Neotropical Endophytic Fungi: Diversity, Ecology, and Biotechnological Applications 3030535053, 9783030535056

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Luiz Henrique Rosa  Editor

Neotropical Endophytic Fungi

Diversity, Ecology, and Biotechnological Applications

Neotropical Endophytic Fungi

Luiz Henrique Rosa Editors

Neotropical Endophytic Fungi Diversity, Ecology, and Biotechnological Applications

Editor Luiz Henrique Rosa Departamento de Microbiologia Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais Belo Horizonte, Minas Gerais, Brazil

ISBN 978-3-030-53505-6    ISBN 978-3-030-53506-3 (eBook) https://doi.org/10.1007/978-3-030-53506-3 © Springer Nature Switzerland AG 2021, Corrected Publication 2021 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

Ancient plant species Ficus gomelleira Kunth & C.D. Bouché (Moraceae) inside the endangered Tropical Rain Forest fragment at Parque Estadual do Rio Doce (PERD), Minas Gerais state, Brazil (Photo credits: L.H. Rosa) v

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Preface

The Neotropical biogeographic region (Fig. 1), also known as Neotropics, includes Central America, the Caribbean, and South America and represents one of the eight biogeographic realms of the Earth’s land surface. The major Neotropical regions include Amazonia, Caribbean, Central America, Central Andes, Eastern South America, Northern Andes, Orinoco, and Southern South America. Neotropics is recognized to shelter a rich biological diversity of animal, plants, and microorganisms. However, these knowledge is insufficient and many species still hidden and unknown by the science. In addition, the Neotropical biodiversity represents an important genetic heritage for humanity able to origin different bioproducts for industrial and agricultural sectors. Neotropics is vast and comprises several distinct biomes and habitats such as seasonally dry forests, arid zones, high-elevation grasslands, young and old mountain systems, and the Atlantic Forests and Amazonia rainforests (Hoorn et al. 2010). In all these biomes shelter rich and diverse biological communities, many of them endemics and endangered.

Fig. 1  Perspective view of the Neotropical realm ranging from Southern Mexico to the Southern portion of South America. (Source: Google Maps)

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Kingdom Fungi includes eukaryotic organisms that are present in nearly all environments on Earth. However, different fungal groups occur mainly in the tropical regions of the World. However, recent polyphasic taxonomic studies proposed new phyla. Tedersoo et al. (2018) proposed the following phyla for Fungi: Aphelidiomycota, Basidiobolomycota, Calcarisporiellomycota, Glomeromycota, Entomophthoromycota, Entorrhizomycota, Kickxellomycota, Monoblepharomycota, Mortierellomycota, and Olpidiomycota. According to Hawksworth (1993), the complexity of tropical forests of the World associated with the high diverse habitats, available organic matter, and warm climate make it a region with large numbers, if not the majority, of undescribed fungal species. Among the different fungal groups, those characterized as endophytes have been call attention in the last decades. Fungal endophytes virtually can be found all plants, including trees, grasses, and herbaceous species. Mostly of the endophytic fungi belong to the Ascomycota and their anamorphic stages, which live asymptomatically in plant tissues (Petrini et at. 1991). Fungal endophytes are recognised as a repository of different bioactive compounds, including novel metabolites of pharmaceutical and agricultural importance. The broad diversity and taxonomic spectrum exhibited by these fungal group make them especially interesting for ecological, evolutionary, and bioprospecting studies programs. Despite the ecological and biotechnological importance of the Neotropical realm, few mycological studies have been performed to date, in special those with focus on the endophytes, perhaps due its extensive size and the diverse biomes. The aim of this book is to present the vanguard aspects of diversity, ecology, and biotechnology of Neotropical fungi. Experts in tropical mycology and chemistry of natural products from several countries (i.e. Argentine, Brazil, Chile, Colombia, Costa Rica, Ecuador, Spain, and United States) describe and discuss fungal occurrence in the different biomes of Neotropics. We are grateful to Joao Pildervasser (Publishing Editor) and Arun Siva Shanmugam and G. Chitra (Production Editor) for their support and patience during the development of this book. We acknowledge the financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG); Ibero-American Programme on Science and Technology for Development (Cyted); Bio-organizaciones Research Group (University Simón Bolívar, Barranquilla-Colombia); Diseño Biosintético de Fungicidas Research Group (University of Cádiz, Spain); the Center for Marine and Limnological Research of the Caribbean CICMAR (Barranquilla, Colombia); Dr. Giorgio Anfuso (University of Cádiz, Spain) for the design of the map; Luis Diego Vargas, Luis Guillermo Acosta, Jorge Blanco, Catalina Murillo, Myriam Hernandez, Silvia Soto, Loengrin Umaña, and Nefertiti Campos, Allan Jimenez and Ana Lorena Guevara. Grant NIH U01 TW007404 supported the data generated at the former National Institute of Biodiversity; authorities from Absolute Natural Reserve Cabo Blanco; Carara, Braulio Carrillo, Guanacaste, Santa Rosa, Palo Verde, Rincón de la Vieja, and Tenorio National Parks, and to CONAGEBIO for granting the sample collecting permits (307-2003-OFAU, R-012-2005-OT-CONAGEBIO, R-CM-

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INBio-03-2006-OT, R-CM-INBio-06-2006-OT, R-CM-INBio-30-2007-OT, R-CMINBio-059-2008-OT); ANPCyT of Argentina for the financial support to our projects (PICT-2018-01593); Mariana Valente for her expert assistance in making the figures; the National Fund for Scientific and Technological Development, FONDECYT Grant Nº 11190754 (Iniciación); Project High Altitude Laboratory ATA #1799 Ministry of Education of Chile; FONDECYT Postdoctoral Grant Nº 3140279 and the INACH G22-11 Grant of Chilean Antarctic Institute (INACH); the Ministry of Environment of Ecuador; Dr Paul Brickle (director), staff and scientists of the South Atlantic Environmental Research Institute (SAERI) and the Falkland Islands Government. Departamento de Microbiologia Luiz Henrique Rosa Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais Belo Horizonte, Minas Gerais, Brazil  

References Hawksworth DL (1993) The tropical fungal biota: census, pertinence, prophylaxis, and prognosis. In: Isaac S, Frankland JC, Watling R, Whalley AJS (Eds) Aspects of Tropical Mycology. Cambridge University Press, UK, pp.265-293 Hoorn C, Wesselingh FP, ter Steege H et al. (2010) Amazonia through time: Andean uplift, climate change, landscape evolution, and biodiversity. Science 330:927-931 Kirk PM, Cannon PF, Minter DW, Stalpers JA (2008) Dictionary of the Fungi, 10th ed. CAB International: Wallingford, UK. Petrini O (1991) Fungal endophyte of tree leaves. In: Andrews J, Hirano SS (Eds) Microbial Ecology of Leaves. Springer, New York, pp.179–197 Tedersoo L, Sánchez-Ramírez S, Köljalg U, Bahram M, Döring M, Schigel D, May T, Ryberg M, Abarenkov K (2018) High-level classification of the Fungi and a tool for evolutionary ecological analyses. Fungal Div 90:135-159

Contents

1 Ecology of Neotropical Endophytic Fungi ��������������������������������������������    1 Camila Rodrigues de Carvalho, Mariana Costa Ferreira, and Luiz Henrique Rosa 2 Diversity, Ecology, and Applications of Epichloë Fungal Endophytes of Grasses in South America��������������������������������   11 Leopoldo J. Iannone, M. Victoria Novas, Patricia D. Mc Cargo, Andrea C. Ueno, and Pedro E. Gundel 3 Trypanocidal and Herbicidal Activities of Endophytic Fungi Associated with Medicinal Plant Lafoensia pacari Living in Neotropical Wetland Pantanal of Brazil��������������������������������   37 Soraya Sander Amorim, Camila Rodrigues de Carvalho, Jéssica Catarine Silva de Assis, Carlos Leomar Zani, Tânia Maria de Almeida Alves, Policarpo Ademar Sales Junior, Marcos Antônio Soares, and Luiz Henrique Rosa 4 Advances in Research on Biodiversity and Bioprospecting of Endophytic Fungi in Chile������������������������������������������������������������������   53 Rómulo Oses-Pedraza, Víctor Hernández, Leonardo Campos, José Becerra, Dánae Irribarren-Riquelme, Paris Lavín, and Jaime Rodríguez 5 Endophytic Fungal Community Associated with Colombian Plants����������������������������������������������������������������������������   93 Hernando José Bolívar-Anillo, Ezzanad Abdellah, Gesiane da Silva Lima, Inmaculada Izquierdo-Bueno, Javier Moraga, and Gabriel Franco dos Santos 6 Fungal Endophytes and Their Bioactive Compounds in Tropical Forests of Costa Rica������������������������������������������������������������  109 Keilor Rojas-Jimenez and Giselle Tamayo-Castillo

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7 What Do We Know About Fungal Endophyte Diversity in a Mega Diverse Country? An Appeal for Increased Conservation and Research��������������������������������������������������������������������  131 Alexandra Narvaez-Trujillo, María R. Marchán-Rivadeneira, Eliana Veloz-Villavicencio, and Carolina E. Portero 8 Diversity, Ecology, and Bioprospecting of Endophytic Fungi in the Brazilian Biomes of Rupestrian Grasslands, Caatinga, Pampa, and Pantanal ������������������������������������������������������������  151 Camila Rodrigues de Carvalho, Alice Ferreira-D’Silva, Soraya Sander Amorim, and Luiz Henrique Rosa 9 Endophytic Fungi Associated with Medicinal Plants of Amazonian Forest��������������������������������������������������������������������������������  177 Eskálath Morganna Silva Ferreira, Tatiana Maracaípe Corrêia, Juliana Fonseca Moreira da Silva, and Raphael Sanzio Pimenta 10 Association of Endophytic Fungi with Ancient Neotropical Plants������������������������������������������������������������������������������������  199 Marina Bahia and Luiz Henrique Rosa 11 Bioprospecting of Neotropical Endophytic Fungi in South America Applied to Medicine��������������������������������������������������  213 Mariana Costa Ferreira, Denise de Oliveira Scoaris, Soraya Sander Amorim, Betania Barros Cota, Emerson de Castro Barbosa, Jaquelline Germano de Oliveira, Carlos Leomar Zani, and Luiz Henrique Rosa 12 Bioactive Compounds Produced by Neotropical Endophytic Fungi Applied to Agriculture���������������������������������������������  257 Débora Luiza Costa Barreto, Rafaela Nogueira de Azevedo, Camila Rodrigues de Carvalho, Mariana Costa Ferreira, Charles Lowell Cantrell, Stephen Oscar Duke, and Luiz Henrique Rosa 13 Neotropical Plant-Associated Endophytic Fungi: A Source of Promising Macromolecules for Use in Biotechnology����������������������  297 Mariana de Lourdes Almeida Vieira and Luiz Henrique Rosa 14 Bioprospecting of Bioactive Secondary Metabolites of Endophytic Fungi of Carapichea ipecacuanha (Rubiaceae), a Neotropical Medicinal Species��������������������������������������  321 Rafaela Nogueira de Azevedo, Mariana Costa Ferreira, Jéssica Catarina Silva de Assis, Policarpo Ademar Sales Junior, Daniela Nabak Bueno Maia, Tânia Maria de Almeida Alves, Markus Kohlhoff, Carlos Leomar Zani, and Luiz Henrique Rosa

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15 Effectiveness of Endophytic Fungi from Baccharis dracunculifolia Against Sucking Insect and Fungal Pathogens ������������������������������������  337 Yumi Oki, Isabela M. Nascimento, Naíla B. da Costa, Renata Aparecida Maia, Jacqueline A. Takahashi, Vany Ferraz, Ary Corrêa Júnior, and G. Wilson Fernandes 16 Bioprospecting of Secondary Bioactive Metabolites Produced by Endophytic Fungi of the Medicinal Piper sp. in the Brazilian Tropical Rain Forest ����������������������������������������������  351 Raissa Hellen da Silva Florindo, Mariana Costa Ferreira, Carlos Leomar Zani, Tânia Maria de Almeida Alves, Policarpo Ademar Sales Junior, Emerson de Castro Barbosa, Jaquelline Germano de Oliveira, Fernanda Ruth França Cavalcanti, Antoniana Ursine Krettli, Isabela Penna Ceravolo, and Luiz Henrique Rosa 17 Diversity of Endophytic Fungi of Empetrum rubrum Vahl ex Willd (Ericaceae): A Medicinal Plant from Austral South America ������������������������������������������������������������������  375 Mariana Costa Ferreira, Pedro Henrique Rodrigues Loureiro, Jéssica Catarina Silva de Assis, Micheline Carvalho-Silva, Paulo Eduardo Aguiar Saraiva Câmara, Diego Knop Henriques, and Luiz Henrique Rosa  Correction to: Advances in Research on Biodiversity and Bioprospecting of Endophytic Fungi in Chile������������������������������������������ C1 Index������������������������������������������������������������������������������������������������������������������  387

Contributors

Ezzanad Abdellah  Organic Chemistry Department, Faculty of Science,University of Cádiz, Puerto Real, Cádiz, Spain Soraya  Sander  Amorim  Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil Marina Bahia  Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil Débora  Luiza  Costa  Barreto  Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil José Becerra  Laboratory of Natural Products Chemistry, Department of Botany, Faculty of Natural and Oceanographic Sciences, Universidad de Concepcion, Concepcion, Chile Hernando  José  Bolívar-Anillo  Microbiology Department, Faculty of Basic and Biomedical Sciences, Universidad Simón Bolívar, Barranquilla, Colombia Paulo Eduardo Aguiar Saraiva Câmara  Departamento de Botânica, Universidade de Brasília, Distrito Federal, Brasília, Brazil Leonardo  Campos  Vicerrectoria de Investigacion y Postgrado (VRIP), Centro Regional de Investigacion y Desarrollo Sustentable de Atacama (CRIDESAT) – Universidad de Atacama, Copiapo, Chile Charles Lowell Cantrell  Natural Products Utilization Research Unit, Agricultural Research Service, United States Department of Agriculture, Oxford, MS, USA Micheline Carvalho-Silva  Departamento de Botânica, Universidade de Brasília, Distrito Federal, Brasília, Brazil Fernanda Ruth França Cavalcanti  Instituto René Rachou, FIOCRUZ-MG, Belo Horizonte, Minas Gerais, Brazil xiii

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Contributors

Isabela  Penna  Ceravolo  Instituto de Pesquisa René Rachou, FIOCRUZ-MG, Belo Horizonte, MG, Brazil Tatiana  Maracaípe  Corrêia  Laboratorio de Microbiologia Geral e Aplicada, Universidade Federal do Tocantins, Palmas, Tocantins, Brazil Betania  Barros  Cota  Instituto René Rachou, FIOCRUZ-MG, Belo Horizonte, Minas Gerais, Brazil Naíla B. da Costa  Departamento de Genética Ecologia & Evolução, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Raissa Hellen da Silva Florindo  Departamento de Microbiologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil Gesiane da Silva Lima  Departamento de Química, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Juliana Fonseca Moreira da Silva  Laboratório de Microbiologia Geral e Aplicada, Universidade Federal do Tocantins, Palmas, Tocantins, Brazil Tânia  Maria  de Almeida  Alves  Instituto René Rachou, FIOCRUZ-MG, Belo Horizonte, Brazil Jéssica  Catarine  Silva  de Assis  Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil Rafaela  Nogueira  de Azevedo  Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Camila  Rodrigues  de Carvalho  Instituto René Rachou, FIOCRUZ-MG, Belo Horizonte, Minas Gerais, Brazil Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Emerson  de Castro  Barbosa  Instituto René Rachou, FIOCRUZ-MG, Belo Horizonte, Minas Gerais, Brazil Mariana de Lourdes Almeida Vieira  Departamento de Química, Centro Federal de Educação Tecnológica de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Denise de Oliveira Scoaris  Fundação Ezequiel Dias, Belo Horizonte, Brazil Jaquelline  Germano  de Oliveira  Instituto René Rachou, FIOCRUZ-MG, Belo Horizonte, Minas Gerais, Brazil Gabriel Franco dos Santos  Departamento de Química, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Stephen Oscar Duke  National Center for Natural Products Research, School of Pharmacy, University of Mississippi, Oxford, MS, USA

Contributors

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G. Wilson Fernandes  Departamento de Genética Ecologia & Evolução, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Vany Ferraz  Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Alice Ferreira-D’Silva  Unidade Superior de Ensino de Feira de Santana, Feira de Santana, Bahia, Brazil Eskálath  Morganna  Silva  Ferreira  Laboratório de Microbiologia Geral e Aplicada, Universidade Federal do Tocantins, Palmas, Tocantins, Brazil Mariana Costa Ferreira  Instituto René Rachou, FIOCRUZ-MG, Belo Horizonte, Minas Gerais, Brazil Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Pedro E. Gundel  IFEVA-Facultad de Agronomía (UBA)/CONICET, Cátedra de Ecología, Buenos Aires, Argentina Diego  Knop  Henriques  Departamento de Botânica, Universidade de Brasília, Distrito Federal, Brasília, Brazil Víctor  Hernández  Laboratory of Natural Products Chemistry, Department of Botany, Faculty of Natural and Oceanographic Sciences, Universidad de Concepcion, Concepcion, Chile Leopoldo J. Iannone  DBBE-FCEN-UBA & INMIBO-CONICET, Buenos Aires, Argentina Dánae  Irribarren-Riquelme  Environmental Microbiology and Biotechnology Unit – Zenobia Group SpA, Chillan, Chile Inmaculada  Izquierdo-Bueno  Department of Biomedicine, Biotechnology and Public Health, Faculty of Marine and Environmental Sciences, University of Cadiz, Puerto Real, Cádiz, Spain Ary  Corrêa  Júnior  Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Policarpo  Ademar  Sales  Junior  Instituto René Rachou, FIOCRUZ-MG, Belo Horizonte, Brazil Markus Kohlhoff  Instituto René Rachou, FIOCRUZ-MG, Belo Horizonte, Minas Gearis, Brazil Antoniana  Ursine  Krettli  Instituto de Pesquisa René Rachou, FIOCRUZ-MG, Belo Horizonte, MG, Brazil

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Contributors

Paris Lavín  Facultad de Ciencias del Mar y Recursos Biológicos, Departamento de Biotecnología; Laboratorio de Complejidad Microbiana y Ecología Funcional, Instituto Antofagasta, Universidad de Antofagasta, Antofagasta, Chile Pedro Henrique Rodrigues Loureiro  Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil Daniela  Nabak  Bueno  Maia  Instituto René Rachou, FIOCRUZ-MG, Belo Horizonte, Minas Gearis, Brazil Renata  Aparecida  Maia  Departamento de Genética Ecologia & Evolução, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil María  R.  Marchán-Rivadeneira  Biotechnology Institute, Ohio University, Athens, OH, USA Patricia D. Mc Cargo  DBBE-FCEN-UBA & INMIBO-CONICET, Buenos Aires, Argentina Javier  Moraga  Department of Biomedicine, Biotechnology and Public Health, Faculty of Marine and Environmental Sciences, University of Cadiz, Puerto Real, Cádiz, Spain Alexandra  Narvaez-Trujillo  Center for Research on Health in Latinamerica (CISeAL) – Plant Biotechnology Research Group, Pontificia Universidad Catolica del Ecuador (PUCE), Quito, Ecuador Isabela M. Nascimento  Departamento de Genética Ecologia & Evolução, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil M.  Victoria  Novas  DBBE-FCEN-UBA & INMIBO-CONICET, Buenos Aires, Argentina Yumi Oki  Departamento de Genética Ecologia & Evolução, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Rómulo Oses-Pedraza  Vicerrectoria de Investigacion y Postgrado (VRIP), Centro Regional de Investigacion y Desarrollo Sustentable de Atacama (CRIDESAT)  – Universidad de Atacama, Copiapo, Chile Environmental Microbiology and Biotechnology Unit  – Zenobia Group SpA, Chillan, Chile Raphael  Sanzio  Pimenta  Laboratório de Microbiologia Geral e Aplicada, Universidade Federal do Tocantins, Palmas, Tocantins, Brazil

Contributors

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Carolina E. Portero  Center for Research on Health in Latinamerica (CISeAL) – Plant Biotechnology Research Group, Pontificia Universidad Catolica del Ecuador (PUCE), Quito, Ecuador Jaime  Rodríguez  Center of Biotechnology, Universidad de Concepcion, Concepcion, Chile Keilor Rojas-Jimenez  Escuela de Biologia, Universidad de Costa Rica, San Jose, Costa Rica Luiz  Henrique  Rosa  Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Marcos  Antônio  Soares  Departamento de Botânica e Ecologia, Universidade Federal do Mato Grosso, Cuiabá, Brazil Jacqueline A. Takahashi  Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Giselle  Tamayo-Castillo  Escuela de Química, Universidad de Costa Rica, San José, Costa Rica Centro de Investigaciones en Productos Naturales (CIPRONA), Universidad de Costa Rica, San José, Costa Rica Andrea  C.  Ueno  IFEVA-Facultad de Agronomía (UBA)/CONICET, Cátedra de Ecología, Buenos Aires, Argentina Eliana  Veloz-Villavicencio  Center for Research on Health in Latinamerica (CISeAL) – Plant Biotechnology Research Group, Pontificia Universidad Catolica del Ecuador (PUCE), Quito, Ecuador Carlos  Leomar  Zani  Instituto René Rachou, FIOCRUZ-MG, Belo Horizonte, Minas Gearis, Brazil

Chapter 1

Ecology of Neotropical Endophytic Fungi Camila Rodrigues de Carvalho, Mariana Costa Ferreira, and Luiz Henrique Rosa

Abstract Endophytic fungi are a diverse group of microorganisms that have ­different effects on the ecology of the host plant in its healthy state and on its evolution. Different hypotheses have been proposed to explain how endophytic fungi manage to infect and often even grow within their hosts without causing visible disease symptoms. The initial hypothesis was that the asymptomatic colonization is based on a balanced antagonism between fungal virulence factors and host defense responses. The more recent proposal is that fungal endophytes, besides maintaining a balanced antagonism with the plant, also maintain a balance with bacterial and other fungal inhabitants of the host. In this chapter, the authors discuss the current knowledge on ecological interaction and signal transduction between Neotropical plants and their endophytic fungal, in addition to some examples of benefits that these fungi can offer to their host plants. Keywords  Fungi · Host plants · Balanced antagonism · Tropical

1.1  Introduction The community of microorganisms associated with a diversity of unique ­environments can be defined as “microbiome” (Gilbert et al. 2014). Identifying and understanding plant microbiomes may contribute to improvement of agricultural practices and development of novel biotechnological approaches for diagnostic and therapeutic purposes (Rosier et  al. 2016). According to Yan et  al. (2019), plants Camila Rodrigues de Carvalho and Mariana Costa Ferreira contributed equally with all other contributors. C. R. de Carvalho (*) · M. C. Ferreira Instituto René Rachou, FIOCRUZ-MG, Belo Horizonte, Minas Gerais, Brazil Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil L. H. Rosa Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil © Springer Nature Switzerland AG 2021 L. H. Rosa (ed.), Neotropical Endophytic Fungi, https://doi.org/10.1007/978-3-030-53506-3_1

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h­ arbor a wide diversity of microorganisms such as fungi, bacteria, algae, archaea, and protists, both within and outside their tissues. Endophytic fungi are among those ­microorganisms that inhabit plant organs that, at some time in their life, can colonize internal plant tissues without causing apparent harm to the host (Petrini 1991). These fungi show a complex relationship with their hosts, which are widely studied in some instances and could be beneficial for the plant. These benefits have been widely studied and could be used in agriculture, medicine, and other industries.

1.2  Endophytic Fungi: Definition Over the years, several definitions of endophytes have been proposed (endo = within; phyte = plant). Originally, the term was introduced by de Bary (1866), who defined endophyte as any organism that can colonize internal plant tissues. However, the most commonly used definition over the years is that proposed by Petrini (1991) as all the organisms inhabiting plant organs that at some time in their life, can colonize internal plant tissues without causing apparent harm to the host. Petrini’s definition includes those endophytic organisms that have lengthy epiphytic phases as well as latent pathogens that may live symptomless in their hosts during a certain time in their life. The term endophyte may include protists (Peters 1991), bacteria (Kobayashi and Palumbo 2000), and fungi (Stone et al. 2000; Carvalho et al. 2012; Ferreira et al. 2017, 2020); most often, it refers to fungi that are isolated most frequently (Strobel and Daisy 2003; Strobel et al. 2004; Tan and Zou 2001). All plants in a natural ecosystem seem to have some interactions with endophytic or some other associated fungi (Rodriguez et al. 2008).

1.3  Balanced Antagonism Hypothesis Compant et al. (2016) reported that despite extensive studies on endophytic communities, the interactions established among the endophytes and their host plants are still poorly understood. Over the last decades, several studies have been conducted with the purpose of drawing hypotheses about the establishment of this interaction. They aimed to understand how fungal endophytes manage to infect and often even grow within their hosts without causing visible disease symptoms (Schulz and Boyle 2005; Kusari et al. 2012; Schulz et al. 2015). The underlying mechanisms of these interactions, such as the influence of environmental, genetic, and phenological factors that may apparently determine the establishment of specific associations of microbial communities found in different plant tissues, are some interesting points that are currently under study (Compant et  al. 2016). Figure 1.1 shows the history of our understanding of the mechanisms of endophytic fungi-host plant interaction proposed by different authors in the last decades.

1  Ecology of Neotropical Endophytic Fungi

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Fig. 1.1 Representative diagrams of evolution of endophytic-host plant interactions. (a) Hypothesis of balanced antagonism proposed by Schulz and Boyle (2005); (b) hypothesis of balanced antagonism proposed by Kusari et al. (2012). a. Balanced antagonism hypothesis is shown. b. Plant disease caused by pathogenic fungi is presented. c. Endophyte-pathogen reciprocity is demonstrated. The question mark (?) indicates that this phenomenon might not be universal, and further research is necessary for verification. d. Endophyte survival strategy is illustrated. e. Balanced synergism is shown. (c) Hypothesis of balanced antagonism proposed by Schulz et al. (2015)

The relationship between endophytic fungi and their host plants is quite complex; it involves interactions with other endophytic species and the influence of biotic and abiotic factors. Although the asymptomatic nature of colonization by an endophyte may lead to its classification as a mutualistic relationship, it is still considered that while these microorganisms are enormously diverse, some of them may be saprobes or opportunistic pathogens (Strobel and Daisy 2003). This hypothesis is reinforced by the fact that some phytopathogens have an endophytic origin, as well as that some of such microorganisms can cause symptomatic infections in an old plant or under stress conditions (Tan and Zou 2001; Firáková et  al. 2007). According to Schulz and Boyle (2005), fungi that are accidental opportunists can usually be found on diverse other substrates and are not specifically adapted to their hosts. For example, some coprophilous species could be detected in plant tissues. According to Kogel et  al. (2006), mutualistic interaction does not mean the absence of plant defense. These relationships require a sophisticated balance between plant defense responses and endophytic nutrient demand. Schulz and Boyle (2005) reported that asymptomatic colonization of plant tissue or organ by an endophyte would result from a balanced antagonistic relationship, where an equilibrium is reached between the microorganism virulence and host defense responses. The hypothesis of the balanced antagonism was initially proposed to demonstrate how an endophyte could prevent the activation of host defense mechanisms, thus

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p­ rotecting itself before being incapacitated by the host’s toxic metabolites, and keeps growing in plant tissues without causing visible manifestations of infection or disease (Fig. 1.1a) (Schulz and Boyle 2005). Host specificity requires a close adaptation between the host plant and the endophyte, suggesting that a mutual influence develops during cohabitation and coevolution over thousands of years (Kusari et  al. 2012). Over time, this association becomes permanently engraved in the genetic material of both organisms, which therefore develop complementary genetic systems. This emphasizes that the relationship between endophytic fungi and their hosts is much more complex than initially assumed (Moricca and Ragazzi 2008). For example, several metabolites produced by endophytic fungi are also found in their host plants, suggesting that an event of horizontal transfer of the genes that regulate synthesis of these compounds might occur from the host plants to the fungi (Venugopalan and Srivastava 2015). The hypothesis established by Kusari et al. (2012) proposes that asymptomatic colonization is an equilibrium of antagonisms between the endophyte and the host. The interaction could occur by the following scenarios: (i) if fungus virulence and plant defense strategies are balanced, the association remains apparently asymptomatic, and this phase is only a transitional period where environmental factors play an important role in destabilizing the established delicate equilibrium; (ii) if the plant’s defense mechanisms completely neutralize the fungal virulence factors, it may die, and in contrast, if the plant succumbs to the fungus virulence, the fungal invasion can lead to a disease. The variability in this interaction would depend not only on the adaptation of the endophytic microorganism to a host tissue or organ but also on such factors as innate endophyte virulence, plant defense responses, and environmental conditions (Fig. 1.1b). Further, plant-endophyte interaction may be not just a balance between fungal virulence and host defense, but a much more complex and precisely controlled interaction. According to Zipfel and Oldroyd (2017), plants are able to perceive the signals emitted by microbes and respond appropriately by activating the plant immune system. Plants rely on the innate immunity to recognize microbial signal molecules, which results in two different defense systems: one is based on the detection of microbe-associated molecular patterns (MAMPs) via cell surface-localized pattern recognition receptors and leads to the MAMP-triggered immunity. The other is mediated by recognition of the molecules produced by microorganisms (termed effectors) via intracellular receptors; it activates effector-triggered immune response (Mendoza-Mendoza et al. 2018; Yan et al. 2019). Kusari et al. (2012) also suggest that the plant is colonized by high diversity of endophyte microorganisms that interact directly or indirectly (fungus/fungus, fungus/bacteria, bacteria/bacteria) and such microbial interactions play an important role in the production of secondary metabolites by endophytes. Schulz et al. (2015) showed that for endophytic fungi to be able to grow and survive, they must maintain a balanced antagonism not only with the host plant but also with the competing microbiota, whether endophytic or pathogenic. These authors suggest that secondary metabolites play an important role in maintaining this balance. The plethora of antibacterial and antifungal

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­ etabolites that endophytic fungi produce is crucial for equilibrating the antagom nisms with microbial competitors, resulting in a compatible multipartite symbiosis (Fig. 1.1c). Thus, the balanced antagonism hypothesis postulates that a balance exists between plant defensive responses into endophytes and the toxic effect of endophytes on their host plants. Endophytes resist the host defense mechanisms, avoid being incapacitated by the toxic metabolites, and survive within their hosts without causing visible infection or disease symptoms. In this way, plant-endophyte relationships are asymptomatic as long as there is a balanced antagonism between the host defense and fungal virulence (Yan et al. 2019). However, further studies are still needed to better understand the metabolic pathways and signaling mechanisms involved in these interactions.

