micronutrients is intended to partially compensate for their reduced absorption and translocation to the canopy by an HLB-compromised root system. A significant yield response from at least one foliar nutrient program was documented by Stansly et al. (2014) and Tansey et al. (2017). Lowering soil pH raised to high levels by alkaline irrigation water over the years is intended to increase the availability of micronutrients and thus accomplish the same thing (Graham et al., 2013, 2015). Composts increase water-holding capacity, cation exchange capacity and nutritional efficiency in Florida’s sandy soils (Ozores-Hampton et al., 2015).

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mulch will reflect all visible wavelengths but not UV (Croxton and Stansly, 2014). Nevertheless, UV was the most attractive wavelength to ACP reported by Paris et al. (2017). So how then does UV-reflective mulch repel psyllids? A logical explanation is that psyllids fly toward the sky when taking off. However, once in flight they must maintain the source of UV above to stabilize direction, whereas UV from below is destabilizing. The growth and yield benefits observed on young trees grown on metalized mulch are partly due to reduced incidence of HLB as a consequence of reduced ACP (Stansly et al., 2016b). However, more efficient delivery of water, fertilizer and chemical through the obligatory drip irrigation is undoubtedly an additional factor favoring the plastic mulch system. Here then is an example of a strategy conceived to avoid an insect-vectored disease that provided additional horticultural benefits.

14.4.2  Flush management ACP reproduction is totally dependent on young citrus shoots or flush. The transmission of HLB is also greatly enhanced by the presence of ACP nymphs on citrus flush (Lee et al., 2015; Hall et al., 2016). Therefore, reducing the frequency of flushing throughout the year could have a profound effect on ACP populations and HLB transmission. Unfortunately, citrus flushing patterns are largely under the control of temperature and rainfall. The effect of exogenous lAA, GA and cytokinins on bud development varies at different times of the year, suggesting control related to critical levels in the endogenous balance of hormones (Altman and Goren, 1974). However, although practiced in China, there is little or no information on the manipulation of shoot flushing with plant hormones.

14.4.3  Reflective mulch Planting on beds covered with metalized polyethylene mulch, while standard procedure in Florida vegetable production, is a new but promising strategy in citrus (Croxton and Stansly, 2014). The mulch is especially effective at repelling ACP that use visual perception to find the host plant (Paris et al., 2015). It seems that the effect is in response to ultraviolet light, because white

14.4.4  Protected crops Growing citrus under protective screen (CUPS) is another approach to controlling ACP and the ravages of HLB (Schumann et al., 2017). Naturally, costs are high and only justifiable for the small portion of the Florida citrus industry devoted to fresh fruit production. Nevertheless, this could be significant in terms of acreage and crop value. Another disadvantage is vulnerability to Florida’s relatively frequent hurricanes. Time will determine the extent to which CUPS systems become part of Florida citrus production.

14.5  Production Trends Citrus production peaked in Florida during the 1997/8 season at 304 million boxes, of which 244 million boxes (9961 million tonnes) were oranges (FDACS/NASS, 2017). Effects of HLB were felt in the southern part of the state by 2007/8, when statewide production of oranges was still at 170 million 40.8 kg boxes (equivalent to 6940 million tonnes) produced on 218,000 ha with an on-tree value of $1125 million. By the 2015/16 season, production dropped to 81.6 million boxes (3331 million

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metric tons) produced on 157,600 ha with an on-tree value of $948 million. Interestingly, the five southwestern citrusgrowing counties comprising approximately 26% of citrus acreage have lost citrus production at about half the rate of the rest in spite of a poor start for the 3 years of this period (Fig.  14.3). Why this pattern? Higher relative losses early in the HLB epidemic were clearly the results of the movement of the disease from south to north. The southwest (‘Gulf ’ region) has the greatest proportion of large-acreage citrus plantations. The distribution of relatively large farms among few owners has facilitated efficient management and implementation of better vector management and other means of mitigating effects of the disease through improved horticultural practices (Stansly et al., 2014). The Gulf area was first to implement ­voluntary area-wide sprays during the 2008/9 ‘dormant’ (winter) season with virtually 100% compliance (Qureshi and Stansly, 2010; Stansly et al., 2009, 2010; Tansey et al., 2017). Although there is presently limited formal structure to area-wide management, Gulf growers continue relatively aggressive vector control programs

during the dormant season and throughout the rest of the year, as well as innovative practices to optimize growing conditions and minimize plant stress. These practices have allowed most Gulf growers and others in the state to remain profitable despite a harsh economic climate. Statewide citrus production has continued to drop, with 68.8 million boxes (2.81 million tonnes) of oranges harvested in 2016/17 valued at $781 million, the lowest since the 2004/5 hurricane-affected season (Fig. 14.3). However, early estimates of the 2017/18 crop foretold significant increases over previous years, creating hope that the industry had finally turned around. These hopes were dashed when Hurricane Irma ripped through the citrus belt south to north on 10 September 2017. Nevertheless, prices were the highest they had ever been so there was hope for the future.

Acknowledgement Thanks to E. Grafton-Cardwell for a helpful review.

100% SW Florida

Statewide-SWF

Compared to 2007–08

80%

60%

40%

20%

0% 09

2008–

10

2009–

11

2010–

12

2011–

13

2012–

2013–

14

15

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Fig. 14.3.  Citrus production in southwest Florida and state-wide compared with 2007/8.

16

2015–



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References Albrigo, L.G., Attaway, J., Bowman, K., Buker, R.S., Castle, W.S., Hancock, K.W., McCoy, C.W., Muraro, R.P., Rogers, M.E., Ritenour, M.A. et al. (2005) The impact of three hurricanes in 2004 on the Florida Citrus Industry: experiences and lessons learned. Proceedings Florida State Horticultural Society 11, 66–74. Altman, A. and Goren, R. (1974) Growth and dormancy cycles in citrus bud cultures and their hormonal control. Physiologia Plantarum 30, 240–245. Boina, D.R., Meyer, W.L., Onagbola, E.O. and Stelinski, L.L. (2009) Quantifying dispersal of Diaphorina citri (Hemiptera: Psyllidae) by immunomarking and potential impact of unmanaged groves on commercial citrus management. Environmental Entomology 38(4), 1250–1258. Bové, J.M., (2006) Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. Journal of Plant Pathology 88, 7-37. Chen, X. and Stansly, P.A. (2014) Biology of Tamarixia radiata (Hymenoptera: Eulophidae), parasitoid of the citrus greening disease vector Diaphorina citri (Hemiptera: Psylloidea): a mini review. Florida ­Entomologist 97(4), 1404–1413. Childers, C.C. and Rogers, M.E. (2005) Chemical control and management approaches of the Asian citrus psyllid, Diaphorina citri Kuwayama (Homoptera: Psyllidae) in Florida citrus. Proceedings of Florida State Horticultural Society 118, 49–53. Cooper, W.C., Peynado, A., Furr, J.R., Hilgeman, R.H., Cahoon, G.A. and Boswell, S.B. (1963) Tree growth and fruit quality of Valencia oranges in relation to climate. Proceedings of American Society of Horticultural Science 118, 49–53. Coy, M.R., Bin, L. and Stelinski, L.L. (2016) Reversal of insecticide resistance in Florida populations of Diaphorina citri (Hemiptera: Liviidae). Florida Entomologist 99(1), 26–32. Croxton, S.D. and Stansly, P.A. (2014) Metalized polyethylene mulch to repel Asian citrus psyllid, slow spread of huanglongbing and improve growth of new citrus plantings. Pest Management Science 70(2), 318–323. FDACS/DPI (2007) Citrus Health Response Plan (CHRP) State of Florida. Available at: https://www.freshfromflorida.com/content/download/24023/486874/chrp.pdf (accessed 4 February 2020). FDACS/NASS (2017) Florida Citrus Statistics 2015–16. Florida Department of Agriculture and Consumer Services/USDA National Agricultural Statistics Service, Tallahassee, Florida. Graham, J.H., Johnson, E.G., Gottwald, T.R. and Irey, M.S. (2013) Pre-symptomatic fibrous root decline in citrus trees caused by huanglongbing and potential interaction with Phytophthora spp. Plant Disease 97(9), 1195–1199. doi: 10.1094/PDIS-01-13-0024-RE. Graham, J., Gerberich, K., Bright, D. and Johnson, E. (2015) Excess bicarbonate in soil and irrigation water increases fibrous root loss and decline of Huanglongbing-affected citrus trees in Florida. In: Graham, J. and Stelinski, L. (eds) Abstracts from the 4th International Research Conference on Huanglongbing. Journal of Citrus Pathology 2(1), 15. Available at: http://escholarship.org/uc/item/9jw2w9850.1094/ PDIS-01-13-0024-RE. Halbert, S.E. (1998) Entomology section. Triology 37, 6–7. Halbert, S.E., Niblett, C.L., Manjunath, K.L., Lee, R.F. and Brown, L.G. (2002) Establishment of two new vectors of citrus pathogens in Florida. Proceedings of the International Society of Citriculture 9, 1016–1017. Halbert, S.E., Manjunath, K.L., Ramadugu, C., Brodie, M.W., Webb, S. and Lee, R.F. (2010) Trailers transporting oranges to processing plants move Asian citrus psyllids. Florida Entomologist 93, 33–38. Halbert, S.E., Manjunath, K., Ramadugu, C. and Lee, R.F. (2012) Incidence of huanglongbing-associated ‘Candidatus Liberibacter asiaticus’ in Diaphorina citri (Hemiptera: Psyllidae) collected from plants for sale in Florida. Florida Entomologist 95(3), 617–624. Hall, D.G. and Hentz, M.G. (2011) Seasonal flight activity by the Asian citrus psyllid in east central Florida. Entomologia Experimentalis et Applicata 139, 75–85. Hall, D.G. and McCollum, G. (2011) Survival of adult Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae), on harvested citrus fruit and leaves. Florida Entomologist 94(4), 1094–1096. Hall, D.G., Albrecht, U. and Bowman, K.D. (2016) Transmission Rates of ‘Ca. Liberibacter asiaticus’ by Asian citrus psyllid are enhanced by the presence and developmental stage of citrus flush. Journal of Economic Entomology 109, 558–563. Hoddle, M.S. and Pandey, R. (2014) Host range testing of Tamarixia radiata (Hymenoptera: Eulophidae) sourced from the Punjab of Pakistan for classical biological control of Diaphorina citri

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(Hemiptera: Liviidae: Euphyllurinae: Diaphorinini) in California. Journal of Economic Entomology 107(1), 125–136. Hoy, M.A. (2005) Classical biological control of citrus pests in Florida and the Caribbean: interconnections and sustainability. In: Hoddle, M.S. (ed.) Proceedings, Second International Symposium on Biological Control of Arthropods, Davos, Switzerland, 12–16 September, 2005. Publication FHTET-2005-08, USDA Forest Service, Washington, D.C. pp. 237–253. Johnston, N.S. (2018) Dispersal patterns of Asian citrus psyllid (Diaphorina citri Kuwayama) and secondary-­ host interactions. MS thesis, University of Florida. Langdon, K.W. and Rogers, M.E. (2017) Neonicotinoid-induced mortality of Diaphorina citri (Hemiptera: Liviidae) is affected by route of exposure. Journal of Economic Entomology 110(5), 2229–2234. doi: 10.1093/jee/tox231. Lee, J.A., Halbert, S.E., Dawson, W.O., Robertson, C.J., Keesling, J.E. and Singer, B.H. (2015) Asymptomatic spread of huanglongbing and implications for disease control. Proceedings of the National Academy of Sciences 112(24), 7605–7610. Lewis-Rosenblum, H., Martini, X. and Tiwari, S. (2015) Seasonal movement patterns and long-range dispersal of Asian citrus psyllid in Florida citrus. Journal of Economic Entomology 208, 3–10. Manjunath, K.L., Halbert, S.E., Ramadugu, C., Webb, S. and Lee, R.F. (2008) Detection of ‘Candidatus Liberibacter asiaticus’ in Diaphorina citri and its importance in the management of citrus huanglongbing in Florida. Phytopathology 98(4), 387–396. Martini, X., Hoyte, A. and Stelinski, L. (2014) Abdominal color of the Asian citrus psyllid (Hemiptera: Liviidae) is associated with flight capabilities. Annals of the Entomological Society of America 2014, 842–847. Martini, X., Addison, T., Fleming, B., Jackson, I., Pelz-Stelinski, K. and Stelinski, L.L. (2013) Occurrence of Diaphorina citri (Hemiptera: Liviidae) in an unexpected ecosystem: the Lake Kissimmee State Park forest, Florida. Florida Entomologist 96(2), 658–660. Michaud, J.P. (2001) Numerical response of Olla v-nigrum (Coleoptera: Coccinellidae) to infestations of Asian citrus psyllid (Hemiptera: Psyllidae) in Florida. Florida Entomologist 84(4), 608–612. Michaud, J.P. (2004) Natural mortality of Asian citrus psyllid (Homoptera: Psyllidae) in central Florida. Biological Control 29(2), 260–269. Michaud, J.P. and Olsen, L.E. (2004) Suitability of Asian citrus psyllid, Diaphorina citri, as prey for ladybeetles. BioControl 49(4), 417–431. Monzo, C. and Stansly, P.A. (2017) Economic injury levels for Asian citrus psyllid control in process ­oranges from mature trees with high incidence of huanglongbing. PLOS ONE 12(4), e0175333. Monzo, C., Qureshi, J.A. and Stansly, P.A. (2014) Insecticide sprays, natural enemy assemblages and predation on Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae). Bulletin of Entomological Research 104(5), 576–585. Monzo, C., Arevalo, H.A., Jones, M.M., Vanaclocha, P., Croxton, S.D., Qureshi, J.A. and Stansly, P.A. (2015) Sampling methods for detection and monitoring of the Asian citrus psyllid (Hemiptera: Psyllidae). Environmental Entomology 44(3), 780–788. doi: 10.1093/ee/nvv032 National Research Council (2010) Strategic Planning for the Florida Citrus Industry: Addressing Citrus Greening Disease. National Academies Press, Washington, DC. Paris, T.M., Croxton, S.D., Stansly, P.A. and Allan, S.A. (2015) Temporal response and attraction of Diaphorina citri to visual stimuli. Entomologia Experimentalis et Applicata 155, 137–147. Paris, T.M., Allan, S.A., Udell, B.J. and Stansly, P.A. (2017) Wavelength and polarization affect phototaxis of the Asian citrus psyllid. Insect 8, 88. doi: 10.3390/insects8030088. Ozores-Hampton, M., Adair, R. and Stansly, P.A. (2015) Using compost in citrus. Citrus Industry 96(12), 8–11. Qureshi, J.A. and Stansly, P.A. (2007) Integrated approaches for managing the Asian citrus psyllid Diaphorina citri (Homoptera: Psyllidae) in Florida. Proceedings of the Florida State Horticultural ­Society 120, 110–115. Qureshi, J.A. and Stansly, P.A. (2009) Exclusion techniques reveal significant biotic mortality suffered by Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae) populations in Florida citrus. Biological Control 50, 129–136. Qureshi, J.A. and Stansly, P.A. (2010) Dormant season foliar sprays of broad-spectrum insecticides: an effective component of integrated management for Diaphorina citri (Hemiptera: Psyllidae) in citrus orchards. Crop Protection 29(8), 860-866. Qureshi, J.A., Rogers, M.E., Hall, D.G. and Stansly, P.A. (2009) Incidence of invasive Diaphorina citri (Hemiptera: Psyllidae) and its introduced parasitoid Tamarixia radiata (Hymenoptera: Eulophidae) in Florida citrus. Journal of Economic Entomology 102(1), 247–256.



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Qureshi, J.A., Rohrig, E.A. and Stansly, P.A. (2012) Introduction and augmentation of natural enemies for management of Asian citrus psyllid and HLB. Citrus Industry 93(6), 14–16. Qureshi, J.A., Kostyk, B.C. and Stansly, P.A. (2014a) Insecticidal suppression of Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae) vector of huanglongbing pathogens. PLOS ONE 9(12), e112331. Qureshi, J.A., Rohrig, E.A., Stuart, R. J., Hall, D.G., Leppla, N.C. and Stansly, P.A. (2014b) Imported parasitoids for biological control of Asian citrus psyllid. Citrus Industry 95(6), 10–13. Rohrig, E.A., Hall, D.G., Qureshi, J.A. and Stansly, P.A. (2012) Field release in Florida of Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae), an endoparasitoid of Diaphorina citri (Homoptera: P ­ syllidae), from mainland China. Florida Entomologist 95(2), 479–481. Schumann, A., Waldo, L. and Wright, A. (2017) Research update: citrus undercover production systems and whole tree thermotherapy. Citrus Industry 97(11), 8–13. Sétamou, M. and Bartels, D.W. (2015) Living on the edges: spatial niche occupation of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), in citrus groves. PLOS ONE 10(7), e0131917. Stansly, P.A. (2016) Effective and economical spray programs for psyllid control. Citrus Industry 97(12), 16–17. Stansly, P.A., Conner, J.M. and Brushwein, J.R. (2002) Control of citrus leafminer and Asian citrus psylla in sweet orange, 2001. Arthropod Management Tests 27, D10. Stansly, P.A., Arevalo, H.A., Zekri, M. and Hamel, R. (2009) Cooperative dormant spray program against Asian citrus psyllid in SW Florida. Citrus Industry 90, 14–15. Stansly, P.A., Arevalo, H.A. and Zekri, M. (2010) Area-wide psyllid sprays in Southwest Florida: an update on the cooperative program aimed at controlling the HLB vector. Citrus Industry 91, 6–8. Stansly, P.A., Arevalo, H.A., Qureshi, J.A., Jones, M.M., Hendricks, K., Roberts, P.D. and Roka, F.M. (2014) Vector control and foliar nutrition to maintain economic sustainability of bearing citrus in F ­ lorida groves affected by huanglongbing. Pest Management Science 70(3), 415–426. Stansly, P.A., Qureshi, J.A., Stelinski, L.L. and Rogers, M.E. (2016a) Asian citrus psyllid and citrus leafminer. In: Rogers, M.E., Dewdney, M.M. and Vashisth, T. (eds) 2017–18 Florida Citrus Production Guide. UF-IFAS, Gainesville, Florida. Stansly, P.A., Croxton, S. and Sherrod, J. (2016b) Big boost in young tree growth and yield from insecticides and metalized mulch. Citrus Industry 97(3), 20–22. Tansey, J.A., Vanaclocha, P., Monzo, C., Jones, M. and Stansly, P.A. (2017) Costs and benefits of insecticide and foliar nutrient applications to huanglongbing-infected citrus trees. Pest Management Science 73(5), 904–916. Tiwari, S., Mann, R.S., Rogers, M.E. and Stelinski, L.L. (2011) Insecticide resistance in field populations of Asian citrus psyllid in Florida. Pest Management Science 67(10), 1258–1268. Tiwari, S., Killiny, N. and Stelinski, L.L. (2013) Dynamic insecticide susceptibility changes in Florida populations of Diaphorina citri (Hemiptera: Psyllidae). Journal of Economic Entomology 106(1), 393–399. Tsai, J.H. and Liu, Y.H. (2000) Biology of Diaphorina citri (Homoptera: Psyllidae) on four host plants. Journal of Economic Entomology 93, 1721–1725. Tsai, J.H., Wang, J.J. and Liu, Y.H. (2002) Seasonal abundance of the Asian citrus psyllid, Diaphorina citri (Homoptera: Psyllidae) in southern Florida. Florida Entomologist 85(3), 446–451.

