Handbook of Techniques in Plant Histopathology 3031146581, 9783031146589

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João Paulo Rodrigues Marques Marli Kasue Misaki Soares

Handbook of Techniques in Plant Histopathology

Handbook of Techniques in Plant Histopathology

João Paulo Rodrigues Marques  Marli Kasue Misaki Soares

Handbook of Techniques in Plant Histopathology

João Paulo Rodrigues Marques Basic Science Department Faculty of Animal Science and Food Engineering University of São Paulo Pirassununga, São Paulo, Brazil

Marli Kasue Misaki Soares Biological Sciences Department Luiz de Queiroz College of Agriculture University of São Paulo Piracicaba, São Paulo, Brazil

Translation from the Portuguese language edition: “Manual de Técnicas Aplicadas à Histopatologia Vegetal” by João Paulo Rodrigues Marques and Marli Kasue Misaki Soares, © The Authors 2021. Published by Editora FEALQ. All Rights Reserved.

ISBN 978-3-031-14658-9    ISBN 978-3-031-14659-6 (eBook) https://doi.org/10.1007/978-3-031-14659-6 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of 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

Foreword

To understand the conception of this book, it is necessary to know a little about the trajectory of its authors, whom I met at different times in my professional life: Ms. Marli Kasue Misaki Soares, when I was still a recent doctoral student in the area of Plant Anatomy, and Dr. João Paulo Rodrigues Marques, four years before I became a Full Professor at the Luiz de Queiroz College of Agriculture (ESALQ). Ms Soares graduated in Pharmacy, in 1991, from the Methodist University of Piracicaba (UNIMEP), was approved in the competition for the position of technician of the Plant Anatomy Laboratory (LanVeg) of ESALQ, in 1994. Since then, she is the technician responsible for the LanVeg, having completed, in 2013, the Master in Physiology and Biochemistry of Plants, in ESALQ. She is a professional who loves what she does and, therefore, develops her work with enthusiasm, seriousness, and dedication, always seeking to improve her knowledge in the area. I met Dr. Marques in September 2002, when he was still a first-year student in the biology course at UNIMEP. From 2002 to 2012, under my guidance, he was a FAPESP scholar in Scientific Initiation, master’s, and PhD.  His studies always addressed the histopathology of plant-pathogen interaction, having continued in this same line of research in the postdoctoral fellowship, under the supervision of Professor Maria Lúcia Carneiro Vieira of the Department of Genetics of ESALQ. I highlight that, since his scientific initiation, João Paulo has always been interested in histological techniques, researching the literature, and proposing new methodologies. The scientific training of the authors and the years of coexistence at LanVeg allowed the exchange of ideas and laboratory experiences that led to the preparation of this excellent Handbook of Techniques in Plant Histopathology. The handbook has been carefully prepared with detailed descriptions of sample preparation methods for light microscopy, epifluorescence microscopy, and electron microscopy. The illustrations are of excellent quality and assist the reader in interpreting the expected results with the methodologies employed. In conventional sample preparation, the cutting plans are illustrated and the procedures for fixation, dehydration, embedding, sectioning, staining, and mounting of

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microscopy slides are explained. The preparation of fixative solutions, stains, and reagents is described in detail, as well as the purpose of using each one. This is an unprecedented work, whose great differential in relation to other technical books is related to the set of procedures aimed at histopathological analysis, some of them also unprecedented. It describes techniques for routine analysis, immunocytochemistry, as well as fast, efficient, and low-cost techniques for fungal detection. The handbook will be very useful to students and professionals of different specialties and cannot be missing in the laboratories of those dedicated to studying plant-pathogen interactions. Full Professor (MS-6), Department of Biological Sciences Luiz de Queiroz College of Agriculture, University of São Paulo, São Paulo, Brazil

Beatriz Appezzato da Glória

Acknowledgments

We would like to express our thanks to the people who helped in so many ways in the production of this book. First, we thank Prof. Dr. Beatriz Appezzato da Glória for her incentive, trust, and example. Her comments made in the embryonic stages of this work were very much appreciated. We also thank her for the opportunity to conduct most of the techniques in experiments in the Laboratory of Plant Anatomy, Luiz de Queiroz College of Agriculture (ESALQ), University of São Paulo, under her guidance. It is also with honor and gratitude that we received from Professor Beatriz the foreword of this book. To Prof. Dr. Elliot Watanabe Kitajima for the example of professionalism and constant encouragement and stimulus in the study of plant histopathology. To Prof. Dr. Maria Lucia Carneiro Vieira (Department of Genetics, ESALQ) and Dr. Hudson Wallace Pereira de Carvalho (DVTEC, CENA) for their trust and freedom to allow this book to be conducted in parallel with Dr. João Paulo’s PostDoctoral fellowship. The authors also thank Prof. Lilian Amorim and Prof. Marcel Bellato Spósito for the constant support in research projects funded by FAPESP and CNPQ. Dr. Marques is also grateful to the Special Fertilizers and Plant Nutrition Group (GEFEN - Universidade de São Paulo) for creating a favorable and fruitful environment for research. To the University of São Paulo, especially the Luiz de Queiroz College of Agriculture for enabling the development of the experiments over all these years of dedication to research and the Department of Basic Science of the Faculty of Animal Science and Food Nutrition for welcoming Dr. Marques as a Professor. To the different funding and research agencies, São Paulo Reserach Founda­ tion (FAPESP—Processes: 03/05563-1; 06/54926-8; 12/25315-1; 12/25315-1; 16/00118-0), Brazilian National Council for Scientific and Technological Development, Coordination for the Improvement of Higher Education Personnel (PNPD CAPES-001) for funding the research. To the Electronic Microscopy Centers of Editor de Ciências da Vida ESALQ, of the Institute of Biosciences of UNESP in Botucatu, and of the School of Dentistry

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of Piracicaba (FOP), UNICAMP, for the technical support for the development of protocols for transmission electron microscopy. The editorial guidance offered by Mr João Pildervasser and Mrs Sowmya Thodur. Dr. Marques thanks his wife Aricelis, his mother Maria Inês, his father Luiz (in memorian), and his brother Luiz Eduardo for the support and constant encouragement throughout the academic career. The author also thank his newborn son João Miguel for bring hapiness to the family. Ms Soares thanks her parents Misaki Naoyoshi and Yotuko (in memorian), her husband Osvaldo, and her children Guilherme and Isadora for the happy moments and especially in difficult times.

Contents

1

Introduction����������������������������������������������������������������������������������������������    1

2

Conventional Sample Preparation����������������������������������������������������������    3 Collecting and Subdividing Plant Materials����������������������������������������������    3 Fixation������������������������������������������������������������������������������������������������������    3 Types of Chemical Fixatives����������������������������������������������������������������������    6 Acetic Acid��������������������������������������������������������������������������������������������    6 Acetone – CAS No. 67-64-1������������������������������������������������������������������    6 Aldehydes����������������������������������������������������������������������������������������������    6 Ethanol – CAS 64-17-5��������������������������������������������������������������������������    7 Formalin – CAS 50-00-0 ����������������������������������������������������������������������    7 Glutaraldehyde – CAS 111-30-8 ����������������������������������������������������������    7 Osmium Tetroxide – CAS 20816-12-0��������������������������������������������������    7 Uranyl Acetate – CAS 6159-44-0����������������������������������������������������������    8 Fixative Solutions for Light Microscopy ����������������������������������������������    8 Karnovsky’s Solution – Adapted (Karnovsky 1965) ����������������������������    9 Fixative Solutions for Electron Microscopy������������������������������������������   11 Karnovsky Solution��������������������������������������������������������������������������������   11 Buffered Osmium Tetroxide (OsO4)������������������������������������������������������   12 Dehydration������������������������������������������������������������������������������������������������   12 Acetone, Butanol, Ethanol, and Xylol ��������������������������������������������������   12 Ethanol ��������������������������������������������������������������������������������������������������   13 Acetone��������������������������������������������������������������������������������������������������   13 Embedding Media for Light Microscopy��������������������������������������������������   14 Paraffin��������������������������������������������������������������������������������������������������   14 Polyethylene Glycol (PEG)��������������������������������������������������������������������   14 Historesin ����������������������������������������������������������������������������������������������   15 LR-White (London Resin White)����������������������������������������������������������   17 Embedding Media for Electron Microscopy����������������������������������������������   18 LR-White (London Resin White)����������������������������������������������������������   18 Spurr������������������������������������������������������������������������������������������������������   18 ix

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Contents

Sectioning Methods ����������������������������������������������������������������������������������   19 Fresh or Fixed Non-infiltrated Material ������������������������������������������������   19 Sectioning in Cryostat for Fixated Samples������������������������������������������   20 Sectioning Paraffin Blocks��������������������������������������������������������������������   21 Sectioning Plastic Blocks (Historesin and LR-White)��������������������������   21 Sectioning PEG (Polyethylene Glycol) Blocks ������������������������������������   22 Transmission Electron Microscopy�����������������������������������������������������������   22 Trimming the Blocks ����������������������������������������������������������������������������   22 Semi-Fine Sections��������������������������������������������������������������������������������   23 Ultrathin Cuts Sections��������������������������������������������������������������������������   23 3

Techniques for Histopathological Analysis��������������������������������������������   25 Routine Analysis����������������������������������������������������������������������������������������   25 Toluidine Blue����������������������������������������������������������������������������������������   25 Immunohistochemistry������������������������������������������������������������������������������   26 Changes in the Cuticle and Cell Wall��������������������������������������������������������   29 Ruthenium Red and Sudan IV ��������������������������������������������������������������   29 Nile Red and Calcofluor White��������������������������������������������������������������   31 Aniline Blue ������������������������������������������������������������������������������������������   32 Phloroglucin������������������������������������������������������������������������������������������   33 Analyses of Changes in Primary Metabolism��������������������������������������������   35 Detection of Proteins and Lipids������������������������������������������������������������   35 Carbohydrate Detection ����������������������������������������������������������������������������   36 Analyses of Changes in Secondary Metabolism����������������������������������������   38 Detection of Phenolic Compounds��������������������������������������������������������   38 Ferric Chloride ��������������������������������������������������������������������������������������   38 Ferrous Sulfate in Formalin ������������������������������������������������������������������   39 Autofluorescence Detection ������������������������������������������������������������������   40 Detection of Flavonoids ����������������������������������������������������������������������������   41 NEU Reagent ����������������������������������������������������������������������������������������   41 Autofluorescence Detection ������������������������������������������������������������������   42 Detection of Terpene����������������������������������������������������������������������������������   42 NADI Reagent����������������������������������������������������������������������������������������   42 Autofluorescence Detection ������������������������������������������������������������������   44 Detection of Alkaloid��������������������������������������������������������������������������������   44 Dragendorff’s Reagent��������������������������������������������������������������������������   44 Autofluorescence Detection ������������������������������������������������������������������   45 Detection of Reactive Oxygen Species������������������������������������������������������   46 Hydrogen Peroxide – H2O2��������������������������������������������������������������������   46 Superoxide Anion O2−����������������������������������������������������������������������������   47 Tissue Diaphanization After Reaction with DAB or NBT������������������������   49 Crystal Structure Detection������������������������������������������������������������������������   50 Use of Polarized Light ��������������������������������������������������������������������������   50

Contents

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Fungus Detection��������������������������������������������������������������������������������������   51 Light Microscopy��������������������������������������������������������������������������������������   51 Method of Imprinting the Epidermis with Nail Polish��������������������������   51 Bleaching Method Followed by Cotton Blue Staining��������������������������   53 Double Stained with Safranin O and Cotton Blue ��������������������������������   55 Double Stained with Ruthenium Red and Cotton Blue ������������������������   56 Fluorescence Techniques ��������������������������������������������������������������������������   58 Double-Staining with WGA-AF488 and Calcofluor White������������������   58 Analysis of Fungi Expressing GFP��������������������������������������������������������   59 Electron Microscopy����������������������������������������������������������������������������������   60 Conventional Process for SEM��������������������������������������������������������������   60 Osmium Vapor Technique for Scanning Electron Microscopy (SEM)��������������������������������������������������������������������������������   61 Cryofracturing Technique����������������������������������������������������������������������   63

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Detection of Bacteria����������������������������������������������������������������������������������  67 Light Microscopy��������������������������������������������������������������������������������������   67 Fluorescent In Situ Hybridization����������������������������������������������������������   67 Electron Microscopy����������������������������������������������������������������������������������   69 Transmission Electron Microscopy�������������������������������������������������������   69

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Virus Detection������������������������������������������������������������������������������������������  71 Light Microscopy��������������������������������������������������������������������������������������   71 Azure A��������������������������������������������������������������������������������������������������   71 Calcomine Orange and Luxol Brilliant Green ��������������������������������������   73 Fluorescent In Situ Hybridization����������������������������������������������������������   73 Electron Microscopy����������������������������������������������������������������������������������   75 Transmission Electron Microscope��������������������������������������������������������   75

References ��������������������������������������������������������������������������������������������������������   77

Chapter 1

Introduction

Plants are constantly challenged by different microorganisms that try to benefit from the nutrients and shelter that plant tissues can provide. In general, resistance is the rule and disease the exception (Agrios 2005; Pascholati 2011). Resistance can be qualitative or quantitative and is directly linked to an intricate mechanism of perception, signal processing and defense response with different signaling pathways, gene expression, protein synthesis and activity culminating in structural and biochemical changes in order to contain infection or delay colonization (Camargo 2011; Pascholati 2011). From a histopathological point of view, defense can occur by the presence of constitutive (preformed or passive) or induced (postformed or active) structural and biochemical characters that occur at different moments of the interaction between plant and pathogen (Pascholati 2011; Marques et al. 2018). Despite the continuous efforts to understand plant resistance in different pathosystems, plant defense is not always analyzed from the histological point of view. Besides the alterations in the plant tissues, the histopathological studies also have the purpose of evidencing the structures and localization of the phytopathogenic organisms providing valuable details of the penetration and colonization process. To this end, distinguishing the pathogen from plant cells has been a subject of wide use in the academic environment and here some methods will be presented that make it possible to distinguish pathogens from plant cell structures in bright field, fluorescence and electron microscopy. It is also emphasized that the analysis of the plant-­ pathogen interaction and the biochemical and structural barriers require the use of different instruments and techniques that can be simple and fast, in the case of a preliminary analysis, or more elaborate techniques that require the use of more sophisticated instruments and previous training. An important recommendation refers to thoroughly analyzing the symptoms of the disease under study, i.e. understanding the structural differences between injured and adjacent tissues. The work of Marques et al. (2010) may become a model to be followed throughout the analysis, because the authors investigated the lesions of

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 J. P. Rodrigues Marques, M. Kasue Misaki Soares, Handbook of Techniques in Plant Histopathology, https://doi.org/10.1007/978-3-031-14659-6_1

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1 Introduction

leaves and fruits infected by the citrus leprosis virus focusing on the different histopathological characteristics of each sector of the lesion. Throughout this work, different techniques are described in bright field, fluorescence and electron microscopy highlighting the importance of choosing the procedures according to the need and resources available to the researcher. It should be added that this Handbook of Techniques does not aim to investigate the operation of different types of microscopes, but to guide the student and researcher in the search for the best procedures to investigate the biological phenomenon of biotic stress in plants. Reagent or dye codes are provided as colorimetric indices (C.I.) or CAS number (www.cas.org). Some methods presented here can also be applied to investigate the effect of abiotic stress on plant tissues.