1.4  E  ndophytic Fungi: Classification, Benefits, and Interaction Rodriguez et al. (2009) published an important study where they classified endophytic fungi in two major groups according to differences in their evolutionary origins, plant hosts, taxonomy, and ecological functions, as follows: C-endophytes or clavicipitaceous endophytes, which infect some grasses, and NC-endophytes or nonclavicipitaceous endophytes, which can be isolated from asymptomatic tissues of nonvascular plants, ferns and allies, conifers, and angiosperms. C-endophytes (endophytes of grasses) may constitute a monophyletic clade with the fungal family Clavicipitaceae (Ascomycota) (Clay and Schardl 2002) that are fastidious in culture and are limited to some cool- and warm-season grasses (Bischoff and White 2005). Traditionally, this group of endophytes could be found within plant shoots, where they form systemic intercellular infections, and may be transmitted vertically through seeds and horizontally (Rodriguez et  al. 2009). According to Clay and Schardl (2002), the grass/endophyte associations are based primarily on the protection of the host from biotic and abiotic stresses. Furthermore, the endophyte infection can provide some benefits, such as enhanced drought tolerance, photosynthetic rate, and growth, as well as defense of the host plant against pests, herbivores, and plant pathogens. NC-endophytes are highly diverse fungi that predominantly belong to the Ascomycota phylum, while some minor species belong to Basidiomycota. They may include at least three distinct functional classes based on the host colonization patterns, mechanism of transmission between host generations, in planta biodiversity levels, and ecological role (Rodriguez et  al. 2009). Rodriguez et  al. (2009) describe the host range and tissue colonized by different classes. They show that Classes 2, 3, and 4 have broad host ranges; however, Class 2 endophytes may grow in above- and underground tissues (roots, stems, and leaves), Class 3 endophytes are restricted to aboveground tissues, and Class 4 is restricted to roots. Colonization of

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host tissues (in planta colonization) also differs: Class 3 endophytes form highly localized infections, while Class 2 and 4 endophytes are capable of extensive tissue colonization. When the in planta diversity is analyzed for each class of endophytes within a host plant’s organs or tissues, the diversity of Class 2 endophytes is usually low; the one of Class 3 endophytes is extremely high; and in contrast, the diversity of Class 4 endophytes is unknown (Rodriguez et al. 2009). The authors still report that differences in in planta biodiversity of Class 2 and 3 endophytes may reflect the variability in transmission patterns: although both classes are transmitted horizontally, Class 2 endophytes are also transmitted vertically. Transmission of Class 4 endophytes is only horizontal. An important trait of the Class 2 endophytes is related to the fitness benefits. Only the Class 2 endophytes have shown the ability to confer habitat-specific stress tolerance to the host plants (Rodriguez et al. 2008, 2009). According to Rodriguez et al. (2009), endophyte-conferred fitness benefits are defined as habitat-adapted if the benefits are the result of habitat-specific selective pressures such as temperature, pH, and salinity or as nonhabitat-adapted if the benefits are common among endophytes regardless of habitat. As reported by Busby et al. (2015), the colonization of plants by fungal endophytes has beneficial effects, which are mainly represented by the increase of the plant resistance to biotic and abiotic stresses. Few studies reported the beneficial relationship between the Neotropical plants and their endophytic fungi host. Some examples reported in the literature are ­discussed below. According to Pietro-Souza et al. (2020), endophytic microorganisms are instrumental for metal remediation, or they could assist their hosts in phytoremediation processes. Root endophytic fungal assemblages of Polygonum acuminatum Kunth. and Aeschynomene fluminensis Vell., collected in Poconé, Mato Grosso, Brazil, in areas characterized as wetland were studied by Pietro-Souza et al. (2017). These plants were collected in places with and without mercury (Hg+2). The authors observed higher endophytic fungi in hosts in soil contaminated with mercury. Besides, the frequency of colonization, the abundant distribution of taxa of endophytic fungi, and the structure and community function were influenced by mercury contamination. When the strains were identified, the authors observed a predominance of Sordariomycetes for hosts collected in contaminated areas and an abundance of Dothideomycetes in uncontaminated areas, and the order of Pleosporales was proportional and larger in all analyzed communities. The selected strains of endophytic fungi were evaluated in host growth promotion in the presence of mercury. In short, the seeds of A. fluminensis were germinated in vases, the seedlings were posteriorly transferred to new vases, and then, the selected strains were inoculated. After a period of cultivation in the absence of mercury, doses of Hg+2 were applied at the final concentration of 120 mg kg−1 of Hg+2. The authors suggested that the use of mercury-tolerant fungal strains positively influenced the growth of A. fluminensis in mercury contamination conditions since nine strains promoted the growth of the host with growth promotion efficiency (GPE) (dry biomass and length) greater than that obtained for uninoculated plants and those without addition of mercury to the substrate. The authors concluded that the inoculation of

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A. ­fluminensis with certain strains of stress-tolerant endophytic fungi contributes to colonization and establishment of the host and may be promising microorganisms in bioremediation programs. Pietro-Souza et al. (2020) examined the capacity to promote mercury bioremediation of 30 mercury-resistant endophytic fungi isolated from the A. fluminensis and P. acuminatum root systems in their previous study (Pietro-Souza et al. 2017). Among the tested strains, Aspergillus sp. A31, Curvularia geniculata P1, Lindgomycetaceae P87, and Westerdykella sp. P71 remediated more than 97% of Hg+2 added to culture media and were selected to evaluate for mercury bioremediation and bioaccumulation in vitro, as well as for growth promotion of A. fluminensis and Zea mays in the presence or absence of the metal. Those endophytic fungi strains removed up to 100% of mercury from the culture medium in a species-­ dependent manner, and they promoted A. fluminensis and Z. mays growth in substrates with or without mercury. According to the authors, the increase in the host biomass correlated with the reduction in soil mercury concentration due to the metal bioaccumulation in host tissues and its possible volatilization. The soil mercury concentration was decreased by 7.69% in A. fluminensis plants inoculated with Lindgomycetaceae P87 + Aspergillus sp. A31 and by 57.14% in plants inoculated with Lindgomycetaceae P87. The authors report that the resistance mechanisms of mercury volatilization and bioaccumulation in plant tissues mediated by these endophytic fungi can contribute to bioremediation programs. According to Vergara et  al. (2017, 2018), the use of dark septate endophytic (DSE) fungi to promote plant growth can be beneficial to agriculture, and these organisms are important allies in the search for sustainable agriculture practices. Vergara et al. (2017) investigated the effects of inoculation with the DSE fungi isolates A101, A104, and A105 on nutrient recovery efficiency, nutrient accumulation, and growth of tomato plants (cv. Santa Clara I-5300) fertilized with organic and inorganic N sources, Canavalia ensiformis (L.), and ammonium sulfate, respectively. The effects of inoculation were evaluated under greenhouse conditions where all the three isolates colonized the root tissue of tomato plants and promoted tomato growth without causing symptoms of apparent disease, using finely ground C. ensiformis-­15N as the organic N source. Tomato plants exhibited significant increases in aboveground dry biomass, plant height, and leaf number, relative to the uninoculated treatment. Besides, the 15N, N, P, K, Ca, Mg, Fe, Mn, and Zn contents increased. In contrast, the only positive effects observed in the presence of an inorganic N source were fertilizer-K recovery efficiency, content of K, and leaf area when inoculated with the fungus A104. The authors concluded that, especially when an organic N source is used, tomato plants inoculated with DSE fungi acquired macro- and micronutrients more efficiently, resulting in increased plant growth. Vergara et  al. (2018) studied the contribution of four dark septate fungi (A101, A103, A104, and A105) to the absorption of nutrients by rice plants and their ensuing growth. The four isolates were inoculated to the rice plants. The colonization was significant by all of the fungi, mainly the isolate A103 (Pleosporales) that increased the fresh and dry biomass of the shoots and the number of tillers per plant, amino-N, and soluble sugars as well as the N, P, K, Mg, and S contents in c­ omparison with the control treatment.

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1.5  Conclusion In Neotropical biomes, endophytic fungi can have different effects on the ecology of their host plant in its healthy state and during its evolution. They help the host plant to adapt to the environment and to establish tolerance to biotic and abiotic stresses. Such microorganisms should be widely explored for better characterization of their species diversity, interactions with host plants, and biotechnological applications, among others. However, further ecological studies have to be conducted to understand the impact of endophytic fungal assemblages associated with rich plant communities inhabiting the Neotropical biomes.

References Bischoff JF, White JF (2005) Evolutionary development of the Clavicipitaceae. In: Dighton J, White JF, Oudemans P (eds) The fungal community: its organization and role in the ecosystem. Taylor & Francis, Boca Raton, pp 505–518 Busby PE, Ridout M, Newcombe G (2015) Fungal endophytes: modifiers of plant disease. Plant Mol Biol 90:645–655 Carvalho CR, Gonçalves VN, Pereira CB, Johann S, Galliza IV, Alves TMA, Rabello A, Sobral MEG, Zani CL, Rosa CA, Rosa LH (2012) The diversity, antimicrobial and anticancer activity of endophytic fungi associated with the medicinal plant Stryphnodendron adstringens (Mart.) Coville (Fabaceae) from the Brazilian savannah. Symbiosis 57:95–107 Clay K, Schardl C (2002) Evolutionary origins and ecological consequences of endophyte symbiosis with grasses. Am Nat 160:99–127 Compant S, Saikkonen K, Mitter B, Campisano A, Mercado-Blanco J (2016) Editorial special issue: soil, plants and endophytes. Plant Soil 405:1–11 De Bary A (1866) Morphologie und Physiologie der Pilze, Flechten und Myxomyceten. Hofmeister’s handbook of physiological botany, vol II. Engelmann, Leipzig Ferreira MC, Cantrell CL, Wedge DE, Gonçalves VN, Jacob MR, Khan S, Rosa CA, Rosa LH (2017) Diversity of the endophytic fungi associated with the ancient and narrowly endemic neotropical plant Vellozia gigantea from the endangered Brazilian rupestrian grasslands. Biochem Syst Ecol 71:163–169 Ferreira MC, de Assis JCS, Rosa LH (2020) Diversity of endophytic fungi associated with Carapichea ipecacuanha from a native fragment of the Atlantic Rain Forest. S Afr J Bot 57:1–5 Firáková S, Sturdíková M, Múcková M (2007) Bioactive secondary metabolites produced by microorganisms associated with plants. Biologia 62:251–257 Gilbert JA, Jansson JK, Knight R (2014) The earth microbiome project: successes and aspirations. BMC Biol 12:69 Kobayashi DY, Palumbo JD (2000) Bacterial endophytes and their effects on plants and uses in agriculture. In: Bacon CW, White JF (eds) Microbial endophytes. Marcel Dekker, New York, pp 199–236 Kogel K-H, Franjken P, Huckelhoven R (2006) Endophyte or parasite what decides. Curr Opin Plant Biol 9:358–363 Kusari S, Hertweck C, Spiteller M (2012) Chemical ecology of endophytic fungi: origins of secondary metabolites. Chem Biol 19:792–798 Mendoza-Mendoza A, Zaid R, Lawry R, Hermosa R, Monte E, Horwitz BA, Mukherjee PK (2018) Molecular dialogues between Trichoderma and roots: role of the fungal secretome. Fungal Biol Rev 32:62–85

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Moricca S, Ragazzi A (2008) Fungal endophytes in Mediterranean oak forests: a lesson from Discula quercina. Phytopathology 98:380–386 Peters AF (1991) Field and culture studies of Streblonema macrocystis new species (Ectocarpales, Phaeophyceae) from Chile, a sexual endophyte of giant kelp. Phycologia 30:365–377 Petrini O (1991) Fungal endophytes of tree leaves. In: Andrews JH, Hirano SS (eds) Microbial ecology of leaves. Springer, New York, pp 179–197 Pietro-Souza W, Mello IS, Vendruscullo SJ, Silva GF, Cunha CN, White JF, Soares MA (2017) Endophytic fungal communities of Polygonum acuminatum and Aeschynomene fluminensis are influenced by soil mercury contamination. PLoS One 12:1–24 Pietro-Souza W, Pereira FC, Mello IS, Stachack FFF, Terezo AJ, Cunha CN, White JF, Li H, Soares MA (2020) Mercury resistance and bioremediation mediated by endophytic fungi. Chemosphere 240:1–12 Rodriguez RJ, Henson J, Volkenburgh EV, Hoy M, Wright L, Beckwith F, Kim Y-O, Redman RS (2008) Stress tolerance in plants via habitat-adapted symbiosis. ISME J 2:404–416 Rodriguez RJ, White Jr JF, Arnold AE, Redman RS (2009) Fungal endophytes: diversity and functional roles. New Phytol 182:314–330 Rosier A, Bishnoi U, Lakshmanan V, Sherrier DJ, Bais HP (2016) A perspective on inter-kingdom signaling in plant–beneficial microbe interactions. Plant Mol Biol 90:537–548 Schulz B, Boyle C (2005) The endophytic continuum. Mycol Res 109:661–686 Schulz B, Haas S, Junker C, Andrée N, Schobert M (2015) Fungal endophytes are involved in multiple balanced antagonisms. Curr Sci 109:39–45 Stone JK, Bacon CW, White JF (2000) An overview of endophytic microbes: endophytism defined. In: Bacon CW, White JF (eds) Microbial endophytes, vol 30. Marcel Dekker, New  York, pp 3–30 Strobel G, Daisy B (2003) Bioprospecting for microbial endophytes and their natural products. Microbiol Mol Biol Rev 67:491–502 Strobel G, Daisy B, Castillo U, Harper J (2004) Natural products from endophytic microorganisms. J Nat Prod 67:257–268 Tan RX, Zou WX (2001) Endophytes: a rich source of functional metabolites. Nat Prod Rep 8:448–459 Venugopalan A, Srivastava S (2015) Endophytes as in vitro production platforms of high value plant secondary metabolites. Biotechnol Adv 33:873–887 Vergara C, Araujo KEC, Urquiaga S, Schultz N, Balieiro FC, Medeiros OS, Santos LA, Xavier GR, Zilli JE (2017) Dark septate endophytic fungi help tomato to acquire nutrients from ground plant material. Front Microbiol 8:1–12 Vergara C, Araujo KEC, Alves LS, Souza SR, Santos LA, Santa-Catarina C, Silva K, Pereira GMD, Xavier GR, Zilli JE (2018) Contribution of dark septate fungi to the nutrient uptake and growth of rice plants. Braz J Microbiol 49:67–78 Yan L, Zhu J, Zhao X, Shi J, Jiang C, Shao D (2019) Beneficial effects of endophytic fungi colonization on plants. Appl Microbiol Biotechnol 103:3327–3340 Zipfel C, Oldroyd GE (2017) Plant signalling in symbiosis and immunity. Nature 543:328–336

Chapter 2

Diversity, Ecology, and Applications of Epichloë Fungal Endophytes of Grasses in South America Leopoldo J. Iannone , M. Victoria Novas Andrea C. Ueno , and Pedro E. Gundel

, Patricia D. Mc Cargo

,

Abstract  Epichloë fungal endophytes are a conspicuous group of fungi (Ascomycota, Hypocreales, Clavicipitaceae) that form persistent symbiosis with certain cool-season grasses (Pooideae) worldwide. The symbiosis is not vital for the plants, but it seems to be associated with fitness benefits, a basic condition for being favorably selected. Epichloë endophytes infect systemically green tissues, and while sexual stages reduce fertility of the host plant, their asexual forms persist asymptomatically through generations by means of vertical transmission (from plant to seeds). Host plants are endowed by a suite of fungal alkaloids that can be toxic to livestock (such as ergot alkaloids and lolitrem B) or protect plants against herbivorous insects (lolines and peramine). Mainly studied in the Northern Hemisphere, where species with sexual or asexual stages are found, Epichloë in South America appears to present its own characteristics. Only asexual vertically transmitted Epichloë has been detected in South America from Venezuela to Argentina. Although research in genetic biodiversity of Epichloë fungi in South America is in the dawn and mostly restricted to Argentina, we know that most of the endophytes from South America evolved from hybridization events among species from the Northern Hemisphere not found in this region. Interestingly, a few strains or fungal species are associated to more than one host grass species, which contrasts with what is known from the Northern Hemisphere. Regional surveys of grass-­ endophyte associations indicate that some environmental conditions promote the symbiosis while others don’t (e.g., aridity). However, variation among closely related plant species also evidences phylogenetic constrains. Fungal endophytes are being used in breeding programs of forage crops with two main goals: (i) replace those wild toxic endophytes and (ii) inoculate endophytes that protect host plants against agricultural plagues (as agents of biological control). The knowledge of the

L. J. Iannone (*) · M. V. Novas · P. D. Mc Cargo DBBE-FCEN-UBA & INMIBO-CONICET, Buenos Aires, Argentina A. C. Ueno · P. E. Gundel IFEVA-Facultad de Agronomía (UBA)/CONICET, Cátedra de Ecología, Buenos Aires, Argentina © Springer Nature Switzerland AG 2021 L. H. Rosa (ed.), Neotropical Endophytic Fungi, https://doi.org/10.1007/978-3-030-53506-3_2

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diversity of Epichloë fungi, the host grasses they infect, and the ecophysiological impact on plant fitness opens a big potential to advance eco-friendly tools for the development of a more sustainable agriculture. Keywords  Endophytic fungi · Distribution · Neotropical · Taxonomy

2.1  Introduction 2.1.1  O  rigin and Life History of Asexual Epichloë Fungal Endophytes Grasses, as most plants on earth, host microorganisms that during part or the entire life cycle grow asymptomatically within their tissues and are commonly termed “endophytes” (Hyde and Soytong 2008; Wilson 1995). Different fungal species of the genera Acremonium, Alternaria, Epicoccum, Phoma, Phomopsis, and Stemphylium (König et al. 2018; Sánchez Márquez et al. 2008, 2010; Zabalgogeazcoa et al. 2013) have been found growing endophytically in grasses as well as in other plant groups (D’Jonsiles et al. 2019; Suryanarayanan 2013; Vega et al. 2010). Most of them colonize via spores and leaf tissues of seedlings and adult plants establishing local or systemic infections. A particular group of fungal endophytes are those in the genus Epichloë (Clavicipitaceae, Hypocreales, Ascomycota) which show a close coevolutionary relationship with certain species of cool-season grasses in the subfamily Pooideae (family: Poaceae) (Schardl 2010; Schardl et al. 2008). That is the reason why fungi of the genus Epichloë are commonly known as “the fungal endophytes of grasses.” Apart from being a model system to study species interactions in ecology and evolution, grass endophytes are important due to their presence in two of the most economically important forage and turf species (i.e., tall fescue (Festuca arundinacea) and perennial ryegrass (Lolium perenne L.)) as well as in wild grasses of grasslands around the world. During vegetative stages of the host, Epichloë fungal endophytes grow systemically in the intercellular spaces (the apoplast) of aerial plant tissues. In the apoplast, the fungus obtains simple sugars and amino acids for nutrition (Kuldau and Bacon 2008). The fungal hyphae colonize the apical meristems and grow firmly attached to the cell walls of the host. The growth in the meristems provides accession to elongation zones of new leaves and lateral buds that will originate new vegetative or reproductive tillers. As plant cells enlarge, the hyphae are stretched out and grow intercalary (Christensen et al. 2008). As a result, the endophyte grows coordinately with the host without causing a defensive response of the plant. In parenchymal tissues, hyphae are non-branched and parallel oriented to the longitudinal axes of host cells. The mechanisms that regulate the hyphal growth and systemic colonization of the plant without causing an antagonist reaction have been thoroughly studied and include intercalary growth, balanced ROS production, and secretion of small

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p­ eptides (see, e.g., Scott et al. 2018). Even though these endophytes grow mostly endophytically, epiphytic mycelia with potential to produce conidia have been also detected (Craven et al. 2001; Moy et al. 2000; Tadych et al. 2014; White et al. 1996). The mechanisms by which endophytes can grow between epidermal cells and cross throughout the cuticle have been carefully studied by Becker et al. (2016). The species of Epichloë differ in their life cycles and reproductive strategies, which determine the mechanisms by which these endophytes colonize new plants. These differences become evident when the host plants enter in the reproductive phase. In those species that can accomplish sexual reproduction, the hyphae of the endophyte grow epiphytically and profusely forming a stroma with conidiogenous cells and conidia on the phylloplane of reproductive tillers (culms) of the host plant. The development of the stroma arrests the expansion of the flag leaf preventing the emergence of the inflorescence; thus, flowers will not be formed in this culm. Limited to some Epichloë species, this is considered the pathogenic manifestation of the fungus, a syndrome known as “choke disease” (Bucheli and Leuchtmann 1996; Kirby 1961). The conidia formed in the stroma may act like spermatia, and female flies of the genus Botanophila (Anthomyiidae, Diptera) that feed, oviposit, and defecate on the stroma transport conidia from one stroma to another. Slugs that feed on the stroma and the ascospores are also mechanisms for transferring spermatia among stromata (Alderman and Rao 2008; Hoffman and Rao 2014). When conidia are deposited in a stroma of the opposite mating type, fertilization takes place and perithecia with ascospores are formed. When the perithecia mature, the stromata become bright yellow (Fig. 2.1a, b). The ascospores are forcibly ­discharged, and when they reach a host plant, the spore germinates producing conidia that will germinate and colonize a new healthy plant.

Fig. 2.1 (a, b) Picture of Dactylis glomerata grass exhibiting the stromata or choke disease caused by Epichloë typhina. (c, d) Endophyte mycelium growing in culture media. (e, f) Detail of fungal endophyte mycelium and conidia in culture

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Some Epichloë species do not reproduce sexually, and stromata are never formed (or if formed, perithecia are not produced). These fungal endophytes, the vertically transmitted endophytes of grasses, present a life cycle tightly associated to that of the host plant (Fig.  2.2). These endophytes pass from mother plant to seeds by growing hyphae into flowers and ovaries of host plants (Philipson and Christey 1986). Once in the seed, the endophyte mycelium is found in the embryo, aleurone layer, or coats (Liu et al. 2017; Zhang et al. 2017). After germination, endophytic mycelium is then found in leaf tissues of young seedlings. During plant vegetative stages, each tiller can be colonized by the endophyte. During culm elongation and reproductive structure appearance (either spikes or panicles), the endophyte can colonize each flower and developing ovary (Liu et al. 2017; Sugawara et al. 2004;

Fig. 2.2  Schematic drawing showing a simplified life cycle of a grass plant with the stages (seed, seedling, vegetative and reproductive tillers) and vital rates (germination, tillering, flowering, and fecundity). Below each plant stage, it is explained where the fungal endophyte is in the different plant parts, and with the curly brackets, the eventual failures in the endophyte vertical transmission are highlighted (mechanism by which an endophyte-symbiotic plant can produce nonsymbiotic progeny)

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Zhang et al. 2017). In this way, the asexual endophytes are vertically transmitted to the next generation via the seeds of the host plant (Fig. 2.2). Despite the high synchronization between the fungus and grass, the endophyte can be lost at several steps in the plant growing cycle (Gundel et al. 2011). Failures in colonization of tillers and/or flowers can lead to imperfect vertical transmission, and thus, endophyte-symbiotic plants can produce endophyte-free seeds (Afkhami and Rudgers 2008; Gagic et al. 2018). On the other hand, an endophyte-symbiotic seed can give rise to an endophyte-free seedling due to the anticipated death of the fungus (Fig. 2.2). The longevity of the endophyte in the seeds depends among other factors on the temperature and humidity during seed dormancy or storage. High temperatures and relative humidity significantly reduce the survival of the endophyte in the seeds (Gundel et al. 2009, 2010, 2012; Hill and Roach 2009; Rolston et al. 1986; Welty et al. 1987; Wheatley et al. 2007). Accordingly to the life cycle pattern, (Rodriguez et al. 2009; White 1988) established three types of associations. Type I associations are those established by some Epichloë spp. that develop stromata in all the culms of the host plants; thus, these plants will be unable to produce seeds, as occurs in the association between E. typhina and Dactylis glomerata (Fig. 2.1a, b). In the type II associations, the stromata are formed in some of the culms; thus, the host plant yet produces seeds. Type III associations involve those Epichloë species that do not produce stromata on the host plant and are vertically transmitted via seeds. Even though the ascospores are responsible of horizontal transmission, it has been proved that conidia produced on the phylloplane of asymptomatic plants could be transported by water drops and are able to infect new plants. Most of Epichloë species can grow in standard culture media as potato dextrose agar or malt extract agar; thus, they can be isolated from superficially disinfected tissues of the host plant. In culture, they present slow-growing colonies with whitish mycelium and reverse tan (Fig. 2.1c, d). Conidiogenous cells are phialidic and solitary and produce one to four allantoid, reniform, or uncinated conidia (Fig. 2.1e, f). A remarkable feature of most of the asexual Epichloë is their evolutionary origin. First sequence analyses of nuclear genes revealed the presence of two or three alleles in most of the studied genes (Craven et al. 2001; Gentile et al. 2005; Moon et  al. 2004; Tsai et  al. 1994). The phylogeny of each allele of different genes of an asexual species relates it with a different sexual species suggesting that asexual species evolved from hybridization events among endophytes from different parental species. Nowadays, genome analyses indicate that most of the hybrids are allopolyploids since they retain most of the genome of each ancestor (Campbell et al. 2017). At the present time, the known Epichloë endophytes associated with native grasses in South America do not form stromata and reproduce asexually by growing agamic hyphae into the seeds (Iannone et al. 2012a). Because of this, we will focus hereafter on vertically transmitted fungal endophytes of grasses.

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2.1.2  Fitness Effects in an Intimate Relationship The frequency of endophyte-symbiotic individuals greatly varies across species populations and environment (Semmartin et al. 2015). The symbiosis persistence relays mostly on the mutual benefits between partners and the efficiency with which the fungus is transmitted to progeny (Gundel et al. 2008, 2011). Benefits for vertically transmitted endophyte fungi are clear, as they obtain nutrition, protection, and a way of dispersion via the host seeds. Despite that endophyte-conferred benefits on the host fitness have been widely documented (for a review, see Clay and Schardl 2002), the effects can be variable as they are controlled by genetic and environmental factors, hence leading to the idea of the mutualism-parasitism continuum (Rodriguez et al. 2009; Saikkonen et al. 1998). Historically, the symbiosis between grasses and Epichloë was considered a defensive mutualism, since the endophyte produces four main classes of alkaloids that are deterrents or cause toxicity to herbivores (Clay 1988; Clay and Schardl 2002; Saikkonen et al. 2013). The alkaloids lolines and peramine are mainly active against invertebrate animals. Alternatively, the indole-diterpenes (i.e., lolitrem B) and ergot alkaloids (i.e., ergovaline) are tremorgenic or produce metabolic disorders in vertebrate animals such as sheep, livestock, and horses (Philippe 2016; Saikkonen et al. 2013; Schardl et al. 2013a). With exception of peramine, the genes involved in the biosynthesis of the alkaloids are arranged in clusters. Different total or partial deletions in these clusters are observed among Epichloë species or even among strains of the same species (Schardl et al. 2012, 2013b; Shymanovich et al. 2014), which determine differences in alkaloid profile and in the level of toxicity of the plant. In many cases (most prominent in domestic agronomic grasses), the association with Epichloë also provides the host plants with increased growth, higher seed production, and tolerance to abiotic stress factors such as drought (Clay and Schardl 2002; Gundel et al. 2013; Iannone et al. 2012b, 2017; Iannone and Cabral 2006; Novas et al. 2003; Vignale et al. 2013). Interestingly, it has been also well demonstrated that under certain environmental conditions, the endophyte can become a cost for the grass (Faeth et al. 2004; Faeth and Sullivan 2003). Therefore, the outcome of grass-endophyte symbiosis is most likely to be context dependent.

2.1.3  S  pecificity and Maintenance of the Grass-Endophyte Association In general, each species of Epichloë is associated with one or a group of closely related species of grasses. On the other hand, each host species is associated with one or a few species of Epichloë. These patterns suggest the existence of certain host specificity and a coevolution between the host and its endophytes. Studies on the coevolution of plants and endophytes suggest that the symbiosis between Epichloë and grasses arose early in the evolutionary radiation of the Pooideae

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(Schardl et al. 2008). Additionally, a similar co-evolutive pattern can be appreciated among the Epichloë that is horizontally transmitted and their host species (Schardl et  al. 1997). The same co-evolutive pattern is not easy to detect among asexual hybrid endophytes, which are largely more diverse than those sexual or asexual haploid Epichloë; it could be explained by events of endophyte species jumping among host grass species (Craven 2012). Host plants are usually infected with one strain of Epichloë, and if they are coinfected, different endophytes segregate in different tillers, or in the end, just one endophyte will prevail in the entire plant (Christensen et al. 2000; Soto-Barajas et al. 2019; Wille et al. 1999). It has been proposed that in these plants, simultaneous infection by two Epichloë fungi opens the opportunity for new hybrid to appear by means of hyphal anastomosis. If this hybrid outperforms the asexual haploids (Selosse and Schardl 2007) or if it is more efficient in being transmitted through the seeds, it would be fixed and spread. Symbiosis specificity – which seems to work at population and species level – can be evidenced by low capability of establishing a stable interaction in terms of endophyte persistence (which include transmission) and effects on plant fitness (Gundel et al. 2010; Saikkonen et al. 2004). Host specificity has been mainly studied through artificial inoculations, crossing endophytes from one host into another (either population or species) but also through controlled cross-pollination experiments (Gundel et al. 2012; Leuchtmann 1992; Piano et al. 2005; Saikkonen et al. 2010). Fungal endophytes can be artificially cross inoculated by injection of mycelium in meristems of seedlings or callus in tissue culture (Christensen 1995; Latch and Christensen 1985). The success and the effects of the cross inoculations on plant fitness depend highly on the phylogenetic proximity of the plants from where the endophyte was isolated and the plant on which the endophyte will be inoculated (Gundel et  al. 2012; Oberhofer and Leuchtmann 2014; Saikkonen et  al. 2010; Shymanovich and Faeth 2019).

2.2  D  iversity and Evolutionary Origin of Epichloë Fungal Endophytes in South America The discovery of endophyte-infected grasses in South America dates back to the beginning of the twentieth century, when Rivas and Zanolli studied the “tembladera” syndrome, an intoxication in domestic cattle grazing on the grass Festuca fiebrigii in northern Argentina. They discovered a fungus that was growing and producing conidia inside the plants (then, an endophyte). Although without providing a formal description of the species, they associated the presence of this fungus with the toxicity of the plant and named it Endoconidium tembladerae (Rivas and Zanolli 1908). Unfortunately, Rivas and Zanolli’s discovery remained almost ignored until the beginning of the nineties when Dr. D. Cabral and collaborators started a systematic study of the fungal endophytes of grasses in Argentina.

18

L. J. Iannone et al.

Grasses of the subfamily Pooideae are widely spread in South America, with more than 900 species from Venezuela to Tierra del Fuego. In tropical regions from Venezuela to Bolivia, they are commonly found at high altitudes (over 2500m a.s.l.) along the Cordillera de los Andes. In the Southern Cone of South America, including “Argentina, Uruguay, Southern Brazil, Paraguay, and Chile,” Pooideae is the dominant subfamily of the Poaceae and has been recognized as 691 species of cool-­ season grasses (Biganzoli and Zuloaga 2015). In this part of the subcontinent, the Pooideae spreads from the Atlantic to the Pacific oceans (Fig. 2.3). The study of endophytes of grasses in South America is mostly restricted to Argentina and Uruguay. Up to now, 45 taxa of grasses native to South America (Fig. 2.3; Table 2.1) have been detected to be associated with Epichloë endophytes (Bertoni et al. 1993; Iannone et al. 2012a; Lugo et al. 1998), a number that represents less than 10% of the species of Pooideae described for the region. Although Epichloë-associated grasses are more frequently found along or close to the Cordillera de los Andes, many host species inhabit in subtropical forests of the Selva Paranaense, grasslands in the Pampas and Patagonia, and seashores of Argentina and Uruguay (Fig.  2.3). Sexual stromata of Epichloë have not been detected in South America; thus, the detection of endophytes must be achieved by microscopic analysis of the endophyte in seeds and parenchymatic tissues of the potential host species. This result strongly suggests that the Epichloë species associated with grasses from South America reproduce only asexually and that endophyte fungi are transmitted from the mother plant to the seeds. The asexual condition of the Epichloë from South America is also evidenced by phylogenetic analyses of sequences of nuclear genes. In general, most of the isolates bear two alleles from each gene, and each allele is phylogenetically associated with one species that presents sexual reproduction (Fig. 2.4). These sexual species are considered to be the evolutionary ancestors that, by means of interspecific hybridization, gave place to the endophytes from South America. Hybridization events between E. festucae and E. typhina subsp. poae gave origin to E. tembladerae and E. pampeana. Hybridization events between E. typhina subsp. poae and a common ancestor of E. amarillans and E. baconii established the hybrid E. cabralii. In other cases, only one allele associated with E. typhina subsp. poae was detected, suggesting that some endophytes evolved by the loss of sexual reproduction as is the case of E. typhina subsp. poae var. aonikenkana (Mc Cargo et al. 2014). In spite of these clear different evolutionary histories, the different endophytes cannot be easily differentiated by its morphological characteristics in culture. Slight differences could be found in the growth rate and in the size of the conidia and conidiogenous cells (Fig. 2.1e, f), but these characters are very variable, even among strains of the same species. Thus, differences in the morphological characteristics are not consistent enough to clearly differentiate species, and the identification of Epichloë species in South America mostly relies on phylogenetic analyses. As mentioned above, the association between the endophyte and the host is highly specific, and in general, one host species is associated with one or two species of Epichloë. However, one species of Epichloë could be associated with more than one host species. Particularly, E. tembladerae is the most ubiquitous asexual

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Fig. 2.3  Map of the Neotropical ecoregion, which includes the south of North America, Caribbean islands, and Central and South America, showing grass species identified for harboring Epichloë fungal endophytes. The numbers connect the grass species with the site of collection. The species are just listed in alphabetical order

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Table 2.1  List of grass species, their site of collection (country), and the associated Epichloë fungal endophyte. Argentina (Arg.), Brazil (Bra.), Chile (Chi.), Colombia (Col.), Ecuador (Ecu.), México (Mex.), Paraguay (Par.), Peru (Per.), Venezuela (Ven.), and Uruguay (Uru.). Countries in “bold” indicate the fungus was isolated, “underlined” indicates that the observation was made on herbarium specimens, and “plain font” indicates countries where they are present but have not been studied Case Grass species 1 Briza paleapilifera 2 Bromus auleticus

Country Arg. Arg., Bra., Uru.

3 4

Bromus brachyanthera Bromus pictus

Arg., Bra., Uru. Arg., Chi.

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

Calamagrostis alba Calamagrostis bogotensis Calamagrostis ecuadoriensis Calamagrostis sclerantha Calamagrostis tarmensis Festuca argentina Festuca breviglumis Festuca dissitiflora Festuca fiebrigii Festuca fimbriata Festuca hieronymi Festuca horridula Festuca linigluma Festuca magellanica Festuca parodiana Festuca sp. Festuca simplisiuscula Festuca superba Festuca tucumanica Festuca ulochaeta Festuca weberbaueri Hordeum comosum Melica macra Melica stuckertii Phleum alpinum Poa alopecurus subsp. alopecurus Poa alopecurus subsp. fuegiana Poa alopecurus subsp. shuka Poa bergii Poa bonariensis Poa calchaquiensis Poa durifolia

Arg., Bra., Uru. Col., Ven. Ecu. Per. Arg. Arg. Mex. Arg. Arg. Arg., Bra., Par., Uru. Arg. Arg. Arg. Arg. Arg. Arg. Arg. Arg. Arg., Bra., Par. Arg. Arg., Chi. Arg. Arg. Arg., Chi. Arg., Chi. Arg., Chi. Arg., Chi. Arg. Arg., Uru. Arg. Arg.

Fungal endophyte Epichloë tembladerae E. tembladerae Epichloë pampeana Epichloë sp. E. tembladerae Epichloë typhina subsp. poae E. tembladerae

Epichloë sp. E. tembladerae

E. tembladerae Epichloë sp. E. tembladerae Epichloë sp. E. tembladerae E. tembladerae Epichloë sp. Epichloë sp.

E. tembladerae E. tembladerae E. tembladerae-E. cabralii

E. tembladerae E. tembladerae E. tembladerae E. tembladerae (continued)

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Table 2.1 (continued) Case 37 38 39 40 41 42 43 44 45

Grass species Poa holciformis Poa huecu Poa gymnantha Poa lanigera Poa lanuginosa Poa ligularis Poa lillioi Poa plicata Poa spiciformis

Country Arg. Arg. Arg., Chi. Arg., Bra., Uru. Arg. Arg. Arg. Arg. Arg.

46

Polypogon elongatus

Arg., Uru., Bra.