15 

Area-wide Management of Asian Citrus Psyllid in Texas

Mamoudou Sétamou* Texas A&M University-Kingsville, Citrus Center, Weslaco, Texas, USA

15.1 Introduction The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), is a known vector of the bacterial pathogens Candidatus ­Liberibacter spp., causal agents of the deadly ­citrus greening disease or huanglongbing (HLB) (da Graça, 1991; Bové, 2006). ACP was first reported in the US in the state of Florida in 1998 (Halbert, 1998), but has since invaded all citrus producing states in the country. In Texas, ACP was first reported in Hidalgo County on hedges of orange jasmine in September 2001 (French et al., 2001), but has subsequently spread throughout the state and by 2016 all citrus producing counties were infested. D. citri is a highly prolific insect that reproduces exclusively on young flush shoots of its host plants (Grafton-Cardwell et al., 2013), although adult ACP can feed and survive on many plant parts (Sétamou et al., 2008). As citrus trees sequentially produce flush cycles throughout the year starting from the spring flush, ACP population dynamics is intimately related to flush cycles (Sétamou and Bartels, 2015). In Texas, citrus is produced mainly for the fresh fruit market, and fruits sold on the fresh market are at least five times more valuable than those processed for juice. Because of the

need to provide cosmetically appealing fruits, the citrus production goal is not only to preserve crop yields, but also most importantly to produce larger fruits with few, if any, blemishes. For several decades, insects, mites and diseases have been the most important plant health problems affecting citrus in Texas (Sekula, 2009). Traditionally, citrus pests and diseases are controlled with control decisions made on a grove-to-grove or block-to-block basis by individual growers. Historically, commercial citrus groves have been using a sustainable integrated pest management (IPM) program emphasizing biological control for management of sucking insect pests, including whiteflies, blackflies, soft and armored scales and citrus mealybugs. However, pesticides are widely used against phytophagous mites and diseases in Texas citrus (Sekula, 2009). Three to four seasonal and well-timed applications of pesticides are routinely made per season. Occasionally, broad-spectrum insecticides were used as part of the management of insect pests, although such non-selective insecticides have been implicated in reduction of natural enemies leading to secondary pest outbreaks. This traditional IPM program provided satisfactory pest and disease control in commercial groves until the invasion of ACP in 2001 (Sekula, 2009), but the situation has changed

*  Email: [email protected]

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© CAB International 2020. Asian Citrus Psyllid: Biology, Ecology and Management of the Huanglongbing Vector (eds. J.A. Qureshi and P.A. Stansly)



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with the detection of HLB in Florida in fall 2005 (Halbert, 2005) and in Texas in 2012 (Kunta et al., 2012). Increased pest status of D. citri has led to significant changes in citrus pest management in Texas as it has elsewhere (Sétamou et al., 2012a). Adult ACP are highly mobile (Sétamou et  al., 2012b) and most contact insecticides have limited persistence (Childers and Rogers, 2005). Therefore, traditional uncoordinated grove-level pesticide application can only achieve short-term control. Consequently, frequent pesticide applications must be made by individual growers to achieve satisfactory D.  citri ­control, with the attendant economic and environmental costs. To be effective, management of mobile pests must be done on an area-wide basis and a coordinated fashion to target the total pest population in a region (Knippling, 1979). This has been one of the three pillars set forth by the National Research Council to mitigate the spread and incidence of HLB (NRC, 2010). Demonstration of this concept in the commercial citrus producing area of Texas was initiated in 2007 as a 3-year grower participatory trial. Goals were to improve understanding of ACP population fluctuations in relation to citrus phenology in Texas, and to determine the best timing for insecticide applications that ­prevent successful reproduction of D. citri. Following these participatory trials, Texas citrus growers were offered in early 2010 a proactive area-wide integrated management system (AIMS) targeting D. citri populations. AIMS relied on the use of two regional coordinated dormant sprays in November and February, respectively; plus individual grove sprays with judiciously selected insecticides at the onset of flush cycles during the active growing season, and an area-wide monitoring program to report on ­ ACP densities and infestation levels. Grower educational meetings coupled with field visits were instrumental in the rapid adoption of the new concept of AIMS that resulted in substantial r­ eductions of ACP populations in commercial groves in Texas. This chapter reviews factors regulating ACP populations in Texas, and discusses the steps in the development and ­ ­implementation of its area-wide management program in the context of a multi-pest control approach.

15.2  Citrus Production in Texas 15.2.1  Commercial citrus Citrus is an important crop in Texas, grown both as a commercial crop and as residential trees. In 2016, approximately 240,000 t of fresh and processed citrus were harvested from 9915 ha for a total value of $80,749,000 (USDA-NASS, 2017). However, new plantings have been under way in the past few years and the total acreage planted in commercial citrus is ca. 11,330 ha. Commercial citrus is dominated by the red grapefruit, which represents 65% of the total acreage. Three grapefruit varieties, namely ‘Rio Red’, ‘Ruby Red’ and ‘Henderson’, are grown, but the former is by far the most common. Sweet oranges, including the early-maturing (Marrs and Navel), mid-season (Pineapple) and late-­ maturing (Valencia) oranges, represent 30% of total acreage, and the remaining 5% are made of more acidic citrus (limes and lemons) and other citrus species. Although about 200 ha of zipper-­ skin mandarin groves (cv. Satsuma) are grown in Brazoria, Chambers, Galveston, Jefferson and Orange counties along the Gulf Coast of Texas (M. Sétamou, unpublished results), commercial citrus groves are concentrated in the three southern-most counties in the Lower Rio Grande Valley (LRGV) of Texas (Fig. 15.1). These commercial groves are interspersed with residential areas, creating a complex agro-urban system in which D. citri control must be adapted to the constant exchange of adults between the two habitats. Due to the economic importance of citrus in the LRGV, the area-wide ACP management program concentrated initially in south Texas.

15.2.2  Dooryard citrus Citrus trees are abundant and commonly found in dooryards in south Texas. During a survey of 700 houses, we recorded that 62% of residences had at least one of nine citrus tree species, comprising Citrus sinensis (sweet orange), C. paradisi (grapefruit), C. aurantiifolia (Mexican lime), C. latifolia (Persian lime), C. limon (lemon), C. reticulata (mandarin), C. paradisi × C. reticulata (tangelo),

236

M. Sétamou

Zapata

Willacy Cameron

Starr Hidalgo

US 281

HIGHWAY 186

HIGHWAY

107

Y1 06

FM 508

HW

Exp 83 / 12

US

77 HWY 100

Varietal Legend Grapefruit Lemon Mix of Citrus Lemons and Limes

Orange Tangelo Tangerine

Fig. 15.1.  Distribution of commercial groves in south Texas.

C. japonica (kumquat), and C. aurantium (sour orange). As in commercial groves, grapefruit ­ (31.3%) and sweet orange (38.1%) were the dominant citrus species recorded in residential areas in south Texas (Fig. 15.2). The number of citrus trees per backyard ranged from one to 30, with a mean of 3.8 and a median of 3. Using the number of household units from 2015 tax data, the number of residential citrus was estimated to be 938,300 trees, equivalent to about 2500 ha (using a standard planting density of 373 trees

per hectare) of citrus groves scattered throughout south Texas. The abundance and diversity of residential citrus species affect the population dynamics of D. citri. In fact, unlike commercially grown trees that exhibit the same phenology within a grove, asynchronous flush cycles are commonly observed on residential citrus trees. In addition, these trees remain largely unmanaged and thus may harbor high numbers of D. citri. Arredondo (2009) reported that residential citrus constitutes reservoirs



Area-wide Management of Asian Citrus Psyllid in Texas 237

0.7%

0.7%

0.1%

0.4% Sweet orange

17.9%

Grapefruit 38.4%

Lime Lemon

10.6%

Tangerine Kumquat Tangelo 31.2%

Sour orange

Fig. 15.2.  Species composition of residential citrus in south Texas.

of D. citri that could serve as sources of infestations for commercial groves. In a mark–release–­ recapture study using fluorescent dust, we observed a constant exchange of ACP between the urban habitat and adjacent commercial groves (M. Sétamou, unpublished results). For one psyllid moving from an adjacent grove to backyard citrus, five psyllids moved from backyard trees to a commercial grove (Fig. 15.3) (M. Sétamou, unpublished results). Hence, residential citrus trees play more a role of source than sink of ACP for commercial groves in south Texas.

15.3  ACP Population Fluctuations in Relation to Citrus Tree Phenology Population fluctuations of D. citri both in commercial groves and residential areas are intimately related to citrus phenology (Arredondo, 2009; Sétamou and Bartels, 2015). In south Texas, D. citri is found year-round in citrus groves. D. citri overwinters as adults mostly at the underside of mature citrus leaves from November to February and initiates the first ­ ­generation of the year by laying eggs on newly ­produced flush shoots during the first flush cycle in mid-February/March. Although D. citri overwinters as adults in south Texas, immatures have been recorded on limes and lemons during

the winter (November to early February) as climatic conditions allowed for new flush shoot development of these citrus species (Arredondo, 2009). It therefore appears that the most limiting factor of D. citri reproduction during the winter in south Texas is the availability of young flush shoots. One generation of D. citri is generally completed per flush cycle, but up to two generations can be recorded during the fall flush in late September/October (Sétamou and Bartels, 2015). D. citri populations gradually increase from the first flush cycle of the year with subsequent flush cycles during the active citrus growing ­season until the last flush cycle of the year in September/ October, when its populations are at the highest (Flores et al., 2009; Sétamou and Bartels, 2015). However, due to the high the summer temperatures and the short duration of the summer flush cycle, D. citri densities during the summer flush cycle are lower than during spring and fall flush cycles (Sétamou and Bartels, 2015). Although many factors, including tree age, citrus variety, grove location, tree position in the grove and grove care practices such as i­rrigation and pruning or hedging, significantly affect ACP populations, Sétamou and Bartels (2015) reported that the presence of young shoots was the most important factor determining densities of all life stages of D. citri. At the onset of a flush cycle, D. citri are immigrants into the grove with many adults coming in for reproduction. It is as if 

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M. Sétamou

Grove to Urban 18.8%

Urban to Grove 29.7%

Grove to Grove 81.2% (a)

(b)

Urban to Grove 21.4% Urban to Urban 70.3%

Grove to Urban 78.6% (c)

Fig. 15.3.  Change of habitat by D. citri at the interface of commercial groves and urban citrus in mark–release–recapture studies. (A) Marked D. citri released in groves and recaptured in urban area or groves. (B) Marked D. citri released in urban areas and recaptured in urban area or groves. (C) Percentage of D. citri recaptured in habitat different from released one.

the grove turns the green light on for psyllid recruitment, whereas at the end of flush cycle most adults leave the groves, similar to the red light being turned on within the grove. Four to six flush cycles are generally recorded in mature commercial groves (Fig.15.4), but young plantings of up to 4 years old produce new flush shoots more frequently. Consequently, young plantings harbor significantly more ACP than mature groves (Sétamou and Bartels, 2015). Similarly, in a study conducted in 2007 prior to the implementation of area-wide management in south Texas, higher D. citri populations were recorded in sweet orange compared with grapefruit groves using yellow sticky traps (Fig. 15.5) (M. Sétamou, unpublished results). D. citri also exhibited strong border effects in its niche occupation strategy, with outer groves in a cluster and border trees within a grove harboring more ACP (Sétamou and Bartels, 2015). In residential areas, Arredondo (2009) also recorded significant variations in D. citri populations. Lime and lemon trees were the most infested and harbored significantly higher numbers of ACP relative to sweet orange and grapefruit. Among the four citrus evaluated in residential areas, D. citri populations were lowest on grapefruit trees. It is not known whether host plant preference per se, flush shoot maturation or flush shoot physical or chemical characteristics, or a combination of these factors, contribute most to the observed differential ACP densities between citrus species. However, tissue softness (Sétamou et al., 2016a) and developmental stage (Cifuentes-­ Arenas et al., 2018) of flush shoots is a key

­determining factor for acceptability by D. citri for oviposition. Also, citrus flush shoots exhibit strong ontogenetic variation in both their physical characteristics and chemical contents d ­ uring maturation (Sétamou et al., 2016a; C ­ ifuentesArenas et al., 2018).

15.4  Development and Implementation of ACP Area-wide Integrated Management System (AIMS) in Commercial Groves 15.4.1  Development of AIMS Most commercial citrus in Texas is grown according to standards and procedures of worldwide Global Good Agricultural Practices (GLOBALG.A.P.). Because fruits are produced mainly for both domestic and export fresh markets, citrus growers voluntarily follow these standards to ensure that their crops meet the worldwide certification requirements. Hence, an integrated system is being used for the management of the many diseases and arthropod pests affecting citrus in the subtropical climate of south Texas. In light of the absence of effective natural enemies (Sekula, 2009; Maoz et al., 2014), mite pests and citrus diseases are mostly controlled using chemical miticides and fungicides, respectively. Growers mostly rely on natural control achieved by predators and parasitoids for the management of many sucking insect pests, including citrus blackflies, whiteflies, mealybugs,



Area-wide Management of Asian Citrus Psyllid in Texas 239

Jan

Feb

H A R V E S T

Mar

Apr

May

Jun

Jul

Aug

Sep

Oct

Nov

Vegetative Flush Bud Break

Flowering and Fruit Set

Fruit Development and Expansion

Root Flush

Maturation Root Flush

Dec H A R V E S T

Fig. 15.4.  Phenology of mature citrus in south Texas. 8

Mean # D. citri adults/trap

7

Sweet orange

Grapefruit

17-Jun

17-Aug

6 5 4 3 2 1 0 17-Apr

17-May

17-Jul

17-Sep

17-Oct

17-Nov

2007 Fig. 15.5.  Comparative populations of Diaphorina citri adults in grapefruit and sweet orange in Texas prior to the area-wide management. Ten pairs of groves were evaluated, with each pair having adjacent grapefruit and sweet orange groves and receiving the same grove care.

and various armored and soft scales. However, in case of outbreaks of these secondary pests as observed in recent years due to broad-spectrum insecticides being used for ACP control, many growers resort to synthetic chemical or organic insecticides for the control of these sucking ­insect pests. ACP control tactics were developed to be integrated into ongoing IPM programs in commercial groves. In Texas, approaches to ACP management can be categorized as both proactive and therapeutic. Proactive ACP control focuses on reducing populations during the overwintering period and also preventing adults from successfully reproducing by controlling at the onset of a flush cycle. Proactive ACP sprays during the overwintering period are termed ‘dormant sprays’. Dormant sprays were first developed in Florida (Stansly et al., 2009; Qureshi and ­Stansly,

2010), and successfully tested under Texas conditions. To ensure that ACP populations are reduced over the entire citrus growing region, two coordinated dormant sprays, one in November and another in early February, are recommended in south Texas. An additional third proactive coordinated spray is recommended mid to late August, just prior to the September flush cycle. Traditionally, September is a rainy month in south Texas, and almost all groves have a flush cycle in September. Thus, to prevent a dramatic increase in D. citri populations due to the abundance of young shoots suitable for reproduction in groves during that flush cycle (Sétamou and Bartels, 2015), a coordinated proactive areawide spray has been recommended to growers since 2014. These three coordinated area-wide sprays (November, February and August) are complemented with well-timed proactive sprays

240

M. Sétamou

Texas has been to prevent successful reproduction on young expanding flush shoots (Sétamou and Alabi, 2018). Because organic insecticides are not very persistent for the control of D. citri (Qureshi et al., 2015), two coordinated sprays are recommended for organic growers within a 2-week period for each coordinated spray in conventional groves, one at the beginning and another at the end of each defined spray window (Fig. 15.6B). For young plantings, in addition to foliar sprays, soil-applied systemic insecticides (e.g. neonicotinoids or anthranilic diamide) are

at the onset of flush cycles or therapeutic sprays when any immatures are detected on expanding flush shoots during the active growing season at individual grove level, thus defining the yearlong ACP control program in Texas (Fig. 15.6A). Due to the fact that ACP grove colonization starts from border trees where most of its populations are recorded (Sétamou and Bartels, 2015), border sprays are sandwiched between whole-grove sprays to prevent ACP population establishment in groves (Sétamou and Alabi, 2018). Hence, the focus of D. citri control in

(A) Timing of psyllid sprays in conventional citrus groves in Texas

Nov

Dec

Jan

Feb

Mar

Apr

Whole Spray

Whole Spray

Whole Spray

As determined by scouting

May

Jun

Jul

Aug

Whole Spray

Dormant Spray-Coordinated

Dormant Spray-Coordinated

Oct

= Border Spray

Whole Spray-Coordinated

Border sprays sandwiched

Coordinated whole grove sprays

Sep

(B) Timing of psyllid sprays in organic citrus groves in Texas

Oct

Nov

Dec

Jan

Feb

Mar

Apr

May

Jun

Jul

Aug

Whole Spray

2-wk apart

Whole Spray

Whole Spray-Coordinated Whole Spray-Coordinated

Whole Spray

2-wk apart

Whole Spray

Whole Spray

2-wk apart

Whole Spray Whole Spray

2-wk apart

Whole Spray

Dormant Spray-Coordinated

Dormant Spray-Coordinated

2-wk apart

Sep

Fig. 15.6.  Insecticide spray timing and approach in conventional and organic mature groves for D. citri control in Texas.



Area-wide Management of Asian Citrus Psyllid in Texas 241

recommended to ensure effective ACP control, given these young trees produce new flush shoots more frequently (Fig. 15.7). The main thrust of ACP control in commercial groves in Texas is application of insecticides at the right moment. As ACP control is done mainly to mitigate HLB, it is important that lowest possible populations are present when citrus is the most vulnerable to bacterial infection. Such an approach holds its justification from the fact that young developing flush shoots not only regulate ACP biotic parameters, abundance and population fluctuations (Sétamou and Bartels, 2015; Cifuentes-Arenas et al., 2018), but also enhance CLas acquisition (Sétamou et al., 2016b) and transmission (Hall et al., 2016). In addition, newly infected young shoots can become infectious within 10–15 days after infection (Lee et al., 2015), and thus an important source of bacterial inoculum for acquisition by D. citri, especially at the nymphal stage (Ammar et al., 2016). Hence, preventing D. citri feeding and successful reproduction on young shoots is critical for effective HLB mitigation via vector control. Despite the presence of generalist predators and of the nymphal parasitoid Tamarixia radiata in commercial groves in south Texas, D. citri populations are mainly controlled with insecticides. Most are applied as foliar sprays with the exception of young plantings where systemic insecticides are soil-applied via chemigation or as soil drench. Conventional citrus growers have relied heavily on neonicotinoid (imidacloprid, thiametoxam, clothianidin), pyrethroid (beta-­ cyfluthrin, cyfluthrin, fenpropathrin, zeta-­ cypermethrin), carbamate (carbaryl, oxamyl), organophosphorus (chlorpyrifos, phosmet), insect growth regulator (diflubenzuron, buprofezin) insecticides, and several other newly developed insecticides with different modes of action, including

a butenolide (flupyradifurone), two mitochondrial electron transport inhibitors (METIs) (tolfenpyrad, fenpyroximate), and an anthranilic diamide (cyantraniliprole), among others. In organic groves, growers rely mostly on spray oils (e.g. JMS stylet oils, Suffoil-X, Superior spray oil), botanicals (e.g. Trilogy (Neem oil), Pyganic or Evergreen (pyrethrum), surfactants like Oroboost) and insecticidal soaps (e.g. M-Pede, Des-X) and kaolin spray. With the exception of the two coordinated dormant sprays, these proactive and therapeutic applications are directed at a variety of pests and diseases in addition to ACP by using tank mixes of different active ingredients. Integration of ACP control into ongoing citrus pest management programs has proved to be cost effective by reducing the number of passes and may also preempt secondary pest outbreaks. With the new concept of coordination of sprays by most growers in south Texas, flare-­ ups of secondary pests can also be anticipated and prevented by incorporating suitable pesticides for mitigating these risks. The only downside of a multi-target pest control is that, to be effective, it must be conducted as ground sprays with relatively high spray volumes of at least 935 l/ha (100 gallons per acre) or more, thus excluding the use of low-volume spray methods (aerial or ultra-low volume (ULV)). Spraying with sufficient water volume is critical in south Texas to prevent quick evaporation due to the warm weather and mostly clear days. As D. citri is a diurnal insect mostly active during the day (Sétamou et al., 2014), recommendations are also made to perform spray applications in the evening or at night when adults are not flying. The duration of ACP control in citrus after a spray application varies with application

Foliar sprays as needed (other active ingredients than systemic used in soil-drench; fungicides, miticides)

Soil-drench application of systemic insecticides every two months Thiamethoxam

Jan

Feb

Imidacloprid

Mar

Apr

Cyantraniliprole

May

Jun

Clothianidin

Jul

Aug

Imidacloprid

Sep

Oct

Fig. 15.7.  D. citri control program in young plantings (up to 4 years old) in Texas.