Chapter 2

Conventional Sample Preparation

Collecting and Subdividing Plant Materials Collecting the samples for the preparation of histopathological slides is the great importance. In this step, care should be taken to avoid those tissues with disease symptoms are altered by handling of the material (e.g. tweezers). In addition to diseased samples, always collect material normal, disease-free tissues of age to compare changes. It is considered an essential step to have samples closer to healthy tissue, without lesions or stress symptoms. The number of repetitions is essential. It is suggested that a minimum of three to four samples be collected from symptomatic and normal tissues. The sample must be identified, labeling the vials with pencil and indicate the name of the species, date and place of collection, and the name of the collector, After collection, it is best to place it immediately in the fixative solution. If this is not possible, the samples must be kept in a humid place for the shortest possible time, inside humidified plastic bags and, if necessary, rehydrated before fixation. The crucial point in the harvest process is to know how to separate the samples according to section plan (Fig. 2.1). This question should be directly associated with its object of study, for example, to confirm the presence of secretory ducts (Marques et al. 2010) or the presence of tyloses along xylem vessel elements (De Micco et al. 2016) longitudinal sections should be performed in addition to cross sections.

Fixation The fixation is the most important stage in the samples processing because it is when cell death occurs, preserving the tissue as close as possible to natural living conditions. Thus, the histopathological symptom can be correctly analyzed without © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 J. P. Rodrigues Marques, M. Kasue Misaki Soares, Handbook of Techniques in Plant Histopathology, https://doi.org/10.1007/978-3-031-14659-6_2

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2  Conventional Sample Preparation

Fig. 2.1  Methods of subdividing botanical organs. Cross and longitudinal sections of leaves and stems. The leaves can be sectioned paradermic

Fixation

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the interference of flaws in the technique (artifacts) resulting from preparation of the material. If this step is performed without care, it is likely that there will be detachment of the plasma membrane and consequent plasmolysis, being an undesirable phenomenon for histopathological analyses. The ideal is to work with small samples (up to 1 cm2 for light microscopy and up to 3 mm2 for transmission electron microscopy), which facilitate the penetration of the fixative, which should be selected for the material and technique to be performed. The rule is: smaller tissues samples are the best samples! For electron microscopy, it is recommended to cut the sample in a drop of fixative, preferably on a dental wax plate and in an exhaustion hood (Fig. 2.2a). After removing the portion of the tissue of interest from its organ of origin, the sample is can be kept for few seconds in a drop of buffers and then transferred to a vial containing fixative. The volume of fixative should be at least 10 times the size of the sample as indicated in Fig. 2.2. The samples require the use of a desiccator coupled to a vacuum pump to remove the air from the plant tissues, facilitating the penetration of the fixative. This step is critical. If it is done without due care the sample will

Fig. 2.2  Appropriate setup for harvest tissue to be prepared for light and electron microscopy. (a) Subdivide into small fragments (10–12 mm2) on a dental wax. Arrows indicate that the plant sample may remain in drop containing buffer (arrowhead) and then transferred to vials with fixative (arrow). (b) Samples subject to vacuum pump. (c) Fixation is complete when samples sink into the fixative (arrow). Avoid use tweezers as it can cause tissues crushing

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accumulate air and thus the fixative will not have access to the internal tissues of the structure, promoting structural damage to the tissues.

Here a rule applies: poorly fixed tissues generate poor results that negatively influence the histopathological description.

Types of Chemical Fixatives In addition to chemical fixation using alcohols, aldehydes or acetic acid, fixation can be physical, such as cryofixation and air drying. Each fixation method has its own purpose and depends on the equipment and resources available. In this context, we chose to present only chemical fixation due to its wide use and possibility to work at room temperature. Listed below are those that are routinely used for histopathological analyses:

Acetic Acid This acid is one of the most used components in fixatives, because it infiltrates, hardens the tissues and can promote turgescence of the cytoplasm neutralizing the plasmolyzing effect of other reagents such as chromic acid, ethanol and formalin.

Acetone – CAS No. 67-64-1 This reagent presents high hygroscopicity and produces less retention of water in the plant tissues additionally the extraction of lipids is higher. Acetone is miscible with epoxy resins and is not recommended in acrylic resins, such as historesina and LR-White

Aldehydes Different combinations of aldehydes and buffers are used as primary fixatives. These solutions present different speeds of penetration in tissues, preserving protein structures and stabilizing nucleoproteins. The most commonly used aldehyde fixatives are formalin and glutaraldehyde.

Types of Chemical Fixatives

7

Ethanol – CAS 64-17-5 It is a more efficient fixative when combined with formalin or chloroform and acetic acid than when used alone. Pay attention that ethanol itself can wash hydrophobic substances.

Formalin – CAS 50-00-0 It is the most used fixative in histopathology, both alone or mixed with other fixatives, with the buffered fixative being superior. Some factors may interfere in the quality of the fixation, such as the speed of penetration in the tissues, pH and temperature. As it is a smaller molecule, the speed of penetration of formalin is 5–10 times greater than that of glutaraldehyde and osmium tetroxide. This factor must be taken into account when selecting the size of the sample to be fixed. Paraformalin is the methanol-free formalin a white powder sparingly soluble in water.

Glutaraldehyde – CAS 111-30-8 Due to its molecular size and protein-binding property, this reagent penetrates more slowly than formalin. However, it confers better stabilization of cellular components and tissues due to its dialdehyde nature and also by cross-linking with proteins.

Osmium Tetroxide – CAS 20816-12-0 It is used as a post-fixation agent mainly to make lipid substances insoluble in the passage by dehydration (Santos et al. 2007). It has a low penetration rate of about two millimeters deep and therefore should be applied in small samples, as is the case of transmission electron microscopy. It is noteworthy that this reagent is very toxic and volatile and should always be handled in an exhaustion hood, wearing gloves and long-sleeved lab coat.

8

2  Conventional Sample Preparation

Uranyl Acetate – CAS 6159-44-0 Uranyl acetate is commonly used in the process of contrasting ultrafine sections for transmission electron microscopy. In the case of the use of uranyl acetate used in block, usually in the process of post-fixation, its action is given to the phosphate group of various molecules such as nucleic acids and phospholipids (Huxley and Zubay 1961). According to Sesso (2011), in the presence of phosphate and cacodylate, uranyl precipitates. Therefore, care should be taken to wash the samples well in distilled water if the previous step was post-fixation of the samples with osmium tetroxide diluted in phosphate or cacodylate buffer.

Fixative Solutions for Light Microscopy Carnoy (Berlyn and Miksche 1976) Reagents: Ethanol 100% Glacial acetic acid Chloroform

600 ml 100 ml 300 ml

FAA – Formalin, glacial acetic acid and ethanol (Berlyn and Miksche 1976, p. 30) Reagents Ethanol 95% Distilled water Glacial acetic acid Formalin 37%

90 ml 35 ml 5 ml 10 ml

It is one of the most used fixatives in plant anatomy. This fixative can harden the material when the sample remains in the solution for a long time.

FAA – Formalin, acetic acid and alcohol (Johansen 1940, p. 41) Reagents Ethanol 50% or 70% Glacial acetic acid Formalin 37%

90 ml 5 ml 5 ml

Types of Chemical Fixatives

9

FPA – Formalin, propionic acid and ethanol (Berlyn and Miksche 1976, p. 30) Reagents Ethanol 95% Distilled water Propionic acid Formalin

50 ml 35 ml 5 ml 10 ml

Karnovsky’s Solution – Adapted (Karnovsky 1965) Because it is a complex fixative solution it consists of five steps. This solution originally uses sodium cacodylate buffer, but it has been replaced by phosphate buffer because it is less toxic than cacodylate. Our experience of more than two decades shows that there is no problem in replacing the buffer. However, the use of phosphate buffer should be avoided in transmission electron microscopy. Step 1 – Sodium Hydroxide 40% Reagents Sodium hydroxide Distilled water, make up to

4 g (4 tablets) 10 ml

Preparation Add the sodium hydroxide gradually to the water, taking care never to reverse the order. Step 2 – Formaldehyde 4% Reagents Paraformaldehyde Sodium hydroxide 40% Distilled water

8 g 5–8 drops 200 ml

Preparation Add 8 g of paraformaldehyde in 200 ml of distilled water heated to 60 °C and keep it stabilized at this temperature during the whole process. Then add some drops of sodium hydroxide 40% (+/− 8 drops) until the liquid becomes translucent and stir until all paraformalin is dissolved.

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Step 3 – 0.2 M Phosphate Buffer (PBS) – pH 7.2 Reagents Solution A (sodium phosphate, monobasic, 0,2 M) Monobasic sodium phosphate monohydrate Distilled water, make up to

6,9 g 250 ml

Solution B (sodium phosphate, bibasic, 0,2 M) Anhydrous dibasic sodium phosphate Distilled water, make up to

14,2 g 500 ml

Preparation Check the pH of solution A and adjust the pH to 7.2 with solution B. Step 4 – Glutaraldehyde 1% in 0.1 M Phosphate Buffer pH 7.2 Reagents Glutaraldehyde 25% 0,2 M phosphate buffer, pH 7,2 Distilled water Final volume

20 ml 240 ml 240 ml 500 ml

Preparation Add the distilled water and 0.2 M phosphate buffer, then add the glutaraldehyde. Step 5 – Glutaraldehyde, Formaldehyde, Phosphate Buffer, 5:3:2 (v/v) Reagents Glutaraldehyde 1% in 0.1 M phosphate buffer. pH 7.2 0,2 M phosphate buffer, pH 7,2 Formaldehyde 4%

500 ml 300 ml 200 ml

Note: Keep the fixative in the refrigerator in a dark, unopened, labeled bottle. The pH of the solution in step 5 is 7.2

Types of Chemical Fixatives

11

Preparation Follow step 2 of Karnovsky’s solution. Add phosphate buffer (for preparation see step 3 of Karnovsky’s solution).

Fixative Solutions for Electron Microscopy Glutaraldehyde in cacodylate buffer Step 1: Preparation of the cacodylate buffer Reagents: Sodium cacodylate Distilled water q.s.p. (quantity sufficient for preparation)

4,28 g 100 ml

Preparation Dissolve the sodium cacodylate in distilled water. To adjust the pH of the solution, add 0.2 N HCl until the pH reaches 7.25. Step 2: Preparation of the fixative Reagents Glutaraldehyde 25% (ampoule) 0,2 M sodium cacodylate buffer at pH 7,25 Distilled water Total solution

10 ml 20,8 ml 52,5 ml 83,3 ml

Note: Glutaraldehyde is stored in amber glass bottle in the refrigerator. At the time of use wait to thaw. It is recommended to store the vial in the refrigerator (4 °C) overnight before use so that it thaws in time. Be careful because it is toxic

Karnovsky Solution Reagents Glutaraldehyde 25% (ampoule) Paraformaldehyde 20% (ampoule) 0,2 M sodium cacodylate buffer at pH 7,25 CaCl2 0.1 M Distilled water Total solution

10 ml 10 ml 25 ml 1 ml 54 ml 100 ml

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2  Conventional Sample Preparation

Note: This solution is stored in amber glass bottle in the refrigerator. Be careful because it is toxic

Buffered Osmium Tetroxide (OsO4) Step 1: Preparation of the 2% stock solution – Toxic product! Preparation To dissolve the osmium tetroxide crystals the ampoule should be broken inside an amber flask with distilled water, previously boiled, at room temperature. Wait about 15–24 h before using. Observation Store in a dry and well-ventilated place. Do not store the vials in the refrigerator, as the vapors may darken their interior. If the solution becomes dark it should be discarded. Step 2: Dilution for use When fixing the samples dilute in 0.1 M cacodylate buffer pH 7.2 (1:1). Uranyl acetate Reagents Sucrose Uranyl acetate Distilled water

13,3 g 0,5 g 100 ml

Preparation Dissolve the sucrose in distilled water, then add the uranyl acetate. Note  Work with great care, as it is a radioactive product. The uranyl acetate solution must be kept in the refrigerator and in the dark. Always use gloves and fume hood.

Dehydration Acetone, Butanol, Ethanol, and Xylol Since embedding process do not accept or accept little water, it is necessary to replace the water contained in the tissues with dehydrating agents such as acetone, butanol, ethanol, and xylol

Dehydration

13

Uses: Paraffin-embedded materials. Procedure 1. After fixation, samples can be passed directly into 70% ethanol (4 h) and follow dehydration in 80%, 90% and 100% ethanol (2  h) which should be passed at least twice in each ethanol concentration. 2. At the third ethanol change, drip a few drops of erythrosine + clove oil (30 min). 3. Then they should be transferred in progressive mixtures of ethanol and xylol in the proportions of 3:1, 1:1 and 1:3, pure xylol and finally to xylol and chloroform in the proportion 9:1 with the flask sealed for 2 h each step. Observation The process of ethanol exchange for xylol is called diaphanization and is necessary for the paraffin to penetrate the tissues, since it is insoluble in ethanol. In this phase, it is essential to work in the exhaustion hood or at least in a place with plenty of ventilation and away from flames, due to the high toxicity and inflammability of xylol. Contact with the skin should also be avoided as much as possible.