Fungal endophyte E. tembladerae E. tembladerae E. tembladerae E. tembladerae

E. tembladerae Epichloë sp. E. tembladerae

species of Epichloë in the world, being present in at least 20 grass species in Argentina (Table  2.1) and in two species from the United States (Charlton et  al. 2014; Iannone et  al. 2012a; Mc Cargo et  al. 2014). Some host species could be associated with different endophytes, and many of the grasses associated with E. tembladerae (e.g., Bromus auleticus, Bromus pictus, and Phleum alpinum) are also associated with another endophytic species (Table  2.1), although different endophytes coinhabiting in the same individual plant have not been detected. Even though asexual endophytes are considered to be exclusively vertically transmitted, some characteristics of the endophytes from South America are not consistent with this assumption. The presence of hybrid endophytes whose parental species are not present in the region, the wide host range of E. tembladerae, and other seed-transmitted endophytes lead us to consider the existence of events of horizontal transmission of endophytes between sympatric host species. Conidia formed on the epidermis of several host species (e.g., Poa spiciformis, Bromus auleticus) would suggest the occurrence of this strategy (Iannone et  al. 2009; White et al. 1996). Some of the host species associated with E. tembladerae are toxic to cattle. Festuca argentina and Poa huecu in Patagonia and Festuca fiebrigii in the north of Argentina cause some disorders known as “huecu” and “tembladera” in sheep, horses, and donkeys (Cabral et  al. 1999; Rivas and Zanolli 1908). Interestingly, other forage species associated with E. tembladerae such as Bromus auleticus, Bromus pictus, Hordeum comosum, or Poa spp. have not been reported to produce toxicosis in domestic animal. These observations would suggest the existence of toxic and nontoxic strains of E. tembladerae; however, genetic characterization indicates that E. tembladerae isolates from toxic and nontoxic grasses do not have genes for ergot alkaloids or lolitrem B biosynthesis (Iannone et al. 2012a) and these alkaloids have not been detected in any of the toxic grasses from Argentina (Pomilio et al. 1989; Rivas and Zanolli 1908; Scuteri et al. 1992).

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Fig. 2.4  Phylogenetic tree of calM gene of Epichloë fungal endophytes isolated from grasses from Argentina. Boxes in different colors highlight the different endophytes. The names in the branches indicate the host plant from where the endophyte was isolated. The numbers in the nodes represent bootstrap support and probabilities of each node

2.3  Ecology of Grass-Endophytes Symbiosis Determining selection forces – either biotic or abiotic – that drive endophyte incidence in populations has been central to understand the ecology and evolution of the grass-Epichloë symbiosis. The most common experimental approaches are of at

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least one of the following types: (i) field surveys searching for associations between the endophyte incidence (proportion of endophyte-symbiotic plants in a population) and a given environmental variation factor either discrete (e.g., with or without herbivory) or continuous (e.g., aridity or precipitation) and (ii) controlled condition experiments where the performance of endophyte-symbiotic and endophyte-free plants is compared under different conditions of a given environmental factor (e.g., herbivory, drought). When performed together, the combination of both approaches provides, occasionally, robust results.

2.3.1  A  ssociations Between the Endophyte Incidence and Environmental Conditions As we stated in the previous section, many grass species from a wide range of environments are associated with Epichloë fungal endophyte species (Iannone et  al. 2011, 2015). It offers a variety of model systems to study the role of this symbiosis in the adaptation of plants to different ecological settings and/or environmental conditions. In this section, we summarize the current knowledge regarding the ecology of the grass-Epichloë symbiosis in South America. Patagonia encompasses 60 million ha in southern Argentina covering an extensive area of South America. Within this region, fungal endophytes of grasses are preceded by a negative reputation since Epichloë tembladerae has been associated to the “huecu” disease in sheep and cattle grazing on Festuca argentina (Speg.) Parodi and Poa huecu Parodi (Cabral et al. 1999; Parodi 1950). However, the fungal endophyte symbiosis in many other Patagonian grasses seems not to be related to animal toxicity (Iannone et al. 2011; Novas et al. 2007; Wilson 2007). Nonetheless, the confirmation of toxicity requires a combination of genetic and biochemical analyses to determine the alkaloid genes in the fungus and alkaloid concentration in the plant that have, in addition, to be complemented with animal bioassays. Despite the alkaloids, the real threat for grazing animals will depend on the abundance a given grass represents in the whole vegetation community or as part of its diet. The association between the incidences of Epichloë fungal endophytes in natural populations of Bromus pictus Hook. f. (Bromus setifolius) and abiotic or biotic factors was explored in two contrasting ecological frames. Firstly, a survey was conducted in the Andes Mountains near Las Leñas town, Mendoza province (35°12´ S/69°45´ W), between 2000 and 4000 meters above sea level. While the endophyte incidence was not associated with the altitude, the incidence was higher in those populations located in areas with high abundance of leaf-cutting ants (Acromyrmex sp.) (Fig. 2.5) (White et al. 2001). Considering that E. tembladerae was found to produce two different alkaloids (ergovaline and peramine), which are known to be toxic for herbivores, it was suggested that the endophyte presence would dissuade the ants from attacking the plants. Thus, it provided an evidence of the defensive mutualism hypothesis (White et al. 2001). In another work on B. pictus populations

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Fig. 2.5  Summary picture highlighting the main environmental factors or bioclimatic variables driving the symbiosis incidence for the studied native grass species and Epichloë fungal endophytes

in Southern Patagonia, the association between endophyte incidence and environmental variables and soil conditions was explored (Novas et al. 2007). The study included 36 sites along a northwest-southeast 400 km transect, covering the conspicuous ecological areas of the region (Novas et  al. 2007). The incidence of Epichloë endophytes in the surveyed populations ranged from 0% to 100% (none and all individuals infected, respectively), showing a positive and significant correlation with mean annual precipitation (Fig.  2.5). The incidence did not show an association with the analyzed soil parameters. The factor herbivory was not included in the analysis as neither ants nor other herbivorous insects were observed during the work period. In Tierra del Fuego island, the southernmost region of Patagonia, the association between Epichloë incidence in natural populations of Phleum alpinum L. and Poa spiciformis Hauman and Parodi and environmental variables was explored (Novas et  al. 2007). Phleum alpinum and P. spiciformis are perennial grasses with no

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records of toxicity to livestock. Phleum alpinum plants were sampled in 30 sites along a southwest-northeast transect, including the Nothofagus forests, grasslands, Chiliotrichum dense scrub, and “vegas.” The incidence of Epichloë endophytes in populations of Ph. alpinum varied from 0% to 100%. The ecological analysis revealed that populations with lower endophyte incidence were found in community stands dominated by evergreen forests of Nothofagus pumilio, humid areas characterized by the highest mean annual precipitation in the transect (Novas et al. 2007). In the case of P. spiciformis, all the populations presented plants associated with Epichloë, and the incidence ranged from 40% to 100%. The association between endophyte incidence and plant communities was negatively correlated with the abundance of Empetrum rubrum (Fig. 2.5; Novas et al. 2007), a species indicative of acidic and infertile soils (Collantes et al. 1989). Although endophyte incidence diminished in these stressful soils, P. spiciformis is one of the scarce grasses that grow in those poor habitats and is one of the principal taxa consumed by sheep throughout the year (Posse et al. 1996). At least for Ph. alpinum, the environmental conditions that promotes the symbiosis seem to support the idea that fungal endophytes improve plant tolerance to drought. Based on a previous survey which covered several species (Vila-Aiub et  al. 2001), 14 wild populations of Hordeum comosum J. Presl were sampled in northwestern Patagonia to explore the incidence of Epichloë in association with environmental variables (Iannone et al. 2015). The Epichloë incidence ranged from 0% to 100%, and while endophyte-free populations were restricted to a humid meadow and grassy steppes from the Sub-Andean district, the populations with the higher proportion of endophyte-infected individuals were collected in shrub steppes (Fig.  2.5). The results evidenced a negative correlation between endophyte incidence and the mean annual precipitation (Iannone et al. 2015). The endophyte species E. tembladerae associated to H. comosum (Iannone et al. 2015) is predicted to synthetize the alkaloids peramine and terpendole C (Yi et al. 2018). Although this can imply that the endophyte confers protection to this highly preferred grass by herbivores, there is no study so far that confirmed the previous expectation based on genetic analysis. In fact, the endophyte incidence in H. comosum populations subjected to intense grazing regimes was low compared to those in moderate or ungrazed conditions (Hernández-Agramonte and Semmartin 2016). An endophyte-mediated increased tolerance to drought in some plant species is suggested to be behind the positive association between symbiosis incidence and aridity (Lewis et al. 1997; Malinowski and Belesky 2019). This would be explained by the patterns of endophyte incidence in relation to precipitation for H. comosum and Ph. alpinum (Iannone et al. 2015; Novas et al. 2007). However, opposite patterns have been described for the grass B. pictus (Novas et al. 2007) and for H. comosum in a new exhaustive study (Casas et al. 2015). The tendency to present a very low level of – or not at all – fungal endophytes in populations located in sites with very low mean annual precipitation (80 5

˂5 ˂5 – ˂5

>80

–

–

9.1

–

–

– 11.6

–

1.2 ˂2

– ˃1

>16 ˃1

>9.1

–

–

–

96 ± 4

–

11394

–

72 ± 8

11369

99 ± 2

Percentage Percentage reduction IC50 over T. cruzi growth CC50 on reduction of T. cruzi in new cultivation at (20 μg mL−1)b mouseL929 cellc Selectivity indexd – – 27.8 ˃80 ˃2.9

UFMGCBa 11339e

3  Trypanocidal and Herbicidal Activities of Endophytic... 43

UFMGCBa Lp 9 bark Lp 12 bark Lp 13 bark Lp 14 bark Lp 22 bark Lp 23 bark Lp 28 leaf Lp 28 bark Lp 29 bark Lp 30 leaf Lp 30 bark Benzonidazolef

Percentage reduction of T. cruzi 80 ± 5 63 ± 7 64 ± 9 64 ± 1 55 ± 3 65 ± 1 68 ± 14 97 ± 2 85 ± 3 87 ± 3 96 ± 4 –

Percentage reduction in new cultivation 97 ± 5 96 ± 2 97 ± 1 96 ± 2 100 ± 1 94 ± 1 – 82 ± 3 82 ± 11 – 79 ± 5 –

IC50 over T. cruzi growth at (20 μg mL−1)b 7.5 7 7.7 ˂5 11.3 8.6 – 14.1 14.5 – 9.2 1

Lp Lafoensia pacari specimen, − = not assayed a UFMGCB = Federal University of Minas Gerais collection of microorganisms and cell b Extract concentration that inhibits 50% of Trypanosoma cruzi growth (IC50) c Cytotoxic concentration that reduces viability of the L929 cells by 50% (CC50) d CC50 L929 cell/IC50 parasite, assay performed on MTT, in Vero cells e New fungus cultivation f Positive control

Fungal species

Table 3.1 (continued) CC50 on mouseL929 cellc 40 40 40 40 40 40 – >80 >80 – >80 625 Selectivity indexd 5.3 5.7 5.2 ˂8 3.5 4.6 – >5.7 >5.5 – >8.7 625

44 S. S. Amorim et al.

3  Trypanocidal and Herbicidal Activities of Endophytic...

45

Acanthospermum australe (Asteraceae), a medicinal plant native to the Brazilian savanna. Two substances, ophiobolin K and 6-epi-ophiobolin K, isolated from this endophyte displayed trypanocidal activity against T. cruzi with IC50 values of 13 and 9.62 μM, respectively. However, these compounds showed a relatively low selectivity index against the parasite and were cytotoxic to the host cells. In the case of A. aculeatus, the fungal genome has been recently sequenced by the Joint Genome Institute; however, none of its genes have yet been correlated with the production of active metabolites (Petersen et al. 2015). Despite this fact, it has already been shown that A. aculeatus is able to produce important secondary metabolites including aculeacine AG (antibiotics and antifungals); CJ-15,183 (squalene synthase inhibitor and antifungal agent); aspergillusol A (α-glucosidase inhibitor); secalonic acids A, D, and F (toxins); asperparaline A; cytotoxic aculeatusquinones A–D; and okaramines H and I (okaramine alkaloids) (Ingavant et al. 2009). According to our knowledge, the current work is the first report of the species A. aculeatus producing trypanocidal metabolites. In relation to the other two bioactive taxa, it has been reported that the Valsariaceae family contains species of Myrmaecium that are able to produce secondary metabolites (Jaklitsch et al. 2015) and a species of the genus Coniothyrium, isolated from Pinus wallichiana (Himalayan Blue Pine) that showed activity against Candida albicans with IC50 of 17  μg  mL−1 (Qadri et  al. 2014). However, no trypanocidal activity was reported for these taxa. Chagas disease affects approximately seven to eight million individuals, with 50,000 new cases diagnosed each year in Latin America, North America, and Europe. It is estimated that more than 90 million individuals are at risk of infection with the disease causative agent T. cruzi (Coura and Dias 2009; WHO 2014; Vazquez et  al. 2015). Conventional treatment is based on benzimidazole and nifurtimox, drugs developed over a century ago, which have strong adverse effects, such as loss of appetite, vomiting, polyneuropathy, and dermopathy. Many patients give up on the treatment because, in addition to the strong side effects of these drugs, there is a need for long-term therapy (Guedes et  al. 2011). In addition, benzimidazole and nifurtimox are more effective in the acute phase of the disease, when the parasite remains in an extracellular blood form, and have low efficacy against intracellular forms of T. cruzi during the chronic phase of the disease (Muellas-Serrano et al. 2002). The results obtained in the present study are promising for the investigation of new bioactive molecules isolated from the three taxa as their trypanocidal activity was reproduced after recultivation. Since there are no reports of A. aculeatus being a producer of trypanocidal metabolites and the fungal species of Valsariaceae sp. UFMGCB 11294 and Coniothyrium sp. UFMCCB 11339 have probably not been described yet, these fungi are an especially attractive target for the discovery of trypanocidal substances. Fourteen isolates displayed strong and selective herbicidal activity against both monocotyledonous (A. schoenoprasum) and dicotyledonous (L. sativa) targets with 100% inhibition of the seed germination (Table 3.2). The isolates included A. aculeatus, Chaetomiaceae sp., Diaporthe sp. 4, D. insconspicua, Lasiodiplodia sp. 1,

Fungi Aspergillus aculeatus A. aculeatus A. aculeatus A. aculeatus A. aculeatus A. aculeatus A. aculeatus Beltroniella sp. Chaetomiaceae sp. Colletotrichum sp. 2 Colletotrichum sp. 2 Colletotrichum sp. 2 Diaporthe insconspicua D. insconspicua D. insconspicua S. insconspicua Diaporthe sp. 4 Lasiodiplodia sp. 1 Lasiodiplodia sp. 1 Lasiodiplodia sp. 1 Lasiodiplodia sp. 1 Lasiodiplodia sp. 1 Lasiodiplodia sp. 1 Lasiodiplodia sp. 2 Lasiodiplodia sp. 2

Herbicidal activity Lactuca sativa Allium schoenoprasum – 5 ± 0 – 5 ± 0 5 ± 0 5 ± 0 5 ± 0 5 ± 0 5 ± 0 5 ± 0 5 ± 0 5 ± 0 5 ± 0 5 ± 0 3.5 ± 0.5 3.5 ± 0.5 5 ± 0 5 ± 0 – 3 ± 0 – 5 ± 0 – 4 ± 1 – 4 ± 1 3.5 ± 0.5 4.5 ± 0.5 – 5 ± 0 5 ± 0 5 ± 0 5 ± 0 5 ± 0 5 ± 0 5 ± 0 5 ± 0 5 ± 0 – 3 ± 0 – 5 ± 0 – 4 ± 1 5 ± 0 5 ± 0 – 3 ± 0 5 ± 0 5 ± 0 UFMGCBa 11433 11435 11436 11432 11250 11256 11264 11311 11313 11331 11332 11333 11294 11208 11277 11270 11279 11439 Lp 1 leaf Lp 22 leaf Lp 29 leaf NC PC SC

Fungi Penicillium sp. 1 Penicillium sp. 3 Penicillium sp. 4 Penicillium wotroi Phyllosticta sp. 1 Phyllosticta sp. 1 Phyllosticta sp. 1 Phyllosticta sp. 1 Phyllosticta sp. 1 Phyllosticta sp. 1 Phyllosticta sp. 1 Phyllosticta sp. 1 Valsariaceae sp. NI Neofusicoccum sp. NI NI NI – – – – – –

Herbicidal activity Lactuca sativa Allium schoenoprasum – 4 ± 1 – 5 ± 0 3.5 ± 0.5 4.5 ± 0.5 5 ± 0 4.5 ± 0.5 5 ± 0 5 ± 0 – 5 ± 0 – 5 ± 0 3 ± 0 3 ± 0 3 ± 0 – – 3.5 ± 0.5 – 4 ± 1 – 4 ± 0 5 ± 0 5 ± 0 5 ± 0 5 ± 0 – 5 ± 0 – 5 ± 0 – 3 ± 0 5 ± 0 5 ± 0 – 3 ± 0 – 3.5 ± 0.5 – 3 ± 0 0 ± 0 0 ± 0 5 ± 0 5 ± 0 0 ± 0 0 ± 0

Lp Lafoensia pacari specimen, NC negative control, PC positive control, SC solvent control a UFMGCB = Culture of Microorganisms and Cells from the Federal University of Minas Gerais. The qualitative estimate of phytotoxicity was evaluated by using a rating scale of 0–5, where 0 = no effect and 5 = no growth or no germination of the seeds. Plant targets: L. sativa = Lactuca sativa (lettuce) and A. schoenoprasum = Allium schoenoprasum (chive)

UFMGCBa 11205 11215 11369 11409 11410 11412 11413 11357 11430 11207 11221 11316 11237 11245 11282 11321 11322 11213 11394 11395 11399 11418 11419 11209 11397

Table 3.2  Herbicidal activities of extracts obtained from cultures of fungal endophytes species isolated from Lafoensia pacari 46 S. S. Amorim et al.

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Lasiodiplodia sp. 2, Phyllosticta sp. 1, and Valsariaceae sp. Several phytotoxins, such as phyllosinol, have been previously identified in Phyllosticta species (Wikee et al. 2011). In the present study, eight extracts from Phyllosticta showed herbicidal activity for both or only one of the studied plants. Species of this genus have frequently been isolated as endophytes and have been considered to be excellent sources of novel lead bioactive compound structures (Rodrigues-Heerklotz et  al. 2001). Diaporthe spp. are known as producers of secondary metabolites with the potential for herbicidal and anti-herbivory activity (Ash et  al. 2010; Andolfi et al. 2015). Five isolates of A. aculeatus showed total inhibition of both A. schoenoprasum and L. sativa seed germination, and two isolates selectively inhibited only the chive seed germination. Four Penicillium specimen showed herbicidal activity, and Penicillium sp. 4 UFMGCB 11436 and P. wotroi UFMGCB 11432 inhibited the germination of the seeds of both plant models used in the assay. Khattak et  al. (2014) have shown that crude ethyl acetate extracts from Aspergillus and Penicillium have phytotoxic activity against Lemna minor (water lentil) and may also delay seed germination of the weed Silybum marianum. Zhang et al. (2013) isolated a bioactive compound from the endophytic fungus A. fumigatus obtained from the stem bark of a medicinal plant Melia azedarach. The compound was named brevianamide F and represented a potentially new class of broad-spectrum herbicides as it showed greater activity than the positive control of glyphosate herbicide. Brevianamide F also inhibited the turnip (Raphanus sativus) root and amaranth (Amaranthus mangostanus) seedlings growth. Brazil is one of the largest consumers of agrochemicals in the world, and glyphosate is the most commercialized herbicide in the country (Brazil, IBAMA 2018). Glyphosate-based herbicides are currently among the most widely used agricultural chemicals in the world (Gianessi and Reigner 2006). The impact of this class of herbicides on modern farming practices is undeniable and includes the development of resistant crop varieties as well as environmental and ecological implications (Gilbert 2013). The use of natural herbicides in place of traditional chemical herbicides can provide a large number of environmental and socioeconomic benefits. It could favor better management and lead to the reduced impact of agriculture on the environment as well as on human health. Moreover, it could contribute to the control of the weeds that are resistant to current herbicides. In our study, we obtained extracts from the A. aculeatus UFMGCB 11413 and Valsariaceae sp. UFMGCB 11294 fungi, which displayed antiparasitic activity and inhibited the germination of all lettuce and chive seeds. These results point to the promising potential of these fungi for the future isolation of substances with herbicidal properties.

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3.7  Conclusion The present study has contributed to the knowledge about the community of endophytic fungi associated with Brazilian medicinal plants and allowed them to be considered as a potential source of bioactive metabolites. The obtained results confirm the importance of medicinal plants present in the rarely studied Brazilian Neotropical environments, such as the Pantanal wetlands, which are potential reservoirs of known and still unknown endophytic fungi. These as yet undiscovered symbionts are likely to produce bioactive secondary metabolites, which could be used in the future for agricultural and therapeutic purposes.

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Chapter 4

Advances in Research on Biodiversity and Bioprospecting of Endophytic Fungi in Chile Rómulo Oses-Pedraza , Víctor Hernández, Leonardo Campos, José Becerra, Dánae Irribarren-Riquelme, Paris Lavín, and Jaime Rodríguez

Abstract  In this chapter we review the current advances in ­bioprospection, ecology, diversity, and potential applications on endophytic fungi “(endobiomes).” These fungi associated with endemic plant species of Chile or economically important crops in agroforest ecosystems. Chile has many endemic species, as it is considered a biogeographical island, i.e. it borders in the north to the hyper-arid Atacama Desert, in the east to the high mountains of the Andes, in the west to the Pacific Ocean, and in the south to the freezing Antarctic continent. It features one of Earth’s most spectacular ecological, topographic, and climate gradients that encompasses a multiplicity of “field laboratories.” Its biodiversity is recognized as an important hot spot, and is the The original version of this chapter was revised. The correction to this chapter is available at https://doi.org/10.1007/978-3-030-53506-3_18 R. Oses-Pedraza () Vicerrectoría de Investigación y Postgrado (VRIP), Centro Regional de Investigación y Desarrollo Sustentable de Atacama (CRIDESAT) – Universidad de Atacama, Copiapó, Chile Environmental Microbiology and Biotechnology Unit – Zenobia Group SpA, Chillán, Chile e-mail: [email protected] V. Hernández · J. Becerra Laboratory of Natural Products Chemistry, Department of Botany, Faculty of Natural and Oceanographic Sciences, Universidad de Concepción, Concepción, Chile D. Irribarren-Riquelme Environmental Microbiology and Biotechnology Unit – Zenobia Group SpA, Chillán, Chile L. Campos Vicerrectoría de Investigación y Postgrado (VRIP), Centro Regional de Investigación y Desarrollo Sustentable de Atacama (CRIDESAT) – Universidad de Atacama, Copiapó, Chile P. Lavín Facultad de Ciencias del Mar y Recursos Biológicos, Departamento de Biotecnología; Laboratorio de Complejidad Microbiana y Ecología Funcional, Instituto Antofagasta, Universidad de Antofagasta, Antofagasta, Chile J. Rodríguez Center of Biotechnology, Universidad de Concepción, Concepción, Chile © Springer Nature Switzerland AG 2021, Corrected Publication 2021 L. H. Rosa (ed.), Neotropical Endophytic Fungi, https://doi.org/10.1007/978-3-030-53506-3_4

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focus of many studies and conservation efforts, due to a high degree of endemism and uniqueness. The necessity to perform research on endophytic fungi is of significant relevance as it directly contributes to the conservation of endophytic fungi biodiversity, and gives insight host–endophyte interactions, especially in different environmental gradients. Additionally, these studies can open the doors to a wide range of potential uses and/or modulations of endophytic fungal microbiomes with the aim to enhance the health and productivity of plants in agricultural and forestry, e.g. the endophytic fungi could promote plant growth or biologically control diseases caused by phytopathogenic microorganisms. These endophytic fungi also represent a valuable bioresource for prospecting secondary metabolites for biotechnological purposes. In Chile, bioprospecting studies on endophytic fungi are just as scarce as the ecological studies on them, however, the few that have been conducted show very promosing results. This highlights the need to thoroughly study endophytic fungus, and conserve its unique and extreme ecosystems in Chile. Keywords  Chile flora · Endemism · Agroforest ecosystems · Endophytic fungi · Environmental gradient · Biodiversity · Ecology · Applications

4.1 

Introduction

4.1.1  C  hile as Natural Laboratory and Its Potential for Endophytic Fungi Research Chile is a large country with a very diverse geography and unique features in South America. It covers 75 million hectares; and is approximately 4.300 kilometers from north to south with an average width of 180 kilometers. It is considered a biogeographical island because it is, isolated by the hyperarid Atacama Desert on the north, the Antarctic ice on the south, the Andes Mountains in the east, (up to 6.960 meters above see level; m.a.s.l.), and the Pacific Ocean in the west, In this large environmental gradient, species distribution is the result of constrasting selective pressures imposed by climatic conditions (e.g. day/night temperatures variations, evaporationrainfall rates), or geo-morphology and mineral composition of parent materials (Risarcher et al. 2003; Dorador et al. 2013). These environmental conditions facilitate the formation of various types of (longitudinal) climate gradients e.g. arid to temperate and polar, along the country, and steep latitudinal gradients with large elevation differenced, from tops of the Andes to sea level at the coast. These steep changes over short distances result in the formation of diverse bioclimatic landscapes (“mosaic effect”), ranging from favorable in the coastal area and valleys to extreme in the high mountain areas. In total 25 different climates are recognized for continental Chile,

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Fig. 4.1  Map of macrobioclimates (*) and representative landscapes of Chile. Symbology for macrobioclimates (*): orange = tropical; red = Mediterranean; brown = temperate; green = sub-­ Mediterranean temperate; blue = Antarctic. (a) “Hornitos” beach, II Region of Antofagasta; (b) desert blossom, Huasco Valley, III Region of Atacama; (c) “Chañaral de Aceituno” National Marine Reserve, III Region of Atacama; (d) “Santa Gracia” National Reserve, IV Region of Coquimbo, semidesert ecosystem; (e) Isla Riesco – Patagonia forest – XV Region of Magallanes and Chilean Antarctic and sub-Antarctic ecosystems; (f) Laguna Verde 4328 m.a.s.l., high altitude Puna Plateau, III Region of Atacama; (g) “Pan de Azúcar” National Park, desert ecosystem, III Region of Atacama; (h) “La Campana” National Park, Mediterranean ecosystem, V Region of Valparaíso; and (i) Nahuelbuta NationalPark, Sub-mediterranean temperate, IX Región of Araucania, (j) Antarctica Peninsula and Antarctic ecosystem, Isla Lagotellerie (*) adapted from Luebert and Pliscoff (2006)

based on climatic and geographical criteria (Sarricolea et al. 2017; Fig. 4.1). In at least 14 of these different climates soils have been described. (Casanova et al. 2013). Chile still possesses pristine ecosystems with little human intervention, and a diversity of climatic conditions (Braun et al. 2017; Garces-Voisenat and Mukherjee 2016).There are 30 types of terrestrial ecosystems in Chile including those of n­ atural (terrestrial, continental and insular aquatic, marine, coastal and ­oceanic islands) and anthropogenic origin (Martínez-Tilleria et al. 2017a, b). Chile has many protected islands destined to protect unique ecosystems with high levels of endemism, e.g.

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Chañaral Island, Choros Island, Damas Island, La Rinconada, Cabo de Hornos, Archipiélago Juan Fernández, San Félix and San Ambrosio Islands, and Salas and Gómez Islands (Comision Nacional de Medioambiente 2009). Additionally, has a large diversity on other aquatic ecosystems, displaying a spatial heterogeneity of great complexity (Figueroa et al. 2009; Hauenstein et al. 2005). It has an extensive coastline with ecosystems influenced by the Humboldt Current (Daneri et al. 2000; Escribano and Hidalgo 2000; Ortiz et al. 2015), a great diversity of rivers and their basins (Peredo-Parada et al. 2011), coastal wetlands, river deltas high altitude wetland ecosystems (“bofedal”) in the northern part of the Andes, and large lakes in the south, mainly in Patagonia. The Chilean biodiversity is characterized by the biogeographical isolation of the country which is the driving force of the high degree of endemism and exclusiveness when comparing Chilean flora with the flora of other countries with similar climates. The flora de Chile includes over 4.500 taxa (Marticorena 1990; Rodríguez et al. 2018; Zuloaga et al. 2008) and is extraordinary, because it contains the highest percentage of endemisms (ca. 50%) in South America (Cowling et  al. 1996; Marticorena 1990). It contains four endemic families and 83 endemic genera (Moreira-Muñoz 2011), of which 67 genera inhabit continental Chile and 16  the islands. The high biodiversity of flora in arid ecosystems (Squeo et al., 2001), as well as the high levels of floral endemism concentrated in central part of the country, are important attributes that converted Chile in one of the 25 “biodiversity hotspots” (Myers et al. 2000). This status is currently threatened by anthropogenic climate change (Fuentes-Castillo et al. 2020). More than 75% the flora of the Juan Fernández archipelago has status vulnerable and highly threatened, mainly due to introduction of alien invasive species from the continent (Swenson et al. 1997). However, the levels of endemism and vulnerability of Chilean flora should not only be considered in national conservation efforts, but also the future (bioprospecting) research of endophytic fungi related to these rare plants. Endophytic fungi are defined as symbiotic microorganisms capable of migrating into plant endosphere, colinizing healthy plant tissues (e.g. seeds, leaves, twigs, stems, wood, bark, petioles, fruits, flowers), inter-and/or intracellularly, and persisting for the whole or part of the cycle without Strobel have been studied not only in natural ecosystem but also in anthropogenic communities. Endophytic fungi can be beneficial for their host producing a range of natural product, activating abiotic/biotic host stress responses, triggering plant defense, and improving plant fitness (Rodriguez et al., 2009; Singh et al., 2011). They are ubiquitous, diverse and context-dependent, revealing the crucial of both the identify of host plants and the geographic location in which plants occurs (Saikkonen et al., 2007; Arnold et al., 2007). It has been suggested that they can influence the distribution, ecology, physiology, and biochemistry of the host plants (Arnold et al. 2007; Busby et al. 2016). In recent years, great attention have been made to endophytic fungi due of its ability to produce novel bioactive secondary compounds which are of pharmaceutical, industrial and agricultural relevance. Given the great diversity of terrestrial and aquatic ecological systems in combination with high degree of endemic flora species associated with ecosystems, it is expected that Chile contains a hidden diversity of (endemic) endophytic fungi as

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reported previously (Giauque & Hawkes 2013; Millberg et al., 2015; Giauque et al., 2016; Daniels et al., 2018). Due to the wealth of environmental gradients and climates in Chile we expect to find changes along these gradients in the diversity or assembly of fungal communities. The communities are expected to contain species with special morphological and/or remarkable physiological adaptations to e.g. cold, salt, UV radiation, or the ability to produce bioactive compounds with biotechnological potential. In Chile, research, development, and innovations using endophytic organisms are still emerging; however, it offers great opportunities and new challenges for the exploration with target not only in different environments but also in different phototrophs not only trees, shrubs, and herbs but also macrophytes, seaweeds, and seagrass (Sridhar 2019).