Cyantraniliprole

Nov

Dec

242

M. Sétamou

methods and type of insecticide (Stansly et al., 2012). Shorter residual controls are generally obtained with reduced-volume spray methods such as low-volume sprays with fogger (Stansly et al., 2014). The higher-volume spray applications with ground rig applicators provide the longest residual control of D. citri for the same insecticides. Many systemic (neonicotinoids, butenolide, sulfoximine) and organophosphorus (chlorpyrifos) insecticides have an effective action period of an entire flush cycle (or 21–28 days) against D. citri. Other insecticides (mostly contact insecticides) exhibit shorter residual control (15–21 days: M. Sétamou, unpublished results). Most organic insecticides have a relatively shorter duration of ACP control (10–14 days) as compared with conventional insecticides (Qureshi et al., 2015). Systemic insecticides applied to the soil in young plantings (up to 4 years old) are released slowly with time and provide longer residual control of 2 months or longer. However, they require up to 7 days after application for trees to accumulate concentrations effective against D. citri in the sandy loam soil of south Texas (Sétamou et al., 2010). These soil-applied systemic insecticides are suitable for protecting young plantings and constitute the backbone of ACP control in newly established groves. To prevent potential resistance development, growers are recommended to intersperse soil applications of these systemic insecticides, with foliar sprays of an insecticide with a different mode of action (Fig. 15.6). 15.4.2  Implementation and grower education Field demonstration of any new concept generally facilitates its adoption by stakeholders. Hence, grower participatory trials were established in 2009 at eight conventional and two ­organic citrus groves to demonstrate the effectiveness of area-wide integrated management system (AIMS) for ACP in south Texas. Collaborating growers conducted all spray applications while project scientists from TAMUK Citrus ­Center provided specific recommendations on the pesticides to be used either as stand-alone or in tank mixes for ACP or multi-target pest control, as well as timing of spray applications based on

pre-established pest thresholds. Any ACP immatures (eggs or nymphs) detected on a sample of 40 expanding flush shoots or 20% of flush shoots infested with adults based on either visual observation or tap sampling are used as spray triggers. Organized field visits during the growing season allowed the citrus grower community to evaluate outcomes of this integration of ACP control into current IPM programs. Field visits were coupled with targeted educational meetings with the goal of raising grower awareness of the potential impact of D. citri and HLB on the citrus industry, and the need for initiating proactive ACP control. Data on D. citri and other citrus pest populations were collected by project personnel and provided to participating growers in a timely manner every 2 weeks. Due to the fact that grove care operations (irrigation, fertilization, hedging and pruning) varied from one grove to another, there was no synchrony of tree phenology during the active growing season from March to August. Thus, it was nearly impossible to coordinate spray applications among the different participating groves in the demonstration project during this active growing period. However, from mid-August to February almost all groves exhibited phenological synchrony. Hence, three coordinated sprays were applied plus additional sprays as needed (Fig. 15.6). The AIMS program was well received by most growers from its onset in January 2010. Commercial citrus acreage under AIMS gradually increased from 57% in 2010 to 92% in 2016 (Fig. 15.8), but approximately 8% of commercial acreage in south Texas has still not implemented AIMS, despite intensive outreach efforts. These groves mostly belong to small-scale growers who either do not want to pay the additional costs for all ACP sprays ($150–$200), or have no spray equipment to implement the program during the prescribed timeframe.

15.4.3  Sampling and monitoring of D. citri populations Transmission of CLas occurs when infective ACP adults move to a non-infected tree. Both D. citri nymphs and adults are capable of acquiring and transmitting the bacterium (Lee et al., 2015; Ammar et al., 2016), but adult movement



Area-wide Management of Asian Citrus Psyllid in Texas 243

100 90 80

% Acreage

70 60 50 40 30 20 10 0

2010

2011

2012

2013

2014

2015

2016

Year

Fig. 15.8.  Percentage of total citrus acreage under area-wide integrated D. citri management system in Texas, from the inception of the program in January 2010.

is n ­ecessary for tree-to-tree transmission to occur within a grove or between groves. Nymphs are more efficient than adults at acquiring the bacterium, and adults emerging from these CLas-positive nymphs remain infective their entire life (Ammar et al., 2016). Effectiveness of ACP spray programs can be evaluated by monitoring population trends using methods such as tap sampling or trapping of adult populations on trees and observations of the presence of nymphs on expanding flush shoots. Information collected during monitoring is also used to make decisions about spray applications for ACP control. To implement IPM of D. citri, it is necessary to monitor adult and nymphal populations. Effective IPM strategies require a full understanding of the biology and life history of a pest within an ecosystem (Dent, 1991). The most frequently used techniques for determining adult densities of D. citri include trapping (Flores et al., 2009; Hall, 2009; Hall et al., 2010), stem tapping (Hall and Hentz, 2010) and visual observation (Sétamou et al., 2008), but trapping with yellow sticky cards or ACP traps has been shown to be the most sensitive method (Miranda et al., 2018). Due to the fact that ACP exhibits strong border effect in its niche occupation in groves, recommendations are made to monitor its populations on trees along grove borders (Sétamou and Bartels, 2015). This border sampling strategy results in an overall inflation of D. citri densities in the grove, but it allows for timely spray application by growers. Sampling is done once every 2 weeks

by visually examining or tapping five flush shoots per tree for a total of 16 to 20 trees per grove (four to five trees per each border), or by deploying four yellow sticky cards per grove, one at each corner of the grove. Traps are hung directly on the outer canopy of trees at ca. 1.5 m high and evaluated every 2 weeks in situ, but replaced every 4 weeks. Action thresholds of ten D. citri adults per trap have been proposed to growers to implement spray application when no new flush shoots are present in the grove in Texas. However, detection of any single infested flush shoot with immatures (eggs or nymphs) when young expanding flush shoots are present leads to control action. Hence, the phenological stage of trees is considered in making ACP spray recommendations in Texas. From 2010 to 2012, yellow sticky traps were used for monitoring D. citri populations. A total of 62 sentinel commercial groves were evaluated every 2 weeks by the Texas Citrus Pest and Disease Management Corporation (TCPDMC). Tap sampling was adopted for ACP sampling from 2013 to 2016 as a more rapid monitoring method (Hall and Hentz, 2010; Monzo et  al., 2015), and the number of groves monitored was increased to 110. Mean numbers of adult D. citri showed a gradual decrease with time, indicating an overall reduction of ACP populations on an area-wide basis (Fig. 15.9). It was also apparent that the inclusion of a third coordinated spray in late August from 2014 was effective at preventing fall peaks previously observed in D. citri populations in south Texas.

244

M. Sétamou

90 80

Mean D. citri/48 taps

70 60

2013 2014 2015 2016

50 40 30 20 10 0 1/15 1/29 2/12 2/26 3/12 3/26 4/9 4/23 5/7 5/21 6/4 6/18 7/2 7/16 7/30 8/13 8/27 9/10 9/24 10/8 10/22 11/5 11/19 12/3 12/17

Fig. 15.9.  Population fluctuations of D. citri adults in commercial citrus groves (n = 110) showing reduction in the pest densities as a result of the implementation of area-wide integrated management systems in Texas.

15.5  ACP Control in Minimally Managed and Abandoned Groves Trees in abandoned groves hardly survive after 3  years under the climatic conditions of south Texas. Our observations showed that these trees are generally covered with vines, thus making the habitat unsuitable for ACP feeding and reproduction. However, recently abandoned and minimally managed groves continue to produce new flush shoots and are suitable feeding and reproduction sites for D. citri. In general, the TCPDMC encourages owners of abandoned or minimally managed groves to remove and destroy trees with the incentive of maintaining their agricultural tax exemptions for 5 years thereafter. For these groves, ACP populations can only be controlled by natural enemies. D. citri is attacked by a large number of natural enemies, which consist mostly of generalist predators (e.g. coccinellids, chrysopids, syrphids) and entomopathogenic fungi (e.g. Hirsutella, Isaria) and the nymphal parasitoid T. radiata. It is believed that T. radiata was initially introduced into south Texas along with its ACP host before the introduction and mass releases of the Pakistani strain from 2010 onward (Flores and Ciomperlik, 2017). The efficacy of entomopathogenic fungi is dependent on weather conditions, with relatively high rates of infection (about 30%) recorded in

late August to October, mainly during the fall flush cycles when weather conditions are moist and warm (A. Chow, 2017, personal communication). Very high rates of predation on D. citri nymphs by coccinellids have been observed in south Texas. In exclusion studies conducted in 2006 and 2009, we observed that with up 90% of D. citri nymphs were devoured by various predators in organic groves mainly during the spring flush cycles. The impact of these predators on ACP populations ­varied greatly with time of the year, with lower predation rates (< 20%) recorded after spring (M. Sétamou, unpublished results).

15.6  ACP Control in Urban Settings Traditionally, the abundant urban citrus trees remain largely unmanaged in Texas with no pest control. At best, residential trees received tree care programs such as irrigation, fertilization and/or pruning – operations that stimulate flush shoot growth and consequently enhance ACP populations. An effective and sustainable area-wide control program must target ACP populations in all settings where host plants are present. In light of the fact that residential citrus has been shown to play more the role of source than sink of ACP for commercial groves (Fig. 15.3), a pilot project was conducted in 2012 to evaluate



Area-wide Management of Asian Citrus Psyllid in Texas 245

was evaluated at 8.6% in 2010 prior to releases and increased subsequently from 32% in 2011 to 82% in 2014, stabilizing around 40% in 2015 and 2016 (Flores and Ciomperlik, 2017).

15.7  Nursery Regulations to Mitigate ACP In addition to effective ACP control and removal of HLB infected trees, planting of ‘clean nursery trees’ was another component of the threepronged approach recommended by the National Academy of Sciences for HLB management (NAS, 2010). Production of citrus nursery stock in ACP-exclusion enclosures coupled with insecticide applications have been recommended to prevent exposure of nursery trees to bacteriferous D. citri and further mitigate the risk of nurseries acting as sources of CLas spread. ­Towards this end, the Texas Department of Agriculture (TDA) in collaboration with the citrus industry and the research community in Texas put in place new regulations focused on transitioning citrus nursery owners from the traditional open-field production nurseries to ­ ­enclosed insect-resistant structures in October 2013, and to prevent movement of outside nursery stock into the commercial citrus zone of south Texas. Nevertheless, trees budded prior to the enactment of the new regulation by the TDA were initially ‘grandfathered’ into giving nurserymen a grace period of 1 year to comply with the new regulation. However, detection of HLB-­ infected trees in an open-field nursery in April 2014 (Alabi et al., 2014) led to an immediate

250 Treated

200

Untreated control

150 100 50

Fig. 15.10.  Effectiveness of residential ACP control with insecticides.

01 2 /2 23 10 /

/2 23 9/

/2 8/ 23 Date

01 2

01 2

01 2 /2 7/ 23

01 2 /2 6/ 23

/2 5/ 23

/2 4/ 23

01 2

0

01 2

Mean # D. citri/trap

the outcome of treatment of all urban citrus trees with insecticides in two adjacent residential blocks. All citrus trees within the first block were treated with insecticides for ACP control, while trees of the second neighborhood were used as untreated control. Insecticides registered for home use were selected and applied by a licensed private applicator. A total of six foliar and six soil-drench applications were made during the year. Foliar sprays were made within a short time span (3 days maximum) to ensure that D. citri were controlled in a coordinated fashion in the residential block. Five sentinel sweet orange trees were selected within each block for ACP monitoring. One yellow sticky card was hung on the outer canopy of each sentinel tree and replaced every 2 weeks. Insecticide treatments significantly reduced but did not eliminate ACP populations. Mean numbers of D. citri caught on sticky cards were 90% fewer than from untreated trees (Fig. 15.10). Despite such a high efficiency, the cost of insecticide treatment in urban settings ($6 per tree per application for a total of $36 per year) was too high and prohibitive to allow for large-scale implementation. Thus, the Texas citrus industry turned to biological control for ACP management in urban areas. Two rearing facilities were established, one by APHIS-PPQ and another by TCPDMC for mass production of T. radiata. On average, about 50,000 parasitoid adults were released every month from July 2010 to March 2014, increasing to 200,000 per month from April 2014 onward. Post-release assessment confirmed establishment of T. radiata throughout urban areas of south Texas. Percentage parasitism of D. citri nymphs by native T. radiata

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M. Sétamou

the onset, but established groves may immediately benefit from ACP reduction provided by windbreak through the enactment of screen barriers along grove edges. In Texas, preliminary data have shown a 60–70% reduction in ACP population in citrus orchards with perimeter fencings (COPF) of 3–3.7 m (10–12 ft) with 15.8 Perspectives ACP-resistant screen. These screen fences mimic the effect of ACP-­excluding structures used in The ACP problem in citrus is due to the bacter- nurseries and under citrus under protective ium and the disease it causes. Whereas ACP structures (CUPS). It is well established that citrus row orientapopulations can be effectively managed in nursery settings through the combination of tion affects penetration of wind into groves as ACP-exclusion structures and insecticides, cit- well as tree exposure to sunlight and hence the rus growers rely almost solely on insecticides for uniformity of light distribution within the tree the control of D. citri in commercial groves. canopy. Thus, row orientation can affect the Systemic insecticides such as neonicotinoids microclimate in groves and potentially the suit­ and butenolides rotated with sprays of different ability of trees for ACP colonization. D. citri exmodes of activity constitute the backbone of hibits positive phototaxis (Sétamou et al., 2014); ACP control in young plantings, while only fo- its developmental parameters are affected by liar applied insecticides are mostly available for temperature (Hall et al., 2013), and its flight bethe management of this insect pest in mature havior is dependent on wind speeds (M. Sétagroves. Groves in Texas and other states are pri- mou, unpublished results). At our latitude in marily open-field habitats that are largely inter- south Texas where citrus is commercially grown mingled with urban settings where abundant (25º 85' to 26º 45'), horticultural recommendaand unmanaged ACP host plants that serve as tions are for north–south orientation. Coinciconstant sources of ACPs and of the bacterium dentally, groves with rows oriented north–south are present. Therefore, effective and sustainable had the lowest ACP infestations and populations ACP management requires the use of a multi-­ relative to any other orientation (east–west, tactic approach. Management strategies that ex- northeast–southwest and southeast–northwest) ploit the ecological behavior of D. citri need to be (M. Sétamou, unpublished results). At present, despite the amount of research developed and integrated into ACP management carried out to find odorants attractive to D. citri programs. D. citri is often found in aggregated distri- adults (Patt and Sétamou, 2010; Aksenov et al., butions, with populations congregating along 2014; Coutinho-Abreu et al., 2014; Zanardi grove borders. This border effect in ACP niche et al., 2018), there are no semiochemicals availoccupation is at least partially due to grove col- able to manipulate the behavior of ACP in onization starting from border trees (Sétamou groves. The strong response of D. citri adults to and Bartels, 2015). Hence, border management visual cues (Flores et al., 2009; Hall et al., 2010; via insecticide sprays or physical barriers pre- Sétamou et al., 2014), can be exploited by develventing initial ACP incursion can be critical oping attract-and-kill devices that could be steps in protecting the groves (Sétamou and Ala- ­deployed along grove borders. Yellow and lime-­ bi, 2018). Martini et al. (2015) reported that green devices impregnated with insecticides trees adjacent to windbreaks had lower popula- can be deployed on border trees to lure and kill tions of ACP. However, D. citri movement in D. citri. As D. citri is important primarily due to its groves is a bidirectional one regulated by flush cycles. When new flush cycles are developing, CLas transmission ability and less as a direct pest there is a recruitment of incoming adults (‘green of citrus, management approaches need to intelight on’), while emigration of adults (‘red light grate the relationships between vector and on’) is observed at the end of flush cycle (Séta- pathogen. The ability of D. citri to acquire (Sétamou and Bartels, 2015). Nevertheless, new cit- mou et al., 2016b) and to transmit CLas (Hall rus plantings can incorporate live windbreaks at et  al., 2016) is significantly enhanced by the and total ban of all open-field nurseries. Currently, all citrus nursery trees must be produced in TDA-certified structures to ensure production of clean trees as an integral component of HLB mitigation efforts in Texas.



Area-wide Management of Asian Citrus Psyllid in Texas 247

presence of young expanding flush shoots. Moreover, D. citri is an efficient vector when the bacterium is acquired in the nymphal stage (Ammar et al., 2016). Thus, one important strategy in managing D. citri would be to prevent adult feeding and successful reproduction during flush cycles via the use of efficacious insecticides. Between flush cycles, ACP populations can be controlled with softer materials such as mineral oils or kaolin that have proven effective

at reducing adult ACP populations or at preventing feeding, or simply by conducting border sprays as incorporated into the year-long season control program in Texas (Fig. 15.6). Citrus trees are evergreens that are attacked by a diverse group of insects, mites and pathogens. To be sustainable, ACP management cannot be a stand-alone program. It must be integrated into ongoing pest and disease management program as a multi-pest strategy.

References Aksenov, A.A., Martini, X., Zhao, W., Stelinski, L.L. and Davis, C.E. (2014) Synthetic blends of volatile, phytopathogen-induced odorants can be used to manipulate vector behavior. Frontiers in Ecology and Evolution 2, 78. doi: 10.3389/fevo.2014.00078. Alabi, O.J., Kunta, M., Dale, J. and Sétamou, M. (2014) Survey and detection of ‘Candidatus Liberibacter asiaticus’ in a citrus nursery facility in South Texas. Plant Health Progress 15, 184–188. doi:10.1094/ PHP-RS-14-0028.Huanglongbing. Ammar, E.-D., Ramos, J.E., Hall, D.G., Dawson, W.O. and Shatters R.G. Jr (2016) Acquisition, replication and inoculation of Candidatus Liberibacter asiaticus following various acquisition periods on Huanglongbing-infected citrus by nymphs and adults of the Asian citrus psyllid. PLOS ONE 11(7), e0159594. doi: 10.1371/journal.pone.0159594. Arredondo, I.M.J. (2009) Abundance and population dynamics of Asian citrus psyllid Diaphorina citri ­Kuwayama (Hemiptera: Psyllidae) as affected by flush shoots in different host plants. MSc ­Thesis, Texas A&M University-Kingsville. Bové, J.M. (2006) Huanglongbing: a destructive, newly emerging, century-old disease of citrus. Journal of Plant Pathology 88, 7–37. Childers, C.C. and Rogers, M.E. (2005) Chemical control and management approaches of the Asian citrus psyllid, Diaphorina citri Kuwayama (Homoptera: Psyllidae), on Florida citrus. Proceedings of Florida State Horticultural Society 118, 49–53. Cifuentes-Arenas, J.C., de Goes, A., de Miranda, M.P., Beattie, G.A.C. and Lopes, S.A. (2018) Citrus flush shoot ontogeny modulates biotic potential of Diaphorina citri. PLOS ONE 13(1), e0190563. doi: 10.1371/journal.pone.0190563 Coutinho-Abreu, I.V., Forster, L., Guda, T. and Ray, A. (2014) Odorants for surveillance and control of the Asian citrus psyllid (Diaphorina citri). PLOS ONE 9(10), e109236. doi: 10.1371/journal.pone.0109236. da Graça, J.V. (1991) Citrus greening disease. Annual Review of Phytopathology 1991, 109–136. Dent, D.R. (1991) Insect Pest Management. CAB International, Wallingford, UK. Flores, D. and Ciomperlik, M. (2017) Biological control using the ectoparasiotid, Tamarixia radiata, against the Asian citrus psyllid, Diaphorina citri, in the lower Rio Grande Valley of Texas. Southwestern Entomologist 42, 49–59. Flores, D., Hall, D.G., Jenkins, D.A. and Sétamou, M. (2009) Abundance of Asian citrus psyllid on yellow sticky traps in Florida, Puerto Rico, and Texas Citrus Groves. Southwestern Entomologist 34, 1–11. French, J.V., Kahlke, C.J. and da Graça, J.V. (2001) First record of the Asian citrus psylla, Diaphorina citri Kuwayama (Homoptera: Psyllidae), in Texas. Subtropical Plant Science 53, 14–15. Grafton-Cardwell, E.E., Stelinski, L.L. and Stansly, P.A. (2013) Biology and management of Asian citrus psyllid, vector of huanglongbing pathogens. Annual Review of Entomology 58, 413–432. Halbert, S.E. (1998) Entomology section. Triology 37, 6–7. Halbert, S.E. (2005) The discovery of huanglongbing in Florida. In: Proceedings 2nd International Citrus Canker and Huanglongbing research workshop. Florida Citrus Mutual, Orlando, Florida, p. 50. Hall, D.G. (2009) An assessment of yellow stick card cards as indicators of the relative abundance of adult Diaphorina citri in citrus. Journal of Economic Entomology 102, 446–452. Hall, D.G. and Hentz, M.G. (2010) Stick card and stem tap sampling protocols for the Asian citrus psyllid (Hemiptera:Psyllidae). Journal of Economic Entomology 103, 541–549.