Ethanol Uses: Materials embedded in historesin and immunolabeling (LR-White). Procedure To infiltrate the samples in historesin, dehydration is made with ethanol in increasing order (10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 100%) at room temperature, 10–15 min per step If the samples are already in 70% ethanol, just follow from this concentration. Large samples can be submitted to dehydration under vacuum pump. If necessary, the samples can be kept in 70% alcohol for weeks or months. For immunolabeling the increasing series with cold ethanol (30, 50, 70, 90, 100, 100%) is used.

Acetone Uses: Materials embedded in Spurr. Procedure For embedding samples in epoxy resins such as Spurr or Araldite, the dehydration is done in acetone (30, 50, 70, 90, 100, 100%) for 15 min in each step and at room temperature.

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2  Conventional Sample Preparation

Embedding Media for Light Microscopy Paraffin There are several companies that produce paraffin for use in plant histology such as Sigma-Aldrich®, Sinth®, Merck®, among others. The prices and quality of paraffin also vary. The sections obtained from paraffin-embedded tissues presents a lower quality comparing to those from historesin. Uses: structural and histochemical analysis, immunohistochemistry and in situ hybridization. Procedure 1. The vials containing the samples in 100% xylene shall be placed in an oven at 35  °C and paraffin wax chips shall be added every day for 4  h to overnight. Beside this step prepare an additional vial with paraffin wax chip melted to be used at step 3. 2. The next step is to transfer the vials to the oven at 60 °C, and continuing add two or three paraffin chips every hour. It is know that the xylene is saturated when the paraffin wax chips no longer dissolve. Then three or four partial changes are then performed, every 4 h, when one-half of the liquid is discarded, and pure melted paraffin wax is added. 3. Then, total paraffin changes are performed every 4 h, in order to discard all paraffin with xylene residues, replacing with pure paraffin. 4. The paraffin block consists in solidifying the paraffin with the samples, which must already be oriented for the sectioning. 5. The paper molds (used to prepare the paraffin blocks) should be dipped in the liquid paraffin to form a protective layer that does not stick to the samples. 6. Heat the paraffin in a water bath or in the oven at 60 °C to liquefy both the new paraffin and the paraffin within the samples. 7. Fill the molds with the liquid paraffin and transfer the plant samples, orienting the material and holding it with a wood stick in the paraffin that will solidify at the bottom. Avoid use tweezers as it can cause tissue crush. 8. Eliminate air bubbles that form around the samples.

Polyethylene Glycol (PEG) Uses: Structural, histochemical, immunohistochemical and in situ hybridization analyses. Procedure 1. Place the PEG pellets in a 100 ml beaker and place in a 60 °C oven to melt.

Embedding Media for Light Microscopy

15

Fig. 2.3  Stages of embedding with polyethylene glycol (PEG)

2. After melting the PEG, leave at room temperature until solidified (Fig.  2.3  – Step 1). 3. Add the same amount of distilled water (PEG:distilled water 1:1) (Fig.  2.3  – Step 2). 4. Place the covered beaker in an oven at 60 °C for 24 h. 5. After this time, keep the beaker open and leave it in an oven at 60 °C until the water is completely evaporated (Fig. 2.3 – Step 3). 6. The block preparation consists in solidifying the PEG with the samples, which must already be oriented for the sectioning. 7. The paper molds should be dipped into the liquid PEG so that it forms a protective layer that does not stick to the samples. 8. Heat the PEG in a water bath or in the 60 °C oven to liquefy both PEG pellets and PEG with the samples. 9. Fill the molds with melted PEG and transfer the samples, orienting the material and keep them at room temperature until they get solidified at the bottom.

Historesin The use of methacrylate resin for plant tissues has been implemented as an embedding medium in substitution to paraffin. It confers better preservation of plant tissues and subcellular structures when samples are sectioned on the microtome and is easier to handle (Marques and Nuevo 2022). One of the problems of using historesin lies in the difficulty of the resin to permeate internal tissues of large samples, this fact can be solved with the polymerization of the resin at low temperature (Paiva et al. 2011). Uses: Structural and histochemical analysis.

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2  Conventional Sample Preparation

Reagents Solution A or infiltration solution (Leica historesin kit®) Activator (envelope) Basic resin

0,5 g 50 ml

Solution B (Prepare at time of use only) Solution A Hardener

15 ml 1 ml

Preparation 1. Dissolve the activator in the basic resin in a magnetic stirrer to prepare the solution A. 2. To prepare the solution B, separate 15 ml of solution A and add 1 ml of hardener in a beaker. 3. Shake the solution B for couple of minutes and then use it. Note  Do not pipette the hardener directly from the bottle. Ideally, add a small amount into a 10 ml beaker and then pipette from it. The remainder that is left in the beaker can be discard. Procedure 1. Immediately after dehydration, an intermediate infiltration is performed by transferring the samples to infiltration solution (Solution A) and 100% ethanol (1:1). 2. Place in the desiccator coupled to the vacuum pump for 5–10 min at least 5 times. 3. Leave in rotation on the vial rotation. 4. Keep the materials in this solution for at least 3 h at room temperature. 5. After this time, add an amount equal infiltration solution (Solution A) and 100% ethanol (2:1). 6. Place back in vacuum for a few minutes, in rotation, for at least 3 h. 7. Infiltration is performed by removing all liquid from the flask and replacing it with 100% infiltration solution (Solution A). The infiltration is completed when samples become translucent and sink to the bottom of the vial (about 24  h). Dense, large samples or samples infiltrated in a refrigerator will require a ­longer time (days or weeks). Submit the samples to vacuum followed by rotation is crucial. If proper infiltration does not occur, the material comes apart or crumble when sectioned and compromises the analysis. Different infiltration times have been tested. Hard or large samples require days or even weeks (lignified samples). Changing the infiltration solution is recommended when using long periods of resin infiltration. 8. Polymerization should be carried out in an fumehood because toxic vapors are released.

Embedding Media for Light Microscopy

17

9. Identify the molds, fill the polyethylene molds (histomold) with solution B and place the fragments of plant tissues according to the desired orientation with the wood stick. Note: Leave stirring the solution B, because this way it takes longer to start polymerizing. Another resource used to delay polymerization is to place the bottle containing solution B under agitation in a bath with water and ice. Avoid use tweezers as it can cause tissue crush. 10. Leave to polymerize for 40–120 min at room temperature (23–25 °C) or place in the oven at 37 °C until the material hardens for 10–15 min. 11. To remove the blocks containing the plant material, press the bottom of the mold to unstick it and pull with tweezers, stick it on a wooden block and leave it in a desiccator with silica until the moment of cutting. Observation The infiltration and embedding are performed with the Historesin standard kit (glycol methacrylate) from Leica®, but resins of other brands may be used, such as Technovit 7100 (EMS®), following the manufacturer’s instructions for preparation.

LR-White (London Resin White) This resin is widely used in immunohistochemistry because it is not very viscous and is miscible resin in ethanol. Because it is a hydrophilic resin, the sections tend to detach from the glass slides, so it is necessary that the slides be treated with an adhesive substance such as Poly-L-Lisine. Uses: Structural, histochemical, immunohistochemical and in situ hybridization analyses. Procedure 1. Infiltration is done by transferring the samples to ethanol 100% and LR-White solution (3:1, 1:1, 1:3, pure). It is recommended to leave the samples at least 3–4 h at each stage and, if possible, to submit them to the vacuum pump. Larger, lignified samples or samples with fungal structures (thick-walled spores) require more time. 2. Polymerization is done by placing the sample and resin in a gelatin capsule avoiding the presence of air and then destined to the oven at 60 °C for 48 h. Slides Preparation For the preparation of slides, it is suggested to use a glass staining vials for histological slides 1 . Immerse the slides in 1% hydrochloric acid (HCl) in 100% Ethanol. 2. Wash the slides 3 times with deionized water. 3. Allow the slides to dry. 4. Immerse the slides in 1:10 poly-Lysin (CAS 25988-63-0) solution in distilled water for 5 min.

18

2  Conventional Sample Preparation

5 . Wash 3 times in water. 6. Dry for 10 min at 60 ° C or overnight at room temperature. Protect the slides against dust deposition.

Embedding Media for Electron Microscopy LR-White (London Resin White) Immunocytochemistry assays require the use of hydrophilic resins and LR-White meets these requirements. In addition, it is an electron-beam stable resin with ultra low viscosity. Procedure 1. After dehydration with ethanol, transfer the samples to LR-White solution in the proportions 2:1 and 1:1 for 3 h each. 2. Then put the sample in the pure LR-White resin and leave it for 24 h. 3. After this time, place the samples in the molds and fill them with resin. 4. Place in an oven at 60 °C for 48 h. Observation The polymerization can be done at low temperature UV cure.

Spurr Spurr resin is used for plant tissues because it penetrates small samples quickly and effectively and is low in viscosity. The resin is easy to prepare and mixes quickly with stirring and rotation. The hardness is adjusted by changing the amount of stabilizer (DER 736). The blocks are of good quality for trimming and the sections are strong and provide some protection to the electron beam so that the sections can be deposited directly onto 200–300 mesh grids without compromising them, but the analysis must be conducted with care. Reagents Reagents comprising the Spurr resin, and the weights used in the composition of the resin. (Source: www.emsdiasum.com/microscopy/technical/datasheet/14300.aspx). ERL 4221 DER DMAE NSA

10,0 g 8,0 g 0,3 g 25,0 g

Sectioning Methods

19

Preparation 1. Place the kit components as indicated by the manufacturer. 2. Weigh the first two components (ERL 4221  +  DER 736) in a 100  ml beaker. Shake slowly to avoid air bubbles forming. 3. Then add the NSA and stir again. 4. Finally, add 15–18 drops of DMAE (with a plastic pipette) until 0.3 g is completed and stir again. If the solution is left over, it can be stored in the freezer until the next use, not forgetting to wrap it with aluminum foil (photosensitive solution). Procedure 1. After dehydration with acetone, transfer the samples to acetone and Spurr solution in the proportions 3:1 and 1:1 for 3 h each. 2. Then put in the ratio 1:3, leave overnight. 3. Then put the sample in the pure resin and leave it for 24 h. 4. After this time, place the samples in the molds and fill them with resin. 5. Place in an oven at 60 °C for 48 h.

Sectioning Methods The materials infiltrated both in historesin and paraffin can be sectioned in the rotary microtome, being more used 5–7  μm for historesin and 10–15  μm for paraffin (Fig. 2.4a) and for the slide microtome thickness between 20 and 60  μm is used (Fig. 2.4b), the sections obtained are uniform (Kraus and Arduin 1997). The microtome room should be at a temperature no higher than 25 °C.

Fresh or Fixed Non-infiltrated Material Sectioning of fresh plant material can be done freehand or with the aid of a sliding microtome (Fig. 2.4b) or in a cryostat or freeze microtome (Nakazono et al. 2003; Marques et al. 2018). In the sliding microtome, samples can be sectioned from 20 to 60 μm. It is recommended that the region to be sectioned is always moistened to facilitate the sliding of the knife. The use of the cryostat for sectioning is a facilitator to obtain sections of 10–50 μm from fresh or fixed samples, enabling histochemical and immunohistochemical analyses. Due to the fast preparation and under low temperature, the preservation of proteins, nucleic acids and chemical compounds in the tissues occurs. For sectioning in the cryostat, the samples are embedded in a water-­ miscible solution tissue freeze medium (OCT), therefore dehydration of the samples or the use of alcohol fixatives such as FAA must be avoided. However, if the samples are in FAA they can be hydrated to phosphate buffer and then submitted to cryoprotection.

20

2  Conventional Sample Preparation

Fig. 2.4  Microtomes used for sample sectioning. (a) Manual rotary microtome. (b) Sliding microtome

Sectioning in Cryostat for Fixated Samples Procedure 1. Fix in buffered 4% formalin – pH 7.2 for 24 h. 2. Wash 3× in 0.1 M sodium phosphate buffer, pH 7.2 for 30 min before placing in sucrose. 3. For cryoprotection, place in 10% sucrose in phosphate buffer for 24 h under vacuum followed by centrifugation at 3500 rpm for 15 min (twice). 4. After centrifuging, transfer to sucrose at a concentration of 15% diluted in distilled water. Keep in this solution for 3–4 days, performing vacuum over the days (6 times for 15  min each) and centrifuging at 3500  rpm for 20  min (eight times). 5. Infiltration in optimal cutting temperature (OCT) composite (Tissue-Tek®  – Sakura Finitek) in 10 × 10 × 5 mm cryomolds (Tissue-Tek®). 6. After adjusting the orientation, the samples are immediately frozen by floating the cryomolds in liquid nitrogen. 7. Blocks should be stored at −80 °C for 1 h and then transferred to −20 °C for 8 h to acclimate the samples to temperature. 8. The OCT blocks are sectioned longitudinally using a − 25 °C cryostat. 9. The collection of the sections is done by the CryoJane Tranfer strip system (CryoJane® – Leica Biosystems). 10. The longitudinal sections collected on the tape are transferred to coated slides (Leica® – Model62800-1X at www.emsdiasum.com). 11. Fixation of the cuts on the blade is done by exposure to a flash of UV light.

Sectioning Methods

21

Sectioning Paraffin Blocks Procedure 1. Samples should be fixed on a wood block (2cm2) with melted paraffin. 2. After reach the room temperature, the block with material should be taken to the microtome to obtain serial sections with thicknesses that can vary between 5 and 20 μm. 3. As the sections form ribbon, roughly 12 cm long, cut the ribbon and place on a black cardboard. 4. Before placing the sections on the slide, check the size of the coverslip that will be used (e.g.: 24x 50 mm slide, place 2 rows with 6 sections). 5. Make cuts in the paraffin tape in the size that will fit on the slide, put a few drops of water on the slide and deposit the sections on the water. 6. Place the slides on a hot plate at 40 °C for some minutes or until they become distended.

Sectioning Plastic Blocks (Historesin and LR-White) 1. Serial sections between 5 and 10 μm thick can be made for historesin (Fig. 2.5). Above this value, historesin starts to detach from the slide. 2. Place next to the microtome a slide with a few drops of water. 3. Before placing the sections on the slide, check the size of the slide that will be used. 4. Place the sections on the slide (resin distends in contact with water). The forceps to pick up the sections must always be clean and dry, because humidity causes wrinkling of the resin. For details see Marques e Nuevo 2022. 5. Place the slide on the hot plate at 40 °C for about 20 min or until dry. During drying, check that the sections are not moving out of place. Observation Slides intended for immunohistochemistry with samples infiltrated material in LR-White should be prepared in advance and coated with a solution to adhere the sections. Read the slide preparation protocol for LR-White resin Chap. 3 of this volume.