4.2  Biodiversity and Ecology of Endophytic Fungi 4.2.1  Terrestrial Ecosystems Piontelli and coworkers (2002) did one of the first studies on the ecology and biodiversity of endophytic fungi in extreme environments in Chile. They investigated the altitudinal distribution of keratinophilic, epiphytic and endophytic fungi from desert soils in Northen Chile, and used microscopic observations in combination with the classical taxonomical approach to identify the fungi. In the xeromorphic ecosystems they worked in, the vegetation is composed of cushion plants, cacti, xerophytic shrubs and grasses. They analyzed several species for the presence of endophytic fungi, e.g., Ephedra breana, Phil. Fabiana imbricata, Ruiz and Pav., Tessaria absinthioides (Hook. & Arn.), Atriplex atacamensis, Phil., Azorella trifurcata, (Gaertn.) Vulpia sp., Festuca sp., Hordeum sp., Stipa sp., and Chaetantera sp. were analyzed among other species. This study used an altitudinal gradient, and had sampling areas at three altitudes 2,200–2,990; 3,000–4,000; and > 4,000 m.a.s.l.), and a time span of two successive seasonal periods. During the time of this study, the presence and composition of keratinophilic, epiphytic, and endophytic fungal communities were determined, using keratinic and vegetal bait or culture-dependent methods. A total of 1111 isolates of endophytic fungi were recovered, from 70 soils and 67 vegetal samples, belonging to 78 genera and 172 species. 18 genera were observed most frequent in both substrata, and accounted for approximately 80% of the total fungal presence. The most prevalent genera of endophytic fungi along the gradient, were Ulocladium Preuss, Penicillium Link, Cladosporium Link, Alternaria Nees, Phoma Sacc, Trichoderma Pers, Chaetomium Kunze, and Fusarium Link. The most frequent species were Alternaria alternata (Fr.) Keissl., Cladosporium cladosporioides, Alternaria chlamydospora Mouch., and Epicoccum nigrum Link. The composition of endophytic fungi community was similar in different nonspecific host plants, and seemsed stable in time. Interestingly, a 62% of species that were classified as epiphytes, were also classified as endophytes. This study highlights the

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potential ecological roles of endophytic fungi, in the eco-nutritional adaptation of host plants to stressful arid environments (deserts). In the high Andes of central Chile, Casanova-Katny and coworkers (2011) studied the positive interactions between cushion plant and associated plant species. They focused on the effects of fungal root symbionts in the associated plants. For that they evaluated the status and abundance of mycotrophy of arbuscular mycorrhiza (AM) fungi and dark septate endophytes (DSE) in plant communities dominated by cushion-forming species Azorella madreporica, at two sites at different altitudes (3,200 and 3,600  m.a.s.l.). The authors hypothesized that in inside cushions, AM and DSE fungi develop better than outside cushions and that difference is expressed in the relative frequency of mycorrhizal structures within plants. The colonization of fungal root symbiont of A. madreporica cushions was significantly different between both sites, i.e. 68% (at 3.200 m.a.s.l.) and 32% (at 3,600 m.a.s.l.), respectively. The presence of fungal root symbionts associated to cushion communities at high altitudes emphasizes their relevance in these environments where plant species benefit from the microclimatic conditions generated by cushion plants and also from well-developed fungal networks. The positive interactions between host plant and endophytic fungi have been studied as an important strategy for survival and growth, in stressful environments e.g. alpine environments. Molina-Montenegro and coworkers (2015) evaluated if the presence of fungal endophytes isolated from the roots of cushion plants Laretia acaulis (Cav.) Gilles & Hook. can play a fundamental role in the stablishment, performance and survival of both native and exotic plant seedlings, which are known to be facilitated by cushion species, at 3.200 m.a.s.l. in the Andes of Central Chile. The aim of this study was to analyze the effect of endophytic fungi in local soil, associated with the roots of the cushion nurse species Laretia acaulis, on the establishment and performance of three speices known to be facilitated by L. acualis: two native (Phacelia secunda J.F.Gmel. and Hordeum comosum J. Presl) and one exotic species (Taraxacum compylodes G.E.Haglund). In a combination of greenhouse and field experiments they found that endophytic fungi had a positive impact on the survival and growth of both native and invasive species. Morever, the biomass accumulation, maximum quantum efficiency (Fv/Fm), and seed production of the invasive species improved, when soils were inoculated with endophytic fungi. This study suggests that root endophytic fungi do not only mediate the facilitation of native and invasive alpine plants, but also inderectly influence the structuring of plant communities in stressful habitats. As already mentioned above, endophytic fungi have been studied because of their positive impact on survival and growth of their host plant in extreme environments. For example. Fardella and collaborators (2014) evaluated the effect of Antarctic fungal endophyte on the survival and water use efficiency in three native xerophytic species, from the north of Chile. They concluded that, 12 months after inoculation with fungal endophytes from Antarctica, the water use efficiency improved when water supplies were reduced in Flourensia thurifera (Molina) DC., Senna cumingii,(Hook. & Arn.) H.S.Irwin & Barn, and Puya berteroniana Mez. These findings support the hypothesis that it is possible to use fungal endophytes to

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improve the eco-physiological performance and survival of different shrubs and trees. Later, Molina-Montenegro and coworkers (2016a) evaluated the direct and indirect nursing effect of the shrub Porlieria chilensis L.M. Johnst, on the native plant species Flourensia thurifera (Asteraceae), Puya berteroniana (Bromeliaceae), and Senna cumingii (Fabaceae) in semi-arid community in northern Chile. Two questions were evaluated. First, whether microclimate modifications, induced by presence of P. chilensis, improved the survival and growth of these native plant species. Second, whether the presence of soil microorganisms, associated with P. chilensis, enhances the survival and growth of these native plant species. To compare the survival and growth of between individuals originating from underneath P. chilensis or without nurse plant, soil from both microsites was used in greenhouse experiments. The native plants were cultivated in soil taken from both microsites, with or without soil microorganisms. The results showed a significant nursing effect of P. chilensis on the tested species through of improvement of climatic (air temperature, soil moisture, and solar radiation) and edaphic conditions (e.g. nitrogen availability), which increased the performance and establishment of these native species significantly. Plant-growth promoting microorganisms appear to play a key role in the restoration of native plant communities. Because of that, rehabilitation initiatives, targeting degraded dryland ecosystems, should also consider restoring beneficial microflora, associated with local species. It has been reported that, plants in arid environments harbor large and diverse communities of root-associated fungi. However, the ecological role they play, in relation to their host plants, has been little explored. Gonzalez and coworkers (2019) investigated the root-associated fungal endophyte community of Prosopis chilensis (Molina) Stuntz, a near-threatened, drought adapted, leguminous tree from northern Chile. They focused on the potential host benefits, associated with endophyte colonization, in terms of physiological processes and plant growth. They sterilized the surface of asymptomatic roots and isolated the endophytic and isolated the endophytic with culture-dependent methods. With sequencing of the 18S rRNA gene Penicillium sp. was (molecularly) identified as one of the main endophytes. Plants inoculated with endophytic fungi showed a higher PSII efficiency (photosynthesis), leaf nitrogen, carhohydrate content, and growth. Endophytic fungi can play a role in protecting their hosts against the attack of herbivores and pathogens. González-Teuber (2016), for example, analyzed the diversity of foliar endophytic fungi of the southern temperate tree Embothrium coccineum J.R.Forst & G.Forst (Proteaceae), and their potential role in plant protection. Different issues were evaluated, such as the diversity of the foliar endophytic fungi community associated with asymptomatic leaves of E. coccineum, the potential relationship between endophytic fungi frequency and host plant resistance in nature, and whether foliar endophytic fungi inhibit the growth of fungal pathogens in vitro. Fungal endophytes were isolated with culture-dependent methods, and (molecularly) identified (based on the sequencing of 18S rRNA gene). The frequency of endophytes and their role in plant protection was evaluated in juvenile plants of E. coccineum using real-

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time PCR analyses to determine the DNA content per plant. The results showed that most of the taxa (from a total of 178 fungal isolates) were considered rare species, while only few of them the fungal endophyte community. The genera Mycosphaerella Johanson, Öfvers. K. (23%) dominated the community, followed by Xylaria Hill ex Schrank (18%) and Diaporthe Nitschke, Pyrenomycete (10%). Penicillium and Colletotrichum Corda occurred at lower frequencies (less than 10%), whereas other genera were found only on rare occasions, with frequencies between 1% and 5%. The percentage of leaf damage showed positive correlation with frequencies of endophytic fungi. The study showed that endophytic fungi were able to inhibit the growth of fungal pathogens in vitro (confrontation assays). Overall, the fungal endophytes associated with leaves of E. coccineum were of high diversity, and the authors showed a positive relationship between fungal endophyte frequencies in leaves and host plant protection in nature. Later in 2020, Gonzalez-Teuber and coworkers studied the influence endophytic fungi colonization and community composition on leaf resistance traits (e.g., cell wall polysaccharides, leaf toughness, flavonoids, anthocyanins, terpenoids and chitinases) in ten dominant trees species (Aextoxicon punctatum Ruiz & Pav., Amomyrtus luma (Molina) D.Legrand & Kausel, Aristotelia chilensis (Molina) Stuntz, Caldcluvia paniculate (Cav.) D.Don, 1830, Embothrium coccineum J.R.Forst. & G.Forst., Eucryphia cordifolia (Cav.), Gevuina avellana Mol., Laureliopsis philippiana (Looser) Schodde, Luma apiculatae (DC.) Burret, Myrceugenia planipes (Hook. & Arn.) O. Berg) in a temperate rainforest in Southern Chile. By using culture-dependent methods in combination with Illumina MiSeq sequencing, they found that leaf resistance traits may influence fungal endophyte colonization and community composition in tree species. The authors suggest that endophytic fungi able to resist the deterrent effects of the structure and chemistry of the leaves might play a role in plant protection. The status of fungal symbiotic associations, in different ecological niches, is relevant information to design protective and integrative conservation plans, e.g. for endangered Orchid species in Chile. Herrera and coworkers (2017) analyzed the endophytic root fungi community from seven terrestrial orchid species, collected in the Andes and the Coastal Range of central-sourthern Chile, to identify potentially interesting symbiotic fungi. Fungal sequences were identified through molecular techniques and showed a high diversity of dark septate endophyte (DSE) suggests a potential role in plant establishment. A germination tests showed a remarkable effect of endophytic fungi isolates on different orchids seed; the germination of orchid seeds was promoted but at different efficiences and with low specifity. The genera Tulasnella J. Schröt, Ceratobasidum D.P. Rogers, and Thanatephorus Donk were the most relevant genera of the two recovered fungal groups. Of these genera, Tulasnella, isolated from Chloraea gavilu Lindl., induced the highest seed germination in different species. Adult orchids were colonized abundantly by Rhizoctonialike endophytic fungi, which were identified with PCR-ITS sequencing. This colonization suggests that mycoheterotrophy (e.g. plants parasite on fungi for the acquisition of nutrients) could be acting as a complementary strategy for nutritional

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demands in orchids. Nevetheless, it was not possible to rule out the influence of other beneficial associations with endophytic fungi. The number of studies of fungal endophyte communities has increased in recent years. However, the knowledge about the diversity and composition of endophytic fungi associated with agricultural crops have been explored insufficiently. GonzálezTeuber and coworkers (2017) investigated the fungal endophyte community associated with the roots of C. quinoa growing near the Salt Lake of the Atacama Desert, Chile. They were able to isolate root-associated endophytic fungi from C. quinoa plants, and to identify them using the amplification and sequencing of the internal transcribed spacer (ITS) of fungal genomic DNA. One hundred endophytic fungi were reported, and the isolates were classified into eleven genera and 21 distinct operational taxonomic units (OTUs). Ascomycota was the dominant phyllum Penicillium was the most abundant, genus, followed by Phoma and Fusarium. Recently, Guevara-Araya and coworkers (2020) analyzed the differences in community composition of endophytic fungi between above- and below-ground tissues of Aristolochia chilensis (Bridges ex Lindl.), an endemic specie, perennial creeping herb distributed from Mediterranean-type to arid climates (arid ecosystem - Atacama Desert). They found that the diversity in above-and below-ground tissues of A. chilensis was similar. However, the results showed that dominant endophytic fungi (abundance) in roots were twice as high than in shoots. These results showed that fungal endophyte communities in the above- and below-ground tissue types were significantly dissimilar. Endophytic fungi may positively influence plant resistance to drought. The understanding of stress tolerance mechanisms in plants is vital to develop and improve management strategies, considering that water stress is likely to become an increasingly important factor (particularly in arid climates), that will affect crop production in future climate scenarios. González-Teuber and coworkers (2018) investigated how root endophytic colonization by Penicillium ­minioluteum, Dierckx, an endophytic fungus isolated from quinoa, enhances the ecophysiological performance of its host during extended periods of drought. In greenhouse experiment, plants with and without endophytic fungi were submitted to different watering treatments and plant performance, phtotosynthesis, water-use efficiency and photochemical reactions were examined. When plants were inoculated with endophytic fungi and submitted to the low water treatment, they demonstrated a 40% improvement in root formation in enhancement of physiological performance, mediated by endophytic fungi, during the drought period. This work suggests that the nature of the interaction is positive, but significant when there is a restriction in the input of water. Plant-growth-promoting microorganisms one of the harshest and most stressful habitats form of life on Earth in Antarctica. This is particularly true for the two vascular plants Antarctic pearlwort (Colobanthus quitensis (Kunth) Bartl.; Caryophyllaceae) and Antartic hairgrass (Deschampsia antarctica Desv. Poaceae), which occur naturally in this hostile environment (Lewis-Smith 2003). A study by Ramos and coworkers (2018), focused on identifying changes in hormonal profiles, related to the photo-protective effect (due to endophytes), in C. quitensis. For this they measured plant photochemical efficiency, lipid peroxidation and reproductive biomass along a UV-B radiation gradient. When UV-B radiation increased, plants

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inoculated with endophytes presented higher numbers of flores, higher total biomass, lower lipid peroxidation, but had no changes in the photochemical efficiency, in comparison with non-inoculated plants. Shoot tissues of plants, inoculated with endophytic fungi, showed changes in their levels of salicylic acid, jasmonate, indole-3-acetate and absicic acid when exposed to UV-B radiation. This work suggests that endophytic fungi could be envolved in the trigging of hormonal and physiological mechanisms, which help plants cope with high leveles of UV-B radiation. The association of host plants with endophytic fungi can be a suitable strategy to cope with multiple abiotic stresses, e.g., the application of endophytic fungi, isolated from drought adapted host plants, could enhances ecophysiological performance and yield of crops exposed to drought. Plant-root-associated fungi, Molina-Montenegro and collaborators (2016b) evaluated the effect of the inoculation of fungal endophytes isolated from Antarctic plants. They looked for improvement of the net photosynthesis, water use efficiency, and production of fresh biomass in a lettuce cultivar, grown under different water availability regimes. In addition, they assessed the improvement of biochemical mechanisms and gene expression related with environmental tolerance in presence of fungal endophytes. Their results showed that plants inoculated with endophytic fungi showed higher net photosynthesis, water use efficiency in drought conditions, and consequently an increment in fresh and dry biomass production and in the development of their root system. The plants also had a higher proline concentration, less peroxidation of lipids, and up/ downregulation of ion homeostasis. This work suggests that endophytic fungi could contribuite to stress mitigation of crops, enhancing drought tolerance through biochemical and improvement of nutritional status. The symbiosis of endophytic fungi and vascular plants in Antarctic ecosystems have been hypothesized to be a strategy to cope with harsh environmental conditions. However, little is know about the effect of climate change on these types of positive biological interactions. Torres-Díaz and coworkerset (2016) investigated whether the biological interactions and simulated climate change modulate the ecophysiological performance of Colobanthus quitensis. The main goal of this study was to determine, (1), if changes in abiotic factor such as temperature and water differentially affect the photochemical performance of the plant Colobanthus quitensis (direct effect), and (2), if this environmental changes indirectly affect C. quitensis photochemical performance and biomass accumulation by modifying its association with fungal endophytes (indirect effect). Plants of C. quitensis (from King George Island in the South Shetland archipelago (62°090 S) and Lagotellerie Island in the Antarctic Peninsula (65°530 S)) were placed in growth chambers, with different simulated abiotic conditions, to test the effect of direct climate change. Predictive models of global climate change (GCC) were used to determine the climatic conditions in the chambers. The indirect effect of GCC on the interaction between C. quitensis and fungal endophytes was determined in field trials, in Antarctica . Climate models were used to calculate watering regimes, simulating an increase of precipitation in Antarctica.The results showed that warming (+T) significantly increased plant performance; however, its effects tended to be less than watering (+W) and combined warming and watering (+T +W). Endophytic fungi

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improved plant performance, however, their effect significantly decreased when watering. increased. The work concludes that these Results indicate that future population range and evolution could be predicted, using ecological niche models for different climatic scenarios. Despite that the fungal endophyte associations have been suggested as a possible strategy of Antarctic vascular plants for surviving the extreme environmental conditions, the mechanisms by which this occurs are still far from being understood. Oses-Pedraza and coworkers (2020) evaluated the role of root endophytic Penicillium in nitrogen mineralization and nutrient uptake, as well as their impact on the performance of Antarctic plants. They tested root endophytes, Penicillium chrysogenum and Penicillium brevicompactum isolated from Colobanthus quitensis and Deschampsia antarctica, respectively, for lignocellulolytic enzyme production, nitrogen mineralization, and growth enhancement of their host plants. Both root endophytic Penicillium were characterized as psychrophilic fungi with ability to produce amylase, esterase, protease, cellulase, hemicellulase, phosphatase and urease enzymatic activities mainly at 4 °C. The results showed that rates and percentages of nitrogen mineralization, as well as the final total biomass, were significantly higher in symbiotic C. quitensis and D. antarctica plants. The authors suggest that root endophytes play a key ecological role based not only to breakdown different nutrient sources but also on accelerating nitrogen mineralization, improving nutrient acquisition, and therefore promoting plant growth in Antarctic terrestrial ecosystems. Due to global warming, the climate in Antarctica is likely to change, however, is unclear how this change will alter the impact of endophytic fungi on host plants. Hereme and collaborators (2020) evaluated the role of endophytic fungi on osmoprotective molecules (sugar production, proline, oxidative stress) and gene expression (CqNCED1, CqABCG25, and CqRD22) as well as physiological traits (stomatal opening, net photosynthesis, and stomatal conductance), in individuals of C. quitensis. They did simulation experiments with two groups of C. quitensis: with or without Antarctic endophytic root fungi. Both groups were submitted to different treatments of water availability: limiting water availbility (control) and abundant water availability (treatment), as an increase in precipitation predicted for Antarctica. They found that inoculated plants produced higher levels of sugar and proline, which suggests that endophytic fungi can mitigate oxidative stress. Additionally, the inoculated plants had gene expression involved in drought stress response, and this expression was stronger in the limiting water control than in abundant water availability treatment. Inoculated plants also showed higher net photosynthesis, stomatal opening, and conductance. Overall, this meant that the inoculation of endophytic fungi enhanced drought tolerance in C. quitensis significantly, while there was no significant difference between the inoculated plants and non-inoculated plants when water availability was abundant. Later, Ballesteros and coworkers (2020) analyzed the effect of simulated global warming (in-situ) on endophytic and epiphytic microbiomes of C. quitensis plants. in Antartica, by using a meta-transcriptomic approach. They taxonomically classified and functionally annotated the meta transcriptome of the (endophytic or epi-

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phytic) microorganisms on the leaves of C. quitensis. The reference assembly of C. quitensis, and RNA-sequence libraries were used to determine the differences in the transcriptomic profiles of genes of non-plant species colonizing C. quitensis plants grown under normal and global warming conditions. The analysis of the number of differentially expressed genes suggests that climate change modulates the meta-transcriptome of C. quitensis plants and their associated endophytic and epiphytic microbes. Moreover, the authors found a high proportion of the up and down-regulated genes, belonging mainly to non-plant species. They suggested that these genes had fungal origin. The metatranscriptomic analysis showed that diverse biologically processes presented differentially display in non-plant microorganisms associated with C. quitensis, when global warming was simulated. This work concludes that the plant-microorganisms interactions are modulated by climatic factor and the adaptation of plants in extreme habitats could be partly explained by the role of endophytic and epiphytic microbiomes. Mycoheterotrophic plants are described as non-photosynthetic plant that require special root associations with mycorrhizal and endophytic fungi to overcome nutritional scarcity during germination and the development of seedlings. In 2019, Herrera and coworkers described the microbiological interactions in roots of the non-photosynthetic, mycoheterotrophic plant Arachnitis uniflora Phil. (Corsiaceae), from southern Chile. They used culture-dependent methods to recover endophytic fungi form two different microhabitats of the Coastal Mountain range in sourthern Chile (a Nothofagus Blume-dominated forest and a mixed Peumus boldus Molina, Luma apiculata (DC.) Burret, Cupressus sempervirens L., and Pinus radiata D.Don forest). The authors found new beneficial endophytic fungal associations with A. uniflora plants, from Andean ecosystems, i.e., they measured plant growth-promoting activities in all the isolates. Two hypothesis were tested: (1) fungal diversity in the rhizosphere and endosphere of A. uniflora roots are not restricted to mycorrhizas and more importantly, (2) several endophytic fungi that promote plant growth can be isolated. Their results showed that the sampling sites were significantly different in the fungi associated with the plants. From a total of 410 fungal strains, 144 were endophytic fungi and 266 were rhizospheric fungi. The genus Penicillium was the most abundant microorganism in rhizosphere, whereas pathogenic and saprophytic strains were more frequent inside the roots. The results additionally showed that the fungal strains were weak in phosphate solubilization, but that organic acid exudation and indole acetic acid production are their major mechanisms to simulate plant growth (Fig 4.1).

4.3  Endophytes and Marine Ecosystems The diversity of sponge-associated fungi has been poorly investigated, especially in remote geographical areas like Antarctica. However, Henriquez and collaborators (2014) reported 101 phenotypically different fungal isolates, from 11 sponge samples collected in King George Island, Antarctica. The analysis of ITS sequences revealed that they belong to the phylum Ascomycota. Sixty-five isolates belong

Embothrium coccineum (Proteaceae) Anticura, Puyehue National Park (40.65 S; 72.18 W; 350–400 m.a.s.l.), the Andes in southern Chile/temperate Chenopodium quinoa Willd. Socaire (23°36′00′′ S; (Amaranthaceae) 67°50′60′′ W), situated 3.500 m above sea level and 50 km east of the Salt Lake of the Atacama Desert/tropical

Location/macrobioclimate Plant host Antofagasta (II Region II 23°S Ephedra breana, Fabiana imbricata, L and 68°W L)/tropical Tessaria absinthioides, Atriplex atacamensis, Atriplex microphylla, Atriplex desertícola, Lycium deserti, Tetraglochin sp., Cistanthe celosioides, Trisetum sp., Stipa sp., Distichlis sp., Juncus sp., Luzula sp., Cortaderia sp., Hordeum sp., Adesmia hystrix, Schinus latifolia, Azorella trifurcata, Vulpia sp., Fabiana chilensis, Chaetantera sp., Jaborosa caulescens, and Atriplex oreophila Tedania sp., Hymeniacidon sp., Fildes Bay (62°12´00 S 58_5705100 W)/King George Microciona (Clathria) sp., and/or Crella sp. Island, Antarctica

Table 4.1  Summary of endophytic fungi reported in Chile

Penicillium murcianum, Penicillium minioluteum, Phoma sp., Fusarium sp., Fusarium oxysporum, Alternaria alternata, Penicillium sp., Rhinocladiella similis, Alternaria sp., Bartalinia robillardoides, Cadophora malorum, Coniochaeta sp., Embellisia sp., Fusarium acuminatum, Fusarium avenaceum, Fusarium sambucinum, Fusarium tricinctum, Neonectria macrodidyma, Penicillium brevicompactum, Plectosphaerella sp., and Sarocladium spinificis

Geomyces pannorum, Penicillium polonicum, Penicillium commune, Penicillium solitum, Epicoccum sp., Pseudeurotium, Thelebolus sp., Cladosporium cladosporioides, Aspergillus versicolor, Aureobasidium pullulans, Phoma herbarum, and Trichocladium Mycosphaerella sp., Xylaria sp., Diaporthe sp., Penicillium, and Colletotrichum

(continued)

González-­Teuber et al. (2017)

González-­Teuber (2016)

Henríquez et al. (2014)

Endophytic fungi References Piontelli et al. Penicillium, Cladosporium, Phoma, Trichoderma, (2002) Chaetomium, Fusarium, Alternaria alternata, Cladosporium cladosporioides, Alternaria chlamydospora, and Epicoccum nigrum

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Fundo Maitencillo, Elqui Province (29°58′31″ S; 70°45′52″W), situated 836 m above sea level and 70 km west of the city of La Serena, Chile (semiarid–arid climate)/ Mediterranean Coquimbo Region in Northern Chile (29° 58′ S; 71° 22′ W) (semiarid - arid climate)/ Mediterranean 3200 meter above sea level (m.a.s.l.) in the Andes Mountains in central Chile (33°S) and close to the Valle Nevado ski complex (33°20′ S;70°16′ W), central Chile/ Mediterranean 1200 m.a.s.l, in the El Ing enio, Cajón del Maipo in central Andean Precordillera of Chile (Latitude:3 3° 46´08.76”; Longitude: 70 °16´35.03”

Location/macrobioclimate Maule Region of south- central Chile, the coastal range and Andes range

Table 4.1 (continued)

Molina-­Montenegro et al. (2015

Vidal et al. 2020)

Fusarium, Penicillium, Phialemonium and Trichoderma

Pseudogymnoascus pannorum and Lecanicillium lecanii

Alternaria spp. Aureobasidium spp.

Laretia acaulis (Cav.) Gill. and Hook., Apiaceae (Umbelliferae)

Lithraea caustica (Molina) Hook. & Arn., Acacia caven (Molina) Molina Echinopsis chiloensis (Colla) H.Frie drich & G.D.Rowley

Guevara-Araya et al. (2020)

González-­Teuber et al. (2019a, b)

Aristolochia chilen sis (Bridges ex Lindl.)

References Herrera et al. (2017)

Endophytic fungi Tulasnella sp., Chaetomium globosum, Catenulostroma germanicum, Ceratobasidium sp., Phomopsis columnaris, Thanetophorus sp., Leptodontidium orchidicola, Cadophora sp., Penicillium chrysogenum, and Phialocephala fortinii Penicillium sp. Eucasphaeria capensis, Phomopsis sp., Fusarium oxysporum, Diaporthe sp., Fungal endophyte, Fusarium sp., Alternaria sp., Clonostachys sp., and Ilyonectria sp.

Plant host Chloraea chrysantha, Chloraea bletioides, Bipinnula fimbriata, Chloraea crispa, Chloraea longipetala, Chloraea grandiflora, and Chloraea gavilu Prosopis chilensis (Mol. (Stuntz))

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Penicillium wollemiicola, Rhizoctonia sp., Penicillium spinulosum, Fusarium sp., Penicillium montanense, Ganoderma annulare, Phoma sp., Paraboeremia sp., and Podosphaera sp. Microsphaeropsis olivacea (Bonord.) Hohn

Arachnitis uniflora Phil. (Corsiaceae)

Acremonium bacillisporum, Acremonium strictum, Malbranchea sp., Penicillium chrysogenum, Stegonosporium sp., Triblidiopycnis pinastri, Alternaria alternata, Curvularia tritici, Penicillium chrysogenum, Curvularia protuberata, Cladosporium tenuissimum, Aureobasidium pullulans, Acremonium bactrocephalum, Chaetomium funicola, and Microsphaeropsis olivacea

Penicillium janczewskii

Penicillium chrysogenum and Penicillium brevicompactum

Colobanthus quitensis (Caryophyllaceae) and Deschampsia antarctica (Poaceae)

Pilgerodendron uviferum (D. Don) Florin (Cupressaceae) Prumnopitys andina (Poepp. ex Endl.) Western Andean slopes near Las Trancas, Chillan, region of de Laub (Podocarpaceae) Ñuble Prumnopitys andina (Poepp. ex Endl.) Western Andean slopes near Las Trancas, Chillan, region of de Laub, Austrocedrus chilensis (D. Don) Pic. Serm. and Bizzarri, Ñuble/Mediterranean Araucaria araucana (Mol.) K. Koch., Nahuelbuta National Park/ sub-­Mediterranean temperate Podocarpus nubigena Lindl., Province of Valdivia/temperate Saxegothaea conspicua Lindl., Fitzroya cupressoides (Mol.) I.M. Johnst., and Chiloe Island/temperate Pilgerodendron uviferum (D. Don.) Florin

Antarctic Polish Base “Henryk Arctowski” on King George Island, South Shetland Islands (62°09´44.0´´S; 58°27´56.9´´W)/Antarctic The coastal mountains in Cholchol, region of La Araucanía, southern Chile/ sub-Mediterranean temperate Chiloe Island/temperate Hormazábal et al. (2005) Schmeda-­ Hirschmann et al. (2005) Hormazábal and Piontelli (2009)

Herrera et al. (2019)

Molina-­Montenegro et al. (2016a, b) Oses-Pedraza et al. (2020)

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to the genera Geomyces Traaen, Penicillium, Epicoccum Link, Pseudeurotium J.F.H. Beyma, Thelebolus Tode, Cladosporium, Aspergillus P. Micheli ex Haller, Aureobasidium Viala & G. Boyer, Phoma Sacc., and Trichocladium griseum (Traaen) X. Wei Wang & Houbraken, but 36 isolates could not be identified at genus level. In order to estimate the potential of these isolates as producers of interesting bio-, antimicrobial, antitumoral and antioxidant activities, the authors assayed the fungal culture extracts. The results suggest that fungi associated with Antarctic sponges, particularly the genera Geomyces, could be valuable sources of antimicrobial and anti-tumoral compounds. To our knowledge, this is the first report describing the biodiversity and the metabolic potential of endophytic fungi associated with marine sponges.

4.4  Bioactive Compounds in Endophytic Fungi 4.4.1  Chemical and Biotechnological Approaches from the Study of Fungal Endophytic Species Several authors have addressed research with native species of endophytic fungi. Köpcke et al. (2002) detected (-)-Galiellalactone, a metabolite with promising pharmacological activities in four strains of Galiella rufa (Schwein.) Nannf. & Korf (Sarcosomataceae, Ascomycota) and in two unidentified species (a wood-inhabiting species from Chile and an endophytic isolate from Cistus salviifolius L. from Sardinia, both belonging to the Sarcosomataceae). Galiellalactone is an inhibitor of the transcription factor STAT3 in prostate cancer cells. Despite the efforts made in screening programs, this hexaketide and its precursors have not been found in fungi outside the Sarcosomataceae family. Hormazabal et al. (2005) reported the isolation of seven metabolites with different chemical structures from the endophytic fungi Microsphaeropsis olivacea (Bonord.) Höhn., which was obtained from the native gymnosperm Pilgerodendron uviferum (D.Don) Florin. One of these compounds (derivative 7-hydroxy-2,4-dimethyl-3(2H)-benzofuranone) was new to science. Schemeda-Hirschmann and coworkers (2005) reported the production of secondary metabolites in two endophytic fungi inhabiting the phloem of the Chilean gymnosperm Prumnopitys andina (Poepp. ex Endl.) de Laub: Penicillium janczewskii K.M. Zalessky and the unknown isolate E-3. When these fungi were cultured in a liquid potato-dextrose medium, the secondary metabolites 4-(2-hydroxyethyl) phenol, p-hydroxybenzaldehyde, isochromanone mellein, peniprequinolone and gliovictin were produced. This was the first study that established the production of secondary metabolites in Chilean native gymnosperms by endophytic fungi. Later, Schemeda-Hirschmann and coworkers (2008) reported the isolation of Penicillium janczewskii, (again from the phloem of Prumnopitys andina), which produced two main secondary metabolites, when grown in liquid yeast extract or malt extractglucose broth. This was the first time that these compounds were isolated from this species, and they were identified as the cyclic peptides pseurotin A and cycloas-

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peptide A, by spectroscopic methods. These compounds displayed low cytotoxicity towards human lung fibroblasts with IC ≥ 1000 μm. Additionaly, pseurotin A showed a moderate effect against the phytopathogenic bacteria Pectobacterium carotovorum (Jones 1901) Waldee 1945 (Approved Lists 1980) emend. Portier et al. 2019 and Pseudomonas syringae van Hall 1902, with IC values of 220 and 112 μg ml-1, respectively. Hormazabal and coworkers (2009), reported the first comparative report on the antimicrobial activity of endophytic fungi from Chilean gymnosperms. In this study, thirty-eight endophytic fungi were isolated from stems samples of Araucaria araucana (Molina) K.Koch, Austrocedrus chilensis (D.Don) Pic.Serm. & Bizzarri, Fitzroya cupressoides (Molina) I.M.Johnst., Pilgerodendron uviferum, P. nubigena, P. saligna, Prumnopitys andina, and Saxegothaea conspicua Lindl. Thirteen isolates were identified: Acremonium bacillisporum (Onions & G.L. Barron) W. Gams, A. bactrocephalum W. Gams, A. strictum W. Gams, Alternaria alternata, Aureobasidium pullulans (De Bary) G. Arnaud ex Cif., Ribaldi & Corte, Chaetomium funicola Cooke, Cladosporium tenuissimum Cooke, Curvularia protuberata R.R. Nelson & Hodges, C. tritici S.M. Kumar & Nema, Microsphaeropsis olivacea (Bonord.) Höhn., Penicillium chrysogenum, P. janczewskii, and Triblidiopycnis pinastri. Malbranchea sp. and Stegonosporium sp. were identified at the genus level, according to culture characteristics, colony growth, and conidia morphology. Fourteen isolates were considered “mycelia sterilia”, because they lacked fruit bodies in the culture medium. Crude extracts of liquid cultures were evaluated for antibacterial and antifungal activity, using agar diffusion and by micro dilution assays. Results shown that the extracts of Acremonium bactrocephalum, Microsphaeropsis olivacea, and isolate E-3, inhibited growth of selected pathogenic organisms, indicating their value for further studies.