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Hall, D.G., Sétamou, M. and Mizell, R.F. (2010) A comparison of sticky cards for monitoring Asian citrus psyllid (Diaphorina citri Kuwayama). Crop Protection 29, 1341–1346. Hall, D.G., Richardson, M.L., Ammar, E.D. and Halbert, S.E. (2013) Asian citrus psyllid, Diaphorina citri, vector of citrus huanglongbing disease. Entomologia Experimentalis et Applicata 146, 207–223. doi:10.1111/eea.12025 Hall, D.G., Albrecht, U. and Bowman, K. (2016) Transmission rates of Ca. Liberibacter asiaticus by Asian citrus psyllid are enhanced byt the presence and development stage of citrus flush. Journal of Economic Entomology 109, 558563. Hodges, A.W. and Spreen, T.H. (2012) Economic impacts of citrus greening (HLB) in Florida, 2006/07–2010/11. FE 903. Available at: https://crec.ifas.ufl.edu/extension/greening/PDF/FE90300.pdf. (accessed 1 January 2020). Knippling, E.F. (1979) The Basic Principles of Insect Population Suppression and Management. Science and Education Administration, US Dept of Agriculture, Washington, DC. Kunta, M., Sétamou, M., Skaria, M., Li, W., Nakhla, M.K. and da Graça, J.V. (2012) First report of citrus huanglongbing in Texas. Phytopathology 102, S4.66. Lee, J.A., Halbert, S.E., Dawson, W.O., Robertson, C.J., Keesling, J.E. and Singer, B.H. (2015) Asymptomatic spread of huanglongbing and implications for disease control. Proceedings of the National Academy of Sciences 112(24), 7605–7610. doi:10.1073/pnas.1508253112 Maoz, Y., Gal, S., Argov, Y., Domeratzky, S., Melamed, E., Gan-Mor, S., Coll, M. and Palevsky, E. (2014) Efficacy of indigenous predatory mites (Acari: Phytoseiidae) against the citrus rust mite Phyllocoptruta oleivora (Acari: Eriophyidae): augmentation and conservation biological control in Israeli citrus ­orchards. Experimental and Applied Acarology 63, 295–312. Martini, X., Pelz-Stelinski, K.S. and Stelinski, L.L. (2015) Absence of windbreaks and replanting citrus in solid sets increase density of Asian citrus psyllid D. citri populations. Agriculture, Ecosystems and Environment 212, 168–174. Miranda, M.P., dos Santos, F.L., Bassanezi, R.B., Montesino, L.H., Barbosa, J.C. and Sétamou, M. (2018) Monitoring methods for Diaphorina citri Kuwayama (Hemiptera: Liviidae) on citrus groves with different insecticide application programmes. Journal of Applied Entomology 142, 89–96. doi: 10.1111/jen.12412. Monzo, C., Arevalo, H.A., Jones, M.M., Vanaclocha, P., Croxton, S.D., Qureshi, J.A. and Stansly, P.A. (2015) Sampling methods for detection and monitoring of the Asian citrus psyllid (Hemiptera: Psyllidae). Environmental Entomology 44, 780–788. NRC (National Research Council) (2010) Strategic Planning for the Florida Citrus Industry. Addressing Citrus Greening Disease. National Academies Press, Washington, DC. Patt, J.M. and Sétamou, M. (2010) Responses of the Asian citrus psyllid to volatiles emitted by the flushing shoots of its rutaceous host plants. Environmental Entomology 39, 618–624. Qureshi, J.A. and Stansly, P.A. (2010) Dormant season foliar sprays of broad spectrum insecticides: an effective component of integrated management for Diaphorina citri (Hemiptera: Psyllidae) in citrus orchards. Crop Protection 29, 860–866. Qureshi, J.A., Kostyk, B.C. and Stansly, P.A. (2014) Insecticidal suppression of Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae), vector of Huanglongbing pathogens. PLOS ONE 9(12), e112331. doi: 10.1371/journal.pone.0112331. Qureshi, J.A., Stansly, P.A. and Kostyk, B.C. (2015) Organic insecticides for control of Asian citrus psyllid and citrus leafminer on oranges. Arthropod Management Tests 40(1), D13. doi: 10.1093/amt/tsv037. Sekula, D. (2009) Novel strategies for sustainable management of citrus rust mite, Phyllocoptruta oleivora (Ashmead) (Acari: Eriophyidae) in Texas. MS Thesis, Texas A&M University-Kingsville. Sétamou, M. and Alabi, O.J. (2018) SMART HLB: An ecological approach to improve HLB management. Citrograph 9, 24–27. Sétamou, M. and Bartels, D.W. (2015) Living on the edges: spatial niche occupation of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), in citrus groves. PLOS ONE 10(7), e0131917. doi: 10.1371/journal.pone.0131917. Sétamou, M., Flores, D., French, J.V. and Hall, D.G. (2008) Dispersion patterns and sampling plans for Diaphorina citri (Hemiptera: Psyllidae) in citrus. Journal of Economic Entomology 101, 1478–1487. Sétamou, M., Rodriguez, D., Saldana, R.R., Schwarloze, G., Palrang, D. and Nelson, S.D. (2010) Efficacy and uptake of soil-applied imidacloprid in the control of Asian citrus psyllid and a citrus leafminer, two foliar-feeding citrus pests. Journal of Economic Entomology 103(5), 1711–1719. Sétamou, M., da Graca, J.V. and Prewett, R. (2012a) HLB in Texas: steps and challenges to curb this threat. Citrograph 3, 32–38.



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Sétamou, M., Sanchez, A., Patt, J.M., Nelson, S.D., Jifon, J. and Louzada, E.S. (2012b) Diurnal patterns of flight activity and effects of light on host finding behavior of the Asian citrus psyllid. Journal of Insect Behavior 25, 264–276. Sétamou, M., Sanchez, A., Saldaña, R.R., Patt, J.M. and Summy, R. (2014) Visual responses of adult Asian citrus psyllid (Hemiptera: Liviidae) to colored sticky traps on citrus trees. Journal of Insect ­Behavior 27, 540–553. Sétamou, M., Simpson, C.R., Alabi, O.J., Nelson, S.D., Telagamsetty, S. and Jifon, J.L. (2016a) Quality matters: influences of citrus flush physicochemical characteristics on population dynamics of the Asian citrus psyllid (Hemiptera: Liviidae). PLOS ONE 11(12), e0168997. doi: 10.1371/journal. pone.0168997. Sétamou, M., Alabi, O.J., Kunta, M., Jifon, J.L. and da Graça, J.V. (2016b) Enhanced acquisition rates of Candidatus Liberibacter asiaticus by the Asian citrus psyllid (Hemiptera: Liviidae) in the presence of vegetative flush shoot growth in citrus. Journal of Economic Entomology 109, 1973–1978. Stansly, P.A., Arevalo, H.A., Zekri, M. and Hamel, R. (2009) Cooperative dormant spray program against Asian citrus psyllid in SW Florida. Citrus Industry 90, 14–15. Stansly, P.A., Qureshi, J.A. and Kostyk, B.C. (2012) Comparison of spirotetramat at different spray volumes to some standard insecticides at low volume for control of Asian citrus psyllid and citrus leafminer on oranges. Arthropod Management Tests 37(1), D14. doi:10.4182/amt.2012.D14. Stansly, P.A., Qureshi, J.A. and Kostyk, B.C. (2014) Low volume and standard spray applications of experimental and labeled insecticides against Asian citrus psyllid on oranges: spring, 2013. Arthropod Management Tests 39(1), F1. doi:10.4182/amt.2014. USDA-NASS (2017) US Citrus: Value of Production. Report. US Department of Agriculture, National Agricultural Statistical Service. Available at: https://quickstats.nass.usda.gov/results/A2E7F977-13F13A75-AE99-16395E869BAA (accessed 27 September 2017). Zanardi, O.Z., Volpe, H.X.L., Favaris, A.P., Silva, W.D., Luvizotto, R.A.G., Magnani, R.F., Esperança, V., Delfino, J.Y., de Freitas, R., Miranda, M.P. et al. (2018) Putative sex pheromone of the Asian citrus psyllid, Diaphorina citri, breaks down into an attractant. Scientific Reports 8, 455. doi: 10.1038/ s41598-017-18986-4

16 

Management of Asian Citrus Psyllid in California

Elizabeth E. Grafton-Cardwell* Department of Entomology, University of California, Riverside, California, USA

The commercial citrus industry in California comprised 268,500 acres in 2016/17 producing 3.85 million tons valued at $943 million of which 75% went to the fresh market (NASS, 2017). Asian citrus psyllid, Diaphorina citri Kuwayama, (ACP), was first detected in southern California in 2008 and the putative causal bacterium of huanglongbing (HLB) Candidatus Liberibacter asiaticus (CLas) was first found in a single residential tree in 2012 (Luque-Williams, 2012; Kumagai et  al., 2013). Upon discovery of ACP, California growers formed the Citrus Pest and Disease Prevention Program (CPDPP) to provide assessments for residential citrus treatments and obtained federal funds to survey for HLB throughout the state. The California Department of Food and Agriculture (CDFA) currently surveys for psyllids and symptomatic plant tissue in residential areas and tests samples for CLas using polymerase chain reaction (qPCR). CDFA utilizes a ‘risk-based’ survey that incorporates human population density, demographics, trans­ portation corridors, climatological effects, ACP density and duration, and the location of HLB-­ infected trees for detections (Gottwald et  al., 2014). It is estimated that 60% of residences have one or more citrus trees for a statewide total of 15 million residential citrus trees, thus management of ACP and HLB in residential citrus in addition to commercial orchards is critical

for California. CDFA mandates removal of HLB-­ infected trees and, as of early 2018, these detections have been limited to residential areas of California. California provides a rare example of formal management of ACP and HLB in non-­ commercial citrus. The California citrus industry worked intensively with CDFA in the early days of the ACP invasion to protect the citrus industry from HLB by enacting a series of regulations and voluntary ACP management programs. Recommendations were based on reports that retail nursery citrus and transportation of bulk citrus were key factors in the spread of the psyllid and the disease in Florida (Halbert et  al., 2010, 2012). Budwood protection was increased by requiring mother and increase trees to be confined to screened structures that were carefully monitored by the United States Department of Food and Agriculture (USDA). Nursery trees sold to retail stores were required to be treated with foliar and systemic insecticides prior to shipping (CDFA, 2019a). Measures were implemented to prevent retail nurseries from hosting ACP (Byrne et al., 2016). Trees sold to commercial growers or the general public were required to remain within ­quarantine areas. Central and northern California citrus growers continue to apply voluntary coordinated sprays to locally ‘eradicate’ ACP where not

*  Email: [email protected] 250

© CAB International 2020. Asian Citrus Psyllid: Biology, Ecology and Management of the Huanglongbing Vector (eds. J.A. Qureshi and P.A. Stansly)



Management of Asian Citrus Psyllid in California

yet fully established. Area-wide psyllid-­suppressive treatments to limit the spread of disease are conducted in southern California, where ACP is now endemic. In addition, orchards within quarantine areas must be treated within 14 days of harvest with an approved foliar insecticide or be cleaned of twigs and debris in the field before movement between zones and all bulk citrus loads are required to be tarped (CDFA, 2019b). In spite of these efforts, ACP is now well established in southern California, has invaded parts of the central and northern areas of the state and continues to spread by natural and human-assisted means. Nevertheless, regula­ tory activities that California enacted soon after the first detection of ACP have significantly slowed the spread of vector and disease relative to other citrus growing regions of the USA. Failure to completely stop the spread is attributed to insufficient control of ACP in residential areas and the inability to detect HLB early in the infection process. It takes 9 months to 2 years for a localized bacterial infection to spread throughout the tree sufficiently that it can be effectively sampled to test for CLas. Meanwhile, the psyllid has deposited eggs near where it created a localized bacterial infection that is acquired by the nymphs and subsequently spread to uninfected trees when they become adults (Lee et al., 2015). Few trees in California tested positive for CLas during 2012–2015, and all were restricted to residential citrus in Los Angeles County (Hornbaker and Kumagai, 2016). However, detections of PCR CLas-positive psyllids and trees increased rapidly in 2016–2017 in Los Angeles County, and subsequently in residential Orange and Riverside counties. Disease spread corresponded with the spread of ACP and prevailing winds (Bayles et  al., 2017) and in 2018 threatened commercial citrus in Riverside and San Bernardino counties. ACP management practices in California have evolved in response to regional differences in climate, cultivars, pest pressures and prevalence of HLB (Grafton-Cardwell, 2015; Grafton-­ Cardwell et  al., 2011). The psyllid is well established in many areas of southern California where the climate is uniform and ideal for both the vector and disease. The climate is less hospitable in the prolonged heat of the desert regions of the state and in the central San Joaquin Valley, where extremes of summer heat and ­winter

251

cold harden foliage and so reduce psyllid establishment. Seventy-five percent of the citrus is grown in the central region, which is isolated from the south by the San Gabriel mountain range. ACP is found sporadically in the central region on yellow sticky trap cards and rarely found infesting citrus foliage, even in residential areas. Because of these differences, ACP/HLB management programs are described separately for different regions of the state. Growers use local Task Forces, Pest Control Districts, and/or grower-organized psyllid management areas (PMAs) as vehicles to coordinate treatments. PMAs are groups of 20–35 neighboring growers that form a communication network for responding to ACP and HLB ­ (Grafton-Cardwell et  al., 2015). PMAs are thus similar to the Citrus Health Management Areas (CHMAs) of Florida (CREC, 2019). Recommendations for the timing of insecticide treatments are based largely on the experience of Florida and Texas indicating the value of supressing overwintering adult psyllid populations (dormant sprays) and treating during periods of major flushing (Qureshi and Stansly, 2010; Sétamou et al., 2016). Studies of insecticide efficacy for ACP control indicate that many active ingredients are effective following direct contact with the insect (Grafton-Cardwell et al., 2013; Qureshi et al., 2014, 2017; Bethke et al., 2015; Boina and Bloomquist, 2015). However, insecticides that leave persistent residues are needed to control nymphs as they emerge from eggs tucked within foliage and to protect trees from incursions of ACP adults. Residual control by foliar insecticides has proved longest for the pyrethroids and thiamethoxam and shortest for insecticides approved for organic production (Bethke et  al., 2014, 2015; Tofangsazi et  al., 2016, 2017a, b, 2018). These results, as well as observations of ACP population responses to insecticides applied to commercial orchards, have shaped ACP management strategies in various regions of California as described below.

16.1  Management of ACP and HLB in Southern California The strategy for ACP management has changed in southern California since the first discovery of ACP in 2008 in residential areas of San Diego

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and Los Angeles counties. Initially, the CDFA mandated insecticide treatments in an attempt to eradicate the pest. ACP spread rapidly in spite of the mandatory program. Residential citrus treatments then became voluntary and eventually Tamarixia radiata Waterston and later Diaphorencyrtus aligarhensis (Shafee, Alam & Agarwal) parasitoids were released for ACP population reduction where insecticide treatments were not feasible (Hoddle et  al., 2015; Kistner et al., 2016). Although T. radiata rapidly established throughout southern California, ­incidence of parasitism during a 2012–2014 ­survey of residential citrus averaged less than 5% (Kistner et al., 2016) with disruption by ants as the apparent impediment to better results. Rearing costs are currently too high to mass release sufficient wasps for commercial citrus, although augmentative releases of T. radiata in Florida as well as Mexico and Brazil (Parra et al., 2016) suggest a potential to expand the release program beyond residential areas in California.

In 2016, research was initiated to study the impact of insecticide treatments on ACP populations in 178 orchards in four commercial citrus growing regions of southern California: Ventura County on the coast, the Imperial and Coachella Valleys in the desert, the San Diego and Temecula areas, and San Bernardino and Riverside counties (Fig. 16.1). Citrus in all of these areas is generally infested with ACP. Growers have been organized into ‘area-wide’ insecticide treatment programs in which contiguous citrus acreage is treated with ACP-effective insecticides over a 3-week period during winter and fall. However, the various regions apply different management strategies with varying levels of success, as ­described below. The hot, dry climate of the Imperial and Coachella valleys in the southeast (Fig. 16.1) hardens foliar flush and therefore limits psyllid reproduction and establishment (Sétamou et  al., 2016). The area-wide program in this region consists of broad-spectrum insecticides (primarily

San Joaquin Valley

Riverside/San Bernardino Ventura

HLB Quarantine ACP Quarantine Commercial Citrus

Coachella/Imperial

San Diego

Fig. 16.1.  Citrus growing regions in California (orange) apply different ACP management strategies. ACP quarantines (blue lines) and the HLB quarantines (red lines) as of May 2018 are shown. ACP commercial citrus sampling areas are shown outlined in green.



Management of Asian Citrus Psyllid in California

pyrethroids) applied during December–­January and primarily foliar thiamethoxam during August–­September. Growers generally make additional applications of ACP-effective insecticides that also control pests such as citrus thrips, Scirtothrips citri (Moulton). Commercial orchards in these valleys tend to be clustered and separated from urban areas and so ACP pressure from residential areas is relatively low. Additionally, CDFA staff apply a ‘buffer’ treatment of one to two insecticides to residential citrus within 400 m of the commercial citrus to reduce re-infestation from urban areas. Residential citrus trees are treated with soil-applied systemic imidacloprid and a foliar pyrethroid (beta-cyfluthrin), except at bloom when the latter is omitted to protect bees. Voluntary participation in these treatments is 82–100% for growers conducting area-wide treatments and 95–100% for the voluntary residential program. Psyllid populations were low to undetectable (2% of orchards) and did not persist in these two Valleys during June–November 2017 (Table 16.1). The greatest challenge in this region is the arrival of ACP and HLB from Mexico, where ACP is widespread (Torres-­Pacheco et al., 2013). The climate is milder in San Bernardino County and the remainder of Riverside County compared with the desert valleys. Consequently, citrus trees produce new leaf flush most of the year, providing ideal conditions for ACP survival and reproduction. These conditions are also ideal for biological control of mites, thrips and scale pests, and so growers are not accustomed to spraying insecticides. Tree age, concerns regarding broad-spectrum insecticide disruption

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of natural enemies, economics and other factors influence the level of participation in ACP control programs which varied from null to 100% for PMAs in this region. ‘Soft’ insecticides such as abamectin, spinetoram and spirotetramat were often choices to preserve natural enemies during the grower participatory portion of the program but proved ineffective to control ACP in fall. This was exacerbated by limited (56– 100%) participation in the voluntary residential ACP control program. ACP was detected in the Riverside city/San Bernardino region at 95% of the sample sites, 90% of which had > 1 nymph per flush shoot during one or more sampling periods during June–November 2017 (Table 16.1). An additional fall treatment was added to the management program starting in 2018. HLB-­ infected trees have been detected and removed from several residential locations in Riverside County. Lack of adequate psyllid control and the presence of HLB in the region puts commercial citrus at high risk for infection. Prior to the arrival of ACP, San Diego and Temecula area citrus was historically characterized by low pest pressure. In some regions, small orchards are owned by ‘weekend’ ranchers, many of whom have little concern for pest ­management. Furthermore, organic orchards are common and organic insecticides show little residual activity, resulting in demonstrably higher ACP populations compared with conventional groves. Nevertheless, growers in some portions of the San Diego/ Temecula citrus area work together to apply ­coordinated treatments in winter (December– January) and fall (August–September) and

Table 16.1.  Scouting of 178 commercial citrus orchards every two weeks for ACP nymphs on 50 flushes/ orchard indicating psyllid and HLB pressure in southern California during June–November 2017.

Number of orchards scouted Sites with ACP nymphs on any date Sites with > 1 nymph/flush on one or more sample dates Area-wide treatments First treatment Second treatment Third treatment HLB-infected tree removals from residential citrus as of May 2018

Coachella/ Imperial

San Diego/ Temecula

Ventura

Riverside/ San Bernardino

50 10% 2%

40 48% 23%

48 100% 79%

39 95% 90%

Jan–Feb Petal fall Aug–Sep

Jan–Feb Petal fall Aug–Sep

Dec–Mar Summer Aug–Nov

Nov–Dec Summer Aug–Sep

0

0

0

3

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s­uccessfully control ACP. Grower choice of ACP-effective treatments for other pests are applied between the coordinated treatments. ACP was present in this area during June–­November 2017 at 48% of sampled sites, with 23% having > 1 nymph per flush on one or more sample dates (Table 16.1). Challenges for this region include variable participation in treatments by growers, numerous small citrus orchards, the short residual efficacy of organic treatments and the threat of HLB from adjacent Mexico. The majority of California’s lemons are grown in the Ventura area, where the mild coastal climate and low pest pressure have engendered an abundance of natural enemies for decades. The primary pest in this region is bud mite Aceria sheldoni (Ewing), which causes distortion in new leaf shoots. The frequent flushing of lemons provides continuous oviposition sites for psyllids. Ventura growers apply coordinated area-wide ACP treatments in winter (­January–March) and fall (August–November) and grower-choice ACP-effective treatments for other pests at other times. An extensive urban– agricultural interface similar to Riverside and San Bernardino complicates spraying, with the additional difficulty that spray equipment is ­insufficient to cover citrus acreage within a 2–3-week time frame. Therefore, coordinated treatments progress one region at a time across the county over several months. The result is that some applications are made too far ahead or behind the appearance of new flush. The incidence of ACP in Ventura was 100% of orchards during June–November 2017, and 79% of sites had densities of > 1 nymph per flush on one or more sample dates (Table 16.1). An additional fall treatment was added to the management program starting in 2018. The most effective treatments in this region have been pyrethroids and thiamethoxam, and the least effective treatments abamectin and the organic insecticides. Challenges for this region are the intense urban– agriculture (urban-ag) interface, i­mproving the timing of treatments, and increasing the number of fall treatments to exert a greater effect when psyllid densities are highest. The overarching challenges for the southern California region are the influence of residential citrus, the short residual effect of organic and some selective conventional insecticides, the ineffectiveness of early HLB detection and the

growing presence of HLB. Growers currently apply an average of three to five ACP-effective insecticides for ACP and other pests per year, far below the number of treatments applied in ­Brazil and in Florida prior to HLB becoming widespread. Costs of production are high in California and growers are hesitant to use broad-spectrum insecticides because of the problems of insecticide resistance and disruption of natural enemies. It is very difficult to convince California growers to initiate ACP treatments before HLB has been found in commercial citrus. These challenges of ACP control will be tested to their fullest when HLB reaches the first commercial citrus orchard.