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2  Conventional Sample Preparation

Fig. 2.5  Samples processing steps for analysis under light microscope considering plant tissues embedding in historesin

Sectioning PEG (Polyethylene Glycol) Blocks Procedure 1. Sections are made on a slide microtome (Fig. 2.4b) 10–20 μm thick (less than 20 μm is ideal). 2. The knife can be made of steel or high-profile disposable knife. 3. The slides must be clean and previously prepared with a thin layer of Haupt’s adhesive (Haupt 1930) for better adherence of the sections on the slide. 4. Place on the hotplate to dry the adhesive. 5. After sectioning, collect the section with fine-tipped forceps and place into a container with distilled water to make the sections float. 6. Dip the slide under the section so that the section adheres to the slides. Carefully remove the slide from the water. 7. Place the slide on the hot plate until completely dry.

Transmission Electron Microscopy Trimming the Blocks To obtain the ultrathin sections we need to do several previous procedures, such as roughing the blocks (trimming), then produce semi-fine sections to check if the material is well fixed and infiltrated and to see the region of interest. Trimming can be done in two ways, manually with the aid of a razor blade under a stereomicroscope or with appropriate trimming equipment. Trimming is first done on the side of the block and then on the other four sides until a trapezium is formed.

Transmission Electron Microscopy

23

Semi-Fine Sections Semi-fine sections (300–500 nm) are normally made in the ultramicrotome with the aid of a glass knife (0.6 cm), transferred to a histological slide with a few drops of water and placed on the hot plate. The sections may be stained with toluidine blue (Feder and O’Brien 1968; Sakai 1973), mounted in water, and covered with a glass slide for analysis (Fig. 2.6). After finding the region of interest, the excess material and resin must be removed. And check if the cutting face is the size of a pencil tip.

Ultrathin Cuts Sections The block trimming to obtain ultra-thin cuts is essential. A block with a larger or disproportionate cutting region can generate cuts with defects and uneven thickness, compromising the analysis. The ideal ultrathin sections are 70  nm thick (silver color). After distending the sections with vapors of chloroform, collect on the 300–400 mesh grids to be contrasted (Fig. 2.6). The angle of inclination of the knife should be 5 degrees.

Fig. 2.6  Processing steps of samples embedded in epoxy resin (Spurr) for analysis by transmission electron microscope

Chapter 3

Techniques for Histopathological Analysis

Routine Analysis Toluidine Blue C.I. 52040 (Feder and O’Brien 1968; Sakai 1973) As toluidine blue is a cationic metachromatic dye (O’Brien et al. 1964; Feder and O’Brien 1968), it allows a general analysis of the tissues, helping in the posterior direction to the histochemical stain’s procedures (Kraus and Arduin 1997; Figueiredo et al. 2007). Therefore, toluidine blue may be considered routine for histopathological analyses. Uses: Embedded plastic sections and less frequently on fresh material. Reagents Anhydrous bibasic sodium phosphate Citric acid Toluidine blue Distilled water

14,2 g 9,6 g 0,5 g 1000 ml

Preparation Weigh out the sodium bibasic phosphate and dilute in distilled water. Add citric acid, measure the pH (it must be between 4.0 and 6.0) and add toluidine blue.

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 J. P. Rodrigues Marques, M. Kasue Misaki Soares, Handbook of Techniques in Plant Histopathology, https://doi.org/10.1007/978-3-031-14659-6_3

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Techniques for Histopathological Analysis

Procedure 1. Leave in the dye for 5–7 min. 2. Wash in distilled water 2–3 times or until contrast is noted. The historesin colour should be a very weak blue. 3. Air dry at room temperature for 24 h and mount on synthetic resin. Results Due to its polychromatic nature, toluidine blue, at the pH that was recommended (between 4.0 and 6.0), stains different cell structures of diverse chemical composition and exhibit a gradient of tonality ranging from purple to blue green (Fig. 3.1a–c). Generally, the fungal wall, plant walls and contents of pectic nature stain in reddish-purple, lignifed walls and phenolic compounds turn greenish. Different histochemical test can be directed after analysis with toluidine blue.

Observation If the staining becomes too strong, immerse the slides in warm water until the excess dye is removed. In step 3, the drying of the slides can be accelerated by placing them in the oven at 37 °C for a few hours or drying them on the hot plate.

Immunohistochemistry (Ruzin 1999; Willats et al. 2001) This technique is one of the most important in the detection of specifc antigens on the cell walls, proteins and enzymes. It makes use of the specifc property of antibodies to bind to specifc regions of specifc molecules, enabling their histolocalization. Immunohistochemistry can be performed on conventionally processed material (paraffn or plastic resin) or after cryostat sectioning of frozen samples. Here it is worth mentioning that hydrophilic resins, as is the case of LR-White, are the most recommended, as epoxy or glycol methacrylate-based resins reduce the detection reaction of antigens from plant tissues and cells and, therefore, are not recommended. The choice of method depends on the resources available to the researcher. For antigen detection there are many possibilities. The detection can be direct or indirect, with or without the use of fuorescent molecules, and the signal can be amplifed or not. Methods for antigen detection are shown in Fig. 3.2. In general, permeability of tissues or cells to the antibody is required. Among the great diversity of antibodies, those associated to cell wall carbohydrates commercialized by the company Plant Probes (www.plantprobes.net) stands out. Furthermore, there are other possibilities such as the immunostaining of

Immunohistochemistry

27

Fig. 3.1 Toluidine blue, pH 4.0–6.0, for routine histopathological analyses. (a) Cross section of the sorus of the fungus Sporisorium scitamineum on sugarcane. Note the sporogenous hyphae (SH) and intracellular hyphae (arrows) in purple, the fundamental parenchyma (FP) and phloem (PH) in blue, and the vessel element wall (VE) greenish. (b) Stigma surface of sweet orange tree with papillose cells (PC) in blue, pollen grain wall (PG) greenish and hyphae of the fungus Colletotrichum acutatum in purple (arrows). (c) Longitudinal section of grapevine stem infected with the fungus Elsinoë ampelina. Note in the region of the lesion a greenish shade (*) that indicates the accumulation of phenolic compounds. CT Cortex, EP Epidermis

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Techniques for Histopathological Analysis

Fig. 3.2 Antibodies and immunolabeling reaction. Both primary and secondary antibodies can be used. Antibodies can be conjugated to fuorophores that will generate fuorescence at different wavelengths. They can also be highlighted using enzymes that will degrade the substrate providing a chromogenic product such as DAB (3,3′-diaminobenzidine) or NBT (tetrazolium nitroblue). The markings can be direct or indirect. In the latter case, it is necessary to use a primary antibody that will bind to the antigen present in the plant cell, followed by a secondary antibody, conjugated to a fuorophore or enzyme, which will bind to the primary antibody, showing its location. In some cases, the signal can be amplifed using a biotinylated secondary antibodies

proteins associated to the metabolism of the plant cell or even proteins present in the cell nucleus. Procedure 1. Fix the plant tissue in 4% paraformaldehyde in 0.1 M sodium cacodylate buffer pH 7.2 for 24 h. 2. Wash the fxed material in 0.1  M sodium cacodylate buffer pH  7.2 (2–3 exchanges, 15 min/per step). 3. Dehydrate in ethanol series (30, 50, 70, 90, and 100%), 15 min min/per step. 4. Pre-infltrate with the resin in a 1:1 solution of LRWhite* and absolute ethanol. overnight. 5. Infltrate with pure resin (2 changes every 8 h). 6. Transfer to gelatin capsules and take care when orienting the material. 7. Polymerize in oven at max 50 °C for ~8 h or at 4 °C overnight under UV. 8. Preparation of the slides with adhesive solution (poly-L-lysine – page 24). 9. Make the sections 1–5 μm thick on a rotating microtome. 10. Block tissue hydrolytic enzymes with phosphate buffer: bovine serum albumin (5%). 11. Incubate with the primary antibody in the correct dilution phosphate buffer (previous tests must be done)**. 12. Wash the primary antibody in phosphate buffer**.

Changes in the Cuticle and Cell Wall

29

13. Incubate with fuorophore-conjugated secondary antibody*** diluted in phosphate buffer. 14. Wash the slide. 15. Analysis under epifuorescence**** or confocal microscope. Observation The use of glycol methacrylate resin should be avoided. Pay attention to the buffer that must be used for diluting the antibody. Variations may occur. It is recommended to always read the manufacturer’s instructions. * LRWhite is a comercial brand, pay atteption to the manufacturer’s instruction. **It is important to check with the company that produced the antibody the best buffer to use. ***The antibody dilutions and the reaction time are variable. More than one staining can be performed. **** The choice of flters for epifuorescence should respect the excitation and emission wavelength of the fuorophore. This fact is determining for the detection of the antibody conjugated to the fuorophore.

Changes in the Cuticle and Cell Wall In the presence of pathogens, plants tend to modulate their cell walls to prevent pathogen penetration or restrict fungal expansion into tissues. Therefore, the wall has been recognized as an important physical and chemical barrier in the plant immunity process (Malinovsky et al. 2014), which should be observed and investigated in detail because it can be interpreted as a constitutive or induced barrier (Marques et  al. 2018). In this perspective, histochemical studies should be performed aiming to elucidate the possible parietal alterations that resistant plants may perchance present. Here, we present some methods that can be useful in understanding the defense processes linked to the cell wall.

Ruthenium Red and Sudan IV C.I. 77800 and C.I. 26105 (Chamberlain 1932; Miller 1968; Marques et al. 2016a, b) Cuticular alterations are frequently observed in plants infected by fungi. The use of double staining facilitates the visualization of these alterations, making it possible to detail the changes in the cuticle. Uses: Fresh or plastic-embedded tissues.

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Techniques for Histopathological Analysis

Reagents Solution A: Ruthenium red Distilled water, make up to

0,0025 g 10 ml

Solution B: Sudan IV Ethanol 95% Glycerin

1 g 100 ml 40 ml

Preparation 1. Dilute ruthenium red (ruthenium red, CAS 11103-72-3,) in distilled water. Store in amber fask and refrigerate. 2. Dissolve Sudan IV (CAS 85-83-6) in ethanol by heating to boiling point for a few hours. Allow to decant for 24  h. After this time, 60  ml of supernatant is removed from this solution and 40 ml of glycerin is added. Allow to decant again for 24 h and remove only the supernatant. Filter under vacuum using a flter fask. Procedure 1. Transfer the sections on watch glass, drip solution A and wait 2  min. In historesin, place a few drops on top of the sections, and wait 1–3 min. 2. Wash the sections in distilled water. 3. Place in solution B and wait 20–30 min at 40C. On historesin, drip in solution B, cover with coverslip and wait 20 to a few hours. 4. Assemble in 60% glycerin.

Results The pectin (hydrophilic substance) of the cell wall stains pink purple by reaction with ruthenium red, while the cuticle, which has cutin and waxes (lipophilic substance) shows orange coloration (Fig. 3.3a). Observation If no reaction is observed with Sudan IV, heat the slide on a 40 °C hot plate until a reaction is obtained. In the case of historesin, placing the coverslip before heating prevents the formation of dye granules.

Changes in the Cuticle and Cell Wall

31

Fig. 3.3 Staining methods for detecting cuticle and cell wall. (a) Sweet orange (Citrus sinensis L.) petal after inoculation with Colletotrichum acutatum. Cuticle stained orange by Sudan IV and cell walls pink after reaction with ruthenium red. (b) Vine (Vitis labrusca L.) leaf after staining with Nile Red and Calcofuor White and analyzed under epifuorescence microscope. The cuticle appears in red while the cell walls in blue. CT Cuticle, EP Epidermis, VB Vascular bundle, ME Mesophyll, CW Cell wall, SP Spongy parenchyma, PP Palisade parenchyma

Nile Red and Calcofuor White CAS 7385-67-3 and CAS 4404-43-7 (Greenspan et al. 1985; Hughes and McCully 1975; Trese and Loschke 1990; Leite et al. 2013; Marques et al. 2016a, b) Uses: Fresh or plastic-embedded tissues Reagents Solution A: Calcofuor White (C.I. 40621) Distilled water

0,01 g 100 ml

Solution B: Nile red (CAS 7385-67-3) Acetone 100%

0,001 g 10 ml

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Techniques for Histopathological Analysis

Preparation 1. Dissolve Nile red in acetone. Store in amber bottle and refrigerate. 2. Dilute Calcofuor White in distilled water. Store in amber bottle and refrigerate. Filter the dye before use. Procedure 1. For freehand sections, follow the steps of the bleaching technique before stain. To prepare the slide with historesin sections and sections already bleached, drip a drop of Nile red on the slides and cover with coverslip to avoid evaporation. Wait for 5 min. 2. Wash in 100% acetone and then in acetone and distilled water (1:1). 3. Wash the sections in distilled water (1 min). 4. Drip Calcofuor White over the sections and leave for 1–3 min. 5. Wash in distilled water (30 s twice). 6. Assemble in distilled water or glycerol. 7. For observation under the epifuorescence microscope, the flter used for Nile red is I3 (Ex: 450–490/ Em:515) or N2.1 (Ex: BP 515–560 / Em: LP590) and for Calcofuor White is A (Ex: 325–375/ Em 435–485). Results Lipophilic substances (droplets) and cutin fuoresce orange-red and cellulose fuoresces blue white (Fig. 3.3b). Nile red fuoresces in the red spectrum.

Observation Coloring can be done separately.

Aniline Blue CAS 28631-66-5 and C.I. 42755 (Smith and McCully 1978a, b; Ruzin 1999) Aniline blue is a fuorochrome used for the detection of callose, which is a polysaccharide with β-1,3-glucan bonds. Callose deposition is very important in studies of plant defense mechanisms, and may be associated with papillae or hyphae encapsulation, in the pore obstruction of the phloem sieve plate (Marques et al. 2017) or in the plasmodesmata. Uses: Fresh or plastic-embedded tissues Reagent Aniline blue 0.5 M phosphate buffer pH 8.5

0.005 g 100 ml

Changes in the Cuticle and Cell Wall

33

Preparation Dissolve the aniline blue in phosphate buffer. Procedure 1. For freehand sections follow the steps of bleaching technique (page 62) 2. Stain the sections for 20 min. 3. Wash the sections in distilled water for 1 min. 4. Deposit the stained sections on a slide and drip a few drops of distilled water, then place the coverslip. 5. Observe under fuorescence microscope under UV light. Filter A (Ex: 325–375/ Em 435–485) Leica can be used®.