4.5  Endophytic Fungi in Agroecosystems 4.5.1  L  atent Infections and Its Impact on Pathogenic Potential Risk The biological cycle of endophytic fungi in host plants is affected by different environmental factors. Therefore, it becomes difficult to identify latent pathogenic stages and to establish the pathogenic potential or risk associated. For example, Neonectria galligena (Bres.) Rossman & Samuels is a phytopathogen that causes “Nectria canker”. It exhibits a wide spectrum in host range diversity, being recorded on tree and shrub species, but is particularly prevalent in commercial apple (Malus sylvestris (L.) Mill.) and pear (Pyrus communis L.) plantations, all over the world. It is also problematic on a range of forest hardwood species causing a reduction in log quality, value and inevitable loss in merchantable timber volume. In 2002, Langrell reported the development of a species-specific PCR protocol for the detection of N. galligena from wood of young apple trees and/or propagating materials. He designed specific, primers for N. galligena, and compared the ITS

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regions 1 and 2 of 32 isolates of diverse origins, including 4 from Chile, with sequences from 7 other nectriaceous species: Neonectria ditissima (Tul. & C. Tul.) Samuels & Rossman Nectria coccinea (Pers.) Fr., N. coccinea var. Faginata M.L. Lohman, A.M.J. Watson & Ayers, N. punicea (J.C. Schmidt) Castl. & Rossman, N. fuckeliana C. Booth, N. cinnabarina (Tode) Fr., and N. radicicola Gerlach & L. Nilsson. Further adaptation of this approach allowed for semi-quantitative competitive PCR, enabling the determination of target fungi in infected lignified tissues. This work highlights the relevance to develop a fast, sensitive, reliable and accurate identification and quantification protocol for this latent pathogen, that does not only infect apple propagation material, but also others host plant. Apple (Malus domestica Borkh) is a commercially important crop and the second most planted fruit tree (35.937 ha, representing 11% of the total fruit crops) in Chile. Moldy core (MC) disease has a high prevalence (16 to 46%) in the fruit, especially in susceptible cultivars. MC occurs primary in apple cultivars with open sinuses and is difficult to find in cultivars with close sinuses. Alternaria spp. have been associated with MC, in Chile. Elfar and coworkers (2019) conducted research which aimed to (1) to identify the species of Alternaria and other filamentous endophytic fungi associated with apparently healthy flowers and fruits of apples, (2) to study the ability of Alternaria spp. to colonize carpels in apple fruits, and (3) to study the population dynamics of species of Alternaria in the flowers and fruits of susceptible and resistant apples. The methodology included the sampling and surface disinfection of apparently healthy flowers and fruits at six growth stages, from the pink bud to the mature fruit. They treated the susceptible ‘Oregon Spur’ and resistant ‘Granny Smith’ apples during two growing seasons. Seven fungal genera were detected colonizing flowers and the fruit of both cultivars. Small-spored Alternaria spp. were the primary species identified throughout all the growing stages. Independently of the growth stages, sepals, stamens and carpels of flowers were similarly colonized by Alternaria spp. in the susceptible (‘Oregon Spur’) and resistant (‘Granny Smith’) apple cultivars. However, in fruits of 4–6 cm in diameter, big differences in the frequency of Alternaria spp. were observed between susceptible (55%) and resistant apples (1%). At least five Alternaria spp. were identified at species level, using a plasma membrane ATPase molecular marker, namely, A. alternata, A. arborescens E.G. Simmons, A. limoniasperae E.G. Simmons, A. tenuissima (Kunze) Wiltshire in sect Alternaria, A. kordkuyana Poursafar, Gannibal, Ghosta, Javan-Nikkhah, & D.P. Lawr. in sect. Pseudoalternaria and Alternaria sp. in sect. Infectoriae. All endophytic Alternaria isolates, from sepals, stamens and carpels, and from apparently healthy flowers and fruits were pathogenic, producing MC symptoms in apples. In this study, the authors suggested that apparently healthy flowers and fruits may act as an important inoculum source of Alternaria spp. for MC infections, and also suggest that control strategies against MC using fungicide applications should be applied at flowering, from pink bud onwards. Chile is a major exporter of table grapes (Vitis vinifera L.) and wines, and regional viticulture has expanded considerably in recent years. The rapid growth of this industry has been accompanied by the demand for nursery plants (biological materials) and specially for grafted grapevines. However, the quality of the available prop-

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agation material has declined and problems associated with the production practices of grafted grapevines have been detected, such as (1) the inhibition of the basal callus formation, (2) the decrease in root emission, (3) poor formation of the grafting callus and graft failures, and (4) symptoms of incompatibility. Petri disease is recognized as the vascular endophytic fungal disease related to the decreasing quality of these propagation plant materials. Díaz and coworkers (2009) reported the effects of endophytic fungi Phaeomoniella chlamydospora (W. Gams, Crous, M.J. Wingf. & Mugnai) Crous & W. Gams and Phaeoacremonium aleophilum W. Gams, Crous, M.J. Wingf. & Mugnai on grafted grapevines on the major rootstock used for wine grapes in Chile. In this study, cuttings of five grapevine rootstocks (Vitis vinifera) were wounded and immediately inoculated with suspensions of either P. chlamydospora, P. aleophilum or a mixture of both species. The presence of these endophyte fungi decreased the quality of each of the five rootstocks. Among the rootstocks investigated in this study, 1103P and 101-14 MG were less susceptible to the infection caused by Pa. chlamydospora and Pm. aleophillum.

4.5.2  Occurrence of Endophytic Fungi in Host Grasses In 1989, Piontelli and Toro conducted a preliminary study on the fungal associations in forage grasses. They analyzed the presence of fungal endophytes in seeds of five grass species, from central and south Chile. Four of them presented a high incidence of fungal infection, and the authors were able to isolate two fungi belonging to the genera Acremonium (Acremonium sp. A, a new isolate, and Acremonium sp. B). Festuca arundinacea Schreb. (“Kentucky 31”), was for 79,3 % infected; Lolium multiflorum Lam. (“Tama”), for 86,5%; Dactylis glomerata L. for 18,51%; while Lolium multiflorum, from the central zone, showed an infection. Morgan-Jones and coworkers (1990), described the occurrence of a new anamorphic fungal endophyte of the genera Acremonium (part of the section Albo-lanosa endophytes of grasses) inindividuals of symptomless Dactylis glomerata host plants, in grasslands in southern Chile. This study included descriptions and illustrations of the intercellular habits of this endophytic fungus in seed, stem, leaf tissues and other parts, but the fungus had the aggressive potential to become intracellular and seed-transmitted. On the base of a detailed review of morphological features, the fungus was classified as a new species and was taxonomically described as Acremonium chilense Morgan-Jones et White. In comparison with others Balansieae micro conidial anamorphs fungi, A. chilensis showed a rapid growth in culture and produced a lot of conidia in vitro. At the same time, an endophytic fungus from the genus Pseudocercosporella was isolated from the forage grass Trichachne insularis (L.) Nees from Brazil, Argentina and Chile (White et al., 1990). The fungal endophyte belonged to the genus Pseudocercosporella. However, it was an unknown species, for which the name P. trichachnicola J.F. White was proposed. The fungus, which occurs in symptomless grass, is not known to sporulate in vivo, as its morphological characteristics

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were described in vitro. The study suggests that a mutualistic association may have evolved, probably from a previously pathogenic condition, between the fungus and its host. The endophyte has a widespread distribution, with high frequency in temperate to subtropical climates. It occupies a predominantly intercellular position within host tissue, although some intracellular hyphae occasionally occur. The fungus inhabits leaf sheaths, culms and seeds and is seedtransmitted. Butendieck and coworkers (1994), studied the introduction of a new ryegrass cultivar infected with a fungal endophyte, to improve resistance to insects in Southern Chile. A trial to measure if intake and milk production had changed in Holstein cows, due to this new cultivar, was designed and implemented. Perennial ryegrass (Lolium perenne L.) cultivars Santa Elvira and Embassy were used as the only feed supplied. Two groups of cows were randomly assigned to 2 groups of cows. In the beginning, the two groups grazed both cultivars and afterwards one of the groups, was individually fed indoors with cultivar Santa Elvira. The average initial milk production was similar in both treatments. After a week, the cultivar Embassy was offered to cows in both groups. The intake declined and the milk production dropped in the group that had been fed Santa Elvira before. In the other group the milk reduction was lower. The clinical symptoms of ryegrass staggers became evident in both groups, however were more severe in the indoor fed group, where two cows presented downer cow syndrome. After changing the forage to a mixture of ryegrass cv Nui and white cover (Trifolium repens L.) cv Huia, the intake and production returned within 12 days to former levels. In 1995, Galdames reported the presence of the endophytic fungi Acremonium coenophialum Morgan-Jones & W. Gams in plants and seeds of tall fescue (Festuca arundinacea), using microscopic analysis. Data collected from seeds of the varieties K-31, Fawb and Manade, showed that only K-31 had a high infection level (71-90%). The authors furthermore studied 15 meadows located in the IX Region of Chile (Curacautin, Victoria, Temuco and Gorbea), and found infested plants in 12 of them, with an infestation level ranging from 40 to 100%. The study suggests that since K-31 has been the most sown variety for long time, it is very probable that the endophyte is present in the majority of tall fescue pastures in Chile. Sepúlveda and coworkers (1996) described eight cases of fescue toxicosis in cows due to ingestion of fescue pasture, infected with high levels of the endophytic fungus Acremonium coenophialum. Clinical symptoms such as excitement, hyperesthesia and lameness were observed. Subsequently the cows presented necrosis of the distal part of the legs and loss of the phalanx. Later in 1997, Cruz and coworkers reported reproductive problems related to fescue toxicosis in mares in Chile. They described episodes of outbreak of abortion, agalactia and weak foals in a thoroughbred breeding farm in south - central Chile. In this study of 96 pregnant mares, who grazed on tall fescue, 11 aborted and 6 presented agalactia. Additionally, a high percentage of weak foals was observed. Acremonium coenophialum was isolated from tall fescue, suggesting that the reproductive problem observed were caused by the presence of the endophyte. All these studies point out the need to test every new cultivar, before it is marketed.

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Parra and coworkers (2013) investigated the potential to use cattle pastures (Festuca arundinacea), infected with non-toxic, “friendly” fungal-endophyteinfected (E+), as a strategy to reduce horn fly loads in cattle. They evaluated the possible bio-insecticide effect on the larvae of horn fly Haematobia irritans Linnaeus, 1758, a bloodsucking ecto-parasite of pastured cattle, and the major pest of livestock in North and South America and Europe. A decrease in fly-load was observed when cattle grazed on tall fescue infected with the friendly endophytic fungi, compared to endophyte-free pastures. The infestation of horn fly load decreased when the percentage of endophytes, present in the different pastures, increased (0 to 100%). However, two groups of animals with significant differences in the fly-load (high and low fly-load) were observed in the same herd. The authors additionally determined a bio-insecticide effect of cattle dung, for horn fly larvae (80%), from animals fed E+ tall fescue. These results are very valuable for pasture management. as it provides preliminary understanding of the role of cattle pasture diet as a part of an integrated pest management strategy. In 2017, Parra and coworkers studied the incidence of Listronotus bonariensis Kuschel, 1955 (Argentine stem weevil). This is an important pest in pastures, especially in ryegrass (Lolium sp.) pastures, compromising grassland productivity. For over a decade there have been no reports on the presence of this weevil in southern Chile, however, the results of two years of prospecting showed that this weevil is currently causing damage to the ryegrass pastures. Chemical control has limited effectiveness when dealing with adult insects and negative effects on the environmental, and it is toxic for pollinators, when applied close to the flowering period. Therefore, the main currently strategy to control the weevil is based on the use of cultivars of Lolium infected with an endophyte fungus from the Neothypodium genus. However, the consuming of ryegrass (Lolium perenne L.) infected with endophytic fungus Neotyphodium lolii (Latch, Christensen, and Samuels) Glenn, Bacon, and Hanlin., produced the ryegrass staggers syndrome in cattle, sheep, and horses. This syndrome is caused by a neurotoxic substance Lolitrem-B, produced by endophytic fungi. The increasing use of ryegrass pastures in southern Chile made a detection and quantification method necessary (Moyano et al. 2009). Ryegrass samples without the endophyte were freeze-dried, and kept in darkness at -18 ºC. A pure Lolitrem-B standard was added, and the toxin was later extracted with a mixture of chloroform-methanol (2:1), purified in manually prepared silica gel 60 columns. The recovery was 96.6 to 99.9%, on average and the coefficient of variation (CV) ranged between 0.9 and 5.9%. Quantification was done by high-performance liquid chromatography (HPLC) and fluorescence detection (quantification limit 0.05 mg kg ). This method of determining neurotoxin Lolitrem-B in ryegrass samples was reported to be fast, inexpensive, accurate, repeatable and more importantly the first implemented in Chile.

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4.6  Endophytic Fungi for Plant Disease Control Fungal endophytes have shown the potential for the biological control of diseases. Barra-Bucarei and coworkers (2019), compiled information on the importance of entomopathogenic fungi (endophytes) in their potential application as biological control for diseases in tomatoes. Results showed the endophytic colonization of tomato by native strains of Beauveria bassiana (Bals. Criv.) Vuill., Metarhizium anisopliae (Metschn.) Sorokin and Metarhizium robertsii J.F. Bisch., Rehner & Humber. All the evaluated strains showed some level of inhibition of pathogens and hence have some potential as management tool against plant diseases. These entomopathogenic fungal endophytes (EFEs) can colonize some part plants more efficiently than others, which might consequently influence the level of plant protection against different phyto-pathogens. Botrytis cinerea Pers. causes substantial losses in tomato and chili pepper crops, worldwide. Barra-Bucarei and coworkers (2020) used native strains of the entomopathogenic fungus B. bassiana to determine its ability to endophytically colonize tomato and chili pepper plants. The antifungal activity of the endophytic fungi, and the mitigation of the negative effects caused by B. cinerea were evaluated in these crops. Roots were drenched in B. bassiana, and the endophytic colonization capacity in roots, stems, and leaves was determined. The antifungal activity was evaluated in vitro. After plant roots were drenched in the endophyte, leaves were inoculated with the pathogen. The authors found ten native strains of endophytes which could colonize tomato tissue, and eight which could colonize chili pepper tissue. All strains showed significant high antifungal effects against B. cinerea (30–36%), and strains RGM547 and RGM644 showed the lowest percentage of the surface affected by the pathogen. Recently, Vidal and coworkers (2020) isolated and identified endophytic fungi with antifungal activity against the plant pathogenic fungus B. cinerea isolated from plants from Central Andean Precordillera of Chile. Three endophytic fungi with antifungal activity against B. cinerea were isolated from native and endemic plants. Two Alternaria spp., were isolated from Lithraea caustica (Molina) Hook. & Arn., and Acacia caven (Molina) Molina, whilst Aureobasidium spp., was isolated from Echinopsis chiloensis (Colla) H.Friedrich & G.D.Rowley. The authors concludes that the antifungal activity is mediated by diffusible and volatile compounds which reduced mycelial growth, sporulation and conidia germination of B. cinerea.

4.7  Endophytic Fungi of Forest Trees The bioconversion of organic matter by wood-decaying fungi play an important role in global carbon and nitrogen cycling in all ecosystems. The colonization and degradation of wood as well the attack of cell components is mediated by enzymatic and non-enzymatic mechanisms displayed by fungi. The understanding of the role of fungal endophytes in wood biodegradation, including invasiveness, colonization

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strategies and degrading mechanisms, will help to understand the carbon cycling in forests ecosystems. Additionally, endophytic fungi could potentially be used for biopulping and wood pretreatment in the bioethanol production (Rodríguez et al. 2011). Oses and coworkers (2006) isolated wood-inhabiting fungal endophytes from the Chilean tree species Drimys winteri J.R.Forst. & G.Forst. and Prumnopitys andina, and evaluated them for lignocellulolytic enzymes production and wood biodegradation. Four endophyte fungi were isolated: in D. winteri a basidiomycete was identified as Bjerkandera sp., and a Deuteromycete classified as Mycelia sterilia (Dw-2), whilst in P. andina P. andina an unidentified Basidiomycete (Pa-1), and a Mycelia sterilia (Pa-2) were found. In vitro, the Basidiomycetes displayed positive reaction to phenoloxidase (PO) and cellulase, but did not show iron-reducing activity. Both Mycelia sterilia showed a weak reaction to the cellulase and iron-reducing assay, but did not show PO activity. After treating wood chips of D. winteri with Bjerkandera sp. they presented increased weight loss and component loss after 45 days: weight 13.3±1.5%; total lignin 13.2±1.2%; glucan 16.9±4.4%; polyoses 22.6±3.8%; and extractives 16.0±1.7%. For Pa-1, weight and component losses were: weight 5.6±0.0%; total lignin 8.0±0.6%; glucan 7.0±0.3%; polyoses 9.0±0.5%; and extractives 7.7±0.1%. These results indicated, for first time, the ability of isolated fungal basidiomycete endophytes to develop a non-selective whiterot wood decay pattern. Oses and coworkers (2008) conducted a survey of fungal endophyte associated with xylem in presumably healthy trees. They reported the isolation of wood fungal endophytes from surface sterilized core samples of Prumnopitys andina, Podocarpus salignus D.Don, Drimys winteri and Nothofagus obliqua (Mirb.) Oerst. Five basidiomyectes (Inonotus sp., Bjerkandera adusta (Willd.) P. Karst. and three unknown strains, two ascomycetes (Xylaria sp. and Bipolaris sp.), and one anamorphic strain were detected. Xylaria sp., and Bjerkandera adusta were the most frequent fungal isolates. Ultrastructural changes in biodegraded wood core samples were characterized by scanning electron (SEM), transmission electron (TEM), and light microscopy. The authors discovered the presence of latent infections along the parenchyma rays, indicating fungal colonization and distribution. Results showed simultaneously the decay of all wood components (characterized by the thinning cell-walls from the cell lumen to the middle lamella) and erosive wood degradation (typical for the non-selective white rot biodegradation pathway). The results from the SEM, TEM and light microscopy allowed them to demonstrate the natural incidence of latent infections in woody hosts, and to visualize the spreading of colonization and their ability to degrade wood under suitable conditions. Slippers and coworkers (2009) analyzed the fungal family Botryosphaeriaceae with the focus on the diversity of the assemblage of Eucalyptus trees in (non-) native environments. Members of this family cause endophytic fungal infections in leaves and bark of various trees, including Eucalyptus, apparently persisting as latent pathogen for extended periods of time. These endophytic fungi cause many different disease symptoms on Eucalyptus species, which experience stress, such as stem and branch cankers and dieback (a condition in which a tree or shrub begins to die from the tip of its leaves or roots backwards). Given their cryptic, endophytic nature the

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fungi are easily overlooked when moving seeds and plants around the world. This review clarifies that Botryosphaeria dothidea (Moug. ex Fr.) Ces. & De Not. and Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips rarely infect Eucalyptus, but that, Aplosporella yalgorensis K. Taylor, Barber & T. Burgess, B. mamane D.E. Gardner, N. parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips, N. eucalyptorum (Crous, H. Sm. ter & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips, N. eucalypticola (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips, N. australe (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips, N. macroclavatum (T.I. Burgess, Barber & Hardy) T.I. Burgess, Barber & Hardy, N. andinum (Mohali, Slippers & M.J. Wingf.) Mohali, Slippers & M.J. Wingf., N. mangiferae (Syd. & P. Syd.) Crous, Slippers & A.J.L. Phillips, Dichomera eucalypti (G. Winter) B. Sutton, N. versiforme (Z.Q. Yuan, Wardlaw & C. Mohammed) Crous, Fusicoccum ramosum Pavlic, T.I. Burgess & M.J. Wingf., Pseudofusicoccum stromaticum (Mohali, Slippers & M.J. Wingf.) Mohali, Slippers & M.J. Wingf., P. adansoniae Pavlic, T.I. Burgess & M.J. Wingf., P. ardesiacum Pavlic, T.I. Burgess & M.J. Wingf., P. kimberleyense Pavlic, T.I. Burgess & M.J. Wingf., Lasiodiplodia crassispora T. Burgess & Barber, L. gonubiensis Pavlic, Slippers & M.J. Wingf., L. pseudotheobromae A.J.L. Phillips, A. Alves & Crous and L. rubropurpurea T. Burgess, Barber & Pegg infect Eucalyptus more commonly. The authors noted that different species dominate on Eucalyptus in different countries/continents, e.g. in South Africa and Chile, species such as N. parvum, N. eucalyptorum and N. eucalypticola are most common, despite the presence of N. ribis and N. australe on related hosts such as Syzygium. Considering their endophytic nature and occurrence in asymptomatic tissues, together with the patterns of distribution and pathogenicity, the most probable explanation is that the Botryosphaeriaceae were accidentally moved internationally in germplasm samples. Therefore, this review highlights the relevance of monitoring both the rare and the more common endophytic species for increases in their incidence, especially as an increase in stress is expected due to climate change. Moreover, the characterization of the overlap of endophytic species in native and introduced Eucalyptus and other hosts are required for a better understanding of the fungal infection process. The cryptic nature of some endophytic fungi makes it difficult to distinguish species or their pathogenic potential (latent pathogen phase). This hinders to monitor their presence, identify their origin, and determine their host and geographical range (Slipper and coworkers 2009). Table 4.2 summarises the endophytic fungi reported as latent pathogens in agroforest ecosystems of Chile. Therefore, it is extremely challenging to proactively manage potential events of invasions. This is even more true for latent pathogens where, due to their asymptomatic life stage (or endophytic stage), disease symptoms may not be apparent on infected plants. Sakadalis and coworkers (2013), reported the characterization of the diversity and geographical distribution of the cryptic species in the Neofusicoccum parvum – N. ribis species fungal pathogen complex. The aim of this work was to characterize the host, and geographical distribution of the individual species, the origins of the pathogens, and their spreading patterns, in order to understand the patterns between host and latent pathogen. Polymorphic microsatellite markers were used to characterize the distribution and the diversity

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world-wide of the most common species in the complex, N. parvum. All sequence data for the N. parvum–N. ribis complex, available from GenBank and private databases, were reinterpreted considering a current phylogenetic framework for this species complex. Results showed that of the 169 verified N. parvum isolates, 38 were isolated from Australia, 2 from Chile, 6 from China, 16 from Colombia, 14 from Hawaii, 2 from Indonesia, 14 from New Zealand and 77 from South Africa. The majority of isolates came from Eucalyptus sp. The study concludes that there is an unconstrained and frequent movement of latent pathogens or ­endophytes between the countries. The most widespread species was N. parvum, and it was reported in the majority of the hosts studied in the N. parvum – N. ribis complex. The authors suggest that the current dispersal of N. parvum and its sister species is probably due to repeated introductions of plant material, with Eucalyptus and Vitis vinifera being the two most prominent candidates for material transfer. In 2016, Gundale and collaborators characterized the root fungal endophyte communities associated with Pinus contorta Douglas ex Loudon, which originates from north-western North America (USA and Canada), and which has been introduced in several countries of the Southern Hemisphere, where it has become highly invasive (e.g. Chile and New Zealand), and across northern Europe (e.g. Scotland, Sweden, and Finland), where it typically achieves higher growth rates than the native Pinus sylvestris L. The study aimed to explain the enhanced performance of P. contorta in new environments, and concluded that their soil microbial communities may be different and provide greater benefits than microbial communities encountered in the soil around native species. The authors used a molecular analysis technique (pyrosequencing) to compare the root fungal endophyte communities associated with P. contorta and phylogenetically similar and dissimilar tree species (i.e. P. sylvestris in Europe and Nothofagus spp. in the Southern Hemisphere) in their native and introduced environments. The results showed that P. contorta- endophytic fungi interactions are plastic, and the association with different fungal communities in native and introduced environments is feasible. This study also indicates that endophytic fungal communities, associated with introduced plants, can assemble through different mechanisms, e.g. by associating with existing fungal communities of phylogenetically close species, or through reassembly of co-introduced and co-invading fungi. The authors suggest that the identification of different endophytic fungal communities in a plants new environment could be key step to understand how soil biota may impact growth and how invasive a species can become. Ortiz and collaborators (2019) identified genes involved in plant growth promotion and metal response, through analyzing the transcriptomic response of Eucalyptus globulus Labill., inoculated with Chaetomium cupreum L.M. Ames. The aim of this study was to identify genes involved in plant growth promotion and metal stress response. This provided important molecular information to understand plant–microbe–metal interactions and contribute to new strategies that can be used in phytoremediation processes. A total of 393.371.743 paired-end reads were assembled into 135.155 putative transcripts. The authors found that 663 genes sig-

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Table 4.2  Summary of endophytic fungi as latent pathogen agroforest ecosystems Location Chile (central)

Chile (south-central)

Plant host Malus sylvestris and Pyrus communis (tree) Vitis vinifera L.

Chile (south-central)

Eucalyptus sp. (tree)

Pucón, Chile (south)

Dactylis glomerata L. (grass) Trichacne insularis (grass)

Region IX of Chile (Curacautin, Victoria, Temuco, Gorbea) Region of Bío-Bío, Araucania, Los Ríos, and Los Lagos (south Chile) Nahuelbuta range (37°35¨S; 73°10¨W) Termas de Chillán (36°52¨S 71°52¨W)

Endophytic fungi Neonectria galligena

Phaeomoniella chlamydospora and Phaeoacremonium aleophilum Neofusicoccum parvum, Neofusicoccum parvum, Neofusicoccum eucalyptorum, and Neofusicoccum eucalypticola Acremonium chilense

Pseudocercosporella trichachnicola

Acremonium coenophialum Festuca arundinacea var. K-31

Disease/ pathology “Nectria canker”

References Langrell (2002)

“Petri disease”

Díaz et al. (2009)

Canker and Slippers et al. dieback (2009) and Sakalidis et al. (2013) Ryegrass staggers syndrome Ryegrass staggers syndrome Ryegrass staggers syndrome

Morgan-Jones et al. (1990) White et al. (1990) Galdames (1995), Sepulveda et al. (1997) and Cruz et al. (1997) Parra et al. (2017)

Lolium sp.

Neothypodium

Ryegrass staggers syndrome

Prumnopitys andina, Podocarpus saligna, Nothofagus obliqua, Drimys winteri Malus domestica Borkh

Basidiomycetes PA-1, Bipolaris sp., Inonotus sp., Xylaria sp., Basidiomycetes NO-1, Bjerkandera adusta, Basidiomycete DW-1, and anamorphic sp. DW3

Wood decay fungi/stain sapwood fungi

Alternaria alternata, Alternaria arborescens, Alternaria limoniasperae, Alternaria tenuissima, Alternaria kordkuyana, and Alternaria sp.

Moldy core Elfar et al. (2019) (MC) disease

Oses et al. (2006, 2008)

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nificantly changed their expression in the presence of C. cupreum: 369 genes were up-regulated and 294 were down-regulated. The results showed that these differentially expressed genes (DEGs) encoded for metal transporters, transcription factors, stress and defense response proteins, and were involved in auxin biosynthesis and metabolism. The inoculation of C. cupreum in E. globulus enhanced its tolerance to metals and promoted growth (Table 4.2).

4.8  Potential Uses and Applications Stinton and coworkers (2003) isolated the endophytic fungi Gliocladium sp. from the Patagonian Eucryphiacean tree (Eucryphia cordifolia Cav., known locally as ‘‘ulmo’’), and identified it on the basis of its morphology and aspects of its molecular biology. The endophytic fungus produced a mixture of volatile organic compounds (VOC’s) lethal to plant pathogenic fungi such as Pythium ultimum Trow and Verticillium dahliae Kleb. An experiment showed that other pathogens were inhibited by these volatiles. Some of the volatile bioactive compounds exuded by Gliocladium sp. (1-butanol, 3-methyl-, phenylethyl alcohol and acetic acid, 2-phenylethyl ester, as well as various propanoic acid esters) are also produced by Muscodor albus Worapong, Strobel & W.M. Hess, a well-known volatile antimicrobial producer. Therefore, this study was the first that showed that the production of these selective volatile antibiotics by endophytic fungi is not exclusively confined to the Muscodor spp. However, the primary volatile compound produced by Gliocladium sp. was 1,3,5,7-cyclooctatetraene or annulene, which is an effective inhibitor of fungal growth. Strobel and colleagues (2008) reported a new endophytic fungus, Gliocladium roseum Bainier (NRRL 50072), isolated from Eucryphia cordifolia (ulmo) in Patagonia, Chile. This endophytic fungus was able to growth on oatmeal-based agar medium, under microaerophilic conditions, and produced a series of volatile hydrocarbons and hydrocarbon derivatives, which could be detected with solid-phase microextraction (SPME)-GC/MS. G. roseum was able to produce an extensive series of acetic acid esters of straight-chained alkanes, including those of pentyl, hexyl, heptyl, octyl, sec-octyl and decyl alcohols. It also produced other hydrocarbons, including undecane, 2,6-dimethyl; decane, 3,3,5-trimethyl; cyclohexene, 4-methyl; decane, 3,3,6-trimethyl; and undecane, 4,4-dimethyl. Volatile hydrocarbons (including heptane, octane, benzene, and some branched hydrocarbons) were produced when grown on a cellulose-based medium, or an extract of the host plant. The volatile organic compounds were quantified with a proton transfer mass spectrometry (PTR-MS) technique. This technique indicated a level of organic substances in the order of 80 parts per million by volume (p.p.m.v.) in the head space above the oatmeal agar medium after 18 days. Scaling of the results from the

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PTR-MS profile quantified the acetic acid heptyl ester at 500 p.p.b.v. Subsequently the amount of each compound in the GC/MS profile could be estimated; all yielded a total value of about 4.0 p.p.m.v. The hydrocarbon profile of G. roseum contained a number of compounds which are normally associated with diesel fuel, and therefore the volatiles of this fungus were named ‘myco-diesel TM. The extraction of liquid cultures of the fungus revealed the presence of numerous fatty acids and other lipids. Stadler and Schulz (2009) speculated on the ecological role of volatiles in endophyte–host relationships, and their potential applications in energy production. In 2010, Strobel and co-workers, reported the isolation of endophytic fungi Ascocoryne sp., from Eucryphia cordifolia (Patagonia, Chile), that produced hydrocarbon derivatives. It was classified as Clonostachys rosea (Link) Schroers, Samuels, Seifert & W. Gams (Gliocaldium roseum) strain C-13 = NRRL 50072, primarily based upon its morphological features. The fungus produced slimy clumped conidia on verticillated conidiophores and redpigmented particles in culture. However, DNA sequence analysis (ITS rDNA) suggested a 99% similarity with Ascocoryne sarcoides (Jacq.) J.W. Groves & D.E. Wilson, rather than to fungi assigned to the group of Gliocladium-like anamorphs. Comparative genetic, biological and morphological studies confirmed that the anamorphic stage of an authenticated field-collected culture of Ascocoryne sarcoides (Griffin et al. 2010) AV-70 shares many of the same morphological, and genetic features with NRRL 50072, but the latter is unable to produce synnematal masses, unless it is grown on a proper substrate. These data suggest that NRRL 50072 is most closely related to the asexual stage of A. sarcoides, and this result refined its taxonomy to A. sarcoides. Serial transfer of A. sarcoides resulted in major cultural changes in the fungus, especially in the production of aerial hyphae, pigment production and the number of synnemata being formed. This report set an appropriate taxonomic framework for further molecular biological and biochemical work on NRRL 50072 and related fungi. Griffin and coworker (2010) reported the production of a variety of medium chain and highly branched volatile organic compounds (VOCs) by Ascorine sarcoides NRRL 50072. Volatile organic compounds (VOCs) have been highlighted for their potential as fuel alternatives and are collectively termed myco-dieselTM (Strobel et al 2008). The novelty of this study was the comparison of A. sarcoides with closely related, commercially available, Ascocoryne strains and their capabilities to produce VOC. DNA sequencing established a high genetic similarity between NRRL 50072 and each Ascocoryne isolate, consistent with its reassignment as Ascocoryne sarcoides. VOCs detected from the cultures consisted of short- and medium chain alkenes, ketones, esters and alcohols, and several sesquiterpenes. The efforts to imitate the highly branched medium-chain-length alkanes production of NRRL 50072 with commercially available Ascocoryne strains were unsuccessful. Ascocoryne strains NRRL 50072 and CBS 309.71 produced a more diverse range of volatiles than the other isolates tested. The study confirmed the production of 30

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products, which expanded the list of VOCs produced by the genus Ascocoryne. CBS 309.71 showed enhanced production compared with other strains when grown on cellulose agar. Collectively, the members of the genus Ascocoryne produced of over 100 individual compounds, and a third of the short- and medium-chain compounds are also produced when cultures were grown on a cellulose substrate. This comparative production analysis could facilitate future studies to identify and manipulate the biosynthetic machinery responsible for production of individual VOCs, several that could potentially be used as biofuels. Gianoulis and coworkers (2012), did a genomic characterization of the endophytic fungus A. sarcoides. The genes involved in cellulose degradation were described, using transcriptomic and metabolomic approaches. They hypothesized that pathways for the biofuel production pathways would be present and also found that the production of potential biofuel metabolites by this fungal endophyte is possible, when grown on cellulose-based medium. However, the specific genetic pathways needed for this production were still unknown and the lack of genetic tools made traditional reverse genetics difficult. They identified almost 80 biosynthetic clusters, including several previously found only in plants. Additionally, many transcriptionally active regions, outside of genes, showed condition-specific expression. This offered more evidence for the role of long non-coding RNA in gene regulation. This study offered one of the highest quality fungal genomes and, to our knowledge, is the only study that thoroughly annotated and transcriptionally profiled the fungal endophyte genome. The analyses and datasets obtained in this research provide the genomic foundation for a model endophyte system. Mallette and coworkers (2012) reported the production of volatile hydrocarbon by Ascocoryne sacroides during its growth cycle. At distinct time points the composition of the gas-phase was measured, using proton transfer reaction-mass spectrometry (PTR-MS) and head space solid phase microextraction (SPME) techniques with gas chromatography-mass spectrometry (GC-MS). The PTR-MS ion signal revealed temporal resolution of the volatile production, while the SPME results revealed distinct identities. The quantitative PTR-MS results showed that volatile production was dominated by ethanol and acetaldehyde, while the concentration of the remainder of volatiles consistently reached 2,000 p.p.b.v. The measurement of alcohols from the fungal culture by the two techniques correlated well. Interesting, the “myco-fuel” compounds included nonanal, 1-octen-3-ol, 1-butanol, 3-methyl- and benzaldehyde. Abiotic comparison of the two techniques demonstrated SPME fiber bias toward higher molecular weight compounds, making quantitative efforts with SPME alone impractical. But together, PTR-MS and SPME GC-MS were shown to be valuable tools for characterizing compound production in microbiological sources. Strobel (2014) discussed the discovery of endophytic fungi that produce volatile organic compounds (VOCs), when growth on agricultural wastes substrates. The fungal chemistry is best defined as hydrocarbon and hydrocarbon-like, and these compounds have a potential use as ‘green chemicals’ and fuels. Their study discusses

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the discovery of the first fungal producer of ‘Myco-diesel’ TM. The authors also mentioned many other examples of fungi making these VOCs and some of the novel methods that have been developed to study the fungal production of hydrocarbons. Their work concludes with a discussion if commercial scale up is feasible, and if it could, solve the world’s need for liquid fuels. Acuña-Rodríguez and coworkers (2019), evaluated the effect of microbial-consortium, containing plant growthpromoting rhizobacteria (PGPB), and endo-symbiotic fungi, isolated from Antarctic vascular plants, on saline stress tolerance in different crops. In a greenhouse experiment, they compared non-inoculated plants without saline stress, non-inoculated plants subjected to saline stress (200 mM NaCl), inoculated plants with microbial consortium without saline stress, and inoculated plants subjected to saline stress. The aim of this study was not only the evaluation of the impact of microbial consortiums in crops experiencing saline stress, but the authors additionally searched for clues on the mechanism of the stress tolerance. Parameters such as percentage of plant survival, eco-physiological performance, lipid peroxidation, proline accumulation and NHX1 antiporter gene expression were measured. Also, roots, shoots and total biomass were obtained as a proxy of yield and productivity. Crops such as lettuce, onion and tomatoes inoculated with the Antarctic extremophiles showed an increment of fitness and survival related traits when exposed to salt in comparison with the non-inoculated plants. Even though saline stress negatively impacted all measured trait, inoculated plants were less affected. In controlled osmotic conditions, there were no differences in proline accumulation and lipid peroxidation between inoculation treatments. However, in control salinity the quantum yield (Fv/Fm) was higher in inoculated plants after 30 and 60 days. The parameters Fv/Fm, proline accumulation and NHX1 expression were higher under osmotic stress, whilst lipid peroxidation was lower in inoculated plants compared to un-inoculated individuals. When experiencing saline stress, the final biomass of inoculated plants was similar in comparison with plants without stress. The article indicates that microbial consortia containing Antarctic microbes can lessen the physiological impact of saline stress in crops. It also points out that extreme environments can be great sources of beneficial microbes, with potential application. Later, Later Molina-Montenegro and coworkers (2020) evaluated the beneficial effect of Antarctic root endophytes on the individual performance and final yield of two crop species, Lactuca sativa L. and Solanum lycopersicum L. growing under salt stress conditions in field trial. In order to get an insight into the underlying mechanisms which increase salt tolerance, authors assessed whether the endophytemediated improvement of host plant performance (under sodium stress) is related with enhanced individual physiological processes, such as photosynthesis, water use efficiency, or upregulation of vacuolar NHX H+/Na+ antiporters. The results showed that the improved energy production goes hand in hand with an improved pathway for vacuolar Na+ sequestration, suggesting a potential capacity of the Antarctic endophytes.