16.2  Central California ACP and HLB Management The San Joaquin Valley region continues to experience very low psyllid trap captures 6 years after the first detection in 2012. Sporadic ACP captures on yellow sticky trap cards and sporadic infestations of trees are characteristic of this region. Growers rely on CDFA to monitor sticky cards, report infestations in their groves and direct growers to respond to finds with treatments meant to eradicate ACP locally. The strategy is to treat all commercial citrus trees with one to two insecticide treatments within 800 m and all residential citrus trees within 400 m of an ACP find. Both residential and commercial citrus treatments are voluntary. The actual choice of insecticide is left to the grower as long as it is ACP-effective (University of California IPM citrus pest management guidelines) (UC ANR, 2019). Organic growers are asked to apply two insecticide treatments for each conventional insecticide treatment because of their shorter residual effect. Residential trees are treated with a foliar pyrethroid and systemic imidacloprid as described above. ACP is found periodically outside of the 800 m treatments applied around trap finds in the San Joaquin Valley, indicating a wider area of infestation. The San Joaquin Valley Task Force therefore recommends that growers should treat a larger area in a coordinated fashion. The treated area (4000–40,000 acres (1600–16,000 ha)) depends on the acreage of contiguous citrus and



Management of Asian Citrus Psyllid in California

ACP distribution. Applications are timed for a period when most growers are treating for a common pest such as citrus thrips. The recommendation is for all growers to treat within a 2–3-week period with a psyllid-effective insecticide. ACP remained undetected for many months following coordinated treatments applied to five San Joaquin Valley citrus grower areas during 2015–2017. Biological control of traditional pests is more difficult to achieve in the San Joaquin Valley region because of greater extremes of summer heat and winter cold. This climate tends to synchronize pest populations such as California red scale Aonidiella aurantii (Maskell) and citricola scale Coccus pseudomagnoliarum (Kuwana), limiting parasitoid activity to certain months of the year and excluding natural enemy activity in winter. In addition, two insecticide applications are required to control Fuller rose beetle Naupactus cervinus Boheman, for navel oranges exported to South Korea. The common choice for these treatments is foliar thiamethoxam, which is also very effective against ACP. It is likely that the harsher climate and the routine sprays for citrus thrips, scales and Fuller rose beetle have had a significant impact on establishment of ACP. The San Joaquin Valley also benefits from the clustering of citrus acreage in more rural communities compared with southern California, reducing the urban-ag interface. In addition, an ongoing program in southern San Joaquin Valley citrus to protect nearby grape crops from glassy-winged sharpshooter Homalodisca vitripennis (Germar) uses pyrethroids and neonicotinoids that aid control of ACP. The greatest risk in the San Joaquin Valley is inadvertent transport of ACP (and eventually CLas) from southern California. Aggressive training programs for field workers, packinghouses, growers, pest control advisors, Master Gardeners and mass media outreach combined with quarantine restrictions have helped to reduce but not eliminate this threat.

16.3  Statewide Summary Efforts continue to set up or realign Pest Control District (PCD) activities to manage ACP with the understanding that continued spread of HLB will require intensified grower response in the

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near future. For example, the Kern County (San Joaquin Valley) PCD provides funding to remove citrus trees in rural residential areas where psyllids have chronically reappeared. Tree removal of uninfected trees in residential areas of southern California where HLB has been confirmed is encouraged. The citrus industry is also actively supporting research to develop one or more early detection techniques (EDTs) to find and rogue out trees that are in the early stages of CLas infection (LeVesque and McRoberts, 2017). EDTs under study include canines trained in CLas detection, a volatile organic compound sniffer, testing of leaf surface microbial communities, assays of metabolites produced as a host plant response and proteins produced by the bacteria that circulate throughout the tree. Some of these studies could also lead to genetic engineering of the plant to reduce citrus susceptibility to CLas infection. Sampling methods for ACP utilized by other states (Hall and Hentz, 2010; Monzo et  al., 2015) have been modified for California situations. Low-density populations of ACP in central California orchards are detected using yellow sticky cards placed by CDFA employees at a rate of one trap per 10 acres and replaced every 2 or 4 weeks, depending on the time of year. Additional traps are placed at higher density around trap finds to better define the region of coordinated treatments. In southern California where ACP is well established, monitoring psyllids is the responsibility of grower-hired Pest Control Advisors (PCAs). Their reports include the number of nymphs per flush (when flush is available) sampled on two to four borders and a central row of each orchard (Sétamou and ­Bartels, 2015). Treatment is recommended at a threshold of 0.5 nymphs per flush. This threshold is likely higher than that set for Florida, which bases its threshold on tap samples of adult psyllids (Monzo and Stansly, 2017); however, in the absence of the disease in commercial orchards and given the potential for disruption of natural enemies, it is difficult to convince growers to further intensify spray programs. ­ The ­desired level of suppression is more readily achieved in the desert and the central valley compared with other areas of southern California, for reasons discussed above. The goal for California ACP management is to reduce psyllids to extremely low densities and thus slow the spread of HLB, allowing time

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for the research community to develop better disease management tools and eventually a cure for the disease. The difficulties in achieving this goal are physical, sociological and economic and, as a result, grower participation varies around the state, but progress has been

made in slowing the spread of the disease relative to other regions around the world. It remains to be seen whether this strategy will provide the desired result of maintaining the viability of the California citrus industry in the face of HLB.

References Bayles, B.R., Thomas, S.M., Simmons, G.S., Grafton-Cardwell, E.E. and Daugherty, M.P. (2017) Spatiotemporal dynamics of the Southern California Asian citrus psyllid (Diaphorina citri) invasion. PLOS ONE 12(3), e0173226. doi: 10.1371/journal.pone.0173226. Bethke, J., Whitehead, M., Morse, J., Byrne, F., Grafton-Cardwell, E., Godfrey, K. and Hoddle, M. (2014) Organic pesticide screening at the Chula Vista insectary. Citrograph 5(2), 44–51. Bethke, J., Whitehead, M., Morse, J., Byrne, F., Grafton-Cardwell, E., Godfrey, K., Hoddle, M. and Corkidi, L. (2015) Screening conventional insecticides against adult ACP. Citrograph 6(4), 48–55. Boina, D.R. and Bloomquist, J.R. (2015) Chemical control of the Asian citrus psyllid and of huanglongbing disease in citrus. Pest Management Science 71, 808–823. doi: 10.1002/ps.3957. Byrne, F.J., Daugherty, M.P., Grafton-Cardwell, E.E., Bethke, J.A. and Morse, J.G. (2016) Evaluation of neonicotinoid insecticides for the management of the Asian citrus psyllid Diaphorina citri on containerized citrus. Pest Management Science 73(3), 506–514. doi: 10.1002/ps.4451. CDFA (2019a) ACP treatments. Available at: http://phpps.cdfa.ca.gov/PE/InteriorExclusion/pdf/acptreatments.pdf (accessed 27 November 2019). CDFA (2019b) ACP grower information. Available at: http://phpps.cdfa.ca.gov/PE/InteriorExclusion/pdf/ acpgrowerinformation.pdf (accessed 27 November 2019). CREC (2019) Citrus Health Management Areas. Citrus Research and Education Center, University of ­Florida, Lake Alfred, Florida. Available at: http://www.crec.ifas.ufl.edu/extension/chmas/index.shtml (accessed 27 November 2019). Gottwald, T., Luo, W. and McRoberts, N. (2014) Risk-based residential HLB/ACP survey for California, Texas and Arizona. Citrograph 5(2), 53–58. Grafton-Cardwell, E.E. (2015) The status of citrus IPM in California. In: Sabater-Munoz, B., Moreno, P., Pena, L. and Navarro, L. (eds) Proceedings of the XIIth International Citrus Congress, Nov 18-23, 2012, Valencia, Spain. Acta Horticulturae 1065(2), 1083–1090. Grafton-Cardwell, B., Morse, J. and Taylor, B. (2011) Asian citrus psyllid treatment strategies for California-­ Arizona. Citrograph 2(5), 5–10. Grafton-Cardwell, E.E., Stelinski, L.L. and Stansly, P.A. (2013) Biology and management of Asian citrus psyllid, vector of the huanglongbing pathogens. Annual Review of Entomology 58, 413–432. doi: 10.1146/annurev-ento-120811-153542. Grafton-Cardwell, B., Zaninovich, J., Robillard, S., Dreyer, D., Betts, E. and Dunn, R. (2015) Creating psyllid management areas in the San Joaquin Valley. Citrograph 6(4), 32–35. Halbert, S.E., Keremane, L.M., Ramadugu, C., Brodie, M.W., Webb, S.E. and Lee, R.F. (2010) Trailers transporting oranges to processing plants move Asian citrus psyllids. Florida Entomologist 93(1), 33–38. doi: 10.1653/024.093.0104. Halbert, S.E., Keremane, M., Ramadugu, C. and Lee, R.F. (2012) Incidence of huanglongbing-associated ‘Candidatus Liberibacter asiaticus’ in Diaphorina citri (Hemiptera: Psyllidae) collected from plants for sale in Florida. Florida Entomologist 95(3), 617–624. doi: 10.1653/024.095.0312. Hall, D.G. and Hentz, M.G. (2010) Sticky trap and stem-tap sampling protocols for the Asian citrus psyllid (Hemiptera: Psyllidae). Journal of Economic Entomology 103, 541–549. doi: 10.1603/EC09360. Hoddle, M.S., Bistline-East, A., Hoddle, C.D. and Lewis, M. (2015) Enlisting a second natural enemy species for ACP biocontrol. Citrograph 6(2), 58–63. Hornbaker, V. and Kumagai, L. (2016) HLB detections in San Gabriel. Citrograph 7, 24–28. Lee, J., Halbert, S.E., Dawson, W.O., Robertson, C.J., Keesling, J.E. and Singer, B.H. (2015) Asymptomatic spread of huanglongbing and implications for disease control. PNAS 112(24), 7605–7601. doi: 10.1073/ pnas.1508253112.



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LeVesque, C. and McRoberts, N. (2017) Comparative study of early detection techniques: Texas 2 study. Citrograph 8(2), 44–47. Luque-Willams, M.J. (2012) California’s response to the first detection of HLB. In: Sabater-Munoz, B., Moreno, P., Pena, L. and Navarro, L. (eds) Abstract of the XIIth International Citrus Congress, Nov 18–23, 2012, Valencia, Spain, pp. 196–197. Kistner, E.J., Amrich, R., Castillo, M., Strode, V. and Hoddle, M.S. (2016) Phenology of Asian citrus psyllid (Hemiptera: Liviidae) with special reference to biological control by Tamarixia radiata, in the residential landscape of southern California. Journal of Economic Entomology 109, 1047–1057. doi: 10.1093/jee/tow021. Kumagai, L.B., LeVesque, C.S., Blomquist, C.L., Madishetty, K., Guo, Y., Woods, P.W., Rooney-Latham, S., Rascoe, J., Gallindo, T., Schnabel, D. and Polek, M. (2013) First report of Candidatus Liberibacter asiaticus associated with citrus huanglongbing in California. Plant Disease 97(2), 283. Monzo, C. and Stansly, P.A. (2017) Economic injury levels for Asian citrus psyllid control in process ­oranges from mature trees with high incidence of huanglongbing. PLOS ONE 12(4), 30175333. doi: 10.1371/ journal.pone.0175333. Monzo, C., Arevalo, H.A., Jones, M.M., Vanaclocha, P., Croxton, S.D., Qureshi, J.A. and Stansly, P.A. (2015) Sampling methods for detection and monitoring of the Asian citrus psyllid (Hemiptera: Psyllidae). Environmental Entomology 44(3), 780–788. doi: 10.1093/ee/nvv032. NASS (2017) Citrus Fruits 2017 Summary. National Agricultural Statistics Service, US Department of ­Agriculture, Washington, DC. Parra, J.R., Alves, P.G.R., Diniz, A.J.F. and Vieira, J.M. (2016) Tamarixia radiata (Hymenoptera: Eulophidae) × Diaphorina citri (Hemiptera: Liviidae): mass rearing and potential use of the parasitoids in Brazil. Journal of Integrated Pest Management 7, 1–11. Qureshi, J.A. and Stansly, P.A. (2010) Dormant season foliar sprays of broad-spectrum insecticides: an effective component of integrated management for Diaphorina citri (Hemiptera: Psyllidae) in citrus orchards. Crop Protection 29(8), 860–866. doi: 10.1016/jcroppro.2010.04.013. Qureshi, J.A., Kostyk, B.C. and Stansly, P.A. (2014) Insecticidal suppression of Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae) vector of huanglongbing pathogens. PLOS ONE 9(12), e3112331. doi: 10.1371/journal.pone.0112331. Qureshi, J.A., Kostyk, B.C. and Stansly, P.A. (2017) Single and multiple modes of action insecticides for control of Asian citrus psyllid and citrus leafminer. Hortscience 52(5), 732–735. doi: 10.21273/HORTSCI11726-17. Sétamou, M. and Bartels, D.W. (2015) Living on the edges: spatial niche occupation of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae) in citrus groves. PLOS ONE 10(7), e0131917. doi: 10.137/journal.pone.0131917. Sétamou, M., Simpson, C.R., Alabi, O.J., Nelson, S.D., Telagamsetty, S. and Jifon, J.L. (2016) Quality matters: influences of citrus flush physicochemical characteristics on population dynamics of the Asian citrus psyllid (Hemiptera: Liviidae). PLOS ONE 11(12), e0168997. doi: 10.1371/journal.pone.0168997. Tofangsazi, N., Grafton-Cardwell, B., Rogers, B. and Ferro, E. (2016) Fall insecticide treatments for ACP in southern California. Citrograph 7(4), 50–54. Tofangsazi, N., Grafton-Cardwell, B. and Ferro, E. (2017a) ACP control in southern California: whole o ­ rchard studies of the efficacy of grower-applied spring insecticides. Citrograph 8(3), 58–61. Tofangsazi, N., Grafton-Cardwell, B. and Ferro, E. (2017b) Fall 2016 Pauma grower control of ACP. Citrograph 8(4), 70–75. Tofangsazi, N., Morales-Rodriguez, A., Daugherty, M.P., Simmons, G. and Grafton-Cardwell, E.E. (2018) Residual toxicity of selected organic insecticides to Diaphorina citri (Hemiptera: Liviidae) and non-­ target effects on Tamarixia radiata (Hymenoptera: Eulophidae) in California. Crop Protection 108, 62–70. doi: 10.1016/j.croppro.2018.02.006. Torres-Pacheco, I., Lopez-Arroyo, J.I., Aguirre-Gomez, J.A., Guevara-Gonzalez, R.G., Yanez-Lopez, R., Hernandez-Zul, M.I. and Quijano-Carranza, J.A. (2013) Potential distribution in Mexico of Diaphorina citri (Hemiptera: Psyllidae) vector of huanglongbing pathogen. Florida Entomologist 96(1), 36–47. doi: 10.1653/024.096.0105. UC ANR (2019) IPM citrus pest management guidelines. Agriculture and Natural Resources, University of California, Davis, California. Available at: http://ipm.ucanr.edu/PMG/selectnewpest.citrus.html (­accessed 27 November 2019).

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Advances in RNA Suppression of the Asian Citrus Psyllid Vector and Bacteria (Huanglongbing Pathosystem) Wayne B. Hunter1*, Sasha-Kay V. Clarke2,4, Andres F. Sandoval Mojica3, Thomson M. Paris4, Godfrey Miles1,3, Jackie L. Metz4, Chris S. Holland5, Greg McCollum1, Jawwad A. Qureshi4,8, John M. Tomich6, Michael J. Boyle,7 Liliana Cano4, Sidney Altman9, Kirsten S. Pelz-Stelinski3 1 USDA, ARS, US Horticultural Research Laboratory, Fort Pierce, Florida, USA; 2 University of the West Indies, Department of Basic Medical Sciences, Biochemistry Section, Kingston, Jamaica; 3Florida Atlantic University Harbor Branch, Fort Pierce, Florida, USA; 4University of Florida, Institute of Food and Agricultural Sciences, Indian River Research and Education Center, Fort Pierce, Florida, USA; 5Maverick Biologicals, Inc., Fort Pierce, Florida, USA; 6Kansas State University, Department of Biochemistry and Molecular Biophysics, Manhattan, Kansas, USA; 7Smithsonian Marine Station at Ft. Pierce, Florida, USA; 8University of Florida, Institute of Food and Agricultural Sciences, Southwest Florida Research and Education Center, Immokalee, Florida, USA; 9Yale University, New Haven, Connecticut, USA

The devastation to the US citrus industries from huanglongbing (HLB, or citrus greening disease) is spread during feeding by the Asian citrus psyllid (ACP) Diaphorina citri, Kuwayama, (Hemiptera: Liviidae). The citrus tree pathogen ‘Candidatus Liberibacter asiaticus’ (CLas) is a fastidious alpha-proteobacterium that has invaded all US citrus growing regions, causing severe tree decline and economic losses estimated in billions of dollars. Innovative technologies, like RNA suppression by RNAi, morpholino oligos, or gene editing tools, like CRISPR/Cas9, all provide non-transgenic strategies, as well as transgenic solutions to manage arthropod vectors, pests and pathogens. Innovative breakthroughs that

improve gene editing in psyllids, such as the BAPC-assisted-CRISPR/Cas9 System, enable injection of adult females near their ovaries to produce heritable germline gene editing. This opens the world of gene editing in arthropods and bypasses the need for microinjection of eggs. Current results from researchers report that these methods enable suppression of ACP vectors, their endosymbionts and the Liberibacter pathogens in infected citrus trees. The development of therapeutic treatments for establishing a sustainable citrus industry appears to be on the horizon. Hurdles still exist, such as the timeline and costs associated with passing through regulatory approvals.