Results In fuorescence, callose stained with aniline blue turns blue-green and may be deposited near the cell wall (Fig. 3.4a) or around the hyphae (Fig. 3.4b).

Phloroglucin (CAS 108-73-6) (Ruzin 1999; Marques et al. 2018) Lignin is a complex wall polymer consisting of different subunits (siringyl, guaiacyl and coniferol) from the phenylpropanoid route. It is very important in the plant defense process and can be constitutively present in the tissues or induced after contact with the pathogen (Pascholati 2011; Marques et al. 2018). The composition of lignin varies according to the organ, age and infectious process (Walters 2011). Acidic phloroglucin reacts with the coniferaldehyde groups of lignin generating a red to pink coloration, but reacts weakly to syringyl units which leads to weak reactions of foroglucinol on secondary walls rich in this monomer (Sarkamen and Ludwig 1971). It is emphasized here that fresh samples, whether or not sectioned on slide microtome or cryostat (Marques et al. 2018), are better for reacting with foroglucinol. Samples fxed with Formalin Acetic Acid and Ethanol (FAA) or Karnovsky (page 14) can also be used, but it is suggested to avoid the embedding process in resins. Uses: Fresh or fxed tissues Reagent Phloroglucinol Hydrochloric acid 37% Ethanol 95%

0,50 g 12,25 ml 12,25 ml

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Techniques for Histopathological Analysis

Fig. 3.4 Histochemical tests for the detection of callose (a, b) and lignin (c, d) accumulated in sugarcane cell walls in the vicinity of intracellular hyphae of the fungus Sporisorium scitamineum. (a) Photomicrograph obtained by superimposing bright-feld and fuorescence images of callose stained by aniline blue (arrows). (b) Epifuorescence image. Callose (blue) deposited around intracellular hyphae (IH) or close to the cell wall (arrows). (c, d) Lignifed cell walls (pink) in regions in contact with hyphae of the fungus. CW Cell Wall, EP Epidermis

Preparation 1. Dilluted the phloroglucin in ethanol 2. Dilute the acid in Ethanol with Phloroglucin. Never the other way around. Procedure Deposit the sections on a slide and drip a few drops of the solution and place the coverslip on it. Prefer use a glass pipette. Results The lignin deposited on the cell walls turns reddish pink is. (Fig.  3.4c, d). After 4 h the coloration disappears.

Analyses of Changes in Primary Metabolism

35

Observation The solution contains acid and must therefore be handled with gloves and in a fume hood. Be very careful when analyzing, because the acid in the solution may come in contact with the microscope objective or plate, which can damage the instrument. It is suggested to seal the slide with colorless nail polish before analyzing.

Analyses of Changes in Primary Metabolism Detection of Proteins and Lipids Xylidine Ponceau and Sudan Black B CAS 3761-53-3 and CAS 4197-25-5 (Pearse 1968; Cortelazzo and Vidal 1991) This technique aims to recognize cellular components of proteic and lipophilic nature in plant cells of injured regions. The stains can be performed separately or together providing important data of the distribution of these compounds in the plant cells. to the researcher, which favors the interpretation of cellular alterations. Uses: Fresh or plastic-embedded tissues Reagents Solution A: Glacial acetic acid Distilled water

3 ml 97 ml

Solution B: Xylidine Ponceau (C.I. 16150) Solution A

0,1 g 100 ml

Solution C: Sudan black B (C.I. 26150) Ethanol 70%

1 g 100 ml

Preparation 1. For the preparation of solution A, dilute glacial acetic acid in distilled water. 2. To prepare solution B, dissolve the xylidine Ponceau in solution A. 3. For the preparation of solution C, heat the 70% ethanol to 60 °C and dissolve the Sudan black B. Store in amber bottle and refrigerated. Filter before use.

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Techniques for Histopathological Analysis

Procedure 1. Make sections of materials embedded in historesin. 2. Transfer the sections on glass slides, drip solution B in the historesin sections and wait for 15 min. 3. Wash for 10–30 min with solution A. 4. Wash in distilled water. 5. Drip solution C, cover the slide with a coverslip petri dish the watch glass to prevent evaporation and wait for 5–10 min. 6. Wash carefully the sections by passing them quickly in 50% ethanol and then in distilled water. 7. Mount the slide in distilled water, or entellan.

Result Proteins stain red and lipids stain navy blue to black. In injured regions it is noted that necrotic cells may accumulate lipophilic compounds but are usually surrounded by cells that accumulate protein (Fig. 3.5a, b). The protein may occur as droplets (Fig. 3.5c) or as an amorphous substance in the cytoplasm of the cells. This technique allows evidence that different tissues present cells that accumulate lipids and proteins and in different cellular compartments (Fig. 3.5c).

Observation First stain with xylidine Ponceau and then with Sudan black B, because as Sudan is formulated in ethanol, there may be detachment of the resin from the slide.

Carbohydrate Detection Zinc chloride-Iodine CAS 7646-85-7, 7681-11-0 and 7553-56-2 (Strasburger 1913) The detection of starch grains in plastids is a routine technique, in view of the importance of starch as an energy reserve. As a rule, regions injured by phytopathogens tend to present a reduction of starch in the injured area (Marques et al. 2007). It is worth emphasizing that there is the transient or primary starch and the reserve or secondary starch. The transient starch occurs in photosynthesizing organs and its presence may be associated with physiological conditions.

Carbohydrate Detection

37

Fig. 3.5 Lesion of citrus black spot disease caused by the fungus Guignardia citricarpa submitted to double staining with xylidine Ponceau for proteins (red color) and Sudan black B (dark blue color) for lipophilic compounds. (a) Overview of the lesion showing the accumulation of lipophilic compounds in the epicarp (EC). (b) Detail of the section highlighted in A. Note the epicarp cells stained dark blue and the mesocarp (MC) cells with protein accumulation (arrow). (c) Detail showing the amorphous lipophilic content (*) and protein content in drops (arrow)

Uses: Fresh or plastic-embedded tissues Reagents Zinc chloride Potassium iodide Iodine Distilled water

20,0 g 1,0 g 0,1 g 30,0 ml

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Techniques for Histopathological Analysis

Preparation 1. Weigh the potassium iodide in a small beaker (the reagent reacts with aluminium) and dissolve in water. 2. Add the iodine until dissolved. 3. Slowly add the zinc chloride. Procedure 1. Make sections of fresh materials or follow for historesin inclusion techniques. 2. Drip the reagent solution and wait for 5–7 min. 3. Wash in distilled water. 4. Mount the slide in distilled water or entellan. Results Starch grains present in the plastids occur as bluish black spots. Observation Sections of fresh material tend to react faster. To tell if the reagent is working, check that the flter paper turns black in contact with the reagent.

Analyses of Changes in Secondary Metabolism Detection of Phenolic Compounds Phenolic compounds are commonly observed in different types of plant-pathogen interactions. They are a diverse and complex group and can be evidenced with different techniques. In interactions with pathogens the deposition can occur on the cell walls or inside the cells with accumulation in the vacuole.

Ferric Chloride (CAS 10025-77-1) (Johansen 1940) The ferric chloride test works both on sections from historesin-infltrated material and on non-infltrated material (fresh or fxed). Uses: Fresh or plastic-embedded tissues

Analyses of Changes in Secondary Metabolism

39

Reagents Sodium carbonate Ferric chloride hexahydrate Distilled water, make up to

0,3 g 10 g 100 ml

Preparation 1. Dissolve the ferric chloride in the distilled water and add the sodium carbonate. Store in amber fask and refrigerate. 2. Make freehand sections of fresh materials and place them in distilled water. 3. Transfer the sections to a slide and drip a drop of ferric chloride reagent onto them. 4. Mount with coverslip, waiting 1 or 2 min. For sections of material embedded in historesin, leave the slide in the solution for at least 3 h. 5. Drip a drop of distilled water or 50% glycerin on the edge of the slide and remove the reagent with flter paper. 6. Deposit few drops of distilled water, then place the coverslip. Results Ferric chloride produces a dark brown coloration indicating the presence of phenolic compounds (Fig. 3.6a, b). Observation In some cases, sections obtained from historesin-infltrated material may be kept in ferric chloride overnight or longer.

Ferrous Sulfate in Formalin CAS 7782-63-0 and CAS 50-00-0 (Johansen 1940; Marques et al., 2015) The detection of phenolic compounds can be done by fxing the plant tissues in ferrous sulfate in formalin. After fxation, phenolic compounds are detected in the sections without the need for further staining. Generally, thicker sections are more indicated to facilitate the analysis of phenolic compounds by this method. Uses: Fresh or plastic-embedded tissues Reagents Formaldehyde 37% Ferrous sulphate heptahydrate Distilled water

4 ml 10 g 100 ml

40

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Techniques for Histopathological Analysis

Fig. 3.6 Lesions of the disease citrus black spot caused by the fungus Guignardia citricarpa. (a, b) Positive reaction with ferric chloride, evidenced by the dark brown color (arrows). OC oil cavity, EP epidermis, EC epicarp

Procedure 1. Place the material in the fxative for 48 h. 2. Remove the air from the material with the aid of a vacuum pump. 3. Wash in distilled water doing 4 exchanges of 15 min each. 4. Make freehand or slide microtome sections or go for historesin inclusion techniques. Results The presence of phenolic compounds is indicated by the brown coloration.

Autofuorescence Detection (Dai et al. 1995a; Talamond et al. 2015; Roshchina et al. 2017) The use of autofuorescence is a very practical, fast and effcient method to investigate the presence of secondary metabolites synthesized by plants. Phenols, alkaloids and terpenes are metabolites originated from secondary metabolism and in the course of the plant-pathogen interaction there may be an accumulation of different compounds, which may or may not interfere in the course of the infectious process. The usual methods for sample preparation, such as fxation and dehydration, can remove the secondary metabolites from the tissues, however, in some cases, the metabolites can be visualized after soaking in historesin. The presence of autofuorescence does not rule out the need for other histochemical tests. Uses: Material not embedded (fresh or fxed).

Detection of Flavonoids

41

Procedure 1. Make freehand or slide microtome sections or go for historesin inclusion techniques. 2. Mount the sections in glycerin or distilled water. 3. Observe under UV light fuorescence microscope with emission in the blue range (430–450 nm). Results When observed under UV light phenolic compounds can emit light in the wavelengths of blue and greenish colors. Observation It is recommended that these results be combined with those of histochemical tests. The use of autofuorescence should preferably be done as freshly collected and cut material. The use of fxatives and reagents can extract tissue components.

Detection of Flavonoids NEU Reagent (CAS 524-95-8) (Dai et al. 1995b; Mondolot et al. 2006) Uses: Fresh material Procedure 1. Place the sections in 1% 2-aminoethyl diphenyl borate solution in absolute methanol. 2. Mount in glycerin. 3. Analyze under fuorescence microscope. Results Under UV light favonoids emit fuorescence in the blue wavelength, so long pass band flters that allow the passage of light lengths above 400 nm, such as Nikon’s UV-1 A flter, should be used. Observation The reagent 2-aminoethyl diphenyl borate is toxic, so it is recommended to prepare the slides in a fume hood and wearing gloves.

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Autofuorescence Detection (Dai et al. 1995a; Talamond et al. 2015; Roshchina et al. 2017) Uses: Fresh tissues Procedure 1. Make freehand or sliding microtome sections or go for historesin inclusion techniques. 2. Mount the sections in glycerin or distilled water. 3. Observe under UV light fuorescence microscope with emission in the blue range (430–450 nm). Results The regions that accumulate favonoids when excited with UV light emit fuorescence in the blue light spectrum. Therefore, it is recommended that the epifuorescence or confocal microscope have flters, lasers and detectors that can excite the sample in UV and detect in the blue wavelength.

Observation The use of autofuorescence should preferably be done as freshly collected and sectioned material. The use of fxatives and reagents may extract the tissue components. It is emphasized that the autofuorescence detection technique should be associated with histochemical tests to confrm the presence of favonoids (Neu’s reagent).

Detection of Terpene NADI Reagent (David and Card 1964) One of the most abundant components of essential oil is terpene which is responsible for fragrance. The NADI reagent makes it possible to distinguish essential oils from resin acids by differences in color depending on the pH. Uses: Fresh tissues Reagents Solution A: α-Naphthol Ethanol 40%

0,0005 g 0,5 ml

Detection of Terpene

43

Preparation It can be prepared the day before. Solution B: 0,05 M sodium phosphate buffer pH 7,2 Reagents Monobasic sodium phosphate 0,05 M. Monobasic sodium phosphate monohydrate Distilled water, make up to

0,28 g 40 ml

Sodium phosphate, bibasic, 0,1 M. Anhydrous dibasic sodium phosphate Distilled water, make up to

1,42 g 200 ml

Preparation Check the pH of the dibasic phosphate solution and adjust the pH to 7.2 using the monobasic phosphate solution. Solution C Reagents

Dimethyl--phenylenediamine hydrochloride 0,05 M buffer pH 7,2

0,005 g 0,5 ml

Preparation Weigh the Dimethyl-p-phenylenediamine hydrochloride in the small beaker (do not weigh it on aluminium foil, because it reacts with the paper). It can be prepared the day before. NADI Reagent Reagents Solution A Solution B Solution C

0,5 ml 0,5 ml 49 ml

Preparation Mix the three reagents together at the time of use. Prepare in the dark (photosensitive). Procedure 1. Transfer the sections on watch glass and drip NADI reagent (freshly prepared) for 1 h at room temperature in the dark (e.g. cardboard box). 2. Cover the watch glass to prevent evaporation.

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3. Wash in 0.1 M sodium phosphate buffer, pH 7.2 (Page 15) for 2 min. 4. Mount in phosphate buffer. Results A blue colour indicates the presence of essential oils and a reddish colouration shows the presence of acid resins. When essential oils and resin acids are mixed, a violet to lilac colour is observed.