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4.9  Conclusion and Perspectives The research efforts related with endophytic fungi in Chile have covered different areas of science such as biodiversity, ecology, natural products and promising applications. The first research interest was the isolation and characterization of secondary metabolites and their potential applications (mainly antimicrobial activities). However, since 2014, the focus of these studies changed to the ecology of the fungi and their application in agroecosystems. These studies used a combination of classical taxonomical approach and molecular tools. Most of the endophytic fungus reported in this review have been isolated from 8 gymnosperms and 39 angiosperms from Chile. Of these, the Andean Prumnopytis Phil. contributed with 32 species of endophytes, so far. Although, the isolated metabolites have shown significant biological activity, it is presumed that they can be a promising source of new metabolites with interesting pharmacological applications. For this reason, the strains are, at the moment, studied using new protocols and new techniques for chemical identification. The most interesting observation in this review is that plants inoculated with endophytic fungi have significantly different physiologies in comparison with un-inoculated plants. However, the extent to which these effects influence the ecology of plants in the natural environment remains to be clarified. Endophytic fungi may improve eco-physiological performance, and the biochemical response that promotes growth and survival in different plant species (grasses, shrubs and trees). In addition, the use of endophytic fungi could be a successful strategy for the reintroduction of native species in stressful environments (i.e. in arid and semi-arid areas), and can help in the conservation-preservation of endangered species in Chile (through endophyte-assisted propagation protocols). Likewise, it was demonstrated that endophytic fungi were able to aid their host plants to tolerate stressful conditions (e.g. drought, cold, salinity, UV-radiation, metal contaminations). Additionally, their important role in the context of biological control was shown. Using “omics” techniques, significant advances has been made to provide new information to understand molecular mechanisms (transcriptomic approach) involved in plant–microbe interactions under multiples stressors. This newly gained knowledge could help to improve the adaptation of crops to the new challenges, imposed by anthropogenic global climate change, in the next years. The occurrence of some emergent diseases induced by latent fungal infections have been reported in the native Chilean flora, as well as, forest or agronomic interesting species. These spread of these diseases is probably due to climate change and globalization. To safeguard the phyto-sanitary status of Chile, the forestry industry and agriculture sector should take these facts into consideration and generate protection strategies or reinforce proactive surveillance. Preliminary research highlights the relevance to develop a monitoring program of propagation materials, as well as, fast, sensitive, reliable and accurate identification, and quantification methodologies (based on molecular tools) to detect new or recurrent latent infections,

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Table 4.3  Potential uses and bioactivities of endophytic fungi isolated in Chile Application and uses Biopulping – bioremediationBioethanol

Scale of evaluation Laboratory

Biofuels – (mycodiesel)

Laboratory

Eucryphia cordifolia (a)

Growth promotion – propagation strategies

Greenhouse – field (one season)

Growth promotion – propagation strategies Stress tolerance – drought

Greenhouse

Flourensia thurifera (b), Senna cumingii (b), and Puya berteroniana (b) Prosopis chilensis (a)

Lactuca sativa (b) Nursery – field trial (one season)

Penicillium chrysogenum and Penicillium brevicompactum

Stress tolerance – drought Phytoremediation

Greenhouse

Penicillium minioluteum

Plant host Endophytic fungi Drimys winteri (a) Bjerkandera sp.

Chenopodium. quinoa Willd. (a)

Greenhouse

Eucalyptus globulus (b) Solanum Biocontrol – crop Laboratory lycopersicum (b) protection and and Capsicum greenhouse annuum Acacia Laboratory caven (Molina) Molina Lithraea caustica (Molina) Hook. & Arn. Echinopsis chiloensis (Colla) H.Friedrich & G.D.Rowley Capsicum annuum Stress Greenhouse tolerance – salinity field trial (one (a), Lactuca sativa (b), Allium cepa season) (b), and Solanum lycopersicum (b)

Note: “a” means original host; “b” means alternative host

References Oses et al. (2006) Rodriguez et al. 2011) Gliocladium roseum Stinson et al. (2003), Strobel and Ascocoryne et al. (2008), sarcoides and Strobel et al. (2010) Fardella et al. Penicillium (2014) and chrysogenum Molina-­ Penicillium Montenegro brevicompactum et al. (2015) Penicillium sp. González-­ Teuber et al. (2019a, b)

Chaetomium cupreum Beauveria bassiana Alternaria spp. Aurobasidium spp

Penicillium chrysogenum and Penicillium brevicompactum (fungal consortium with rhizobacteria) P. chrysogenum and P. brevicompactum (fungal consortium)

Molina-­ Molina-­ Montenegro et al. (2016a, b) González-­ Teuber et al. (2018) Ortiz et al. (2019) Barra-Bucarei et al. (2020) Vidal et al. (2020)

Acuña-­ Rodríguez et al. (2019) MolinaMontenegro et al. (2020)

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pathogens, or new invasive species. These methods will not only assure the quality of materials for propagation purposes (e.g. rootstocks and others biological materials), but also of complete crop plants (e.g. grapevines, eucalyptus, apples). A similar strategy of proactive phyto-sanitary surveillance for emergent pathogens that affect the endemic and native flora de Chile, should be considered in order to identify new pathogens (e.g. the new pathogen that is likely to cause the reduction in slow-growing and iconic plant species, such as Araucaria araucana (Molina) K. Koch; Pérez et al., 2018; Alarcón et al. 2020). Considering these preliminary evaluations, most of them under controlled conditions, and their promising results, it is possible to visualize some potential applications mediated by endophytic fungi species, isolated from the native flora of Chile or from economically important crops (grasses, horticultural crops, forest trees). According to the technological readiness level or TRLs approach (Mankins 1995) is possible to conclude that the maturity of these potential technologies and applications (most of them in TRL2-TRL3) need more research with special focus in the validation in field trials, stability and safety evaluations. Moreover, to successfully apply the concept, a solid knowledge base is needed for the development, and testing of endophytes (Murphy et al 2018). Table 3 shows a summary of the principal uses of endophytic fungi in Chile. One of the first concrete examples, about how to bring the basic concept to commercial levels, was the development of commercial perennial ryegrass (Johnson and Caradus, 2019). The endophytic fungi associate with this ryegrass were isolated from native grasses in Chile in the 70-80s. The second case corresponds to Gliocladium roseum (now classified as Ascocoryne sarcoides), an endophytic fungi isolated from Eucryphia cordifolia (“ulmo”) from Patagonia forests, which is able to produce fungal hydrocarbons (Myco-dieselTM , US9624515 patent). This system and method of producing volatile organic compounds from fungi could potentially be used to obtain biofuel or as an antimicrobial treatment. According to Harrison and Griffin (2020), there are still knowledge gaps to fill about the biodiversity, lifestyle and functioning of endophytic fungi. Firstly, only about 1–2% of known plant species have been studied for their endophytic associations (Strobel and Daisy, 2003) and secondly, most of the plants studied are the terrestrial, whereas aquatic plants (e.g., ocean, lakes, lagoon) are almost completely unstudied (Strobel, 2018). Harrison and Griffin (2020) point out some open questions that should be consider in future research: i) What is the influence of human development on endophyte biodiversity? (e.g., pollution, habitat fragmentation, ecosystem disturbance frequency, abundance of introduced hosts), ii) What is missing in the study of host phylogeny? (host range and endophyte biogeography), iii) What are the effects of tissue type on endophyte assemblages?, iv) What is best way to share information among studies? (e.g. availability of raw and processed sequence data, standard bioinformatic pipeline, scripts). Consequently, a suitable knowledge of patterns of the biodiversity of endophytic fungi across spatial scales and the host phylogeny is strongly required to under-

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stand the evolutionary forces and ecological pressures that shape endophyte assemblages (Harrison and Griffin 2020). Chile is classified as a biodiversity hotspot, as it has a high biological diversity and a high level of endemism in plant species. The country still has pristine and extreme habitats, both on continental and insular Chile. The latter includes species that have not been studied before. Despite the restricted research efforts performed in Chile, there is great potential to generate new knowledge (bioprospection, characterization and conservation of microbial germoplasms) in collaboration with national and international networks in strategic areas such as biodiversity, ecology and biotechnological applications (agriculture, biomedicine and bioremediation).

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Chapter 5

Endophytic Fungal Community Associated with Colombian Plants Hernando José Bolívar-Anillo , Ezzanad Abdellah , Gesiane da Silva Lima , Inmaculada Izquierdo-Bueno and Gabriel Franco dos Santos

, Javier Moraga

,

Abstract  Colombia is included in the group of megadiverse countries, and it possesses the second largest biodiversity in the world. However, the number of bacteria and fungi reported in Colombia seems to be a much lower amount than that was expected. The richness and abundance of microorganisms could make this country as a potential source for obtaining new microorganisms. Endophytes are microorganisms that are found within tissues of plants during at least part of their life cycle without causing disease under any known circumstances, and many of them are considered to be plant growth-promoting organisms. According to the abundant flora observed in Colombia, the search for microorganisms associated with plants (rhizospheres, endophytes, and epiphytes) furnishes a possibility of new fields of research that does not only allow to know its microbial biodiversity but also can also be seen as a source of microorganisms with potential discovery of new bioactive compounds and species that can be employed as biocontrol, as bioremediation, for the production of enzymes, and promotion of plant growth, among others. This chapter embodies endophytic fungi that have been isolated from plants grown in Colombia, emphasizing its biotechnological potential. Keywords  Endophytic · Fungi · Colombia · Biodiversity H. J. Bolívar-Anillo (*) Microbiology Department, Faculty of Basic and Biomedical Sciences, Universidad Simón Bolívar, Barranquilla, Colombia e-mail: [email protected] E. Abdellah Organic Chemistry Department, Faculty of Science, University of Cádiz, Puerto Real, Cádiz, Spain G. da Silva Lima · G. F. dos Santos Departamento de Química, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil I. Izquierdo-Bueno · J. Moraga Department of Biomedicine, Biotechnology and Public Health, Faculty of Marine and Environmental Sciences, University of Cadiz, Puerto Real, Cádiz, Spain © Springer Nature Switzerland AG 2021 L. H. Rosa (ed.), Neotropical Endophytic Fungi, https://doi.org/10.1007/978-3-030-53506-3_5

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5.1  Biodiversity in Colombia Colombia has 1,141,748  km2 of continental and 930,000  km2 of marine area. In other words, it has a territory of 2,071,748 km2, which represents the 0.22% of the earth surface and harbors an estimated 10% of the currently known species (Andrade 2011; Franco and Ruiz 2014). This country is located in northwestern South America within the intertropical convergence (Fig. 5.1), and due to the presence of three mountain ranges and its coasts in the Pacific and Atlantic ocean and for

Fig. 5.1  Geographic location of Colombia

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possessing part of the Amazon rainforest, Colombia has a great diversity of environments and climates, favoring a great biological diversity (Andrade 2011). With its more than 3 million animals and more than 1.8 million plants registered in the Global Biodiversity Information Facility (GBIF) (https://www.gbif.org/country/ CO/about10/06/18), Colombia belongs to the group of 14 countries that has the highest biodiversity index in the world (Franco and Ruiz 2014). Even with the great biodiversity of flora that Colombia exhibits, few published works have explored its endophytic microbiota. A review carried out by Bolivar-­ Anillo et al. (2016) showed that until 2016 only seven genera of plants had been filled with endophytic microorganisms from Colombian plants (Ludwig-Müller 2015; Joe et al. 2016; Karthik et al. 2017; Raina et al. 2018). Within this context, this chapter aims to explore endophytic species from Colombia and to describe the studies that have been conducted on this species in the world, covering the period to 2018. Colombia’s Biodiversity Information System (SiB) reports that the country has 4270 species of orchids, 289 of palms, 1534 of ferns and correlated, 1692 of mosses and correlated, and 87 of Espeletia. These data place Colombia as the second country in the world with the highest biodiversity, the first in orchid species, and the third in palm species (SiB Colombia). It also has 5 biosphere reserves: the National Parks of Seaflower, Ciénaga Grande de Santa Marta, Sierra Nevada de Santa Marta, El Tuparro, and the Andean Belt, as well as 59 natural areas belonging to the National Natural Park System (142,682  km2) and 6 Ramsar wetlands (Rueda-Solano and Castellanos-Barliza 2010; Andrade 2011; Gómez-Camelo et al. 2011; Vilardy et al. 2012; Borsdorf et al. 2013; Hudgson 2016). Due to the peace agreement that ended the armed conflict, which lasted more than 50 years in this country, it is expected that new species of plants, animals, and other organisms could be reported from areas that could not be accessed by researchers before. The study and protection of biodiversity are expected to play a central role in the post-conflict debates on Colombia’s development (Moreno et al. 2016). Although data reported by GBIF and SiB on plants and animals reflect the reality about the biodiversity in Colombia, the numbers that are registered for bacteria and fungi seem to be very low when analyzing the data for microorganisms. Colombia’s fungi and bacteria show 28,276 and 2088 species listed in GBIF, respectively. However, in SiB, we account only 4400 species of fungi and 1193 bacteria. These reported data not only could reflect an underreporting of microorganisms that have been isolated in different Colombian ecosystems but also highlight the potential for the search for new microorganisms and the natural products associated with them (Bueno et  al. 2011; Bravo and Pereañez 2016). In respect of the abundant flora observed in this country, the search for microorganisms associated with plants (rhizospheres, endophytes, and epiphytes) furnishes a possibility of new fields of research that does not only allow to know its microbial biodiversity but also can also be a source of microorganisms with potential for biocontrol, bioremediation, production of enzymes, bioactive compounds, and promotion of plant growth, among others (Amal et al. 2010; Wani et al. 2015; Zheng et al. 2016).

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5.2  Endophytic Fungi Isolated in Colombia Endophytes are microorganisms that are found within the tissues of plants during at least part of their life cycle without causing disease under any known circumstances, and many of them are considered as plant growth-promoting organisms (Ludwig-­ Müller 2015; Le Cocq et al. 2017). Among the more than 300,000 species of plants that exist in the world, it has been established that each one contains at least one endophytic microorganism (Partida-Martinez and Heil 2011; Shahzad et al. 2018). Furthermore, it has been estimated that more than one million different strains of endophytic fungi inhabit these plants, because each individual plant species can be colonized with more than one strains of fungi (Fouda et al. 2015; Strobel 2018). However, only about 1–2% of these plant species described have been studied in terms of their endophytic microbiota (Strobel 2018). Colombia has a complex topography dominated by the Andes mountain range that crosses it from south to north, dividing the country into five major natural regions: Caribbean, Pacific, Amazon, Orinoquia, and Andes, in addition to an insular region formed by the Archipelago of San Andrés and Providencia in the Caribbean and the Gorgona, Gorgonilla, and Malpelo islands in the Pacific, which have a unique flora and fauna (Cifuentes-Sarmiento and Castillo 2016). Despite the wide variety of endemic plants in the country and in their different natural regions, the isolation of endophytic fungi has only been carried out in 12 species of plants: Rosa hybrida, Cattleya percivaliana, Cattleya trianae, Vanilla spp., Bothriochloa pertusa, Coffea arabica, Espeletia grandiflora, Espeletia corymbosa, Phaseolus vulgaris, Cavendishia pubescens, Ionopsis utricularioides, and Psygmorchis pusida, which were sampled in sites located only in 8 (Cundinamarca, Antioquia, Sucre, Magdalena, Casanare, Caqueta, Valle del Cauca, and Caldas) of the 32 departments of the Colombian geography. Some of these endophytic fungi presented biocontrol capabilities, promotion of plant growth, and production of bioactive compounds (Bolivar-Anillo et al. 2016; Parsa et al. 2016). A highly diverse 100 endophytes from E. grandiflora and E. corymbosa were isolated and identified from Páramo region in the Andean Mountain Range. Twenty-­ two isolates were identified as Diaporthe phaseolorum; fifteen as Nigrospora oryzae; twelve as Beauveria bassiana; nine as Fusarium proliferatum; four as Epicoccum nigrum and Eutypella scoparia; three as Scopulariopsis brevicaulis, Chaetomium globosum, and Trichoderma asperellum; two as Aporospora terricola, Cladosporium tenuissimum, Hypoxylon stygium, Leptodontidium orchidicola, and Leptosphaerulina chartarum; and only one as Aureobasidium pullulans, Bipolaris sorghicola, Botrytis fabae, Cladosporium cladosporioides, Coprinellus micaceus, Curvularia oryzae, Eucasphaeria capensis, Paecilomyces sinensis, Paraconiothyrium sporulosum, Pestalotiopsis disseminata, Phoma glomerata, Penicillium commune, Stemphylium vesicarium, Trichoderma atroviride, and Xylaria polymorpha (Miles et al. 2012). Among the isolates, 17 endophytes showed capacity for producing secondary metabolites with antimicrobial activity (cellophane test) against Botrytis cinerea,

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Rhizoctonia solani, and/or Phytophthora infestans. Eight endophytes completely inhibited (100%) the growth of P. infestans. In addition, the results also showed a wide spectrum of biological activity of the fungus Aureobasidium pullulans and a promising biocontrol activity in vivo against Rhizoctonia solani (Miles et al. 2012). Perez et  al. (2012) had reported the isolation of endophytic species from Bothriochloa pertusa (colosoana grass), grown on farms in the district of Sincé and Sucre, abled to solubilize phosphate of root tissues. Since the phenotypic identification, it has been established that a total of 36 fungi belongs to the anamorphic group and 1 to the traditional Zygomycota group. Among the species reported in this study were Penicillium sp., Paecilomyces sp., and Aspergillus sp., the latter being the most abundant, with the species identified as Aspergillus candidus, Aspergillus niger, Aspergillus flavus, and Aspergillus terreus. Coffee is considered one of the most economic assets in Latin America, and it has improved the assessment of some country in this region (Pineda et al. 2019). Coffee was introduced to Colombia more than 100 years ago, and it is actually considered as part of the traditional culture of this country (Velandia 2017). In the last year, Colombian coffee production was estimated in 13.6 million of bags (60 Kg/ bag). This production places Colombia as the third country in the world with the highest production of coffee after Brazil and Vietnam (Arenas-Clavijo and Armbrecht 2018). More than 50% of culture coffee in Colombia are located in four districts (Huila, Antioquia, Tolima, and Cauca), which are located in tropical Andes (Arenas-Clavijo and Armbrecht 2018). Coffee is considered one of the engines of Colombia development. In this regard, the special coffees represent 36% of Colombia exportation. Fungal endophytes in green seeds of Coffea arabica were isolated and identified from green coffee seeds that originated in seven countries. These include five Aspergillus species, three Penicillium species, two Clavicipitaceae species, and one species in each of the following genera: Acremonium, Eurotium, Fusarium, Gibberella, and Pseudozyma. Out of 19 endophytic fungi, only 3 species were isolated from Colombia (Aspergillus tubingensis, Gibberella sp., and Penicillium olsonii) (Vega et al. 2008). Tissues from Coffea arabica, C. congensis, C. dewevrei, and C. liberica collected in Colombia, Hawaii, and Maryland were sampled for the presence of fungal endophytes (Vega et al. 2006). Tissues used for this study included roots, leaves, stems, and various berry parts. DNA sequencing for identification of species was realized from a set of isolates visually recognized as Penicillium. Comparison of DNA sequences with GenBank and unpublished sequences revealed the presence of eleven known Penicillium species: P. brevicompactum, P. brocae, P. cecidicola, P. citrinum, P. coffeae, P. crustosum, P. janthinellum, P. olsonii, P. oxalicum, P. sclerotiorum, and P. steckii, as well as two possibly undescribed species near P. diversum and P. roseopurpureum. Indeed, the species originally isolated in Colombia were P. brevicompactum (leaves), P. brocae (leaves), and P. oxalicum (leaves) (Vega et al. 2006). Penicillium genus produces a variety of important metabolites, and among them, one receives a special attention due its capacity of contamination – the ochratoxin A

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(OTA). This metabolite can lead to several problems due to its possible adverse effect on human health. In a study conducted by Vega and coauthors, ochratoxin A was identified in only four isolates: P. brevicompactum, P. crustosum, P. olsonii, and P. oxalicum (Vega et al. 2006). In addition, endophyte fungi associated with various asymptomatic tissues of Coffea arabica plants in Colombia, Hawaii, Mexico, and Puerto Rico were reported (Vega et  al. 2010). A total of 843 fungal isolates were recovered and sequenced (Colombia, 267; Hawaii, 393; Mexico, 109; Puerto Rico, 74) yielding 257 unique ITS genotypes (Colombia, 113; Hawaii, 126; Mexico, 32; Puerto Rico, 40). The most abundant taxa were Colletotrichum, Fusarium, Penicillium, and Xylariaceae. The dominant genotype isolated in Colombia was Colletotrichum sp., with 25 isolates from four types of tissues. Endophyte’s abundance in Colombian coffee was reported as following leaf (40.4%), followed by crown (22.9%), stems (16.9%), peduncle (10.1%), berry (6.0%), seed (2.2%), and roots (1.5%). All species isolated from different tissues of Coffea arabica plants in Colombia are shown in Table 5.1. Phaseolus vulgaris (beans), from Fabaceae family, is considered one of the main products of the peasant economy, especially in the Andean region (Vélez and Estrada 2018). Phaseolus vulgaris (beans) is a legume of great importance in Colombia due to its characteristics, such as its nutritional value and production (Flórez et al. 2017). According to the National Federation of Cereal and Leguminous Cultivators (Fenalce), bean production in Colombia was approximately 76,000 tons by 2018 (www.fenalce.org/alfa/dat_particular/ar/ar_85439_q_APR_2018_A__defi.pdf). In a recent research, a survey of fungal endophytes in 582 germinated seeds belonging to 11 Colombian cultivars of Phaseolus vulgaris was conducted, which yielded 394 endophytic isolates belonging to 42 taxa (Parsa et  al. 2016). In this study, five isolates of Fusarium sp., three isolates of Cladosporium cladosporioides, and only one isolate each of Acremonium sp., Alternaria sp., Aspergillus ustus, Aureobasidium pullulans, Chaetomium sp., Chaetomium globosum, Cochliobolus lunatus, Colletotrichum lindemuthianum, Curvularia sp., Curvularia affinis, Epicoccum sp., Epicoccum nigrum, Fusarium phaseoli, Fusarium oxysporum, Fusarium solani, Macrophomina phaseolina, Marasmius aff. nigrobrunneus, Neurospora sp., Penicillium commune, Pestalotiopsis sp., Pestalotiopsis microspore, Pestalotiopsis sydowiana, Pestalotiopsis sp., Peyronellaea glomerata, Phaeosphaeriopsis sp., Pleospora sp., Stemphylium sp., Stemphylium solani, Talaromyces aff. verruculosus, uncultured Ascomycete, uncultured Aureobasidium, uncultured endophytic fungus, uncultured Xylariales, and Xylaria sp. were isolated (Parsa et al. 2016). Aureobasidium pullulans was the dominant endophyte, isolated from 46.7% of samples. Also common were F. oxysporum, Xylaria sp., and C. cladosporioides but found in only 13.4%, 11.7%, and 7.6% of seedlings, respectively (Parsa et al. 2016). Orchids are one of the most important ornamental plants in the world, whose characteristics such as morphology, structure, beauty, and physiological properties have impacted both flower growers and scientists (Silva 2015; Hossain et al. 2013). Orchidaceae is one of the largest and most diverse families of flower plants, which comprises more than 850 genera and between 30,000 and 35,000 species, and

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Table 5.1  Fungal endophyte genotypes isolated from various coffee tissues in Colombia Fungal Agaricomycetes sp. 2 Agaricomycetes sp. 3 Agaricomycetes sp. 4 Ascomycota sp. 2 Ascomycota sp. 3 Ascomycota sp. 4 Ascomycota sp. 5 Ascomycota sp. 6 Ascomycota sp. 7 Aspergillus sp. 4 Aspergillus sp. 6 Aspergillus fumigatus Aspergillus oryzae Aspergillus pseudodeflectus Beauveria sp. Beauveria bassiana Botryosphaeria sp. Cercospora sp. Cladosporium sp. 2 Cladosporium sp. 3 Cladosporium sp. 4 Clonostachys cf. rosea Colletotrichum sp. 2 Colletotrichum sp. 5 Colletotrichum sp. 6 Colletotrichum sp. 7 Colletotrichum sp. 8 Colletotrichum sp. 9 Colletotrichum sp. 10 Colletotrichum sp. 11 Colletotrichum sp. 12 Colletotrichum sp. 13 Colletotrichum sp. 14 Colletotrichum sp. 15 Colletotrichum sp. 16 Colletotrichum sp. 17 Colletotrichum sp. 18 Colletotrichum sp. 19 Colletotrichum sp. 20 Colletotrichum sp. 21 Colletotrichum sp. 22

GenBank code EF694649 EF687930 EF672294 EF694665 EF672299 EF672300 EU002914 EU002910 EF672291 EF672304 EF672303 EF634383 EF591304 DQ778908 EF672308 EF672309 EF672312 EF672313 DQ299300 DQ299298 DQ299299 DQ287243 EF672288 EF672286 EF672287 EF672328 EF672284 EF694637 EF687924 EF672322 EF687920 EF672290 EF687923 EF687925 EF672324 EF672325 EF687922 EF672318 EF694640 EF672326 EF687927

Leaf 1 1 – 1 – 1 1 – 1 – – 2 1 – 2 2 – – – 1 1 2 11 1 1 1 2 1 – – – – 2 1 4 1 1 1 4 –

Berry – – – – – – – – – – – – – 1 – 1 – 1 1 2 – – – – – – – – – – – – – – – – – – – – –

Crown 1 – – – – – – 1 – – – – – – – 6 2 – – 1 – – 6 – – – 2 – – 3 – – – – – – 1 – – – –

Peduncle – – 1 – – – – 1 – – – – – – – 1 – – – – – – 3 – – – – – 2 – 1 – – – – – – – – 1 –

Seed – – – – – – – – – – – – – – – 2 – – – – – – – – – – – – – – – – – – – – – – – – –

Stem – – – 1 1 – – – – 2 1 – – – – – – – – – – – 5 – – – 1 – 1 – – – – – – – 1 – – 4 2

Root – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

(continued)

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Table 5.1 (continued) Fungal Colletotrichum sp. 23 Colletotrichum sp. 24 Colletotrichum sp. 25 Colletotrichum sp. 29 Colletotrichum sp. 31 Colletotrichum sp. 34 Exobasidiomycetes sp. Fusarium sp. 2 Fusarium sp. 13 Fusarium sp. 16 Fusarium sp. 17 Fusarium sp. 18 Hymenochaetaceae sp. Hypocreales sp. 1 Neosartorya sp. Paecilomyces sp. 2 Penicillium sp. 1 Penicillium sp. 2 Penicillium sp. 3 Penicillium sp. 4 Penicillium sp. 5 Penicillium sp. 9 Penicillium brevicompactum Penicillium brocae Penicillium crustosum Penicillium olsonii Penicillium oxalicum Petriella sp. Pezizomycotina sp. Phomopsis sp. 1 Phomopsis sp. 5 Phomopsis sp. 6 Phomopsis sp. 7 Phomopsis sp. 8 Phomopsis sp. 9 Phomopsis sp. 10 Phomopsis sp. 11 Phomopsis sp. 12 Phomopsis sp. 13 Pleosporaceae sp.

GenBank code EF687928 EF694638 EF672329 EF672330 EF687919 EF672320 EU009983 DQ778914 DQ778913 EF687916 EF687951 DQ682580 EF694647 EF694654 EF694671 EU049286 EU002901 DQ778916 EF694646 EF694623 EF694624 DQ682589 DQ682592

Leaf – 12 4 4 4 1 – – – 5 – – – – 1 – – – 1 – – 4 3

Berry – – – – 2 – 1 – – – – 2 – – – – – – – – – – –

Crown – – – – 3 – 1 – – – – 7 1 1 – – – – – – – 5 –

Peduncle – – – – 4 – – – 1 2 – 2 – – – 1 – – – 1 – 2 –

Seed – – – – – – – 1 – – – 1 – – – – – – – – – – –

Stem 2 – 6 – – – – – 1 6 – 1 – – – – 1 1 – – 1 1 –

Root – – – – – – – – – – 1 – – – – – – – – – – – –

EF634396 DQ123647 DQ778918 DQ123663 EU002908 EF672296 EU002923 EU002928 EU002926 EU002921 EU002917 EU002922 EU002915 EU002925 EU002927 EU002918 EF687932

1 – – 1 – 1 1 1 1 – – – – 1 – – –

– 1 – – – – – – 1 – – – – – – – –

– 2 – – – – – 1 – 1 1 1 1 – 1 2 1

– – – – – – – – – – – – – – – – 1

– – – – – – – – – – – – – – – – –

– – 1 – – – – – – – – – – – – – –

– – – – 2 – – – – – – – – – – – –

(continued)

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Table 5.1 (continued) Fungal Pleosporales sp. 1 Pseudozyma sp. Schizophyllum sp. 2 Sordariales sp. Sordariomycetes sp. 1 Sordariomycetes sp. 2 Sordariomycetes sp. 3 Sordariomycetes sp. 4 Sordariomycetes sp. 6 Sporobolomyces sp. 5 Stereum sp. 1 Stereum sp. 2 Tilletia sp. 1 Tilletia sp. 2 Trametes sp. 1 Trichoderma sp. 1 Trichoderma sp. 2 Xylaria sp. 1 Xylaria sp. 2 Xylariaceae sp. 5 Xylariaceae sp. 6 Xylariaceae sp. 7 Xylariaceae sp. 8 Xylariaceae sp. 9 Xylariaceae sp. 10 Xylariaceae sp. 11 Xylariaceae sp. 12 Xylariaceae sp. 13 Xylariaceae sp. 14 Xylariaceae sp. 23 Xylariales sp. 1

GenBank code EF672298 EU002890 EU002895 EU002897 EF672297 EF694651 EU002935 EF687956 EU002898 EU009967 EU009970 EU009969 EU009972 EU009971 EU009977 EU009980 EU009981 EU009989 EU009958 EU010005 EU010004 EU009959 EU009998 EF694657 EU009997 EU009999 EU010001 EU010003 EU009992 EU009991 EU009994

Leaf – – – – – – 1 – 1 – 1 – – – 1 – – 1 – 1 1 – 2 – – 1 – 1 1 1 1

Berry – – – – – – – – – – – – 2 – – – – – – – – – – – 1 – – – – – –

Crown – 1 1 – – – – – – – – – – 1 1 – 1 – 1 – – – – 2 – – 1 – – – –

Peduncle – – – – – 1 – 1 – – – – – – 1 – – – – – – – – – – – – – – – –

Seed 1 – – – – – – – – – – 1 – – – – – – – – – – – – – – – – – – –

Stem – – – – 1 – – – – 1 – – – – – 2 – – – – – 1 – – – – – – – – –

Root – – – 1 – – – – – – – – – – – – – – – – – – – – – – – – – – –

– = absence

among them, 70% were epiphytes, 25% are terrestrial, and 5% can grow in different supports (Hossain et  al. 2013). Colombia, with approximately 4270 species of orchids reported, lead the worldwide ranking in terms of biodiversity of these amazing plants (Reina-Rodríguez et al. 2017). Species from genera Vanilla are the most important from an economic point of view, highlighting Vanilla planifolia Jacks. ex Andrews, a source of vanilla flavor that represents 0.75% of the world’s imports of species, about US$ 1.5 billion of world trade in these products (Sasikumar 2010). Vanilla species were distributed in all continents, whose main producing countries are India, Indonesia, Madagascar, Mexico, Reunion Islands, and the Comoros (Bory

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et al. 2008; Sasikumar 2010). Due to its suitable environmental conditions necessary for wild population growth, Colombia is considered a promising source to obtain a large amount of Vanilla germplasm, whose cultivation and commercialization could represent new alternatives for small and medium local farmers. Nevertheless, there are few studies of native Colombian Vanilla species (Lizarazo-­ Medina et al. 2014; Gamboa-Gaitán 2014). Plant tissues from healthy leaves of Vanilla planifolia, V. odorata, and a wild unidentified vanilla were sampled for studying their microbiota (Gamboa-Gaitán 2013). Five morphospecies were isolated, four being species members of the Fusarium genus and one identified as Colletotrichum boninense. The Fusarium species found were F. oxysporum f. sp. loti from Vanilla odorata leaves, F. oxysporum f. sp. melonis and F. solani from cultivated V. planifolia leaves, and Fusarium sp. from a wild unidentified vanilla. Colletotrichum boninense, an anthracnose-­ producing fungus, was found as an endophyte in Vanilla leaves (Gamboa-­ Gaitán 2013). A new study of Gamboa-Gaitán (2014) showed that vanillas have a large repertoire of endophytic bacteria and fungi associated with their tissues, which were sampled from roots, stems, leaves, and fruits, in both cultivated and wild vanilla. As a result of this investigation, more than 60 morphospecies of endophytic microorganisms were found in Colombian vanilla. The fungi species found were Alternaria alternata (leaf and roots of V. planifolia), Arthrographis sp. (roots of Vanilla sp.), A. niger (stems of Vanilla sp.), Bionectria sp. (leaf of V. odorata), Biscogniauxia atropunctata (leaf of Vanilla sp.), Colletotrichum boninense (stems of V. planifolia), C. gloeosporioides (leaf of V. planifolia), Colletotrichum sp. (stems of Vanilla sp.), Diaporthe eucalyptorum (stems of Vanilla sp.), F. oxysporum f. sp. loti (leaf of V. odorata), F. oxysporum f. sp. meloni (leaf of V. planifolia), Fusarium solani (leaf and roots of V. planifolia and fruit of V. calyculata), Fusarium sp. (leaf of V. odorata and stems and roots of Vanilla spp.), Hypocrea virens (stems of Vanilla sp.), Lasiodiplodia venezuelensis (stems of Vanilla sp.), Neofusicoccum sp. (fruit of V. calyculata), Penicillium spp. (roots of V. odorata and leaf and stems of Vanilla spp.), Pestalotia sp. (fruit of V. calyculata and leaf of Vanilla sp.), Pestalotiopsis theae (leaf of Vanilla sp.), Phialocephala sp. (roots of V. odorata and Vanilla sp.), Phomopsis sp. (roots of V. planifolia and Vanilla sp.), Rhizoctonia sp. (roots of Vanilla sp.), Trichoderma harzianum (roots of V. planifolia), Trichoderma sp. (stems of V. odorata and leaf and roots of Vanilla spp.), Volutella sp. (leaf of Vanilla sp.), and Xylaria spp. (roots of Vanilla spp.) (Gamboa-Gaitán 2014). As a complement to the previous study, a new study of endophytic fungi from V. planifolia, V. calyculata, V. odorata, and four unidentified species from the Colombian Chocó region was performed (Gamboa-Gaitán and Otero-Ospina 2016). In this study, 1055 plant fragments totaling 4220 mm2 were sampled, and a total of 525 isolates of 56 morphospecies of endosymbiont microorganisms were found in tissues of the Colombian vanillas. The community density found was 1.3 species per cm2, and most morphospecies were fungi (54 out of 56, 96.43%). Species of endophytic fungi found by Gamboa-Gaitán and Otero-Ospina (2016) and the plant species that were isolated are shown in Table 5.2. Species isolated from sick tissue or as epiphytic fungi are not shown in Table 5.2.