*  Email: [email protected]

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17.1  Introduction: Huanglongbing With no proven treatments to prevent HLB, the pathogen currently threatens citrus production worldwide (Halbert and Manjunath, 2004; Bové, 2006; Manjunath et al., 2008). Globally in citrus there are three Liberibacter species associated with HLB: ‘Candidatus Liberibacter africanus’ (CLaf); ‘Ca. Liberibacter americanus’ (CLam); and ‘Ca. Liberibacter asiaticus’ (CLas) (Bové, 2006; Haapalainen, 2014). In the USA, HLB is caused by the phloem-limited CLas, which affects the phloem load and general nutrient transport within the plant (Wang et al., 2017b). This causes nutrient-deficient symptoms such as root dieback, leaf yellowing, smaller discolored, lopsided and bitter fruits, followed by fruit drop and eventually tree death. Since the discovery of HLB in Florida in 2005, it has spread throughout the southern states, including Texas and California, threatening the sustainability of the industry in the USA (Halbert and Manjunath, 2004; Hall et  al., 2013; Wang et  al., 2017a). Current management strategies to reduce HLB in the USA have primarily relied on insecticide treatments in attempts to suppress psyllid populations, early detection and removal of infected trees, increased nutrients to improve tree health, application of antibiotics and protecting trees under nets (Bové, 2006; Qureshi and Stansly, 2007; Belasque et al., 2010; McGhee et al., 2011; Zhang et al., 2015; Mann et al., 2018). Natural resistance within citrus species has yet to be identified; constant applications of insecticides are too costly for most growers and increase the risk of producing insecticide resistance in psyllid populations. Few options remain that could provide tree protection from HLB (NASEM, 2018). Thus, the citrus industry is looking to citrus breeders to add resistance protection into citrus varieties, or to researchers to develop methods to stop CLas transmission by the psyllid vector (Reese et  al., 2013; Ferrara et  al., 2015; Ramsey et  al., 2015, 2017; Arp et  al., 2016; Ren et  al., 2016; Saha et al., 2017a; Ammar et al., 2018). The psyllid’s development and survival are strongly linked to the types of endosymbionts harbored inside special organs, called bacteriomes (Meyer and Hoy, 2008; Nakabachi et  al., 2013; Ramsey et al., 2017). The microbiome includes a variety of endosymbionts: the primary

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ones being ‘Candidatus Carsonella ruddii’ (Nakabachi et  al., 2006), ‘Candidatus Profftella armatura’, and Wolbachia-Diaphorina (WDi) (Subandiyah et  al., 2000; Kolora et  al., 2015). The WDi was shown to be most similar to other ACP Wolbachia from the region of North India, suggesting this as the point of origin (Saha et al., 2012). Studies also suggest a strong interaction among the pathogen, CLas, and the psyllid’s endosymbionts, which may facilitate efficient acquisition and transmission of the pathogen to citrus trees (­Nakabachi et  al., 2013; Ramsey et al., 2015; Chu et al., 2016; Pelz-Stelinski and Killiny, 2016; Arp et  al., 2017; Kruse et  al., 2017; Mann et  al., 2018). All these microbes also affect the ACP’s physiology and immune systems (Eleftherianos et  al., 2013; Arp et  al., 2016, 2017; Hosseinzadeh et al., 2019). As traditional chemical insecticides fail to suppress ACP populations, due to development of chemical resistance (Killiny et al., 2014), new strategies for management, like biopesticides, RNA interference (RNAi), antisense oligos (­Petrick et  al., 2013, 2016; Scott et  al., 2013; Baum and Roberts, 2014; Roberts et al., 2015; Andrade and Hunter, 2016; Kolliopoulou et  al., 2017; Taning et  al., 2017; Gantz and Akbari, 2018; Sinisterra-Hunter and Hunter, 2018; Zotti et  al., 2018), and biological control agents (Qureshi and Subandiyah et  al., 2000; Stansly, 2007; Qureshi et  al., 2009; Avery et  al., 2011; Hunter et al., 2011; Patt et al., 2015) will continue to be developed to reduce psyllid vectors, their endosymbionts, and the pathogens they transmit (Fig. 17.1).

17.1.1  Asian citrus psyllid and huanglongbing HLB, or citrus greening disease, is one of the most destructive of the global citrus industry. Reports of its impact have been documented for major citrus growing regions of Asia, Africa and the Americas (da Graça et  al., 2016; Wang et al., 2017a). In African countries, CLaf is transmitted by two psyllid species: the African citrus psyllid, Trioza erytreae, and the ACP, D. citri, which has been reported to transmit both CLam and CLas bacteria (Massonié et al., 1976; Lallemand et al., 1986). The HLB pathogens replicate and circulate in the plant host,

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W.B. Hunter et al.

Alba-Tercedor, J; and Hunter, W.B. 2016

Fig. 17.1.  Adult Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae) feeding on citrus (left). MicroCT scanning image showing stylets inside citrus leaf during feeding. Salivary sheath made during probing shown inside the vascular bundle, phloem and xylem (Alba-Tercedor et al., 2017; Alba-Alejandre et al., 2018; Cicero et al., 2018a,b). See digital videos of psyllid anatomy at: www.citrusgreening.org.

phloem and roots, and in the psyllid vector’s tissues, salivary glands and midgut tissues (Xu et  al., 1988; Ammar et  al., 2011a,b, 2016; Ghanim, 2016; Kruse et al., 2017). This replicative and circulative model plays a huge role in the rapid spread and persistence of the disease. Both the adult and nymphal stages can transmit the HLB pathogen (Inoue et al., 2009; Pelz-Stelinski et  al., 2010). Transmission efficiency increases when the pathogen is acquired during the nymphal stages, with adult acquisition being less efficient (Pelz-Stelinski et  al., 2010, 2016; Ren et al., 2016). The citrus greening pathosystem still remains the most difficult problem in the citrus industry after 35 years (Bové, 2006; Wang, 2017a, b), but resistance genes have yet to be identified in citrus varieties (Dutt et  al., 2015; Hao et  al., 2016). A review of the research efforts over the past decade reported that no significant solutions to stop HLB have been developed (NASEM, 2018). However, plant breeding efforts may yet produce several citrus varieties with resistance or tolerance, using improved methods like CRISPR/Cas9 (Bortesi and Fischer, 2015; Jaganathan et al., 2018; Sinisterra-Hunter and Hunter, 2018). Other efforts are focused on reducing the ACP vector populations, or preventing the acquisition and transmission of the bacterial pathogen (Grafton-Cardwell, 2013;

Ramsey et al., 2015, 2017; Taning et al., 2016, 2017; Andrade and Hunter, 2017; Darrington et  al., 2017; Pelz-Stelinski et  al., 2017; Zotti et al., 2018). 17.1.2  Management strategies Management of the HLB pathosystem deals with a complex biological system of organisms which interact, made more difficult with a slow-growing, fastidious bacterial pathogen (Fig. 17.2). Management of ACP in Florida enlisted a comprehensive pest management approach depending first on the heavy application of chemical insecticides (Yamamoto et  al., 2009; Tiwari et al., 2011), on increasing numbers of parasitoid biological control agents (http://biocontrol. ucr.edu/asian_citrus_psyllid.html) and on vigorous trimming of symptomatic branches, or roguing entire trees. Biological control agents (natural enemies or predators of an insect pest) are ­considered safer, more target-specific and self-­ sustaining alternatives to applications of broad chemical insecticides (Grafton-Cardwell et  al., 2013). Entomopathogenic fungi such as Isaria fumosorosea, when evaluated as a biological ­control agent (Avery et al., 2011), resulted in decreased feeding, honeydew production and reduction of ACP in laboratory and glasshouse



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Psyllid Embryo Development MicroCT Anatomy

Psyllid Instar Development

Tritrophic Interactions Citrus–Bacteria –Psyllid

Fluorescence Imaging

Proteomics Enzymes

Pathology, Microbial Plant and Insect

Systems Biology Big Data Analyses

Omics, Pathways

Hunter, W.B. (2019) ARS.

Fig. 17.2.  Systems biology approach for deriving solutions from a comprehensive arthropod vector pathosystem model. Methods of interest focus on final outcomes – citrus trees with tolerance that are productive under an HLB pathosystem. Research topic areas: The citrus tree being central to the need for a sustainable citrus industry. Peripheral to tree improvement are strategies to limit the bacterial pathogen replication and spread, and then strategies to reduce the ACP vector. ‘Systems biology’ approaches thus incorporate broad disciplines like pathology, psyllid biology and -omics studies (transcriptomes, genomes, proteome, metabolome, for all organisms within the pathosystem). Big Data platforms are key to providing applications cluster analyses, across multiple interactions, providing a better understanding of functional genomics, genetic pathways, and the potential key interdiction points for solutions. Outreach is a critical component in today’s rapid information age. Collectively society needs these easy interfaces to increase understanding and acceptance of emerging technologies (Alba-Tercedor et al., 2017; Alba-Alejandre et al., 2018; Cicero et al., 2018a).

trials (Avery et  al., 2011; Hunter et  al., 2011; Moran et al., 2011; Patt et al., 2015). Parasitoids such as Tamarixia radiata Waterson (Hymenoptera: Eulophidae), an ectoparasitoid of the ACP, were imported from Asia and have been released across Florida. A survey done throughout 2006/7 in southern Florida highlighted that parasitism varied, ranging from < 20% during spring and summer to 56% in mid-fall (Qureshi et  al., 2009). California initiated the use of

parasitoids in 2010 (Hoddle and Pandey, 2014). Common predators of the ACP include ladybeetles, syrphid flies, lacewings and spiders (Michaud and Olsen, 2004; Qureshi and Stansly, 2009; Khan et al., 2016). The earliest genetic sequencing from ACP was in 2005 (Hunter et al., 2005a, b). This was followed by the establishment of the International Psyllid Genome Consortium (Hunter et al., 2008, 2009, 2010a; Reese et al., 2013), with the first report on pathogenic

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molecular control strategies to reduce the ACP vector, or the pathogenic bacteria in citrus (Lacey et  al., 2015; Kolliopoulou et  al., 2017; Taning et al., 2017). Traditional plant breeding and genetic plant improvement methods have produced resistant varieties, as sustainable approaches to controlling many plant diseases. Even though these strategies may aid production of HLB tolerance in citrus, the registration of transgenic plants still takes 10–12 years. Regulatory approvals still lag far behind the safety record of methods like genetically modified organisms (GMOs) to produce disease-resistant crops (Ivashuta et  al., 2009, 2015; Bawa and Anilakumar, 2013; Wise, 2013; Wise et  al., 2013; Saurabh et  al., 2014; Younis et al., 2014; Sherman et al., 2015; Brooks and Barfoot, 2016; Petrick et  al., 2016; Ricroch and Hénard-Damave, 2016; ISAAA, 2017). Even so, additional issues beyond cost and time, such as consumer acceptance and beliefs, add

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viruses from field-collected ACP in Florida (Marutani-Hert et al., 2008, 2009; Hunter et al., 2009). The two viruses identified were Diaphorina deformed wing virus (Fig. 17.3), and a Diaphorina reovirus (Marutani-Hert et  al., 2008, 2009). The reovirus was detected in 50% of field-collected adults from citrus in Indian River County, Ft Pierce, Florida. In trials, the virus only had a slight negative effect on psyllid biology, with slower development and a shorter lifespan than when uninfected. Thus, it was proposed for use as a delivery system for ACP suppression. A larger study by Nouri et al. (2015) confirmed these virus reports, and expanded the number of known viruses from ACP, by sequencing collections from the USA (California, Florida, Hawaii) and from China, Brazil, Pakistan and Taiwan. The study identified virus sequences from members of the Bunyaviridae Parvoviridae, Reoviridae and the Picornavirus superfamily. Viral pathogens may provide novel biocontrol, or

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RNA Suppression – Asian Citrus Psyllid Vector and Bacteria

more barriers that will require increased efforts in public education and outreach. 17.1.3  Technologies to protect citrus Advancements in technologies for modifying RNA expression and modifying gene regulation have revolutionized research across all fields of science (Saurabh et  al., 2014; Younis et  al., 2014; Criscione et  al., 2015; Lombardo et  al., 2016; Jaganathan et al., 2018; Sinisterra-Hunter and Hunter, 2018). RNAi is one such technological advancement that has aided functional genomic studies. RNAi uses double-stranded RNA (dsRNA) to modulate gene expression at the post-transcriptional level (Fire et  al., 1998; Elbashir et  al., 2001). The elucidation of the functional mechanism of RNAi, the dsRNA trigger, was considered one of the most important scientific breakthroughs in the past 20 years (Zotti et  al., 2018), being awarded the Nobel Prize in Physiology or Medicine in 2006 (for a historical overview, see Sen and Blau, 2006). Researchers were quick to realize that applications of RNAi may provide improved pest management (Elbashir et  al., 2001; Hannon, 2002; Agrawal et al., 2003; Turner et al., 2006; Baum et  al., 2007; Karl et  al., 2007; Price and Gatehouse, 2008; Bellés, 2010; Huvenne and Smagghe, 2010; Kupferschmidt, 2013). However, as more studies were conducted it became clear that arthropods have a wide range of sensitivity to RNAi susceptibility (Terenius et  al., 2011; Baum and Roberts, 2014; Thakur et  al., 2016; Darrington et al., 2017; Zotti et al., 2018). RNAi functions, applications and solutions in arthropods for use and delivery have been extensively reviewed (Ivashuta et al., 2015; Zotti and Smagghe, 2015; Galay et  al., 2016; Joga et  al., 2016; Kanakala and Ghanim, 2016; Rodrigues and Figueira, 2016; Thakur et  al., 2016; Darrington et  al., 2017; Gundersen-Rindel et  al., 2017; Kolliopoulou et  al., 2017; SinisterraHunter and Hunter, 2018; Vélez and Fishilevich, 2018; Zotti et al., 2018). Since the elucidation of RNAi in the 1990s (Napoli et  al., 1990), it has been used to accelerate crop improvement (­Saurabh et  al., 2014; Younis et  al., 2014; Jin et al., 2015; Lombardo et al., 2016; Bally et al., 2018; Jaganathan et al., 2018). However, development of RNAi products that can be exogenously

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applied for the management of arthropod pests, insect vectors and pathogens have been slow to be commercialized (Baum et  al., 2007; Baum and Roberts, 2014; Christiaens and Smagghe, 2014; Palli, 2014; Zotti and Smagghe, 2015; Joga et al., 2016; Zotti et al., 2018).

17.2  Progress in Psyllid Gene Annotation and Resources To develop gene-based targeting technologies against ACP, accurate gene sequences were critical and an International Psyllid Genome Consortium (IPGC) was established in 2009 (Hunter et  al., 2008, 2009, 2010b). The Asian citrus psyllid genome, DIACI_2.0v, and Official Gene Set, DIACI_OGS_v2, (Saha et  al., 2017a, b) along with Gene Reports for many gene families are openly available (https://citrusgreening.org/ organism/Diaphorina_citri/genome) (Hunter et al., 2009; Saha et al., 2012; Reese et al., 2013; Ferrara et  al., 2015; Arp et  al., 2016; Taning et  al., 2016; Wu et al., 2018). Elucidation of the core genes representing the RNAi pathway of psyllids have been completed (Taning et al., 2016; Saha et al., 2017a).

17.3  Advances in RNAi in Psyllids RNA interference is a natural defense and cell regulatory mechanism that detects dsRNA in the cell and uses fragments of it as a template to degrade complementary mRNA sequences (Fire et  al., 1998). This reduces or suppresses the ­corresponding protein concentration (Dowling et  al., 2016; Lima et  al., 2016; Taning et  al., 2016). Non-transgenic approaches have also been developed for delivery of exogenous dsRNA as topically applied treatments (Hunter et  al., 2011, 2012; Burand and Hunter, 2013; Scott et  al., 2013; Xu et  al., 2015; Joga et  al., 2016; San Miguel and Scott, 2016; Gillet et al., 2017; Mitter et  al., 2017a; Yu et  al., 2017). Exogenously applied dsRNA in water for topical foliar sprays or irrigation (soil-applied) have worked for plant feeding hemipterans (Li et  al., 2015; Andrade and Hunter, 2016; Joga et  al., 2016; Faustinelli et al., 2018). Topically applied dsRNA in carriers like clay nanosheets was shown to

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deliver dsRNA into plants for at least 20–30 days to reduce aphid-transmitted virus (Mitter et al., 2017a, b; Worrall et al., 2019). The first reports of soil-applied dsRNA in water for root absorption by citrus trees, grapevines, basil and okra were by W.B. Hunter (USDA, ARS) (Hunter et al., 2010b, 2012). Field trials with citrus trees 1.5 m tall, which received 400 mg dsRNA in 1 liter of water applied within a 30 cm circle of the root zones of trees or grapevines, also resulted in detection of the dsRNA in the leaves of citrus trees within 3 h post treatment. Leaves tested positive for dsRNA each week for up to 45 days post treatments (Hunter et  al., 2012; Andrade and Hunter, 2016). Starting with an increased concentration at the beginning of the growing cycle permits more dsRNA to be present in plant tissue for a longer period. Increasing persistence is one way to decrease the cost and increase the efficacy of exogenously applied RNAi biopesticides. To analyze soil-applied delivery systems, Hunter (2016–2018) conducted trials with soil-applied dsRNA using a variety of absorbents. The best slow release was a clay absorbed with dsRNA in water mixed into the soil. The study demonstrated detection of dsRNA in plant tissues and insects fed on the plants by root-zone absorption for over 1 year (14 months) (Ghosh et al., 2018). The slow, cyclic release of dsRNA from the clay each time it was watered provided a long-term plant delivery system, thus solving a major hurdle in RNAi treatments (Oil-Dri Corporation of America, Chicago, Illinois: http://www.oildri. com). Multiple types of plants were shown to maintain a constant presence of dsRNA in their tissues, including potted citrus seedlings, ornamentals and vegetables (Ghosh et  al., 2018). This demonstrates one application for exogenously applied RNAi treatments that could be adopted by the industry. Clay granules or pellets absorbed with dsRNA would be faster than producing host plant-expressed RNAi, and would permit altering traits of crops without them being transformed. Thus, crop pest protection or trait modification could be accomplished rapidly, instead of taking decades to develop and register plants that would cost millions of dollars. Soil-applied RNAi treatments to potted citrus seedlings, ornamental plants, field grapevines, citrus trees and grasses, like rice and maize, have consistently shown efficient dsRNA delivery, root absorption, systemic spread and

RNAi suppression of insects and pathogens. Examples include hemipterans, like the glassywinged sharpshooter leafhopper Homalodisca vitripennis (Hunter et  al., 2010b, 2012); plant­ hoppers Nilaparvata lugens (Li et  al., 2013, 2015), the Asian citrus psyllid (Hunter et  al., 2012; Andrade and Hunter, 2016, 2017; Ghosh et  al., 2018; Hunter and Sinisterra-Hunter, 2018), and the potato psyllid Bactericera cockerelli (Hail et al., 2010; Wuriyanghan et al., 2011). For published works and reviews on RNAi in plant-feeding hemipterans and other arthropods, see: Li et al., 2013, 2015; Christiaens and Smagghe, 2014; Zotti and Smagghe, 2015; Andrade and Hunter, 2016; Joga et al., 2016; Zotti et al., 2018. Degradation of ingested dsRNA by nucleases can prevent induced RNAi effects (Allen and Walker, 2012). Significant improvements to overcome nuclease degradation include alterations in the shape of the dsRNA, such as attaching one or two hairpins at the end(s) of the dsRNA, which has been shown to improve resistance to degradation (Allison and Milner, 2014). Reports of improved resistance to nucleases and increased activity by incorporating modified (non-canonical) nucleotides into the dsRNA also works (DuraScribe®T7 Transcription Kit: www. Lusigen.com) (Hunter and Sinisterra-Hunter, 2018; Hunter and Lopez, 2019). After application of exogenous dsRNA it is rapidly degraded by microbes (Dubelman et  al., 2014; Parker et  al., 2019). The modified nucleotides are not degraded as fast, thus the dsRNA concentration inside the plant is increased and available for ingestion by feeding insects (Burand and Hunter, 2013; Scott et al., 2013; Lim et al., 2016; Hunter and Sinisterra-Hunter, 2018). Other methods to deliver increased concentrations of dsRNA may use living microbes, which express hairpins of dsRNA, being virus, bacteria, yeast or fungi (Timmons et  al., 2001; Gonsalves et  al., 2010; Koch et al., 2015; Murphy et al., 2016; Whitten et al., 2016; Kolliopoulou et al., 2017; Whitten and Dyson, 2017; Zotti et  al., 2018). In citrus, Hajeri et al. (2014) reported using a plant-infecting citrus tristeza virus (CTV). The CTV-based RNAi expresses a dsRNA to reduce ACP. The CTV-based vector continues to be developed as an expression vector for citrus as a management tool against psyllids and to reduce huanglongbing (Peng et al., 2018).