Autofuorescence Detection (Dai et al. 1995a; Talamond et al. 2015; Roshchina et al. 2017) Procedure 1. Make freehand or slide microtome sections or go for historesin inclusion techniques. 2. Mount the sections in glycerin or distilled water. 3. Observe under UV light fuorescence microscope with emission in the green range (520–530 nm). Results The terpene accumulating regions when excited by UV light emit fuorescence at wavelengths in the green spectrum (520–530 nm). For this purpose, the use of long band emission flters starting at 400 nm is required.

Observation The use of autofuorescence should preferably be performed with freshly collected samples Fixatives and reagents may extract the tissue components. It is emphasized that the autofuorescence detection technique should be associated with histochemical tests to confrm the presence of terpenes.

Detection of Alkaloid Dragendorff’s Reagent CAS 10035-06-0 7681-11-0 (Svendsen and Verpoorte 1983) Uses: Fresh tissues

Detection of Alkaloid

45

Reagents Solution A: Bismuth nitrate Acetic acid Distilled water

3,87 g 6,25 ml 25 ml

Solution B: Potassium iodide Distilled water

4 g 10 ml

Preparation 1. In an ice bath, dilute acetic acid in water and then add bismuth nitrate. 2. Dissolve the potassium iodide in distilled water. Pour the frst solution little by little over the second. Leave it to stand for a few hours and flter. This stock solution can be stored for 6 months. 3. When using, take 5 ml of this solution and add 10 ml of acetic acid and make up to 100 ml with distilled water. Store away from light. Procedure 1. Transfer the sections on watch glass and drip Dragendorff’s reagent and wait for 5–10 min. 2. Transfer to 5% sodium nitrite solution. 3. Wash in distilled water. 4. Mount in glycerinated gelatin. Results Dragendorff’s reagent stains most of the orange-brown alkaloids.

Autofuorescence Detection (Talamond et al. 2015; Roshchina et al. 2017) Procedure 1. Make freehand or slide microtome sections or go for historesin inclusion techniques. 2. Mounting of the sections in glycerin in distilled water.

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3. Observe under fuorescence microscope under UV light and emission in the green-yellow range (515–560 nm). Results The alkaloid accumulating regions when excited by UV light emit fuorescence at wavelengths in the green-yellow spectrum (515–560 nm). For this purpose, the use of long band emission flters starting at 400 nm is required. Observation The use of autofuorescence should preferably be done as freshly collected and cut material. The use of fxatives and reagents may extract the tissue components. It is emphasized that the autofuorescence detection technique should be associated with histochemical tests to confrm the presence of alkaloids.

Detection of Reactive Oxygen Species Reactive oxygen species (ROS) are molecules commonly present in plants subjected to biotic and abiotic stress and have essential functions in the defense response. Among the different ROS, hydrogen peroxide (H2O 2) and superoxide anion (O 2-) stand out. They are produced in cells challenged by pathogens and are important signalers for other defense responses, such as callose production or programmed cell death (Luna et al. 2011). Presented here are methods for detection of H 2O2 and O 2-.

Hydrogen Peroxide – H2O2 CAS 91-95-2 (Adapted from Daudi and O’Brien 2012) The organic compound 3,3′-diaminobenzidine (DAB) is oxidized by hydrogen peroxide, generating a dark brown precipitate. This precipitate is exploited as a stain to detect the presence and distribution of hydrogen peroxide in plant cells. Uses: Fresh tissues Reagents Phosphate Buffer Solution (PBS).

Detection of Reactive Oxygen Species NaCl KCl Na2 HPO4 KH 2PO4

47 8 g 0,2 g 1,15 g 0,2 g

DAB DAB (3,3′-diaminobenzidine) PBS at pH 4,0 with HCl

50 mg 50 ml

Preparation Prepare the solution immediately before use. Procedure 1. Submerge samples in DAB solution in aluminum foil-wrapped vial. 2. Place in the desiccator coupled to the vacuum pump for air removal (2 times of 15 min each). 3. Keep samples in the dark for 4–12 h*. 4. Wash in PBS buffer 2 × 1 min each 5. Periodically analyze the sample under a magnifying glass or microscope until the reaction is verifed. If necessary sections of the region can be made and then the slide can be mounted in PBS. Results Dark brown precipitate indicates cells or tissues accumulating H2O2. (Fig. 3.7a, c, d).

Observation *The duration of the reaction process with DAB is variable, depending on sample thickness and tissue type. The solution should be prepared minutes before use.

Superoxide Anion O2− CAS 298-83-9 (Adapted from Daudi and O’Brien 2012) The detection of anionic superoxide is performed by Nitroblue Tetrazolium (NBT).

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Fig. 3.7 Sugarcane ((Saccharum spp.) buds inoculated with the smut fungus Sporisorium scitamineum (a–f). Reactive oxygen species histochemical test to detect hydrogen peroxide (H2O2) (a, c, d) with DAB and anion superoxide (O2−) (b, e, f) with NBT. The brown color in the leaf scales (Arrows and asterisks) represent the sites of H2O2 accumulation. The blue-purple regions represents the anion superoxide accumulation sites (f) Cross section of analysis of sugarcane smut sorus showing birefringence on the parenchyma cell walls. (g) Coffea arabica leaf infected with Hemileia vastatrix fungi. Note the presence of calcium oxalate crystals (arrows). PA Parenchyma, TE Teliospores

Detection of Reactive Oxygen Species

49

Uses: Fresh tissues Reagents Nitroblue tetrazolium (NBT) PBS pH 7.2 0.2 M

3,5 mg 1 ml (page 14).

Preparation Dissolve NBT in PBS and keep the solution in the dark until use. Procedure 1. Submerge the plant tissues in the NBT solution in an aluminum foil-wrapped vial. 2. Place in the desiccator coupled to the vacuum pump for air removal (2 times of 15 min each). 3. Keep the samples in the dark for 4–12 h. 4. Wash in PBS buffer 2 × 1 min each 5. Analyze under magnifying glass or microscope. If necessary, freehand sections of the region with the precipitate can be made and then the sections mounted in phosphate buffer or in distilled water. Results Areas that accumulate O2-turn purple (Fig. 3.7b, e, f). Note The solution should be prepared minutes before use.

Tissue Diaphanization After Reaction with DAB or NBT As a rule, this step is done with chlorophyllated tissues such as leaves. After the removal of chlorophyll, the leaf becomes translucent allowing the analysis of the regions accumulating H2O2 and O2−. Non-clorophyllated tissues do not require the diaphanization step (Giordani et  al. 2022). This procedure enables visualization under a stereomicroscope as illustrated by Wu et al. (2016). There are two methods of bleaching: 1. Submerge the sample in the previously heated 100% ethanol:acetic acid:glycerin (3:1:1) solution on a beaker (see Daudi and O’Brien (2012) bleaching method (Fig. 4.1a). 2. Submerge the samples in heated 96% ethanol for 10 min (Wu et al. 2016).

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Table 3.1 Iso- and anisotropic characteristics of cellular components Cellular components Isotropic Primary wall Secondary wall Calcium oxalate crystals Amorphous calcium carbonate inclusions Silica Bodies Protoplasm X Plastids X Starch

Anisotropic X X X X X

X

Crystal Structure Detection Use of Polarized Light (Ruzin 1999, Marques et al. 2017 and 2020) In plants there are different cellular components that present birefringence and, therefore, are anisotropic which enables the analysis under polarized light. Two flters are required to obtain the polarized light: the polarizer, located between the sample and the light source; and the analyzer located between the objective and the ocular. Image contrast arises from the interaction of in-plane polarized light with a birefringent (or doubly refractive) sample to produce two individual wave components that are polarized in planes perpendicular to each other. After passing through the sample, the light components are out of phase, but are recombined with constructive and destructive interference as they pass through the analyzer. Polarized light is a frequently used tool in plant anatomy and can generate quantitative and qualitative data on a wide range of anisotropic structures (Table 3.1). Polarized light is very useful for distinguishing cellular inclusions, silica bodies, in determining the orientation of cellulose microfbrils (Ruzin 1999), and in observing differences in morphology between starch grain types (Cai et al. 2014).

Chapter 4

Fungus Detection

Light Microscopy Method of Imprinting the Epidermis with Nail Polish (Marques 2012) This method can be considered the simplest, quickest and most practical to visualize the presence of spores, germ tubes and even apressoria on the surfaces of vegetative and reproductive organs. For its efficiency, agility and low cost, this method can be used in time-course experiments, allowing a complementary analysis to the scanning electron microscopy. Reagents Phenol Cotton blue (C.I. 42780) Lactic acid Glycerin Distilled water

20 g 0.01 g 20 ml 40 ml 20 ml

Preparation Melt the phenol in the fume hood by mix with lactic acid, glycerin and distilled water. Then dissolve the cotton blue in this solution and filter. There are commercial solutions of cotton blue in lactophenol (#61335 Sigma-Aldrich®), which stain sections of historesin infiltrated material well.

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 J. P. Rodrigues Marques, M. Kasue Misaki Soares, Handbook of Techniques in Plant Histopathology, https://doi.org/10.1007/978-3-031-14659-6_4

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Procedure 1. Apply incolour nail polish on the leaf surface, on the inoculated region with the fungus (Fig. 4.1a, b) and wait 10–20 min. If necessary, apply a second layer over the first and wait for it to dry. 2. Remove the nail polish film using fine-tipped tweezers (watchmaker’s tweezers are recommended) (Fig. 4.1c). 3. Stain the nail polish film with 1% cotton blue for 1  min on a watch glass (Fig. 4.1d). 4. Wash the film in distilled water twice for 1 min in each promoting gentle movements (Fig. 4.1e). 5. Mount the nail polish in distilled water (Fig. 4.1f).

Fig. 4.1  Steps in the preparation of the colorless nail polish film for the visualization of the fungus on the leaf surface. (a) Passing colorless nail polish on the surface inoculated with the fungus. (b) Wait for the colorless nail polish film to dry. (c) Remove the film with the aid of fine-tipped steel (d) Staining the film in cotton blue. (e) Wash the film in distilled water. (f) Mount film between slide and coverslip with distilled water. (g–j) Stages of fungus Colletotrichum acutatum development on sweet orange (Citrus sinensis L.) leaves. (g) Germinating conidia (CO). (h) Formation of apressory (AP) at the end of germ tube (GT). (i) Germ tubes arising from a conidium. (j) Twinning and apressory formation from different conidia

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Result The fungal structures stain in blue and the nail polish impression of the plant epidermis becomes translucent. With this technique it is possible to verify the stages of the infectious process of the fungus such as germination (Fig. 4.1g), the formation of apressoria (Fig. 4.1h), the growth of the germ tube (Fig. 4.1i), as well as counting the number of germinated spores and apressoria formed (Fig. 4.1j).

Observation Better brands of colorless nail polish coat generate more stable films and facilitate handling after removal from the mold. Do not pass more layers of nail polish than necessary, as this can generate a thick film that makes it difficult to see the fungus structures. The film can be stored in a dry place and kept in the dark for a few days.

Bleaching Method Followed by Cotton Blue Staining C.I. 42780 (Marques 2012) The procedure is recommended for fungi that colonize leaves. The method aims to remove the leaf pigments allowing light to pass through and color the fungus, enabling visualization of the fungal structures on the plant. As the samples are submitted to bubbling in a bleaching solution, the first stages of the process of deposition and germination of the fungus can be lost. Uses: Fresh tissues Preparation of the cotton blue solution is described above in the epidermal impression technique. Procedure 1. Collect the inoculated plant region or lesion of interest and immediately submerge in the 100% ethanol: 99.7% acetic acid: pure glycerin (3:1:1) solution previously heated on a plate (see Daudi & O’Brien (2012) bleaching method (Fig. 4.2a). 2. Check the sample for clarification and rinse it quickly in distilled water. 3. Stain with 1% cotton blue for 3–5 min (Fig. 4.2b). 4. Wash in distilled water on watch glass (Fig. 4.2c). 5. Mount in water or 60% glycerin (Fig. 4.2d).

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Result The plant tissues become translucent and the fungal structures stained in blue (Fig. 4.2e–g). The technique makes it possible to visualize the mycelium on the leaf surface (Fig.  4.2e), the presence of conidiophores and conidia (Fig. 4.2f). At higher magnification, the plant stomata and different structures of the fungus can be observed (Fig. 4.2g).

Fig. 4.2  Stages of the leaf bleaching process followed by staining of fungal structures. (a) Bleaching of the leaf lamina in a solution of 100% ethanol:acetic acid:glycerin (3:1:1). (b) Staining of the diaphanized material with cotton blue. (c) Washed samples in distilled water. (d) Mounting of samples between slide and coverslip with distilled water. (e) Overview of the mycelium of the fungus Leveilulla taurica (arrows) on the abaxial surface of pepper leaf (Capsicum annuum L.). (f) Detail of conidiophore (CP) with conidia (CO) at its extremity. (g) Arrow indicates the presence of fungal adhesion bodies next to the epidermal cells. ST Stomata

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Double Stained with Safranin O and Cotton Blue C.I. 50240 and C.I 42780 (Marques et al. 2013) This double staining helps in the detection of fungi present in different plant organs. It is noteworthy that the staining time may vary depending on the fungal species (hyphae size and composition) and the infected plant tissue. This method has been widely employed, generating promising results for the understanding of penetration and colonization. Uses: Plastic-embedded tissues Reagents Safranin O Distilled water

0,25 g 100 ml

Preparation 1. The method for preparing solution A (cotton blue) is described above in the technique for impression epidermis with nail polish. 2. In solution B, dissolve Safranin O in distilled water. 3. Filter all the stain solutions before use Procedure 1. Place the slides in solution A (cotton blue) for 5–7 min. The commercial cotton blue solution can be kept for 40 min at room temperature or for 5 min on a hot plate. After dripping the cotton blue the slides sections should be covered by a petri dish 2. Wash 3 times with distilled water for 1 min to remove excess dye. 3. Stain with solution B (safranin) for 10 s. 4. Wash 3 times with distilled water for 1 min to remove excess dye. 5. The slides can be mounted in water for previous analysis. If preservation is required, the slides may be dried and mounted in Entellan®. Results Cotton blue stains the chitin of the fungal cell wall in blue and safranin O stains the plant cell wall in red (Fig. 4.3a, b). This staining has proven very useful for distinguishing fungal structures inside and outside plant cells (Fig. 4.3b).