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Table 5.2  Endophytic fungi in Colombian vanillas and organs in which they were found Fungi Alternaria alternata Arthrographis sp. Aspergillus niger Bionectria sp. Colletotrichum boninense Colletotrichum gloeosporioides Colletotrichum sp. Cosmospora sp. Diaporthe eucalyptorum Fusarium oxysporum f. sp. loti Fusarium oxysporum f. sp. melonis Fusarium solani Fusarium solani Fusarium sp. Fusarium sp. Hypocrea virens Lasiodiplodia venezuelensis Mycelia sterilia Neofusicoccum sp. Penicillium spp. Penicillium spp. Pestalotia sp. Pestalotia sp. Pestalotiopsis theae Phialocephala sp. Phomopsis sp. Rhizoctonia sp. Trichoderma harzianum Trichoderma sp. Trichoderma sp. Volutella sp. Xylaria spp.

Species Vanilla planifolia Vanilla sp. Vanilla sp. V. odorata V. planifolia V. planifolia V. planifolia, Vanilla sp. Vanilla sp. Vanilla sp. V. odorata V. planifolia V. planifolia V. calyculata V. odorata Vanilla spp. Vanilla sp. Vanilla sp. Vanilla spp. V. calyculata V. odorata Vanilla spp. V. calyculata Vanilla sp. Vanilla sp. V. odorata, Vanilla sp. V. planifolia, Vanilla sp. Vanilla sp. V. planifolia V. odorata Vanilla spp. Vanilla sp. Vanilla spp.

Tissue Leaf, root Root Stem Leaf Stem Leaf Leaf, stem Leaf Stem Leaf Leaf Leaf Root, fruit Leaf Stem, root Stem Stem Leaf, stem, root Fruit, leaf Leaf Stem, root Leaf Fruit Leaf Root Root Root Root Leaf Stem, root Leaf Root

Another work of Vanilla endophytic fungi isolation was conducted by Ordóñez et al. (2012). In this work, the effect of 20 endophytic fungi species on plant growth of V. planifolia inoculated into the substrate was evaluated. Twelve of these fungi were isolated from roots of wild Vanilla sp. found in nine ubications in Colombia (three departments – Antioquia, Sucre, and Valle del Cauca). These species were identified by genome sequencing (ITS region) as three strains of Phomopsis sp.; one of Hypoxylon sp., Xylariacea, Phoma sp., Trichoderma sp., and Bipolaris sp.; and four indeterminate taxa (Ordóñez et al. 2012).

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All isolated species, in addition to six strain of Ceratobasidium group (Rhizoctonia form) from roots of the orchids Ionopsis utricularioides and Psygmorchis pusida (Valadares 2009), were evaluated for their effects on the growth of V. planifolia. The results showed that many endophytic species can be used for their effects on the growth of vanilla. One of the best results was attributed to an unidentified endophyte of Vanilla sp. (named V-10), which presented aerial mass = 3.1382 g (1.8902 for control), plant height = 32.5 cm (15.5 cm for control), leaf area = 210.3125 cm2 (160.5975 cm2 for control), root length = 221.975 cm (109.55 cm for control), and root mass = 0.64 g (0.253 g for control) (Ordóñez et al. 2012). Lizarazo-Medina et  al. (2014) obtained endophytic species from fragments of leaves and roots of Cattleya percivaliana and Cattleya trianae, which are grown in the greenhouse and obtained from the Orchid Society of Colombia (Fredonia-­ Antioquia). The growth of endophytic fungi was observed in 6.7% of the leaf fragments (1200) and stems (1200) analyzed, with C. trianae being having the highest percentage of colonization. Three hundred twenty-three fungi were isolated and classified by phenotypic techniques in fourteen genera and five morphotypes, with the genus Fusarium being the most abundant in root and Colletotrichum and Sclerotium in leaves. Other genera reported for these two species of orchids were Chromelosporium, Exophiala, Gonatobotrys, Monilinia, Nodulisporium, Curvularia, Gloeosporium, Trichoderma, Aureobasidium, Botryotrichum, and Cladosporium. Salgado and Cepero (2005) isolated endophyte fungi from Rosa hybrida leaves grown in the urban sector of Bogotá. From cultivated subframes (560), 16.4% (92) presented colonization by endophyte fungi. Through phenotypic identification, it was established that, among the isolated fungi, Nigrospora oryzae and Xylaria sp. were those that presented the highest number of isolates. Other genera and species of fungi isolated in this study were Acremonium, Alternaria sp., Chaetomium globosum, Nodulisporium sp., Phoma sp., Gliocladium virens, and Cladosporium sp. All these endophytic fungi shown above may have a range of secondary metabolites, which plays an important ecological role for the species that produces it but may also present interesting bioactivities to be explored  (Xu et  al. 2015). In the work conducted by Bills et  al. (1992), an endophytic Phomopsis sp. from living bark of Cavendishia pubescens in Colombia produced paspalitrem A (1) and C (2) when incubated for 21 days without stirring (Fig. 5.2). These compounds represent one example of mycotoxins from Phomopsis spp.

5.3  B  ioprospecting of Endophytic Fungi Isolated from Colombia As a result of the interaction between endophytic fungi and their host plant, it is likely that many species have developed genetic systems that allow the transfer of information between them and their host plant (or vice versa), and in this way, it

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Fig. 5.2  Chemical structure of paspalitrem A (1) and C (2) from an endophytic Phomopsis sp.

leads to produce substances that might have applications in different areas (Sudhakar et al. 2013; Borges et al. 2009; Aly et al. 2010; Selim 2012; Qin et al. 2018). In addition, these interactions could promote the biosynthesis of a wide range of natural products with diverse applications, including anticancer, antifungal, antidiabetic, and immunosuppressant compounds (Borges et  al. 2009; Kusari et  al. 2012; Sudhakar et al. 2013). However, it is necessary to take into account that the production of secondary metabolites by endophytic fungi is influenced by genetic, evolutionary, ecological, and environmental factors (Aly et al. 2010; Roopa et al. 2015; Jia et al. 2016). The diversity of endophytic fungi in a plant will be strongly influenced not only by the characteristics of the plant and the fungus but also by conditions such as the soil, temperature, humidity, precipitation, and geographic location (Giauque and Hawkes 2013; Ding et  al. 2015; Yan et al. 2015; Jia et  al. 2016; Aletaha et al. 2018). The phenomena of climatic variability in Colombia are influenced mainly by the intertropical influence zone, the dynamics of the Pacific and Atlantic oceans, and the Amazon and Orinoco basins, which determine the different rainfall gradients in the country (García et al. 2012). In this term, Colombia has one of the locations worldwide with the highest average annual precipitation (>7000 mm yr.−1), located in the Central Pacific region (Quinto-Mosquera and Moreno-Hurtado 2015). Colombia has a wide range of soils due to large variations of parent materials, climatic conditions, biodiversity, and the physiographic position of the Earth (Garcia-­Ocampo 2012). Due to its topography dominated by the Andes (Western, Central, and Eastern Cordillera), Colombia has regions ranging from plains covered by forest formations to regions with altitudes that reach more than 5000 m (Andrade 2011). All the conditions mentioned provide Colombia with unique conditions that could influence not only the diversity of the endophytic microbiota but also its metabolic capacities. In this sense, the studies of the metabolic profile by endophytic fungi from Colombia can provide a wide range of high value-added compounds.

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5.4  Conclusion In Colombia, with its wide biodiversity of flora, only very few species have been explored in relation to endophytic fungi; therefore, it is expected that, in the next few years, research aimed at the study of endophytic fungi in the country will not only allow us to know even more its biodiversity but also project it as one of the world’s pantries of new natural molecules for medical, pharmaceutical, biotechnological, agricultural, and food industries.

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Franco V, Ruiz J (2014) V informe nacional de la biodiversidad de Colombia ante el convenio de diversidad biológica. Instituto Alexander von Humboldt, Bogotá D.C Gamboa-Gaitán MA (2013) Colombian Vanilla and its microbiota, I. Acta Bot Hung 55:239–245 Gamboa-Gaitán MA (2014) Vainillas Colombianas y su microbiota. II.  Diversidad, cultivo y microorganismos endófitos. Univ Sci 19:287–300 Gamboa-Gaitán MA, Otero-Ospina JT, (2016) Colombian vanilla and its microbiota. III. diversity and structure of the endophytic community. Acta Bot Hung 58:241–256 García MC, Piñeros A, Bernal F, Ardila E (2012) Variabilidad climática, cambio climático y el recurso hídrico en Colombia. Rev Ing 36:60–64 García-Ocampo A (2012) Fertility and soil productivity of Colombian soils under different soil management practices and several crops. Arch Agron Soil Sci 58:S55–S65 Giauque H, Hawkes CV (2013) Climate affects symbiotic fungal endophyte diversity and performance. Am J Bot 100:1435–1444 Gómez-Camelo IV, Gerritsen P, Trujillo F (2011) Reserva de biósfera el Tuparro: Un reto para la conservación de la orinoquia Colombiana. Ambiente y Desarrollo 15:43–65 Hossain MM, Kant R, Vân PT (2013) The application of biotechnology to orchids. Crit Rev Plant Sci 32:69–139 Hudgson R (2016) Reserva de biosfera seaflower, buen destino, rumbo equivocado. Colombia Asuntos Económicos y Administrativos 30:245–255 Jia M, Chen L, Xin HL, Zheng CJ, Rahman K, Han T, Qin LP (2016) A friendly relationship between endophytic fungi and medicinal plants: a systematic review. Front Microbiol 7:906 Joe MM, Devaraj S, Benson A, Sa T (2016) Isolation of phosphate solubilizing endophytic bacteria from Phyllanthus amarus Schum & Thonn: evaluation of plant growth promotion and antioxidant activity under salt stress. J Appl Res Med Aromat Plants 3:71–77 Karthik M, Pushpakanth P, Krishnamoorthy R, Senthilkumar M (2017) Endophytic bacteria associated with banana cultivars and their inoculation effect on plant growth. J Hortic Sci Biotechnol 92:568–576 Kusari S, Hertweck C, Spiteller M (2012) Chemical ecology of endophytic fungi: origins of secondary metabolites. Chem Biol 19:792–798 Le-Cocq K, Gurr SJ, Hirsch PR, Mauchline TH (2017) Exploitation of endophytes for sustainable agricultural intensification. Mol Plant Pathol 18:469–473 Lizarazo-Medina PX, Mendoza-Salazar MM, Gutiérrez-Gallo A (2014) Diversidad de la micobiota endófita de Cattleya percivaliana y Cattleya trianaei cultivadas en invernadero. Actual Biol 37:307–318 Ludwig-Müller J (2015) Plants and endophytes: equal partners in secondary metabolite production? Biotechnol Lett 37:1325–1334 Miles LA, Lopera CA, González S, Cepero de García CP, Franco AE, Restrepo S (2012) Exploring the biocontrol potential of fungal endophytes from an andean Colombian Paramo ecosystem. BioControl 57:697–710 Moreno LA, Andrade GI, Ruíz-Contreras LF (2016) Estado y tendencias de la biodiversidad continental de Colombia. Biodiversidad. Instituto de Investigación de Recursos Biológicos Alexander von Humboldt. Bogotá, D. C., Colombia. 106 p Ordóñez NF, Otero JT, Díez MC (2012) Hongos endófitos de orquideas y su efecto sobre el crecimiento en Vanilla planifolia Andrews. Acta Agron 61:282–290 Parsa S, García-Lemos AM, Castillo K, Ortiz V, López-Lavalle LA, Braun J, Vega FE (2016) Fungal endophytes in germinated seeds of the common bean, Phaseolus vulgaris. Fungal Biol 120:783–790 Partida-Martínez LP, Heil M (2011) The microbe-free plant: fact or artifact? Front Plant Sci 2:100 Perez A, De la Ossa J, Montes D (2012) Hongos solubilizadores de fosfatos en fincas ganaderas del departamento de Sucre. Rev Colombiana Cienc Anim 4:35–45 Pineda JA, Piniero M, Ramírez A (2019) Coffee production and Women’s empowerment in Colombia. Hum Organ 78:64–74

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Qin D, Wang L, Han M, Wang J, Song H, Yan X, Duan X, Dong J (2018) Effects of an endophytic fungus Umbelopsis dimorpha on the secondary metabolites of host-plant Kadsura angustifolia. Front Microbiol 9:2845 Quinto-Mosquera H, Moreno-Hurtado F (2015) Precipitation effects on soil characteristics in tropical rain forests of the Choco biogeographical region. Rev Fac Nac Agron 69:7813–7823 Raina D, Singh B, Bhat AK, Satti NK, Singh VK (2018) Antimicrobial activity of endophytes isolated from Picrorhiza kurroa. Indian Phytopathol 71:103–113 Reina-Rodríguez GA, Rubiano-Mejía JE, Castro Llanos FA, Soriano I (2017) Orchids distribution and bioclimatic niches as a strategy to climate change in areas of tropical dry forest in Colombia. Lankesteriana 17:17–47 Roopa G, Madhusudhan MC, Sunil KCR, Lisa N, Calvin R, Poornima R, Zeinab N, Kini KR, Prakash HS, Geetha N (2015) Identification of taxol-producing endophytic fungi isolated from Salacia oblonga through genomic mining approach. J Genet Eng Biotechnol 13:119–127 Rueda-Solano L, Castellanos-Barliza J (2010) Herpetofauna of Neguanje, Tayrona National Natural Park, Colombian Caribbean. Acta Biolo Colomb 15:195–206 Salgado C, Cepero MC (2005) Aislamiento de hongos endofitos en rosa (Rosa hybrida) en Bogotá, Colombia. Rev Iberoam Micol 22:99–101 Sasikumar B (2010) Vanilla breeding - a review. Agric Rev 31:139–144 Selim KA (2012) Biology of endophytic fungi. Curr Res Environ Appl Mycol 2:31–82 Shahzad R, Khan AL, Bilal S, Asaf S, Lee IJ (2018) What is there in seeds? Vertically transmitted endophytic resources for sustainable improvement in plant growth. Front Plant Sci 9:24 Strobel G (2018) The emergence of endophytic microbes and their biological promise. J Fungi 4:57 Sudhakar N, Thajuddin N, Murugesan K (2013) Plant growth-promoting rhizobacterial mediated protection of tomato in the field against cucumber mosaic virus and its vector Aphis gossypii. Biocontrol Sci Tech 21:367–386 Teixeira da Silva JA (2015) Orchids: advances in tissue culture, genetics, phytochemistry and transgenic biotechnology. Floric Ornam Biotechnol 7:1–52 Valadares RB (2009) Diversidade micorrízica em Coppensia doniana (Orchidaceae) e filogenia de fungos micorrízicos associados a subtribo oncidiinae. Master’s thesis. ESALQ, Piracicaba – Brazil. 98 p Vega FE, Posada F, Peterson SW, Gianfagna TJ, Chaves F (2006) Penicillium species endophytic in coffee plants and ochratoxin A production. Mycologia 98:31–42 Vega FE, Posada F, Aime MC, Peterson, SW, Rehner SA (2008) Fungal endophytes in green coffee seeds. Mycosystema 27:75–84 Vega FE, Simpkins A, Aime MC, Posada F, Peterson SW, Rehner SA, Infante F, Castillo A, Arnold AE (2010) Fungal endophyte diversity in coffee plants from Colombia, Hawai’i, Mexico and Puerto Rico. Fungal Ecol 3:122–138 Velandia-Silva CA (2017) The coffee cultural landscape of Colombia. J World Herit Stud Special Issue, pp 44–50 Vélez AT, Bernal Estrada JA (2018) Common Bean (Phaseolus Vulgaris L.) yield response to chemical and biological fertilization in different localities of Colombia. Rev Fac Nac Agron Medellin 71:8573–8579 Vilardy S, González J, Martín-López B, Oteros-Rozas E, Montes C (2012) Los servicios de los ecosistemas de la reserva de biosfera ciénaga grande de Santa Marta. REVIBEC 19:66–83 Wani ZA, Ashraf N, Mohiuddin T, Riyaz-Ul-Hassan S (2015) Plant-endophyte symbiosis, an ecological perspective. Appl Microbiol Biotechnol 99:2955–2965 Xu GB, He G, Bai HH, Yang T, Zhang GL, Wu LW (2015) Indole alkaloids from Chaetomium globosum. J Nat Prod 78:1479–1485 Yan JF, Broughton SJ, Yang SL, Gange AC (2015) Do endophytic fungi grow through their hosts systemically? Fungal Ecol 13:53–59 Zheng YK, Qiao X, Miao C, Liu K, Chen Y, Xu L, Zhao L (2016) Diversity, distribution and biotechnological potential of endophytic fungi. Ann Microbiol 66:529–542

Chapter 6

Fungal Endophytes and Their Bioactive Compounds in Tropical Forests of Costa Rica Keilor Rojas-Jimenez

and Giselle Tamayo-Castillo

Abstract  We present a glimpse of the diversity of endophytic fungi in the tropical forests of Costa Rica and some examples of bioactive compounds obtained from them. We include the characterization of isolates carried out mainly by the former National Biodiversity Institute as well as data reported in the literature during the last two decades. This work includes the analysis of 427 fungal isolates from 280 plant species (belonging to 107 families). All the isolates were classified as Ascomycota, and within this phylum, they were assigned to four classes, 21 orders, 49 families, and 83 genera. The orders Xylariales, Glomerellales, and Diaporthales were the most abundant while Pleosporales, Xylariales, and Hypocreales the most diverse. The class Leotiomycetes presented the more substantial proportion of bioactive molecules. We showed the positive effect on the addition of unique taxa by increasing the number of sampling sites, seasons, habits of plants, altitudinal range, and plant tissues. Also, we show that endophytes produce compounds chemically and structurally diverse, many of which can be useful for the discovery of new drugs. This work provides valuable insights for bioprospecting of endophytes and also for the understanding of the ecology of these fungi in tropical forests. Keywords  Fungal endophytes · Secondary metabolites · Natural products · Biodiversity · Costa Rica

K. Rojas-Jimenez (*) Escuela de Biologia, Universidad de Costa Rica, San Jose, Costa Rica G. Tamayo-Castillo Escuela de Química, Universidad de Costa Rica, San José, Costa Rica Centro de Investigaciones en Productos Naturales (CIPRONA), Universidad de Costa Rica, San José, Costa Rica © Springer Nature Switzerland AG 2021 L. H. Rosa (ed.), Neotropical Endophytic Fungi, https://doi.org/10.1007/978-3-030-53506-3_6

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6.1  Introduction Fungi are everywhere. The majority are saprobes that decompose organic matter, while many others are specialized to infect living organisms. The fungi that infect and express symptoms in plants are called pathogens, whereas the ones that ­colonize inter- and intracellular spaces of the plant tissues, without developing visible signs of disease, are designated as endophytes (Arnold 2007; Schulz and Boyle 2005; Sieber 2007). The endophytic fungi represent a significant component of plant communities where the benefits of the plant-host relationship is based on a fine-tuned balance between the demands of the invaders and the plant response (Kogel et al. 2006). Fungal endophytes perform important functions for their hosts, some of which include systematic resistance against pathogens, tolerance to drought and heavy metals, reduced herbivory, mineral uptake, and generally enhanced growth (Arnold et al. 2003; Clay and Schardl 2002; Redman et al. 2002; Saikkonen et al. 1998). In return, the fungal counterpart is benefited by the provision of nutrients and buffering from external environmental stresses (Schulz and Boyle 2005). However, it is also possible that some endophytes may become opportunistic and can cause disease after the host has been weakened by some other factors (Lodge et  al. 1996; Sieber 2007). Many of these functions are coupled with the production of biologically active secondary metabolites of medicinal, agricultural, and industrial interest. In this regard, one of the significant milestones that aroused interest in the isolation, cultivation, and characterization of endophytic fungi was the discovery of Taxomyces andreanae, isolated from the bark of Taxus brevifolia, which was capable of producing the billion-dollar anticancer drug Taxol (Stierle et al. 1993; Strobel et al. 1996). Since then, the impetus for investigating the fungal endophytes has grown dramatically (Hyde et al. 2019; Kusari et al. 2012; Maheshwari 2006). The enormous biodiversity of fungal endophytes, estimated in at least 1.3 million species, and their large number of biosynthetic pathways result in an extraordinary potential for producing a wide range of molecules (Arnold et al. 2000; Dreyfuss and Chapela 1994; Kontnik and Clardy 2008). Some commonly used molecules of fugal origin include antibiotics, antimycotics, pesticides, immunosuppressors, cholesterol-­ lowering agents, and anticancer compounds (Deshmukh et al. 2014; Kusari et al. 2012; Strobel and Daisy 2003). Therefore, endophytic fungi are now considered a vast source of biologically active natural products (Tan et  al. 2018; Miller et  al. 2012; Strobel et al. 2004). Endophytes have been isolated from every plant species examined to date, but evident differences in their diversity are shown to occur among latitudes, ecosystems, sites, hosts, and tissue types (Rojas-Jimenez et al. 2016; Arnold et al. 2007; Rodriguez et  al. 2009; Suryanarayanan et  al. 2002; Verma et  al. 2007). For bioprospecting purposes, the larger the biodiversity of ecosystems, plants, endophytes, and molecules, the better. In this regard, tropical forests are considered hot spots of fungal species diversity, containing numerous species and molecules (Arnold and Lutzoni 2007; Smith et al. 2008). In this regard, countries like Costa Rica with a

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tropical location that connects biodiversity of North and South America, influenced by both the Pacific and Caribbean, with varied topography, geology, and climatic conditions, and presenting at least 12,000 species and 220 plant families, provide optimal conditions for prospecting new endophytes and derived bioactive molecules. In this work, we present an analysis of the richness and diversity of cultivable endophytic fungi isolated from tropical forests of Costa Rica, including data generated by the former National Biodiversity Institute together with other data on endophytes published in the literature during the last 20 years. The fungal endophytes were obtained from 280 species and 107 families of plants, sampled from different ecosystems, plant habits, and plant tissues. Also, we reviewed the main bioactive compounds described to date from endophytes isolated from Costa Rica. Besides, we provide a list of the plant hosts for each of the 83 fungal genera isolated, which can be useful in future genus-driven bioprospecting campaigns.

6.2  Study Sites and Plant Sampling This work is based on two primary sources of data. The first data set was generated by the former National Institute of Biodiversity, whose methodology is included. The second data set is a compilation of endophytes from tropical forest of Costa Rica reported in the literature. Endophytes from crops or nonvascular plants were excluded. The data of the first data set is based on a study conducted in eight protected areas of Costa Rica, including the Cabo Blanco Absolute Natural Reserve and the following national parks: Carara, Braulio Carrillo, Guanacaste, Santa Rosa, Palo Verde, Rincón de la Vieja, and Tenorio. These areas comprise a high variety of ecosystems covered by primary and secondary forest, with altitudes ranging from the sea level to the top of volcanoes, and influenced by both the Pacific and the Caribbean (Table 6.1). Together, these areas encompass a large proportion of the plant biodiversity of Costa Rica, represented in life zones such as tropical dry forest, tropical moist forest, tropical wet forest, premontane moist forest, lower montane moist forest, and montane wet forest (Holdridge 1947; Holdridge 1967). The plant collection was performed during 60 field trips that took place from September 2003 to February 2009. A basic characterization of the sampling effort and the endophytes isolated is presented in Fig. 6.1. At each site, several kilometers of natural trails were explored in search of trees, shrubs, herbs, vines, epiphytes, and parasites that were in their flowering stage. This phenological condition limited the amount of plants that could be collected but was strictly necessary for the appropriate taxonomical identification and voucher preservation. Plant samples were placed in polyethylene bags, while the fertile voucher with inflorescences and fruits were adequately mounted. The related information including microhabitat description, associated species, and location coordinates was also registered.

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Table 6.1  General characteristics of sites that originated the plant samples

Site Carara

Life zones Tropical moist forest Braulio Premontane Carrillo moist forest Montane moist forest Montane wet forest Guanacaste Premontane moist forest Santa Rosa Tropical dry forest Palo Verde Tropical dry forest Rincón de la Tropical moist Vieja forest Tenorio Tropical wet forest Cabo Blanco Tropical moist forest La Selvaa Tropical moist forest La Gambab Tropical moist forest

Mean altitude (m) 45

Mean precipitation (mm) 2800

Mean temperature (°C) 27

32–2906

2600–5734

12–25

1062

2500–3000

24

170

1500

28

40

1500–2000

27

841

2000

26

831

4000

24

4

2300

26

37

4365

26

70

4000–6000

28

Geographic coordinates 9°47′46″N; 84°35′41″W 10°09′35″N; 83°58′27″W

10°53′33″N; 85°28′12″W 10°56′2″N; 85°43′45″W 10°20′53″N; 85°20′59″W 10°46′7″N; 85°18′9″W 10°42′6″N; 84°59′43″W 9°35′10″N; 85°5′37″W 18°34′N; 95°04′W 8°70′N; 83°20¨W

Del Olmo-Ruiz and Arnold (2017) Hinterdobler and Schinnerl (2019)

a

b

The initial identification of the plant specimens was performed on site by a trained collector and later confirmed by the botanists of the National Biodiversity Institute. The samples included in this study comprised 107 plant families, representing nearly 48% of the families reported for Costa Rica. All plant collections were carried out with respective permits of the National Authority of the Ministry of Environment (307-2003-OFAU, R-012-2005-OT-CONAGEBIO, R-CM-­ INBio-03-2006-OT, R-CM-INBio-06-2006-OT, R-CM-INBio-30-2007-OT, R-CM-INBio-059-2008-OT).

6.3  Isolation of Fungal Endophytes Plant samples were washed thoroughly in running water, and then, healthy and physically undamaged tissues (three leaves, one petiole, one branch, and one root when possible) were selected and sterilized in the surface with ethanol 70% for

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Fig. 6.1  Distribution of the relative number of fungi isolated according to the main methodological variables considered during the plant sampling and endophyte isolation

2  min and with sodium hypochlorite 5% for 4  min and rinsed twice with sterile deionized water for 1 min. The efficacy of the surface sterilization was verified by imprinting the tissues on media plates (Schulz et al. 1993). Under aseptic conditions, the basal, mid, and distal parts of the tissues were sliced into segments of 2 mm2 and plated on agar medium amended with 120 mg L−1 of chlortetracycline and 120 mg L−1 of streptomycin. From years 2003 to 2006, the solid medium used for culturing was yeast mold agar (Difco, MD, USA), while from 2006 onward, we used potato carrot agar medium (20 g L−1 potatoes, 20 g L−1 carrots, 15 g L−1 agar, pH 7.0). The main reasons for this methodological change were the better expression of some phenotypic characteristics useful for morphotype comparison. Every sample consisted of six plates containing eight tissue segments each, which were incubated at 25 °C with a photoperiod of 8 hours. For up to 6 weeks, the plates were checked regularly for the appearance of endophytes. Each emerging fungus was transferred to a new plate of potato dextrose agar (Difco, MD, USA) amended with the same antibiotics mentioned above. A stringent screening step was performed to discard redundant isolates from the same sample and to maximize the diversity of cultivable endophytes and hence the possible number of secondary metabolites produced. This process was carried out by comparing characteristics

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such as color, texture, shape, border type, mycelial density, presence-absence of secretions, and growth rate. For the later trait, the isolates were classified as slow growers if presented a radial growth of less than 2 mm per day, medium growers with 2–3.5 mm per day, and fast growers with more than 3.5 mm per day. The resulting isolates, generally few morphotypes per sample, were preserved in the National Biodiversity Institute’s culture collection. From each of the families of plants in the collection, one or two fungal isolates were randomly selected for a total of 364. Additionally, a search was conducted in the literature on endophytic fungi isolated from the country, resulting in 64 isolates. In total, we present a glimpse of the diversity of endophytes in Costa Rica based only on these 427 isolates.

6.4  Molecular Analyses and Taxonomic Assignation For the DNA extraction, 400 mg of mycelia were ground with mortar and pestle in liquid nitrogen and further extracted using the DNeasy Plant kit (Qiagen, USA) including a pretreatment step consisting of incubation at 60 °C for 1  hour with 400 μL of the kit’s lysis buffer and 30 μL of Proteinase K (20 mg mL, Sigma Aldrich, USA). The ITS1-5.8S-ITS2 region was amplified using primers ITS1 and ITS4 (White et al. 1990) with the following reaction conditions: 95 °C for 10 minutes, 35 cycles at 94 °C for 1 min, 54 °C for 1 min, 72 °C for 1 min, and additional extension at 72 °C for 10 min. The products were purified using the NucleoSpin Extract II kit (Macherey-Nagel, Germany) according to manufacturer’s protocol. Sanger sequencing was performed at the sequencing facility of the Dana-Farber Cancer Institute at the Harvard University, Boston, Massachusetts, USA. Sequences were assembled using SeqMan program of DNASTAR’s Lasergene 8.0. The taxonomic assignment was performed by comparing the ITS1-5.8S-ITS2 sequences against the UNITE database (https://unite.ut.ee/). Every assignment was verified against the Index Fungorum (http://www.indexfungorum.org) and the MycoBank database (http://www.mycobank.org). Records were appropriately curated when synonyms or current names were identified. The statistical analyses and visualizations were performed in R (R-Core-Team 2015). We used vegan (Oksanen et al. 2014) to calculate alpha diversity estimators and canonical correspondence analysis (CCA), based on several matrices with the abundance distribution of the fungal isolates according to variables such as plant habit, season, growth rate, and site.

6.5  Determination of Bioactive Molecules Data on bioactive molecules produced by fungal endophytes isolated from tropical plants in Costa Rica come from different sources in the literature. Although the methodology is described in each referenced study, in general, the processes for the

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fermentation, chemical isolation, assay determination, and structural elucidation included the following steps: (a) Fungi are fermented in liquid or solid media. (b) Chemical extraction and pre-fractionation are performed by adsorbing the crude extracts onto HP20 beads. Then, beads are washed with water and eluted with different mixtures of ethanol/water. (c) Bio-guided assays. The resulting fractions are screened against a variety of assays (i.e., antimalarial, antibacterial, antifungal, and anticancer). (d) Re-extraction. Fungi considered positive for specific tests are cultured and extracted again. Crude extracts are partitioned with a chromatography column of silica gel and further purified by reverse-phase HPLC. (e) Structural elucidation of molecules. HRESIMS analysis are normally used to determine the molecular formula, while the structures were established from NMR analysis using one- and two-dimensional techniques including proton, carbon, gCOSY, gHMQC, gHMBC, DEPT, and ROESY.  In some cases, the structure determination was complemented using single-crystal X-ray diffraction techniques.