RNA Suppression – Asian Citrus Psyllid Vector and Bacteria

RNAi studies in ACP have demonstrated a strong RNAi response across all life stages (El-Shesheny et  al., 2013; Ramsey et  al., 2015; Andrade and Hunter, 2017; Galdeano et  al., 2017; Killiny and Kishk, 2017; Kishk et  al., 2017; Kruse et al., 2017; Yu et al., 2017; Ghosh et  al., 2018). Bioassays for ACP feeding from treated plant material include whole seedlings, cuttings and leaves (Hunter et al., 2010b, 2012; Ammar et al., 2016, 2018; Andrade and Hunter, 2016, 2017; Raiol-Junior et  al., 2017; Hunter and Sinisterra-Hunter, 2018); leaf disks, artificial diets and sugar solutions (Russell and Pelz-Stelinski, 2015; Ammar et  al., 2016); and topical applications and insect soaking (Killiny et  al., 2014; Killiny and Kishk, 2017; Yu et  al., 2017), all of which, have elicited strong RNAi responses. Since concentration of dsRNA ingested can be linked to RNAi efficacy, methods to determine the concentration of dsRNA delivered into citrus tree leaves or ingested by ACP are beneficial. Quantification methods can use qPCR analyses and standard concentration curves, with T7 primers for dsRNA made with common kits (MEGAscript T7 kit, Thermo-­ Fisher Scientific). This detects full-length dsRNA post treatment, or ingestion (Andrade and Hunter, 2017; Faustinelli et  al., 2018). Other methods have reported using fluorescent probes for labeling the bacterium, CLas (Ammar et al., 2011a, b; Yoon et  al., 2017). One example is quantification of delivered dsRNA into citrus seedlings, which were labeled with the ULYSIS® kit that produces fluorescent G-labeled dsRNA (ULYSIS® Nucleic acid labeling technology, KREATECH Diagnostics). The ULYSIS® kit enables rapid and simple coupling of the Alexa Fluor®dyes to purine bases in nucleic acid polymers. The reliable non-enzymatic method for labeling nucleic acid takes about 15 min to complete (Thermo-Fisher Scientific) (Hunter and Sinisterra-Hunter, 2018). To evaluate this method, G-labeled dsRNA with lengths between 140 and 248 nt were produced. The G-labeled dsRNA can serve as the labeled control for visualization, quantification and as an active dsRNA trigger in RNAi trials. Soil-applied concentrations of 10 μg, 100 μg, 1 mg or 10 mg dsRNA per 100 ml water (pH 7.5–8.0) to potted citrus seedlings (1 gallon pots, or 2.85 l), were visualized using

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confocal microscopy of the vascular bundles in leaves, at 3 and 8 days post soil treatments (Fig. 17.4). Quantification of G-labeled dsRNA in leaves or roots using a three-point concentration dilution of G-labeled dsRNA spiked into 100 μl leaf homogenate (0.4 ng, 0.04 ng, 0.004 ng, and 0.0004 ng) resulted in quantification of G-labeled dsRNA in leaf homogenate down to a concentration of 0.4 pg dsRNA per 1 g of tissue as detected by a BioTEK H-1 Fluorescent plate reader. The RFUs at this concentration averaged three times greater than the tissue blank controls. The results supported ­ previous observations on larger citrus trees and grapevines, where soil-applied dsRNA was ­absorbed by roots and detected within 3 h in the most apical leaves of the canopy, and detected for 30–45 days post treatment with a single dose applied (Hunter et al., 2012; Ghosh et al., 2018; Hunter and Sinisterra-Hunter, 2018; Clarke et al., 2019).

17.4  Progress of RNA Suppression Technologies to Reduce ACP RNA suppression technologies, like RNAi, have been developed to target insects and viruses (Zotti and Smagghe, 2015; Joga et  al., 2016; Zotti et  al., 2018). The first RNAi patent to reduce psyllid vectors was issued in 2016/17 (Hunter et  al., 2017; US 2017/0211082 Al in the USA and Brazil). However, targeting bacterial pathogens requires a different approach, especially in fruit trees like citrus, or the endosymbiotic bacteria of insect vectors, like psyllids (Marutani-Hert et al., 2011; Saha et al., 2012; Morrow et al., 2017). One such technology are antisense oligos, like morpholinos (Sala et  al., 2012; Derksen et  al., 2015; Daly et  al., 2017; Summerton, 2017). Researchers have demonstrated the effectiveness of these oligos, which must be conjugated with a cell-penetrating peptide, phosphorodiamidate morpholino oligomer (PPMO). Morpholinos have modified six-­member morpholino rings in place of the ribose sugar, with phosphorodiamidate linkages that substitute for phosphodiester bonds (Jackson et  al., 2016; Jarrous, 2017; Moulton and Moulton, 2017; Summerton, 2017). These modifications provide significantly improved stability and efficacy

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CORE CELLS Fig. 17.4.  Fluorescent labeled dsRNA in leaf midrib of Madam Vinous sweet orange (Citrus sinensis), showing systemic movement post treatment. Soil-applied, root-absorbed labeled dsRNA was detected in the xylem and phloem of new growth leaves on treated citrus seedlings at 3 days post soil-applied treatments. The results supported previous results on whole-plant trials of citrus trees and grapevines (Hunter et al., 2012). Seedlings were 2-year-old potted seedlings. The control leaf (left): no autofluorescence. The leaf was counterstained with NucGreen® 488 reagent, which is suitable for staining nuclei in fixed-cell preparations (Thermo Fisher Scientific). Treated citrus leaf (right). Midrib cross-section, at 72 h post absorption with Fluoro-tagged dsRNA (ULYSIS® Nucleic Acid Labeling Kit [ThermoFisher Scientific]: Molecular Probes cat#U21650. Imaged on confocal microscope, Smithsonian Oceanic Research Lab, Ft. Pierce, Florida.

(Bennet and Swayze, 2010; Kole et  al., 2012). These oligos have been used in functional genetics in animals (Sawyer et  al., 2013) and microbes (Shen et al., 2009). The PPMO can target bacteria, which cannot be achieved with a dsRNA trigger for RNAi (Derksen et al., 2015; Jackson et  al., 2016; Hegarty and Stewart, 2018; Jani et al., 2018; Nan and Zhang, 2018; Shen and Corey, 2018). Thus, modified antisense oligos have shown many applications and activity against bacteria that are human pathogens (Wesolowski et al., 2011, 2013), as well as fruit tree pathogens, like Erwinia amylovora fire blight in apples (McGhee et  al., 2011, 2012; Patel et al., 2017). These studies support the hypotheses that antisense oligos like PPMO may provide a treatment that could reduce bacterial pathogens, like CLas in citrus (Pelz-Stelinski et al., 2017).

17.5  Non-canonical Nucleotides in dsRNA Increases Persistence and RNAi Activity Historically, the use of synthetic nucleosides (non-canonical) and chemical modifications have provided many advantages to improve activity, stability and delivery of these singlestranded oligos (Ludin et al., 2015; Hegarty and Stewart, 2018; Shen and Corey, 2018). Other modifications also provide advantages when incorporated into antisense oligonucleotide structures. These include modified sugars, modified nucleotides and a variety of chemical modifications, all of which add beneficial properties to improve stability and efficacy (Kurreck, 2003; Bennett and Swayze, 2010; Ludin et  al., 2015; Crooke, 2017; Hegarty and Stewart, 2018; Shen



RNA Suppression – Asian Citrus Psyllid Vector and Bacteria

and Corey, 2018; Verma, 2018). Thus, we hypothesized that modified nucleotide incorporation would increase dsRNA performance in a similar manner. To evaluate the benefits, a low-­ performing dsRNA trigger for arginine kinase, dsRNA-ak, canonical nucleotides, resulted in ~18% ACP mortality (Hunter and Sinisterra-­ Hunter, 2018). The dsRNA-ak (200 nt) was compared when made with two kits (MEG­ Ascript T7 kit, Thermo-Fisher Scientific; and DuraScribe® T7 Transcription Kit). ACP was given feeding access on treated citrus seedlings, which received soil treatments with each form of dsRNA (Hunter and Sinisterra-Hunter, 2018). When fed to ACP, concentrations of 1 and 2 μg canonical dsRNA-ak (MEGAscript T7 kit, ­Thermo-Fisher Scientific) resulted in an average mortality of 18%, with negative control dsRNA-­ CTL averaging 12% at 8 days post feeding access (Fig. 17.5). Incorporation of non-canonical modified nucleotides into the dsRNA (49.6%

­replacement, all ‘C’ and ‘U’ replaced) (DuraScribe® T7 Transcription Kit) resulted in significant increases of mortality on day 8 of 28% and 35%, respectively, for each concentration – an average of 31.5%.

17.6  Delivery Mechanisms In order to improve delivery and activity of ­dsRNA triggers and bypass the aforementioned degradation, abiotic and biotic delivery systems have been developed: liposomes, chitosan nanoparticles, bacteria, yeast and viral expression systems for applications in arthropods (Zhang et  al., 2010, 2015; Hajeri et  al., 2014; Mysore et al., 2014; Whyard et al., 2015; Murphy et al., 2016; Taning et  al., 2016; Zotti et  al., 2018). Promising delivery strategies using cationic ­lipids, dendrimers, peptides, polymers, magnetic

Canonical versus non-canonical dsRNA activity, at two concentrations. Percentage mortality of ACP 8 days post ingestion

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Fig. 17.5.  Incorporation of non-canonical nucleotides into dsRNA increases activity and persistence. Host delivered dsRNA as soil-applied treatments in citrus seedling trees can reduce Asian citrus psyllids. Production of dsRNA that incorporated 45 to 55% non-canonical nucleoside forms substituting both ‘C’ and ‘U’ resulted in increased activity reported as % increase in mortality. A low-performing dsRNA-ak trigger that suppresses mRNA for arginine kinase in ACP would normally result in 18% mortality 8 days post ingestion. The same dsRNA-ak sequence from ACP was made with two diffent kits: the traditional MegaScript® kit using canonical nucleosides, compared with dsRNA-ak made with r­ eplacement of the ‘C’ and ‘U’ with non-canonical nucleosides, which was 49.6% of the total nucleotides (Hunter and Lopez, 2019). Produced using the DuraScribe® T7 Transcription Kit. (Cat. No. DS010910 & DS010925, www. Lucigen.com). Control dsRNA-CSBV made with non-canonical replacements did not produce a significant difference from canonical controls (Hunter and Sinisterra-Hunter, 2018).

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beads, quantum dots or other organic nanoparticles, show their value in improving cell entry (Darrington et  al., 2017; Gillet et  al., 2017; Wong et al., 2017; Jin et al., 2018; Parsons et al., 2018). In the medical fields, as well as in livestock, estimates for therapeutic delivery products are estimated to exceed US$30 billion on the annual market by 2020 (Wong et al., 2017).

17.6.1  Branched Amphiphilic Peptide Capsules Branched Amphiphilic Peptide Capsules (BAPC) are a new class of inert, self-assembling peptide nano-capsular spheres (Phoreus Biotechnology, Inc., Manhattan, Kansas). The peptide-based, self-assembling nano-delivery system provides safe, targeted transport of drugs, plasmids, dsRNA, or oligos to specific tissues and organs with minimal off-target accumulation (Sukthankar et al., 2014). Tomich et al. (2014) described the specific characteristics of BAPC in detail.

17.6.2  BAPC-delivered RNAi system in arthropods

microbial expression, exogenously applied ­treatments to plants as topical sprays or soil treatments (El-Shesheny et  al., 2013; Hajeri et  al., 2014; Killiny et  al., 2014; Russell and Pelz-Stelinski, 2015; Andrade and Hunter, 2016, 2017; Taning et al., 2016; Galdeano et al., 2017; Ghosh et  al., 2017, 2018; Killiny and Kishk, 2017; Kishk et  al., 2017; Kruse et  al., 2017; Yu et al., 2017). However, there is still the need to have a product that can produce repeatable RNAi suppression within each delivery strategy. RNAi biopesticides will then be able to move forward to commercialization as suitable pest management products. Based on the physical properties of BAPC with nucleic acids, Hunter et al. (2018) hypothesized that BAPC mixed with guide RNAs and CRISPR/Cas9 components would result in improved delivery and produce a new method for heritable germline gene editing suitable for injection into adult ovaries of psyllids and other arthropods.

17.7  Gene Editing Strategies in Asian Citrus Psyllids 17.7.1  CRISPR/CAS9 background

RNAi studies, which incorporated BAPC mixed with dsRNA specific to the beetle Tribolium castaneum (Coleoptera), caused significant increase in mortality compared with controls when ingested (Avila et  al., 2018). Studies that used BAPC-RNAi fed to the pea aphid Acyrthosiphon pisum (Hemiptera) also reported improved delivery and increased mortality (Wang et al., 2015). BAPC mixed with two dsRNA triggers fed to T. castaneum in diet resulted in a 50% increase in mortality over controls. Similarly, results post-ingestion were observed when dsRNA triggers were fed to pea aphid A. pisum, resulting in a 20% increase in mortality over controls. The BAPC showed no toxicity when fed to control groups. BAPC ­provides a new delivery system to increase RNAi efficient products. BAPC-RNAi is one of many approaches being used to increase RNAi efficacy in ACP. Many RNAi studies on ACP show efficacy using a variety of delivery methods with successful gene target suppression using oral ingestion, topical application, and host plant-­ ­ delivered dsRNA as transgenic plant expression,

Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-­ associated protein, Cas9, represent an invaluable system for the precise editing of genes across all species (Doudna and Charpentier, 2014; Wang et  al., 2016). Because of their relative ease of use and efficiency, CRISPR/Cas9 methods have become the primary gene editing tool in the life ­sciences, including agriculture. For reviews on gene editing see zinc finger nucleases and TAL effector nucleases (TALENs) (Gaj et al., 2013; Bortesi and Fischer, 2015; Markert et al., 2016). For reviews of the CRISPR/Cas9 system, see ­ Wilson and Doudna, 2013; Boettcher and M ­ cManus, 2015; La Russa and Qi, 2015; Liang et  al., 2015; Dominguez et  al., 2016; Gupta and Shukla, 2016; Wang et  al., 2016; Sun et  al., 2017; ­Jaganathan et  al., 2018. The CRISPR/Cas systems continue to demonstrate broad applications in agriculture, increasing options for the management of arthropod pests, insect vectors and transmitted pathogens of plants, animals and



RNA Suppression – Asian Citrus Psyllid Vector and Bacteria

humans (Chen et al., 2016, 2017; Marshall and Akbari, 2016, 2018; Cui et al., 2017; Sun et al., 2017; Taning et  al., 2017, Gantz and Akbari, 2018; Sinisterra-Hunter and Hunter, 2018). 17.7.2  BAPC-CRISPR/Cas9 delivery system: adult ovaries for heritable germline gene editing (Hemiptera: Diaphorina citri) Hunter et al. (2018) reported a new method for heritable germline gene editing by injecting adult ACP, using BAPC-assisted-CRISPR/Cas9 (BAPCtofect-25™ Kit, Phoreus Biotechnology, Manhattan, Kansas). Injection of the components into ACP abdomens of 5th-instar and adult females, near ovaries, bypasses the need for ­embryonic microinjections, resulting in greater survivorship post treatment. The report described the detection of G2 generation knockout mutants of ACP from a G0 injected adult female (Hunter and Sinisterra-Hunter, 2018). The CRISPR/Cas9 system for site-specific genome editing has been used in a variety of arthropod species (Garczynski et al., 2017; Sun et al., 2017; Taning et  al., 2017; Chaverra-Rodriguez et  al., 2018). However, successful applications of germline editing of arthropods relies on injection of editing components into pre-blastoderm embryos, referred to as ‘embryonic microinjection’ (Kistler et  al., 2015; Heinze et  al., 2017; Taning et  al., 2017). Unfortunately, for many arthropod species, embryo injection techniques do not work, as in ACP there is extreme mortality. Previous research with BAPC resulted in efficient gene-delivered dsRNA and plasmid DNA into insects and animal cell cultures (Sukthankar et al., 2014; Tomich et al., 2014; Barros et al., 2016; Avila et al., 2018). Research with similar proof-of-concept delivery using a peptide was reported by ChaverraRodriguez et al. (2018) in mosquitoes using the P2C peptide, which mediated transduction of the Cas9 plasmid from the female hemolymph into the developing mosquito oocytes. Their technology, termed ‘Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control), was shown to work well within Diptera (mosquitoes). The second system, reported by Wang et  al. (2017b), used microvesicles: extracellular vesicles, known as arrestin domain containing protein 1

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[ARRDC1]-mediated microvesicles (ARMMs). These ARMMs could package and deliver intracellularly a myriad of macromolecules, including the tumor suppressor p53 protein, RNAs, and the genome-editing CRISPR-Cas9/guide RNA complex into mammalian cells. 17.7.3  BAPC-CRISPR/Cas9 psyllid gene selection D. citri has at least two thioredoxins, TRX-1 and TRX-2, with variants in the mitochondria and cytoplasm. Thioredoxin participates in various redox reactions and catalyzes dithiol–disulfide exchange reactions. Thioredoxin 2 (TRX-2) was reported by Yoshida et al. (2005) to be preferred over TRX-1 as a reducing substrate of peroxiredoxin-1 and so was selected. Thioredoxins are central metabolic regulators; they are required for female meiosis and early embryonic development and regulate at least 30 other proteins (Yoshida et  al., 2005; Odile et  al., 2017). The CRISPR-associated protein 9 (Cas9) was purchased, and protocols were gleaned from publications on CRISPR (Bassett et al., 2013; Larson et al., 2013; Kistler et al., 2015; Garczynski et al., 2017; Zhang and Reed, 2017). This was the first report for CRISPR gene knocked-out (KO) in D. citri. The gene thioredoxin-2-like (LOC103521994), sequence ID: XM_008487100.1, was from the DIACI_2.0 Genome (https://citrusgreening.org/ organism/Diaphorina_citri/genome) (Saha et  al., 2017a). The two guide RNAs were designed to direct the Cas9 endonuclease to two sites 556 bp apart (Dharmacon, Inc.). The ACP TRX-2 KO used trials of 30 nymphs (4th and 5th instar) and 20 adult females per trial (co-injection of two sgRNAs,100 ng/μl of each, with 200 ng/μl of Cas9 protein, plus BAPC (0.1 ng/ μl) (Hunter et al., 2018). Insects were injected ventrally, lateral to midline of the abdomen, then transferred to a citrus seedling to mature and/or oviposit. Seven days post treatments, the first cohort of six G0 ACP adults oviposited eggs (G1), which were individually analyzed when they were 5th instars for the TRX-KO (~33% detected, four out of 12 tested). Of the remaining cohort of ACP G1 nymphs validated to have the TRX-KO, eclosion from nymph to adult took 6–8 days longer than mock-injected controls. Adult ACP with TRX-KO had significantly shorter lifespans, on average

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8.5 days, compared with controls, which averaged 16 days. Of the eight G1 nymphs that eclosed to adults, these G1 adults produced G2 eggs. From the G2 eggs that successfully developed into adults, a cohort of six adults was analyzed. One out of the six G2 adults had the knock-out, TRX-KO (~16%), representing successful adult treatment to produce a heritable gene edit in psyllids. While the success rate was low, further optimization of the method will no doubt increase the rate of success for heritable gene edits in psyllids and other hemipterans using this method (Hunter et al., 2018).

17.8  Future Perspective Reviewed here are several emerging technologies and strategies that enable direct targeting of pathogenic microbes, like bacteria in citrus trees, and the critical genes specific to arthropod vectors, like ACP and their endosymbionts (Jackson et  al., 2016; Taning et  al., 2017; Gantz and Akbari, 2018; Hunter and Sinisterra-Hunter, ­ 2018; Jani et al., 2018; Nan and Zhang, 2018). RNAi-based approaches continue to make advances to improve persistence and activity, especially when applied as an exogenous spray or as soil-applied treatments (Li et  al., 2015; Cagliari et al., 2018; Hunter and Sinisterra-Hunter, 2018; Worrall et  al., 2019). Expression systems using microbes, such as viruses, yeasts or bacteria, also provide flexibility in methods to manage crop pathogens or their arthropod vectors (Marshall and Akbari, 2016, 2018; Murphy et  al., 2016; Whitten et al., 2016; ISAAA, 2017; Kolliopoulou et  al., 2017; Whitten and Dyson, 2017). RNA-suppressing biopesticides will soon become common in the protection of crops that are either difficult to transform, such as fruit trees, or that are short-term seasonal crops (Baum and Roberts, 2014; Saurabh et  al., 2014; Abdurakhmonov et  al., 2016; Brookes and Barfoot, 2016; Taning et al., 2018; Zotti et al., 2018). The impact on agriculture and green environments will continue to embrace these tools to provide important human needs, such as food, feeds and fibers (Emani and Hunter, 2013; NASEM, 2016; Sinisterra-Hunter and Hunter, 2018). These advances will also bring innovative products like biofuels, biopharma and biomaterials (Ricroch

et  al., 2016; Jaganathan et  al., 2018; Sinisterra-Hunter and Hunter, 2018). These novel commodities will continue to bring social stability, by increasing jobs, variety of foods and better health through a cleaner, greener environment for an ever-increasing global population.