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Observation This method can be done by applying the dyes separately to test the coloring time. Old dye solutions tends to take more time.

Double Stained with Ruthenium Red and Cotton Blue C.I. 77800 and C.I 42780 (Marques & Nuevo 2022) This method is focused on describing pectin and acid mucilage distribution on plant tissues infected by both biotrophic and necrotrophic fungi. The authors demonstrated that the pectin can encapsulate fungal haustorium and that the method is also useful to verify the effect of necrotrophic fungi on cell wall degradation. Fungus structures can also be observed. Uses: Plastic-embedded tissues Reagents Solution A: Phenol Cotton blue (C.I. 42780) Lactic acid Glycerin Distilled water

20 g 0,01 g 20 ml 40 ml 20 ml

Solution B: Ruthenium red Distilled water, make up to

0,0025 g 10 ml

Preparation 1. Filter all the stains solutions before use Procedure 1. Place the slides in solution A (cotton blue) and leave for 5 min and heat on a hot plate at 45C for 5 min. 2. Remove the excess dye by washing the slide three times in a beaker filled with distilled water 3. Stain with 2 mL of 0.01% ruthenium red in water for 1 min 4. Remove the excess dye by washing the slide three times in a beaker filled with distilled water

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Fig. 4.3  Test with double staining for detection of fungal structures in plant tissues. (a, b) Photomicrographs obtained after staining with cotton blue and safranin O and analyzed in a bright field. (c) Photomicrograph obtained in an epifluorescence microscope after staining with calcofluor white and WGA-AF488. (a) Section of the black spot lesion (hard spot symptom) on sweet orange fruit infected by the fungus Guignardia citricarpa causing the citrus black spot disease. Fungal structures of the fungus are seen in blue on the epicarp. Note that in the pycnidia the hyphae also stain blue. (b) Section of petal inoculated with Colletotrichum acutatum causing citrus flower rot. The fungus in blue is observed in intercellular spaces (arrowheads) and intracellularly (arrows). Host cell walls in (a) and (b) stain red-orange (c) Section of leaf lesion caused by Phakospora euvitis causing grapevine rust. Fungal structures are colored green (arrows) and plant cells blue. Note the distribution of the fungus along the mesophyll. (d–f) Coffea arabica leaves infected with rust fungi (Hemileia vastatrix). The fungus stains in blue and the pectic cell wall in pink (arrow in F). AP Appressorium, OC Oil cavity, EP Epidermis, EC Epicarp, PI Pycnidia; SP Spongy parenchyma, PP Palisade parenchyma, PU Pustule, HY hyphae

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5. Put a drop of distilled water over the sections and cover the sections w coverslip for performing light microscopy analysis. Results The intercelular hyphae of the fungus, haustorium mother cell and hautorium stain in blue and the cell wall in pink (Fig. 4.1d–f).

Fluorescence Techniques Fungal detection using fluorophore-conjugated lectins has some advantages over the brightfield double staining method. One advantage is that WGA (Wheat Germ Agglutinin) lectin binds specifically to the N-acetylglucosamine residues that make up the chitin of the fungal wall. Furthermore, when acquiring lectin one can choose different types of fluorophores, which can emit different wavelengths and can be “counterstained” with other plant wall fluorophores. The aims is to distinguish between the plant cell wall and the fungus wall. The method has favored the study of the colonization process of phytopathogenic fungi in the tissues of different hosts.

Double-Staining with WGA-AF488 and Calcofluor White (C.I. 40621) (Navarro et al. 2019) Uses: Plastic-embedded tissues Reagents Solution A1 (stock solution): Wheat Germ Agglutinin AlexaFluor (WGA) 488 0,1 M phosphate buffer pH 7,2 or distilled water

1 mg 1 ml

Solution A2 (working solution): Solution A 0,1 M phosphate buffer pH 7,2

0,2 ml 0,8 ml

Solution B: Calcofluor White Distilled water

0,01 g 100 ml

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Preparation 1. To prepare Solution A1, dissolve the WGA-AF488 in the buffer0,1 M phosphate buffer pH 7,2, bring to centrifuge gently and collect only the supernatant. 2. To prepare solution A2, dilute the supernatant of solution A1 in phosphate buffer. 3. Solution B must be prepared by diluting calcofluor white in distilled water. Filter the dye before use. Procedure 1. Place in solution A2 and leave for 30 min. 2. Wash once with 0.1 M phosphate buffer pH 7.2 to remove excess dye. 3. Stain with solution B for 1–2 min. 4. Wash in distilled water. 5. Assemble in distilled water and proceed to analysis. 6. For WGA-AF488 use the GFP filter (Ex: 450–490 nm, Em: 500–550 nm) and for Calcofluor White the A4 filter (Ex: 340–380 nm, Em: 450–490). Results The hyphae walls of the fungus fluoresce green with WGA-AF488 and the cell wall fluoresces blue with calcofluor (Fig. 4.3c). When used alone, calcofluor white also reacts on the fungal spores. Observation (a) The stock solution can be stored at −20 °C for at least a month. For short term storage add sodium azide at 2 mM concentration and store at 2–6 °C. Protect from light and avoid repeated thawing and freezing. (b) In step 2, centrifugation aims at removing aggregated proteins and reducing non-specific background staining. (c) In step 3, test several dilutions until the excess fluorescence of WGA AF488 is reduced. (d) If the staining sequence is reversed, it is likely that staining will not occur because Calcofluor can also react with the fungal wall in some cases.

Analysis of Fungi Expressing GFP Fluorescent proteins such as GFP (Green Fluorescent Protein) are synthesized through their expression linked to constitutive genes present in the transformed organisms. In this case, the fungus expresses GFP and it can be located under epifluorescence (EF) or confocal microscopy (CM). Because it is a protein, GFP needs to be analyzed in live cells, i.e., without sample processing. Our focus here is to present a simple roadmap on how to section and mount slides to analyze

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transformed fungi. If the researcher is interested in understanding the procedures to produce modified microorganisms, it is recommended to consult laboratories and research centers specialized in genetic transformation of microorganisms. Procedure 1. Fungal-infected tissues should be sectioned on a slide microtome or free-hand with the aid of a razor blade. 2. Sections shall be deposited on watch glass containing 0.2 M phosphate buffer pH 7.2 (Chapter 2, page 10) immediately after preparation. 3. Select the thinnest sections and transfer them to slides containing phosphate buffer. 4. Cover with coverslip. 5. Analyze under fluorescence microscope or confocal microscope. Results After the fungi are transformed, the fluorescence emission should be verified (Fig. 4.4a). Then, the fungus expressing GFP is inoculated in the plant organ of interest and then observed with the use of the specific filter in the fluorescence microscope (Fig. 4.4a) or, with the specific wavelength laser, in case of using the confocal microscope. Genetically modified microorganisms expressing GFP or other fluorescent protein can be analyzed in vivo without staining the tissue (Fig. 4.4b).

Note  It is suggested at the time of sectioning, to place a dark background under the watch glass to better visualize the sections. GFP is a protein that has at its center a fluorophore with maximum excitation at a wavelength of 488 nm and emission at 510 nm. In case of using other fluorescent proteins (YFP, RFP), study the wavelength required for maximum excitation. Attention should be paid to the fact that plant tissues present autofluorescence at various wavelengths. Therefore, the analysis of tissues without the fungus should be conducted to avoid misinterpretation of the fungus distribution in plant tissues.

Electron Microscopy Conventional Process for SEM (Marques et al. 2018) Uses: Material fixed in FAA or Karnovsky’s Solution

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Fig. 4.4  Photomicrographs obtained after epifluorescence microscopy of the fungus Colletotrichum acutatum (a, b) and the bacterium Xanthomonas citri (C) expressing the green fluorescent protein (GFP). (a) Mycelium and conidia of the fungus. (b) Conidia (CO), germinative tube (GT), apressorium (AP) and hyphae (HP) are observed on sweet orange leaf surface. EP Epidermis

Procedure 1. Fix the samples for 24 h in Karnovsky’s solution 2. Dehydrate in ethyl or ketone series (10, 30, 50, 70, 90 and 100%, 15 min each step), then dry by the CO critical point method2 (Horridge & Tamm 1969). 3. Next, the samples are mounted on aluminum supports (stubs) and gold sputter coated by 180s. 4. Sample ready for SEM analysis. Result Fungal structures such as spores can be easily observed (Fig. 4.6a). Observation During fixation and dehydration, avoid shaking the vials so that the fungus structures remain adhered to the sample surface. The exchange of solutions must be carried out with caution and the deposition of alcohol or acetone must never be placed directly on the samples, but on the inner wall of the container.

 smium Vapor Technique for Scanning Electron O Microscopy (SEM) (May-De-Mio et al. 2006) Uses: Fresh material

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Reagents Solution A: Cacodylate buffer Solution A1: Sodium cacodylate Distilled water

4,28 g 25 ml

Solution A2 (HCl 0.2 N): Hydrochloric acid Distilled water

5 ml 95 ml

Preparation Check the pH of solution A1 and adjust the pH to 7.4 with solution A2. Solution B: Osmium tetroxide Previously boiled distilled water

2 g 100 ml

Preparation To dissolve the osmium tetroxide crystals the ampoule should be broken inside an amber flask with distilled water at room temperature, boiled previously. Wait about 15–24 h before using. Procedure 1. Samples are placed in Petri dish containing moistened filter paper and a watch glass containing solution A and B (1:1). Cover with foil and leave in the fume hood on for 12 h. 2. After this period, replace with pure solution B where they should remain for another 5 h. 3. After this time, remove from solution B and dry in the environment for 30 min. 4. Next, the samples are mounted on aluminum supports (stubs) and gold sputter coated by 180s. 5. Sample ready for SEM analysis. Results Fungal structures such as spores can be easily observed (Fig. 4.6b). However, plant structures may not be well preserved. Observation Be careful when handling osmium tetroxide, as it is an extremely caustic and volatile product. It must be handled in an exhaustion hood and away from the eyes,

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because it can fixate the retina and mucous membranes. To discard the used fixator, it is recommended to put it in a solution of ethanol that, in turn, should be properly discarded.

Cryofracturing Technique (Razera et al. 2019) Cryofracture is a technique (Fig. 4.5a–g) used to study the internal of tissue of the infected plant and the presence of fungi in its interior. It is complementary to the anatomical analysis because it generates an in-depth analysis with higher resolution power due to the use of scanning electron microscopy. Uses: Fixed tissues Procedure 1. Fixation of the samples in Karnovsky solution for 24 h. 2. After fixation, the samples are rinsed with phosphate buffer (Page 15) (2 changes, 10 min each), and it is desirable to cryoprotected them (30% glycerin dissolved in water) in order to avoid ice crystals damage (2 h). 3. After glycerin, the samples are submerged in liquid nitrogen and fractured (cryofracture). 4. The cryo-processed samples are rinsed in distilled water. 5. Acetone series (10, 30, 50, 70, 90 and 100%), 15 min each stage. 6. Drying by the CO2 critical point method. 7. Mounting on aluminum supports (STUBS). 8. Coating with a 30–40 nm gold layer. 9. Sample ready for SEM analysis. Results Fungal structures such as spores, inter- and intracellular hyphae can be easily observed. Observation  The use of cryofracture must be done with great caution and repetitions to avoid the description of artifacts from the fracture.

Transmission Electron Microscopy The technique described below has been employed since 2009 and has good results in fungal-infected tissues (Fig. 4.4c, d). The procedure may be altered depending on the sample. Fungal-infected tissues with profound parietal changes often require a longer infiltration time.

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Fig. 4.5  Stages of the cryofracture. (a) Samples in glycerin 30%. (b–d) Fold the plant tissues in a paper. (e) Deep the paper with the plant samples in the liquid nitrogen for 5 s. (f) Put the sample in a cold iron surface and promote the fracture. (g) Transfer the cryofractured tissue to the distilled water

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Fig. 4.6  Scanning (a–c) and transmission (d, e) electromicrographs illustrating the fungus-plant interaction in different diseases. (a) Coffea arabica L. ‘Catuaí vermelho’ coffee tree leaves inoculated with Hemileia vastatrix processed by the conventional method. (b) Vine (Vitis labrusca L.) ‘Pink Niagara’ leaves infected by Phakopsora euvitis analyzed after osmium tetroxide vapor fixation technique. Note the intact fungus spores (arrow) and plasmolyzed epidermal cells. (c) Cryofracture and scanning electron microscope analysis of sugarcane smut sorus showing intracellular infection of Sporisorium scitamineum in parenchymal cells of sugarcane (Saccharum spp.) (d) Sweet orange (Citrus sinensis L.) petals infected by the fungus Colletotrichum acutatum with subcuticular growth. E. Parenchyma cells of sugarcane (Saccharum spp.) infected by the fungus S. scitamineum. CT Cuticle, EP Epidermis, ST Stomata; FU Fungus, IH Intracellular hyphae, CW Cell wall, GT Germ tube, TR Trichome

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Uses: Fixed material Procedure 1. Fix in 2.5% glutaraldehyde in 0.2 M cacodylate buffer pH 7.2. 2. Wash the samples in cacodylate buffer (3x for 10 min). 3. Post-fix with 1% osmium tetroxide in distilled water for 1–2 h. 4. Rinse in distilled water (3x for 15 min). 5. Place the samples in 5% uranyl acetate in distilled water overnight. 6. Wash the samples in distilled water (3x for 15 min). 7. Dehydrate in increasing series of acetone 10, 20, 30, 40, 50, 70, 90, 100%, 100%, for 15 min at each step. 8. Infiltrate in the steps: Spurr resin and 100% acetone (2:1) for 3 h; Spurr resin and 100% acetone (1:1) for 3 h; Spurr resin 100% overnight. 9. This is followed by polymerization of the resin at 60 °C for 48 h. 10. Remove the blocks and roughen them. 11. Make semi-fine cuts. 12. Section in ultramicrotome and collect the sections on 300–400 mesh screens without film or with collodion film**. 13. For contrasting, deposit the screens containing the sections in drops of 1–3% uranyl acetate solution for 10 min, wash in five drops of distilled water and then pass to lead citrate (Reynolds 1963) for 10–15  min. Wash again in drops of distilled water and dry ***.