6.6  Results This work presents a glimpse of the diversity of cultivable fungal endophytes in different life zones and types of tropical forests in Costa Rica. It contains the description of 427 fungal endophytes isolated from 280 species of vascular plants grouped into 107 families. These endophytes were isolated from various sites, plant habits, altitudes, seasons, and tissues. All the isolates were classified in the phylum Ascomycota. Within this, they were assigned to four classes, 21 orders, 49 families, and 83 genera. A distinctive feature of the population of endophytes was the presence of few highly abundant taxa coexisting with a majority of less represented fungi occurring in a narrower range of hosts, sites, habits, and tissues. Furthermore, we observed that the 20% most common genera represented 77.5% of the total population of endophytes. In Table  6.2, we provide a list of all the fungal genera identified in Costa Rica with the respective plant host species. This information evidences a possible host range of each fungal genus. We observed, for example, that more than half of the isolates were isolated from a single host while nearly 10% of the genera were isolated from more than ten hosts. The most abundant fungal class was Sordariomycetes, while the most abundant orders were Xylariales, Glomerellales, and Diaporthales, which represented, respectively, 24.8%, 21.8% and 15.0% of the isolates (Fig. 6.2). Within these orders, the most common genera were Xylaria, Colletotrichum, and Diaporthe, which were present in a wide variety of plant families, with distinct habits; different sites, seasons, and altitudes; and various tissues. In terms of the number of genera per fungal

Fungal genus Apioclypea Pseudovalsaria Guignardia Neodeightonia Neoscytalidium Phyllosticta

Plant species Bolbitis portoricensis and Elaphoglossum doanense Bactris hondurensis Nephrolepis biserrata and Phlebodium pseudoaureum Bromelia pinguin Bixa urucurana Aspidosperma myristicifolium, Calliandra bijuga, Dendropanax caucanus, Eschweilera longyrachis, Eugenia oerstediana, Goethalsia meiantha, Guapira costaricana, Guarea grandifolia, Guarea sp., Guatteria lucens, Hirtella triandra, Licaria cufodontisii, Podandrogyne decipiens, Rhizophora mangle, Rinorea sylvatica, Sapranthus viridiflorus, Simarouba amara, and Stachytarpheta frantzii Capnodiales Cladosporium Acacia collinsii, Apeiba membranacea, Ardisia revoluta, Davilla kunthii, Guatteria tonduzii, Nephrolepis biserrata, Phlebodium pseudoaureum, Senna pallida, and Vitex gaumeri Lecanosticta Crateva tapia and Schoepfia frutescens Mycosphaerella Dioscorea racemosa, Licania arborea, and Struthanthus cassythiodes Passalora Dalbergia brownei Pseudocercospora Crescentia alata, Licania platypus, Marsdenia sp., and Stromanthe tonckat Pseudocercosporella Brosimum alicastrum Ramichloridium Hirtella racemosa Septoria Acalypha diversifolia, Cissampelos pareira, Cordia sp., Croton schiedeanus, Diospyros digyna, Heliconia tortuosa, Ipomoea sp., Rubus urticaefolius, Semialarium mexicanum, Thouinidium decandrum, and Vismia bilbergiana Chaetosphaeriales Menispora Vochysia guatemalensis Chaetothyriales Cyphellophora Conostegia micrantha and Platymiscium curuense Coryneliales Corynelia Lonchocarpus minimiflorus and Quercus oleoides Diaporthales Aurapex Geonoma hoffmanniana Cytospora Conocarpus erectus and Hypoxis decumbens

Fungal order Amphisphaeriales Boliniales Botryosphaeriales

Table 6.2  List of the orders and genera of endophytic fungi identified in tropical forests of Costa Rica with the respective plant host species

116 K. Rojas-Jimenez and G. Tamayo-Castillo

Erysiphales Eurotiales

Fungal order

Talaromyces Colletotrichum

Oidium Aspergillus Penicillium

Phomopsis

Fungal genus Diaporthe

(continued)

Plant species Ardisia standleyana, Aspidosperma myristicifolium, Asplenium sp., Billia rosea, Brachyonidium sp., Cardamine hirsuta, Cavendishia sp., Clethra formosa, Clethra suaveolens, Clibadium leiocarpum, Conostegia sp., Costus sp., Cyathula prostrata, Dendropanax arboreus, Erythrochiton gymnanthus, Forestiera cartaginensis, Forsteronia spicata, Gaiadendron punctatum, Goethalsia meiantha, Hypericum sp., Inga multijuga, Inga sapindoides, Malvaviscus arboreus, Meliosma donnellsmithii, Miconia tonduzii, Monnina costaricensis, Morella cerifera, Prunus fortunensis, Ruagea glabra, Sapranthus viridiflorus, Sigesbeckia jorullensis, Stylogyne laevis, Virola koschnyi, and Weinmannia fagaroides Asclepias curassavica, Aspidosperma myristicifolium, Bactris hondurensis, Bombacopsis quinata, Capparis discolor, Croton billbergianus, Croton schiedeanus, Cupania livida, Guapira costaricana, Guarea sp., Guazuma ulmifolia, Hirtella triandra, Inga sp., Malvaviscus achanioides, Phryganocydia corymbosa, Protium costaricense, Sapranthus viridiflorus, Sloanea petenensis, Sommera donnellsmithii, Theobroma angustifolium, Virola koschnyi, Virola surinamensis, and Vochysia ferruginea Terminalia catappa Cyclopeltis semicordata Ageratina ixiocladon, Bolbitis portoricensis, Cyclopeltis semicordata, Guatteria lucens, Heisteria concinna, Oleandra articulata, Phlebodium pseudoaureum, and Platymiscium curuense Oxalis frutescens Acalypha diversifolia, Acalypha ferdinandii, Ageratina badia, Alloplectus ichthyoderma, Aphelandra scabra, Ardisia sp., Aspidosperma megalocarpon, Astronium graveolens, Besleria notabilis, Billia rosea, Bolbitis portoricensis, Bougainvillea glabra, Burmeistera cyclostigmata, Bursera simaruba, Carica papaya, Casearia corymbosa, Cecropia peltata, Celtis iguanaea, Cestrum strigilatum, Chomelia spinosa, Clibadium leiocarpum, Coix lacryma-jobi, Crescentia alata, Croton billbergianus, Dendropanax arboreus, Dioscorea standleyi, Dracaena americana, Elaphoglossum doanense, Garcinia madruno, Gaultheria gracilis, Goethalsia meiantha, Guatteria lucens, Guazuma ulmifolia, Heisteria concinna, Hirtella triandra, Hoffmannia sp., Inga spectabilis, Jacaratia dolichaula, Jaegeria hirta, Malpighia glabra, Malvaviscus arboreus, Melicoccus bijugatus, Miconia longibracteata, Mimosa pudica, Myrciaria floribunda, Neomirandea costaricensis, Ocotea sp., Peperomia sp., Piper friedrichsthalii, Piper irazuanum, Pseudobombax septenatum, Psychotria aubletiana, Psychotria horizontalis, Psychotria solitudinum, Pteromischum sp., Quararibea asterolepis, Rauvolfia tetraphylla, Rinorea sylvatica, Schefflera rodriguesiana, Schefflera sp., Simarouba amara, Sloanea petenensis, Sloanea terniflora, Solanum circinatum, Solanum ramonense, Talisia allenii, Terminalia amazonia, Topobea pittieri, Trichilia tuberculata, Vochysia guatemalensis, Witheringia sp., Witheringia coccoloboides, Xylosma oligandra, and Zanthoxylum melanostictum

6  Fungal Endophytes and Their Bioactive Compounds in Tropical Forests of Costa Rica 117

Ophiostomatales Pleosporales

Microascales Microthyriales

Magnaporthales

Hypocreales

Helotiales

Fungal order

Ophiostoma Alternaria

Hydropisphaera Ilyonectria Metarhizium Microcera Nectria Trichoderma Volutella Gaeumannomyces Mycoleptodiscus Ophioceras Pyricularia Lanspora Paramicrothyrium

Fungal genus Plectosphaerella Chalara Cryptosporiopsis Hymenoscyphus Pezicula Acremonium Clonostachys Cosmospora Fusarium

Table 6.2 (continued)

Plant species Malvaviscus achanioides and Piper sp. Asterogyne martiana Ageratina anisochroma Tovomita weddelliana Schefflera systyla Spigelia palmeri Palicourea elata Triplaris melaenodendron Aspidosperma myristicifolium, Desmodium adscendens, Palicourea elata, Palicourea pilosa, Palicourea tomentosa, and Psidium guajava Neomirandea angularis Blakea anomala, Cavendishia complectens, Centropogon granulosus, and Hedyosmum goudotianum Kohleria spicata Dorstenia drakena Capparis cynophallophora and Drymonia coriacea Dicranopygium umbrophilum and Rinorea sylvatica Burmeistera cyclostigmata Gonzalagunia rosea and Phyllanthus sp. Cissus alata, Oleandra articulata, Turnera ulmifolia, Zanthoxylum caribaeum Elaphoglossum doanense, Phlebodium pseudoaureum Mapania assimilis Zanthoxylum melanostictum Bolbitis portoricensis, Cyclopeltis semicordata, Elaphoglossum doanense, Phlebodium pseudoaureum, and Tectaria athyrioides Oleandra articulata and Phlebodium pseudoaureum Psychotria carthagenensis

118 K. Rojas-Jimenez and G. Tamayo-Castillo

Xylariales

Rhytismatales Sordariales

Fungal order

Preussia Pyrenochaeta Rhizopycnis Splanchnonema Coccomyces Chaetomium Corynascus Podospora Spadicoides Annulohypoxylon Anthostomella Arthrinium Coniolariella Daldinia

Delitschia Edenia Massarina Paraconiothyrium Phoma

Fungal genus Asteromella Cochliobolus Corynespora

(continued)

Plant species Erythroxylum macrophyllum Gentlea micranthera Acalypha ferdinandii, Astronium graveolens, Cecropia peltata, Cordia eriostigma, Gmelina arborea, Quararibea asterolepis, Rinorea sylvatica, Sloanea petenensis, and Thouinidium decandrum Alloplectus ichthyoderma Nephrolepis brownii Youngia japonica Dendropanax arboreus, Smilax sp., and Stromanthe tonckat Zanthoxylum fagara, Columnea microcalyx, Polygonum punctatum, Solanum rudepannum, and Stemmadenia pubescens Byrsonima crassifolia and Daphnopsis americana Pachira aquatica Casearia corymbosa, Hedyosmum scaberrimum, Ouratea lucens, and Spondias mombin Bursera simaruba, Caesalpinia platyloba, and Conocarpus erectus Disterigma humboldtii Persea americana Guatteria tonduzii and Terminalia catappa Allophylus racemosus and Chrysophyllum cainito Bolbitis portoricensis Elaphoglossum doanense, Nephrolepis biserrata, and Oleandra articulata Bolbitis portoricensis, Elaphoglossum doanense, Oleandra articulata, and Tectaria athyrioides Palicourea solitudinum, Palicourea tomentosa, and Psychotria tsakiana Aciotis indecora and Begonia semiovata Coccoloba padiformis, Hirtella triandra, Neurolaena lobata, Sapranthus viridiflorus, Trema micrantha, and Urera baccifera

6  Fungal Endophytes and Their Bioactive Compounds in Tropical Forests of Costa Rica 119

Fungal order

Microdochium Muscodor Nemania Pestalotiopsis

Fungal genus Discosia Discostroma Entonaema Eutypa Hypoxylon

Table 6.2 (continued)

Plant species Calophyllum brasiliense, Ichnanthus pallens, and Piptocarpha poeppigiana Inga barbourii Miconia argentea Acalypha ferdinandii and Cordia eriostigma Acalypha diversifolia, Bixa urucurana, Brosimum costaricanum, Cirsium subcoriaceum, Erythrochiton gymnanthus, Ficus insipida, Gleichenia sp., Licania arborea, Oreopanax nicaraguensis, Pouteria reticulata, Quararibea asterolepis, Sloanea petenensis, and Tectaria athyrioides Galinsoga quadriradiata and Sigesbeckia jorullensis Miconia argentea Acalypha sp., Palicourea acuminata, and Phlebodium pseudoaureum Blechnum occidentale

120 K. Rojas-Jimenez and G. Tamayo-Castillo

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Fig. 6.2  Relative abundance of 21 orders of endophytic fungi isolated from tropical forest of Costa Rica

order, the higher diversity was observed in Xylariales, Pleosporales, and Hypocreales that presented, respectively, 14, 13, and 11 genera. The inclusion of a broader spectrum of variables during the design of plant sampling and techniques used for isolation had a positive effect in obtaining higher phylogenetic diversity of fungal groups. For example, we observed that some fungal orders were exclusively obtained when sampling was performed in particular plant habitats (e.g., ferns and shrubs), altitudes (e.g., 2000–3000 m), and types of tissues (Fig. 6.3). A significant number of orders were isolated only from plants collected in the dry season while others only during the rainy season. The inclusion of more sampling sites also had a direct effect on increasing the diversity of fungal groups. Also, we noted that several orders of fungi were exclusively slow growers. We performed canonical correspondence analyses using an abundance table of the different genera to explore the relationships between the endophyte communities and the main variables studied (Fig. 6.4). In general, the analyses showed differences in the composition of the genera for each of the variables evaluated. For example, the endophyte communities in herbs were separated from those of other plant habits. In the same way, the community of fungi from roots was separated from that of other tissues. Each altitudinal range seems to have its particular fungal community composition, while the populations of slow and medium growers were more similar to each other in comparison to the fast growers. In Table 6.3, we show the richness and Shannon diversity values calculated for each category within the main variables studied. Higher values of Shannon diversity were mainly observed in samples collected during the rainy season and in altitudes below 1000 meters. A higher diversity was also observed among slow-growing

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Fig. 6.3  Distribution of the relative abundance of the fungal orders according to the main methodological variables considered during the plant sampling and endophyte isolation

Fig. 6.4  Canonical correspondence analysis of the endophytic communities according to some of the methodological variables considered during the plant sampling and endophyte isolation

endophytes. Regarding plant habit and tissue, higher diversity values were obtained from trees and leaves, respectively. However, these estimations should be taken with care due to inequal sampling. The total diversity of the endophytic fungal communi-

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Table 6.3 Values of the richness (number of fungal genera) and Shannon diversity index determined for different methodological factors influencing the sampling and fungal isolation Variable Season Altitude

Habit

Tissue

Growth rate

Category Dry Wet 0–1000 m 1000–2000 m 2000–3000 m Fern Herb Others Shrub Tree Branch Leaf Petiole Root Fast Medium Slow

n 215 212 316 65 46 41 50 30 111 195 57 314 42 11 90 202 94

Richness 37 68 71 23 11 16 24 17 33 45 28 65 17 7 20 41 36

Shannon 2.74 3.60 3.48 2.39 1.76 2.54 2.71 2.62 2.72 3.01 2.8 3.25 2.51 1.66 2.46 2.64 3.20

ties analyzed at the genus level, and according to the Shannon index, reached a value of 3.38 which is considered moderate to high. Over the past two decades, several studies have described bioactive molecules produced by endophytic fungi isolated from tropical forests of Costa Rica. In Table 6.4, we summarize the main findings, and in Fig. 6.5, we show the chemical structure of the compounds. Up to date, 17 novel bioactive molecules have been described, most of which are metabolites, but also, there are reports of volatile organic compounds and peptides. These molecules have shown positive antibacterial activities, antimicrobial, antifungal, antimalarial, and anticancer. It is essential to highlight that members of Leotiomycetes produced 24% of these bioactive molecules, despite this fungal class representing nearly 1% of the population of endophytes.

6.7  Discussion We showed that the richness and diversity of fungal endophytes in tropical forests of Costa Rica are relatively high and that these organisms constitute a rich source of bioactive compounds. Our results are consistent with those obtained in other studies showing that the majority of endophytic fungi isolated from tropical plants belong to the phylum Ascomycota and that few highly abundant taxa, mostly from orders Xylariales, Glomerellales, and Diaporthales, coexist with a broader diversity of less abundant taxa (Arnold and Lutzoni 2007; Cannon and Simmons 2002; Lodge et al. 1996; Smith et al. 2008; Suryanarayanan et al. 2003). Furthermore, we determined

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Table 6.4  Bioactive compounds from endophytic fungi isolated from tropical forests of Costa Rica Molecules Asterogynins

Biological activity/target Antimalarial PfHsp86 Plasmodium falciparum

Codinaeopsins

Antimalarial P. falciparum

Collutellin A

Immunosuppressive and antimycotic CD4+ T-cell IL2 Botrytis cinerea and Sclerotinia sclerotiorum Antibacterial BIA E. coli BR513 and gram-positive

Cytoskyrins

Cytosporones

Antibacterial Gram-positive

Delitzchianones

Antimalarial P. falciparum

Dicerandrols

Anticancer Cell line RPMI8226

Antibiotic Esters of propanoic acid, 2-methyl-; butanoic acid, 2 Pythium ultimum and 3-methylGuanacastepenes Antibacterial MRSA and VREF

Mirandamycin

Neolambertellins

Antibacterial E. coli, Pseudomonas aeruginosa, Vibrio cholerae, MRSA, and Mycobacterium tuberculosis Anticancer CMG2 inhibition

Fungal isolate/ plant host Chalara alabamensis CR1488E Asterogyne martiana Codinaeopsis sp. CR127A Vochysia guatemalensis Colletotrichum dematium Pteromischum sp.

Cytospora sp. CR200 Conocarpus erectus Cytospora sp. CR200 Conocarpus erectus Delitschia sp. CR237A Alloplectus ichthyoderma Penicillium sp. CR1642D Ageratina ixiocladon Oidium sp. Terminalia catappa Unknown fungus CR115 Daphnopsis americana Septofusidium herbarum 1223D Neomirandea angularis

Reference Cao et al. (2010)

Kontnik and Clardy (2008)

Ren et al. (2008)

Brady et al. (2000a), Singh et al. (2007) Brady et al. (2000c), Singh et al. (2007) Cao and Clardy (2011)

Cao et al. (2012b)

Strobel et al. (2008) Brady et al. (2000b), Singh et al. (2000) Ymele-Leki et al. (2012)

Cao et al. Coccomyces proteae CR252M (2012a) Disterigma humboldtii (continued)

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125

Table 6.4 (continued) Molecules Penexanthones

Pentaketide

Biological activity/target Anticancer Cell lines Dox40, Farage, H929, HT, OPM2, and RPMI8226 Antifungal Candida albicans

Pestalopyrones

Antimalarial P. falciparum

Phomoxanthones

Antimicrobial Bacillus megaterium, Chlorella fusca, Ustilago violacea, and P. oryzae Antimalarial PfHsp86 P. falciparum

Viridiols

6,8-Dihydroxy-3-methyl1H-­isochromen-1-one

Anticancer Cell lines Dox40, Farage, H929, HT, OPM2, and RPMI8226

Griseofulvin and 7-dechlorogriseofulvin

Antifungal

Fungal isolate/ plant host Penicillium sp. CR1642D Ageratina ixiocladon Fusarium sp. CR377 Selaginella pallescens Phomatospora bellaminuta CR1092F Zanthoxylum melanostictum Phomopsis sp. Costus sp.

Chalara alabamensis CR1488E Asterogyne martiana Aurapex penicillata CR1207B Geonoma hoffmanniana Xylaria sp. Whc1 Palicourea marcgravii

Reference Cao et al. (2012b)

Brady and Clardy (2000)

Cao and Clardy (2011)

Elsässer et al. (2005)

Cao et al. (2010)

Cao et al. (2012a)

Hinterdobler and Schinnerl (2019)

that the order Xylariales was both highly abundant and highly diverse, suggesting that this group might be a distinctive inhabitant of the endophytic environments. We demonstrate the positive effect of adding more sites and ecosystems and sampling in both seasons of the year and include various habits of plants, altitudinal ranges, and types of plant tissues. Especially noteworthy was the extension of the incubation period for allowing the appearance of slow-growing fungi. The effects of these methodological variables were observed at the taxonomic level of order but even more conspicuous at the family and genus level. This conclusion is supported by the results of the alpha diversity determinations and the analyses of canonical correspondence, which also showed differences between subcommunities of endophytes within each of the variables studied. This work also acquires a particular relevance for the number and variety of plants considered, which accounted 280 species and 107 families. As a reference, the diversity of vascular plants in North America comprises 235 families (Qian 1998).

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Fig. 6.5  Chemical structure of the main bioactive compounds isolated from fungal endophytes in tropical forests of Costa Rica

The high heterogeneity observed in the assemblages of fungal endophytes of Costa Rica remains to be further elucidated. However, it can be explained by the interaction of reasons, some of which include the following: (a) The fungal mechanisms for dispersal and colonization (Gazis and Chaverri 2010; Rodriguez et al. 2009; Suryanarayanan et al. 2002) (b) The endophyte-host interactions (Lutzoni et  al. 2018; Kogel et  al. 2006; Saikkonen et al. 1998) (c) The fungal life cycle (Chaverri and Gazis 2011) (d) The development of specificities for plant hosts and tissues (Gazis et al. 2016; Arnold et al. 2000; Kumaresan and Suryanarayanan 2001; Sieber 2007; Verma et al. 2007) (e) The endophyte-endophyte interactions (Arnold et al. 2003; Schulz and Boyle 2005) The focus of this work was the description of the diversity of endophytic fungi isolated from the tropical forests of Costa Rica. However, the research that ­originated the data has some limitations. First, the results are based on cultivation-­dependent fungi, which represent a small proportion of the overall diversity. Therefore, it is expected that the differences observed among variables might be even more sub-

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127

stantial, as shown when metagenomic approaches are used (Sun and Guo 2012; Arnold 2007; Jumpponen and Jones 2009; Zuccaro et al. 2003). Second, most studies were conducted primarily for bioprospecting purposes and not to s­ pecifically study ecological characteristics of endophytes. Consequently, the main priority of sampling design was obtaining the most extensive diversity of plants, endophytes, and novel compounds. The strategy is based on the premise that the higher the diversity of fungi, the higher the variety of compounds produced. Still, the inclusion of several variables during sampling and isolation might result in valuable contributions to the understanding of the ecology of fungal endophytes. Moreover, some of the practical lessons learned from our experience with the work of endophytic fungi in tropical forest of Costa Rica are the following: (a) The inclusion of more sampling sites has a substantial effect on the number of taxonomic groups obtained and, consequently, on biodiversity. It is preferable to sample several sites than to sample only one with greater depth. (b) Despite rainy season showing higher diversity, some groups occurred only during the dry season. (c) The most substantial diversity of endophytes was observed in lower altitudes (100 6 ± 0 – – 6 ± 0 – – 2 ± 0 – – 3 ± 0 – – 4 ± 0 – – 19 ± 0 – – 2 ± 0 – – 5 ± 0 – – 5 ± 0 – – 1 ± 0 – – 5 ± 0 – – 0 ± 0 – –

Herbicidal activity (number of seeds) Lactuca Allium Sativa schoenoprasum 1 ± 1 2 ± 1 4 ± 1 2 ± 1 5 ± 0 1 ± 0 5 ± 1 2 ± 1 4 ± 0 1 ± 1 5 ± 0 1 ± 1 4 ± 1 2 ± 1 2 ± 0 5 ± 1 5 ± 1 2 ± 1 2 ± 0 4 ± 1 4 ± 1 2 ± 1 1 ± 1 4 ± 0 2 ± 1 4 ± 1

Table 16.1  Biological activities of plant and endophytic fungi extracts

– – – – – * – – –

0 ± 9 0 ± 8 5 ± 1 8 ± 20 0 ± 0 18 ± 7 0 ± 5 12 ± 2 9 ± 13

– – – – – – – – –

– – – – – – – – –

% inhibition of T. cruzi % inhibition IC504 IC505 SI3 >34.5 * * * – * * * – 100 ± 11 – – – 19 ± 4 – – – 0 ± 0 – – – 0 ± 2 – – – 0 ± 3 – – – 0 ± 2 – – – 0 ± 5 – – – 0 ± 2 – – – 0 ± 1 – – – 0 ± 1 – – – 0 ± 1 – – – – – – – – – – –

SI6 * * – – – – – – – – – – – 13 9 6.6 48 3 90 10 8 29

– – – – – ≥200 – – –

– – – – – 0.275 – – –

% inhibition of P. falciparum % inhibition MLD507 IC504 44 – – 37 – – 97 ≥200 0.6 32 – – 32 – – 42 – – 3 – – 25 – – 10 – – 16 – – 30 – – 12 – – 49 – –

– – – – – ≥727 – – –

SI6 – – ≥333 – – – – – – – – – –

0 ± 0 0 ± 3 2 ± 5 9 ± 6 0 ± 0 0 ± 3 36 ± 4 13 ± 0 23 ± 11 0 ± 12 * 65 ± 3

2 ± 1 1 ± 1 5 ± 0 2 ± 1 2 ± 1 1 ± 1 2 ± 0 1 ± 0 1 ± 0 2 ± 1 3 ± 1 2 ± 1

4 ± 1 4 ± 1 5 ± 0 4 ± 1 4 ± 1 5 ± 0 4 ± 1 5 ± 1 4 ± 1 4 ± 1 5 ± 1 4 ± 1 – – – – – – – – – – * –

– – – – – – – – – – * –

–

15444 15447 15449 15451 15457 15458 15466 15483 15491 15519 15521 15527

–

5 ± 3

2 ± 1

5 ± 0

15443

15401 15403 15416 15423 15429 15432 15440 15442

Fungal sp. Fusarium sp. 2 Fusarium sp. 3 Diaporthe sp. 2 Fungal sp. Pestalotiopsis sp. Fungal sp. Colletotrichum gigasporum Neopestalotiopsis sp. 2 Fungal sp. Xylaria sp. Nectriaceae sp. 1 Diaporthe sp. 8 Fungal sp. Diaporthe sp. 6 Colletotrichum sp. Fungal sp. Lasiodiplodia sp. 3 Fungal sp. Xylariaceae sp. 3 Fungal sp.

% protection of DENV-2 % inhibition EC501 CC502 3 ± 2 – – 30 ± 4 – – 10 ± 13 – – 2 ± 6 – – 32 ± 21 – – 5 ± 9 – – 4 ± 1 – – 2 ± 3 – –

Herbicidal activity (number of seeds) Lactuca Allium Sativa schoenoprasum 5 ± 1 4 ± 2 4 ± 0 1 ± 1 4 ± 0 1 ± 1 2 ± 1 5 ± 0 5 ± 0 1 ± 0 4 ± 1 2 ± 1 4 ± 1 1 ± 1 4 ± 0 1 ± 1

UFMGCBa

Fungi

– – – – – – – – – – * –

–

SI3 – – – – – – – –

0 ± 3 0 ± 1 8 ± 7 2 ± 13 0 ± 8 19 ± 6 11 ± 6 0 ± 2 5 ± 9 0 ± 6 0 ± 6 6 ± 8

0 ± 4 – – – – – – – – – – – –

– – – – – – – – – – – – –

–

% inhibition of T. cruzi % inhibition IC504 IC505 0 ± 1 – – 18 ± 1 – – 0 ± 3 – – 0 ± 0 – – 1 ± 7 – – 3 ± 12 – – 0 ± 8 – – 0 ± 6 – –

– – – – – – – – – – – –

–

SI6 – – – – – – – –

5 50 18 9 15 58 46 0 31 41 4 46

9

– – – – – – – – – – – –

–

– – – – – – – – – – – –

–

% inhibition of P. falciparum % inhibition MLD507 IC504 43 – – 18 – – 17 – – 8 – – 10 – – 7 – – 39 – – 50 – –

(continued)

– – – – – – – – – – – –

–

SI6 – – – – – – – –

15528 15536 15538 15540 15544 15555 15564 15568 15572 15573 15576 15582 15590 15596 15598 15601

Diaporthe sp. 7 Xylaria sp. 1 Fungal sp. Diaporthe sp. 9 Fungal sp. Fungal sp. Diaporthe sp. 10 Fungal sp. Diaporthe sp. 10 Diaporthe sp. 10 Fungal sp. Fungal sp. Fungal sp. Fungal sp. Fungal sp. Chaeto sphaeriaceae sp. Fusarium sp. 4 Lasiodiplodia sp. 4 Diaporthe sp. 10 Fusarium sp. 5 Fungal sp. Diaporthe sp. 10

15603 15607 15609 15611 15612 15615

UFMGCBa

Fungi

Table 16.1 (continued)

1 ± 1 1 ± 1 2 ± 0 5 ± 0 5 ± 0 4 ± 0

2 ± 2 5 ± 0 3 ± 0 3 ± 1 4 ± 1 1 ± 1

Herbicidal activity (number of seeds) Lactuca Allium Sativa schoenoprasum 5 ± 0 2 ± 1 5 ± 1 3 ± 0 1 ± 0 4 ± 1 2 ± 0.5 2 ± 1 4 ± 0 3 ± 1 2 ± 0.5 5 ± 1 2 ± 0.5 3 ± 0 2 ± 0.5 2 ± 2 1 ± 0.5 3 ± 0 0 ± 0 2 ± 1 0 ± 0 2 ± 1 4 ± 1 5 ± 1 2 ± 0 2 ± 0 2 ± 0 3 ± 0 0 ± 0 1 ± 0 5 ± 0 5 ± 0 >100 – – – – –

>10 – – – – –

10.5 – – – – –

76 ± 2 25 ± 27 4 ± 7 1 ± 2 1 ± 1 0 ± 14

–

* * – >8 *

SI3 – – – >10 >2 – –

% protection of DENV-2 % inhibition EC501 CC502 13 ± 4 – – 0 ± 10 – – 15 ± 5 – – 94 ± 16 10 >100 76 ± 10 42 >100 34 ± 4 – – 0 ± 1 – – 0 ± 1 1 ± 7 – – 1 ± 3 1 ± 5 * * * * * * 4 ± 11 – – 72 ± 4 12 >100 * * * 2 ± 10 0 ± 5 1 ± 4 85 ± 31 79 ± 43 89 ± 17

– – – – – –

– – – – – –

% inhibition of T. cruzi % inhibition IC504 IC505 0 ± 5 – – 12 ± 12 – – 0 ± 2 – – 5 ± 0 – – 0 ± 2 – – 0 ± 15 – – 85 ± 57 – – 0 ± 0 – – 97 ± 35 – – 66 ± 34 – – 25 ± 36 – – 46 ± 43 – – 17 ± 10 – – 65 ± 99 – – 0 ± 4 – – 97 ± 63 – – – – – – – –

SI6 – – – – – – – – – – – – – – – – 12 20 79 85 97 83

– – ≥200 ≥200 ≥200 ≥200

– – 3.5 1.4 0.87 2.3

% inhibition of P. falciparum % inhibition MLD507 IC504 0 – – 48 – – 0 – – 0 – – 1 – – 16 – – 79 61.4 1.3 87 ≥200 2.3 91 ≥200 2.5 86 ≥200 5.7 90 ≥200 2.4 84 ≥200 0.053 94 ≥200 0.4 8 ≥200 2.1 1 – – 94 ≥200 0.0071

– – ≥57 ≥ 143 230 ≥87

SI6 – – – – – – 47 ≥87 ≥80 ≥35 ≥85 ≥3774 ≥500 ≥95 – ≥28571

UFMGCBa

15617 15621 15631 15634 15637 15653 15703 15704 15707 15716 15718 15725 15730 15732 15733 15738 15743 15744 15753 15766 15784

Fungi

Hypocreales sp. Fungal sp. Diaporthe sp. 10 Fungal sp. Fungal sp. Diaporthe sp. 5 Fungal sp. Fungal sp. Fungal sp. Fungal sp. Fungal sp. Lasiodiplodia sp. 1 Fungal sp. Fungal sp. Fungal sp. Fungal sp. Lasiodiplodia sp. 2 Fungal sp. Fungal sp. Fungal sp. Fungal sp.

Herbicidal activity (number of seeds) Lactuca Allium Sativa schoenoprasum 1 ± 0.5 4 ± 1 3 ± 2 2 ± 2 2 ± 0.5 2 ± 1 1 ± 1 2 ± 1 0 ± 0 2 ± 1 1 ± 0.5 0 ± 0 4 ± 0 2 ± 1 4 ± 0 3 ± 1 4 ± 0 3 ± 0 3 ± 1 4 ± 1 5 ± 1 2 ± 1 4 ± 1 1 ± 1 5 ± 1 3 ± 2 4 ± 1 2 ± 1 5 ± 1 5 ± 1 4 ± 0 2 ± 0 4 ± 1 2 ± 1 5 ± 1 2 ± 1 5 ± 1 3 ± 1 5 ± 1 2 ± 1 5 ± 0 5 ± 0 % protection of DENV-2 % inhibition EC501 CC502 50 ± 5 – – 5 ± 2.5 – – 1 ± 12 – – 0 ± 7 – – 0 ± 14 – – 70 ± 4 7 >100 44 ± 12 5 >100 6 ± 0 – – 10 ± 5 – – 16 ± 7 – – 0 ± 5 – – 13 ± 6 – – 58 ± 14 – – 4 ± 13 – – 20 ± 6 – – 0 ± 4 – – * * * * * * * * * * * * * * * SI3 – – – – – >15 >22 – – – – – – – – – * * * * *

% inhibition of T. cruzi % inhibition IC504 IC505 0 ± 7 – – 75 ± 87 – – 74 ± 93 – – 82 ± 54 – – 80 ± 92 – – 0 ± 6 – – 0 ± 3 – – 0 ± 4 – – 0 ± 12 – – 0 ± 8 – – 0 ± 3 – – 0 ± 4 – – 0 ± 2 – – 0 ± 3 – – 0 ± 7 – – 0 ± 3 – – 0 ± 1 – – 0 ± 12 – – 0 ± 7 – – 0 ± 4 – – 74 ± 50 3.6