Acknowledgments Research targeting microbes in collaboration with Dr Kirsten Pelz-Stelinski, University of ­Florida, Lake Alfred, Florida. We thank Maria T. Gonzalez, Senior Biological Science Technician, USDA, ARS for CRISPR designs and injections; Salvador P. Lopez, BST, USDA, ARS for method development and qPCR analyses; Jennifer ­ ­Wildonger, BST, USDA, ARS, for bioassay analyses, qPCR analyses, imaging by confocal and light microscopy, USDA, ARS, Ft. Pierce, Florida. Dr Steve Garzinsky, USDA, ARS, Wapato, Washington, for assistance in CRISPR sgRNA design and protocols. The LabEx/USA International program: ­Brazil/USDA, ARS, project coordinator, and Dr Eduardo C. A ­ ndrade, EMBRAPA Cassava and Fruits, Rua Embrapa, s/n, Cruz das Almas, Bahia, Cep 44380-000, Brazil, cooperative research program on RNAi to reduce insect pests of citrus, 2011–2015; Chris Holland, confocal imaging, bioassay trials, ORISE program DOE/USDA. This research was supported in part by an appointment to the Agricultural Research Service (ARS) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the US Department of Energy (DOE) and the US Department of Agriculture (USDA). ORISE is managed by ORAU under DOE contract number DE-SC0014664. All opinions expressed in this paper are the authors’ and do not necessarily reflect the policies and views of USDA, ARS, DOE, or ORAU/ORISE. Research supported in part by: National Institute of Food and Agriculture, USDA, SCRI/Citrus Disease ­Research & Extension. Award #2015-7001623028, Developing an Infrastructure and Product Test Pipeline to Deliver Novel Therapies for Citrus Greening Disease, Lead Dr Susan Brown, Kansas State Univ., Manhattan, Kansas (www. citrusgreening.org); and the NIFA, USDA, 2015 National Institute of Food and Agriculture, USDA, SCRI. Award #2015-10479. Targeting



RNA Suppression – Asian Citrus Psyllid Vector and Bacteria

microbes to control huanglongbing disease of citrus #2016-70016-24782, Lead Dr Kirsten Pelz-Stelinski, University of Florida, Lake Alfred, Florida; Co-PI, Dr Wayne Hunter, USDA, ARS, Ft. Pierce, Florida; Dr Sidney Altman, Yale University, New Haven, Connecticut. The Citrus ­Research Board, 2011–2012, Psyllid Draft Genome initiative; The Florida Citrus Research & Development Foundation, CRDF, 2008-2009. ­ Florida Citrus Production Research Advisory Council, 2008; Drs S. Han, and Shannon Johnson, Los Alamos National Laboratory, B-6 Genome Science, PO Box 1663 M888, Los Alamos, New Mexico 87545, for sequencing Psyllid Genome Consortium 2011–2013. Drs Dan Weaver, Justin Reese, Brandi Cantrell, and Justin McCarthy, Genformatic, LLC, assemblies and analyses; Tom D’Elia, and Helen Koch, Indian River State College, Fort Piece, FL, for training and student annotations; National Agriculture Library, Monica Poelchau, and Christopher Childers, USDA, ARS, management of the psyllid genome working group database, Beltsville, Maryland; AgData Commons, National Agriculture Library, USDA, ARS, Beltsville, Maryland. Dr Surya Saha, and

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team, Boyce Thompson Institute, New York, ­bioinformatics, DIACI 2. Genome consortium, OGS 2016-2019. Thanks to Drs Tom Rutherford and Yasmith Bernal, for product use VERGE Clay pellets, Oil-Dri Corporation of America, Chicago, Illinois (http://www.oildri.com). We thank Dr Hong Moulton’s laboratory at Oregon State University for making PPMOs. We acknowledge the long service, dedication and friendship of Dr Phil Stansly, University of Florida. NOTE: The term huanglongbing (HLB), which means yellow (huang) shoot (long, previously interpreted as ‘dragon’) disease (bing), was unanimously adopted as the official name of the disease by the International Organization of Citrus Virologists (IOCV) at the 13th Conference of IOCV in Fuzhou, China, in 1995 (http://iocv.org/ huanglongbing.htm; accessed 22 February 2018). Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply endorsement or recommendation by the US Department of Agriculture to the exclusion of other suitable products. USDA is an equal opportunity provider and employer.

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Index

ACP = Asian citrus psyllid

abandoned groves  146, 215, 244 abiotic factors regulating population  5–7, 88–91, 182–185, 225–226 see also climate acetic acid  50, 58 acquisition access period (AAP)  114, 116–117 Adalia bipunctata (predatory ladybeetle)  96 aerial spraying  172 Africa  166, 259 aggregations of ACP  35–36, 58 air-blast sprayers  172 alimentary canal  23–26 and CLas infection  118–121, 130 altitude  89, 182, 185 Amblyseius swirskii (predatory mite)  96 amino acids provided by endosymbionts  102 anatomy  1, 12–27 antennae 49–50 antimicrobial agents  108 antisense oligos (morpholinos)  265–266 ants, predatory  192, 196–197 area-wide management  146–147, 174 California  174, 251–255 Florida  224–225, 230 Texas  235, 238–244 Asia abiotic factors affecting distribution  182–185 biological control  190–197 citrus industry  181–182 cultural control  197–198 insecticides  185–190, 198 origin and spread of ACP  179–181 

transmission of CLas 115 Wolbachia 104 Australia  180, 192

bacteriocytes 102–103 beetles (ladybeetles)  92, 96, 193–194, 228, 244 Bergera koenigii (curry leaf)  68 beta-cypermethrin 189 Bhutan  182, 185 billygoat weed  197 biological control Asia 190–197 Brazil 214–216 California  252, 255 commercial production of agents  96, 215–216 endosymbionts  107–108, 109 entomopathogenic fungi  92–93, 190, 214, 244, 260 Florida  91, 92, 94–96, 223, 227–228, 261 and insecticide use  174–175, 228 parasitoids see parasitoids predators  92, 96, 192–197, 228, 244 Texas  244, 245 viruses 262 biopesticides dsRNA 263–265 fungi 214 biosecurity  67, 167 in nurseries  148–149, 223, 245–246 branched amphiphilic peptide capsules (BAPC)  268 and CRISPR/Cas9 269–270 Brazil  167, 210–219 biological control  214–216 examples of successful HLB control  217–219 external actions for control  216–217, 218 285

286 Index

Brazil (continued) insecticides  167, 212–214, 216, 218 monitoring  211–212, 218 transmission of CLas 115

Cacopsylla citrisuga (psyllid)  113 California  167, 174, 250–256 climate  168, 169, 251 San Joaquin Valley  167, 254–255 southern  167–168, 251–254 calling behavior  30, 32–33, 35–36 Candidatus Carsonella rudii  102, 128 Candidatus Liberibacter africanus (CLaf) 113 Candidatus Liberibacter americanus (CLam) 113 Candidatus Liberibacter asiaticus (CLas)  101, 105–106 effect on the vector  127–129 development 7–8 fecundity/fertility  34, 105, 128 feeding patterns  51, 106, 128 immune system  107, 128 proteomics 129–131 and the host plant  126, 126–127 host volatiles  55, 56, 128 host-switching 117 transmission  105–106, 114, 131, 242–243, 259–260 acquisition  114–118, 223–224 horizontal 126–127 infective colonization events  140–143 latent period  118, 140 multiplication 121–122 retention and inoculation  122–126 translocation 118–121 vertical 126 and Wolbachia  103, 129 Candidatus Liberibacter solanacearum (CLso) 116, 117, 121, 122, 124, 127 Candidatus Proftella armature  102–103, 129 China biological control  190, 192, 196 citrus industry  181 distribution and spread  179–180, 182 insecticides  186, 187, 189 resistance 189 transmission of CLas 115 citrus greening disease see huanglongbing citrus industry Asia 181–182 Brazil 210 USA  229–230, 235, 250 citrus red mite  196 CLas see Candidatus Liberibacter asiaticus climate  5–7, 88–90, 168 Asia 182–185 USA  168–169, 170, 224, 251 coccinellids (ladybeetles)  92, 96, 193–194, 228, 244

cold temperatures  6, 88 color of ACP  2–3, 130 attractant/repellent  46–48, 246 perception 43–44 communication between psyllids (vibrational)  2, 30, 32–33, 35–36 with humans (unsuccessful)  81 copulation  2, 30, 34 courtship  30, 32–33, 35–36 CRISPR technology  268–270 crumena 20–21 cultural control  228 flush management  198, 229 foliar nutrition  229 interplanting  56–57, 188, 197–198 physical barriers to ACP movement  147, 175, 198, 229, 246 in nurseries  223, 245–246 planting density/orientation  198, 246 reflective mulches  48, 49, 229 removal of plants  150, 161, 216, 218 replanting 150–151 soil amendments  229 cyantraniliprole 173

developmental biology  4–8, 13, 89 Diaphorencyrtus aligarhensis (parasitoid wasp)  91, 92, 190, 191–192 diaphorin  103, 129 dimethyl disulfide (DMDS)  56–57, 59 distribution and spread abiotic factors  89, 182–185 Asia  179–181, 182–185 Brazil  167, 210 California 251 Florida  142, 147–149, 222–223 local spread  144–147 dooryard citrus see residential areas dormant sprays  91, 93–94, 162, 171, 175, 224, 230, 239

economic injury level (EIL)  161, 226 economic thresholds for ACP management  161–163, 171–172, 174, 226 edge effect  145–146, 172, 174, 224, 243, 246 education of farmers and others  187, 190, 217, 242 eggs  3, 4 favored hosts  80 oocyte development  31 temperature effects on oviposition  5, 89 embryology 13 endosymbionts see symbionts entomopathogenic fungi  92–93, 190, 214, 244, 260



environmental conditions and developmental biology  5–7 and population  88–91, 168–169, 182–185, 225–226 esophagus 24 essential oils, repellent  57, 59 excretory system  26 eye 43–44

feeding behavior  50–52, 58, 260 differences between nymphs and adults  116 effect of CLas infection  51, 106, 128 females acquisition of CLas 117 anatomy  1, 2, 26–27 fertility/fecundity  3, 89 CLas infection  34, 105, 128 longevity  4, 5, 8 see also mating behavior filter chamber  13, 24–25 Florida  168, 222–230 area-wide management  224–225, 230 biological control  91, 92, 94–96, 223, 227–228, 261 citrus industry  229–230 climate  168, 170, 224 cultural control  175, 228–229 economic injury level  226 hurricanes 225–226 insecticides  223, 224–225, 226–227, 228 regulations  148–149, 223 spread  142, 147–149, 222–223 transmission of CLas 115 Wolbachia 104 flush shoots attraction of ACP  2, 34–35 growth periods  5, 90, 168, 223–224 reflectance/color 44 removal of summer flush  198, 229 temperature sensitivity  6 flush transmission of CLas  117–118, 223–224 foliar nutrients  229 freezing  6, 88 fruit, transport of   148 fungi, entomopathogenic  92–93, 190, 214, 244, 260

garlic chive  57 generation number  4–5 genetic engineering of ACP  269–270 of citrus  262–263 CRISPR technology  268–269 of endosymbionts  107–108 genome of ACP  261, 263

Index 287

greenleaf desmodium  197 groves abandoned  146, 215, 244 planting density/orientation  198, 246 spread within/between  144–147 young trees  213, 240–241, 242 see also cultural control guava  56–57, 188, 197–198 gustation  50–52, 58 see also olfaction

health of farmworkers biting ants  197 insecticide use  189–190 heat  6, 88, 89, 184 hemocyanin 130 Hippodamia convergens (predatory ladybeetle)  96 Hirsutella citriformis (fungus)  92 HLB see huanglongbing honeydew 4 horticultural mineral oils (HMOs)  57–58, 59, 172, 187, 188 host plants and CLas transmission  117, 126, 126–127 climate effects  168 height 186 location by ACP olfactory cues  53–59, 60 visual cues  44–49 original (non-citrus) host  68 range of species  69–81 resistance to ACP  69, 79, 80 resistance to HLB  144, 151, 260, 262–263 huanglongbing (HLB)  259 Brazil  210, 219 and CLas-positivity in ACP  141–143 Florida  142, 147–149, 223 incubation period  140, 144, 151 named 271 pathogens  113, 259 see also Candidatus Liberibacter asiaticus resistance  260, 262–263 spread between groves  146–147 spread within an individual tree  143–144 spread within a grove  144–146 symptoms  44–45, 144–145 vectors 113 humidity  6–7, 89–90, 184 hyperparasitoids 192

imidacloprid  51, 169, 188 resistance  189, 227 immune system  106–107, 128, 130 incubation period of HLB  140, 144, 151

288 Index

India climate 184 natural enemies  191 spread of ACP  68, 179, 181 Indonesia  180, 182, 191, 192 infection (acquisition) rates of CLas 114–116 infective colonization event transmission  140–151 inoculation of CLas into host plant  123–126 insecticides  94, 169–174 application method  172–173, 185–186 area-wide management  174, 224–225, 230, 239–245, 252–255 Asia  185–190, 198 and biological control  174–175, 228 border spraying  172, 174, 224, 246 Brazil  167, 212–214, 216, 218 California  167–168, 251, 252–255 choice  169–171, 186, 213, 227, 241 dormant sprays  91, 93–94, 162, 171, 175, 224, 230, 239 economic thresholds for use  161–163, 171–172, 226 Florida  223, 224–225, 226–227, 228 health and environmental problems  189–190 in nurseries  150, 213 reduction of feeding behavior  51 resistance  173, 189, 213, 227 secondary pests  227, 241 systemic (soil drenches)  51, 173, 213, 240–241, 242 targeting chordotonal organs  36 Texas  168, 235, 239–242, 245, 246, 247 young orchards  213, 240–241, 242 interplanting with repellents  56–57, 188, 197–198 Isaria fumosorosea (entomopathogenic fungus)  93, 214, 260

jackfruit 81

kaolin-based particle films  48, 49

labium  13, 21 lacewings (Chrysopidae)  96, 194 ladybeetles (coccinellids)  92, 96, 193–194, 228, 244 leaves of citrus  44–45 life cycle  1–7, 89 light leaf reflectance  44–45 phototaxis  7, 34, 46–48, 90 UV  7, 48–49, 90, 185, 229 limb-tap sampling  157–158, 159, 171, 211 longevity  4, 5, 7, 8

Malaysia  182, 184, 191 males anatomy  1, 31 mating behavior  2, 30–36 Malpighian tubules  26 management  156, 166–167 acoustic devices  36 area-wide management  146–147, 174, 224–225, 230, 235, 238–244, 251–255 Asia 185–198 attract-and-kill  59, 60, 246 biological see biological control California  167, 167–168, 250–256 chemical repellents  56–58, 59, 197–198 cultural see cultural control early-stage invasion  167 economic thresholds  161–163, 171–172, 174, 226 Florida 223–230 host resistance  144, 151, 260, 262–263 insecticides see insecticides late-stage invasion  168 mid-stage invasion  167–168 molecular techniques  258, 262–270 monitoring  156–160, 171, 211–212, 242–243, 255 in nurseries  148–149, 149–150, 213, 223, 245–246, 250 prevention of invasion  167 push–pull strategies  59–60 systems biology  260 Texas  168, 235, 238–247 mating behavior  2, 30–36, 58 methyl salicylate  55, 59, 128 midgut  24–26, 130 mineral oils  57–58, 59, 172, 187, 188 monitoring of ACP populations  36, 156–160, 171 Brazil  211–212, 218 California  250, 255 Texas 242–243 morpholinos (antisense oligos)  265–266 mortality  6, 88, 93, 105 mulch, reflective  48, 49, 229 Murraya spp. 68, 126, 147–149, 222

Nepal  182, 184 nurseries  148, 148–149, 149–150 Brazil 213 California 250 Florida  148–149, 223 Texas 245–246 nymphs  4, 80, 89 and transmission of CLas 114–116, 122, 123



Oecophylla smaragdina (predatory ant)  192, 196–197 olfaction anatomy and physiology  49–50, 52–53 host selection  53–58, 59 management tools  59–60 mate searching  31, 34, 58 repellents/confusants  56–58, 59, 197–198 see also gustation oral region anatomy 13–23 CLas transmission  118, 121, 123 orange jasmine (Murraya paniculata)  68, 126, 147–149, 222 orchards see groves organic citrus  240, 241, 253, 254 origin of ACP  179–180 oviposition  3, 4 favored hosts  80 and temperature  5, 89

Pakistan  6, 181, 182, 189, 189–190 parasitoids  91–92, 94–96, 175 Asia 190–192 Brazil 214–216 California 252 Florida  223, 228, 261 rearing 215–216 Texas  244, 245 paratransgenesis  107–108, 109 pesticides see insecticides phagostimulants  52, 58 phenology  90–91, 237–238 pheromones  2, 31–32, 58 Philippines  181, 184, 191 photoperiod 7 phototaxis  7, 34, 46–48, 90 Phytosanitary Alert System (Brazil)  212 phytosanitary measures  67, 167 in nurseries  148–149, 223, 245–246 polarized light  46 polymorphism  2–3, 130 predators  92, 96, 192–197, 228, 244 preoral orifice  14 prevention of psyllid invasion  167 probing behavior  50–52, 58 proteomics (ACP infected with CLas) 129–131

quarantine  149, 167, 252

rainfall  7, 90, 184 regulation of nurseries (USA)  148–149, 223, 245–246 relative humidity  6–7, 89–90, 184

Index 289

repellents ant secretions  196, 197 plant volatiles  56–57, 59, 197–198 reproductive biology  1–3, 5, 7 anatomy  26–27, 31 and CLas  34, 105 mating behavior  2, 30–36, 58 and temperature  5–6, 89 and Wolbachia 103 residential areas  146–147 Brazil 217 California  168, 250, 252, 253, 254, 255 Florida 147–148 Texas  168, 235–237, 238, 244–245 RNA-based control techniques  108, 258, 263, 270 delivery mechanisms  267–268 exogenously applied dsRNA  263–265 morpholinos 265–266 non-canonical nucleotides  266–267 root growth  168 root infection  143–144 rostrum 16

salivary glands/canals  21–23, 118, 121, 123 sampling methods  157–160, 171, 211–212, 243, 255 Saudi Arabia  89 screens  223, 229, 246 seasonality  90–91, 237–238 sexual transmission of CLas 127 social interactions  35 soil applications dsRNA 263–264 insecticides  51, 173, 213, 240–241, 242 pH amendment  229 South Africa  166 Spiroplasma spp. 121 spirotetramat 170 spread and distribution abiotic factors  89, 182–185 Asia  179–181, 182–185 Brazil  167, 210 California 251 Florida  142, 147–149, 222–223 local spread  144–147 stem-tap sampling  157–158, 159, 171, 211 sticky traps see yellow sticky traps stylet(s) adult 16–18 larval 15–16 replacement  13, 18–20 suction sampling  159 surveillance see monitoring of ACP populations sweep nets  159

290 Index

symbionts  101–102, 259 biocontrol using  107–108, 109 Ca. C. rudii  102, 128 Ca. L. asiaticus see Candidatus Liberibacter asiaticus Ca. P. armature  102–103, 129 commensal (extracellular)  106 defensive function  103, 106–107 Wolbachia  103–105, 108, 129 Sympherobius barberi (brown lacewing)  96

Taiwan 181 Tamarixia radiata (parasitoid wasp)  91–92, 94–96, 175 Asia 190–192 Brazil 214–216 California 252 Florida  223, 228 rearing 215–216 Texas  244, 245 temperature  5–6, 88–89, 168 Asia 182–184 tentorium 16 Texas  168, 234–247 area-wide management  235, 238–244 before ACP invasion  234 biological control  244, 245 climate  168, 170 commercial citrus  235 dooryard citrus  168, 235–237, 238, 244–245 insecticides  168, 235, 239–242, 245, 246, 247 phenology 237–238 Thailand  181, 186–187, 190 thiamethoxam  169, 189 thioredoxin gene knockout  269–270 transgenic organisms see genetic engineering transmission of CLas  105–106, 114, 131, 242–243, 259–260 acquisition  114–118, 223–224 horizontal 126–127

infective colonization events  140–143 latent period  118 multiplication 121–122 retention and inoculation  122–126 translocation 118–121 vertical 126 traps  60, 158, 171, 211, 243, 255 auditory lures  36, 157 chemical lures  59, 157 color  46, 49 Trioza erytreae (African citrus psyllid)  113

urban areas see residential areas USA see California; Florida; Texas UV light  7, 48–49, 90, 185, 229

vibrational communication  2, 30, 32–33, 35–36, 157 Vietnam  181, 188, 196 viruses 262 vision 43–49 visual sampling  158–159, 159–160, 211–212 volatile compounds attractant 53–56 repellent  56–57, 59, 197–198

weaver ant (Oecophylla smaragdina) 192, 196–197 weeds as hosts  81 repellent 197 wind  90, 184, 225–226 windbreaks  147, 198, 246 Wolbachia  103–105, 108, 129

yellow sticky traps  46, 49, 158, 171, 211, 243, 255

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