Results The use of transmission electron microscopy is recommended for analyzing the fungus-plant interaction on an ultrastructural scale. Only with this technique it is possible to verify the formation of the penetration peg, haustorial neck, haustoria, intercellular or intracellular hyphae or the plant responses to the pathogen. See electromicrographs in Fig. 4.6c, d.

Comments * Semi-Fine cuts (Chapter 2, page 23) ** The film to cover the screens can be made with collodion solution 5% in amyl acetate EMS (CAS 628-63-7). ***Uranyl acetate is toxic and must be handled with care in the fume hood. Lead citrate undergoes carbonation easily causing contamination of the sections. Therefore, perform the contrasting within a closed environment such as a Petri dish with some sodium or potassium hydroxide tablets to absorb the carbon dioxide from the environment.

Chapter 5

Detection of Bacteria

Light Microscopy Due to their size, phytopathogenic bacteria are difficult to observe with usual light microscopy techniques. Molecular techniques have been employed using genetically transformed bacteria that express fluorescent proteins by hybridization of nuclear material or by immunolabeling. For molecular methods of bacteria labeling, it is recommended that infected tissues should be fixed with 4% paraformaldehyde in a 0.2 M phosphate buffer, pH 7.2 (Chapter 2, page 9). For this purpose, analyses of samples should be performed under epifluorescence or confocal microscopy. Sample preparation techniques for scanning and transmission electron microscope analysis are also recommended.

Fluorescent In Situ Hybridization (Hilf et al. 2013) This technique consists of detecting a DNA segment of the bacteria by making a specific probe containing a fluorophore that will hybridize with the genetic material of the bacteria. For the confection of the fluorescent probe, specific kits exist, such as those of the companies Life® and Sigma-Aldrich®. The histolocalization of bacteria can also be performed by immunolabeling using specific antibodies, but fluorescence in situ hybridization has the advantage of being more specific and sensitive (Neves & Guedes 2012).

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 J. P. Rodrigues Marques, M. Kasue Misaki Soares, Handbook of Techniques in Plant Histopathology, https://doi.org/10.1007/978-3-031-14659-6_5

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Uses: Fixed tissues Procedure 1. Fix the material in 4% paraformaldehyde in 0.2  M phosphate buffer pH  7.2 (Chapter 2, page 10), overnight at room temperature. 2. Dehydrate in an increasing series of ethanol (70, 80, 95 and 100%), being two exchanges of 1 h in each graduation. 3. Incubate in CitriSolv® Fisher Scientific, Pittsburgh, PA *. 4. Transfer to paraffin at 65 °C. 5. Section on a rotary microtome 10 μm thick. 6. Place the sections on histological slides. 7. Incubate in CitriSolv® and then wash in 100, 95, 80 and 70% ethanol for 10 min at each step and then allow to air dry. 8. Wash in phosphate buffer and air dry at room temperature. 9. Incubate in lysozyme (0.5  mg/ml) in buffer (100  mM Tris-HCl and 5  mM EDTA, pH 8.2) at room temperature for 30 min. 10. Wash 2–3 times in phosphate buffer (Chapter 2, page 10). 11. For hybridization, the fluorophore-labeled probes are diluted to the concentration 1 ng/μl in hybridization buffer (0.9 M NaCl, 20 mM Tris-HCl, 0.01% SDS and 40% formamide) incubated in the dark at 46 °C for 2.5 h or overnight on a hotplate or in a ThermoBrite StatSpin (IRIS). 12. Excess probe should be removed by successive 15-min washes in buffer (0.9 M NaCl and 0.02 M Tris-HCl, pH 7.5) at room temperature. 13. Wash the slides in distilled water and let them air dry. 14. Slides may be mounted in products that ensure longer fluorophore life, such as Prolong Gold anti-fade (Life Technologies®). Slides are incubated at 40  °C overnight. 15. Analyses should be conducted under epifluorescence or confocal microscopy.

Results After hybridization, the genetic material of the bacteria fluoresces according to the chosen fluorophore (see Hilf et al. 2013).

Observation In replacement of CitroSolv®, other products can be used such as Histoclean® and xylene can be used. Be careful when using xylene because it is extremely toxic and therefore should be handled inside the fume hood. Pay attention to use epifluorescence microscopes that contain the filters or confocal microscopy that has specific laser lines to excite the fluorophore used in the probe.

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Fig. 5.1  Photomicrographs obtained after epifluorescence microscopy of Xanthomonas citri (C) expressing green fluorescent protein (GFP) infecting leaf tissues of Citrus sinensis L. Cross section of leaf lesion caused by citrus canker. The bacterium fluoresces green (*) in this superimposed brightfield and fluorescence image (GFP filter ex: 488 nm; em: 510 nm). SP Spongy parenchyma

Analysis of Bacteria Expressing GFP The use of fluorescent bacteria is very useful for their detection in plant tissues. As in fungi, different fluorescent proteins with green, yellow and red fluorescence can be used. This variability in the types of proteins is advantageous for determining the presence of the microorganisms in the tissues, making it possible to distinguish them from the autofluorescence of the plant. Figure 5.1 illustrates the distribution of genetically modified bacteria expressing GFP in citrus leaf tissues. Follow the same protocol for fungal analysis, aiming the study of fresh tissues.

Electron Microscopy Transmission Electron Microscopy (Alves et al. 2009) Uses: Fixed material Procedure 1. Small sample fragments are fixed in modified Karnovsky’s solution, phosphate buffer was exchanged for cacodylate buffer (Karnovsky, 1965) for 24 h. 2. Wash in cacodylate buffer three times for 10 min. 3. Transfer to 1% osmium tetroxide aqueous solution for 1  h at room temperature.

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4. Wash twice in distilled water for 15 min. 5. Transfer to a 0.5% uranyl acetate solution overnight at 4 °C. 6. Dehydrate in increasing series of acetone (30, 50, 70, 75, 80, 90, and 100%) for 10 min at each step. 7. Infiltrate gradually in acetone/Spurr resin (2:1 for 8 h, 1:2 for 12 h and twice in pure resin for 24 h). 8. Place the materials in the molds with pure Spurr resin and polymerize at 70 °C for 24 h. 9. Section in ultramicrotome at 70 nm thickness. 10. Contrast with uranyl acetate and lead citrate for 15 min at each stage. Result The use of transmission electron microscopy localize bacteria has been essential to understand where bacteria occur (Alves, 2003).

Observation Pay attention to the polymerization time, because if it is longer than 48 h, the blocks may become brittle. The embedding time in the pure solution of the Spurr resin is variable.

Chapter 6

Virus Detection

Light Microscopy For the observation of individualized viral particles, whose size is on the order of nanometers, analysis under a transmission electron microscope (TEM) has been recommended. However, epifluorescence (EF) and confocal microscopes (CM) have been useful in localizing viruses and/or viral inclusions in plant tissues. For such analyses by EF and CM, primary specific antibodies have been employed that bind to viral protein coat proteins and are revealed using a secondary antibody conjugated to a fluorophore. The genetic material of the virus (DNA or RNA) can also be detected by means of the FISH (fluorescent in situ hybridization) technique. Here we will discuss the main procedures used in the immunolabeling of viral particles. Moreover, light microscopy techniques to observe viral inclusion are presented.

Azure A (C.I. 52005) (Christie and Edwardson 1977) The method described below allows the observation in light microscopy of viral inclusions in plant cells after staining with Azure A. For this purpose, the choice of the target tissue is critical and proof of the presence of the virus by molecular methods is recommended. Viral inclusions are dynamic and may develop or degrade over time (Christie and Edwardson 1977). In addition, environmental conditions tend to alter the rate of development of viral inclusions. Tissues with severe necrosis and chlorosis make the detection of viral inclusions difficult, so it is necessary to analyze not only the injured areas, but also their adjacencies.

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 J. P. Rodrigues Marques, M. Kasue Misaki Soares, Handbook of Techniques in Plant Histopathology, https://doi.org/10.1007/978-3-031-14659-6_6

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Uses: Fresh material. Reagents Azure A 2-methoxyethanol

0,1 g 100 ml

Preparation Shake until the dye is dissolved and filter before use. Procedure 1. Place the sections to float or immerse in the solution for 10–15 min. 2. To remove excess dye, place the sections in a 95% ethanol solution for about 30 s or until the tissue is colorless. 3. Replace the ethanol solution with 2-methoxyethyl acetate, which interrupts the discoloration process. 4. Carefully remove the sections and place in a drop of Euparal R on a glass slide and cover with a coverslip. Results The staining results are shown in Table 6.1. For further information it is suggested to consult the reference Christie and Edwardson (1977).

Table 6.1  Results obtained after staining cellular constituents with Azure A (C.I. 52005) and with Calcomine Orange (C.I. 29156) and Luxol Brilliant Green (CAS 71799-14-9) (Orange-Green) according to Christie and Edwardson (1977) Cellular Constituents Cell Wall Chromatin Cytoplasm Inorganic crystals Microbodies and microcrystals Nucleolus Nucleoplasm Plastids Starch granules Inclusions

Dyes Azure A Colorless Blue Colorless Colorless Colorless Red violet Colorless Colorless Colorless Red-violet (RNA) Blue (DNA)

Orange/Green Yellow-green Green Yellow-green Colorless Green Green Yellow Green Green Colorless Green to olive brown

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Calcomine Orange and Luxol Brilliant Green CAS 71799-14-9 and C.I. 29156 (Christie and Edwardson 1977) Uses: Fresh material Reagents Solution A: Calcomine orange 2-methoxyethanol

0,1 g 10 ml

Solution B: Luxol brilliant green 2-methoxyethanol

0,1 g 10 ml

Solution C: Solution A Solution B Distilled water

10 ml 10 ml 80 ml

Preparation Dissolve solutions A and B in distilled water. Procedure 1. Place sections to float or immerse in solution C for 10–15 min. 2. Remove excess dye (5–10  s) in 95% ethanol for about 30  s or until tissue is colorless. 3. Replace 95% ethanol with 2-methoxyethyl acetate 4. Carefully remove the sections and place in a drop of Euparal on a glass slide and cover with coverslip. Results The staining results are shown in Table 6.1. For further information it is suggested to consult the reference Christie and Edwardson (1977).

Fluorescent In Situ Hybridization (Kliot et al. 2014)

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The virus can be detected using the fluorescent in situ hybridization (FISH) method, in which the DNA or RNA of the virus is localized via hybridization of the nuclear material based on the preparation of a complementary probe in which nucleotides are conjugated to fluorophores. The probe can be made in the laboratory by use of kits produced by different companies. Uses: Fresh or fixed tissues Preparation 1. Fix the plant tissues in Carnoy overnight at room temperature. If it is not possible to continue the protocol immediately afterwards, the fixed sections can be preserved in absolute ethanol for several days or even weeks at room temperature. Subsequently, infiltrate in paraffin as described in the first chapter. 2. Wash sections in hybridization buffer (20 mM Tris-HCl, pH 8.0, 0.9 M NaCl, 0.01% sodium dodecyl sulfate, 30% formamide) three times for 1  min in each wash. 3. Hybridize the sections with 500 μl of hybridization buffer supplemented with 10  pmol of the fluorescent oligonucleotide probe that is complementary to a sequence in the viral gene. The target of the probe may be viral DNA or RNA. 4. Wash sections in hybridization buffer three times for 1 min in each wash. 5. Mount the whole sections on slides with hybridization buffer supplemented with DAPI (4′,6′-diamino-2-phenyl-indol) (0.1 mg/ml in 1× PBS). Cover with coverslip and seal with nail polish. 6. Analyze under fluorescence or confocal microscope.

Results The detection of the genetic material of the virus will only be possible if there is a previous investigation to know if the epifluorescence microscope has the specific filters to excite and emit wavelengths in the range of the fluorophore used.The filter or laser to be used depends on the fluorophore present in the probe.

Electron Microscopy

75

Electron Microscopy Transmission Electron Microscope (Kitajima and Nome 1999) This protocol can be used as a routine for the localization and analysis of viral particles. With the use of this technique, it is possible to understand the morphology of the viral particles or their inclusions, as well as to analyze the ultrastructural changes of the plant cell caused by the presence of the pathogen. Uses: Samples (1–3 mm2) fixed in Karnovsky’s solution. Procedure 1. Samples are fixed in Karnovsky’s solution (first chapter) for at least 24 h and then washed in 0.05 M cacodylate or phosphate buffer three times (10 min in each wash); the choice of wash buffer depends on the buffer used for fixation. 2. Post-fix in 1% osmium tetroxide diluted in the same buffer of fixation for 30 to 2 h maximum. Perform this step in the fume hood, since it is a toxic and volatile product. 3. Wash with distilled water twice (5 min in each wash). 4. Contrasting/post-fixation with 0.5% uranyl acetate in overnight solution may be performed. 5. Dehydrate for 20 min in each step of increasing series of acetone 30, 50, 70 and 90% and three washes in 100% acetone. 6. Infiltrate in Spurr resin and pure acetone (1:1 ratio) for 3–5 h under stirring. 7. Complete the infiltration with pure resin for one night. In case of samples with woody tissues, the infiltration time can be extended. 8. Place the specimens in silicone molds with pure resin and leave to polymerize in the oven at 50–70 °C for 48 h. 9. For the counterstaining of ultra-thin sections see the methodology indicated for MET analyses for phytopathogenic fungi.

Results The analyses of the samples prepared following the protocol described above allow elucidation of the details of the viral infection process (Fig. 6.1a, b), the shape of the virus, its cytoplasm, as well as the ultrastructural changes of the infected plant cells.

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Fig. 6.1  Transmission electromicrographs. (a) Section of tomato leaf infected by Tomato golden mosaic virus (TGMV). Phloem parenchyma exhibits in its core an inclusion formed by TGMV particles (*). (b) Section of palisade parenchyma cell of tobacco, infected by Tomato spotted wilt virus-TSWV. Viral particles 80–100 nm in diameter occur in the lumen of the endoplasmic reticulum. A dense inclusion (*), formed by viral particle precursors (arrows) occurs nearby. CH Chloroplasts, MI Mitochondria, CW Cell Wall. (Images kindly provided by Prof. Elliot Watanabe Kitajima)

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© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 J. P. Rodrigues Marques, M. Kasue Misaki Soares, Handbook of Techniques in Plant Histopathology, https://doi.org/10.1007/978-3-031-14659-6

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