Double-Staining Method to Detect Pectin in Plant-Fungus Interaction

Jo&#227;o Paulo  Rodrigues Marques; Laura Galvan Nuevo

doi:10.3791/63432

Método de doble tinción para detectar la pectina en la interacción planta-hongo

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Biology

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JoVE Journal Biology

Double-Staining Method to Detect Pectin in Plant-Fungus Interaction

Método de doble tinción para detectar la pectina en la interacción planta-hongo

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06:39 min

February 4, 2022

DOI: 10.3791/63432-v

João Paulo Rodrigues Marques¹, Laura Galvan Nuevo²

¹Faculty of Animal Science and Food Engineering, Department of Basic Sciences,University of São Paulo, ²Laboratory of Nuclear Instrumentation, Center of Nuclear Energy in Agriculture,University of São Paulo

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Overview

This protocol describes a light microscopy method for detecting pectin in coffee-fungus interactions. It allows for the identification of fungal structures and defects in pathogen cell wall polymers, providing insights into plant-fungal interactions.

Key Study Components

Research Area

Plant-fungal interactions
Histopathological studies
Fungal pathogen detection

Background

Significance in diagnosing diseases caused by fungi like coffee rust and cercosporiosis
Need for techniques that do not require expensive equipment
Rapid techniques for clear distinction of plant structures

Methods Used

Light microscopy for pectin detection
Utilizes coffee leaf samples infected with fungi
Double staining method with cotton blue and ruthenium red

Main Results

Identified dark blue staining in fungal hyphae and structures
Demonstrated interactions between Hemileia vastatrix and host plant cells
Highlighted changes in cell wall integrity and the presence of reproductive structures

Conclusions

The study illustrates a reliable method for examining plant-fungal interactions
Contributes to understanding biotrophic and necrotrophic fungal diseases

Frequently Asked Questions

What types of fungi can be analyzed using this protocol?

The protocol is applicable for studying biotrophic and necrotrophic fungi, including specific pathogens like Hemileia vastatrix and Cercospora coffeicola.

Do I need specialized equipment for this experiment?

No, this protocol is designed to be performed using standard light microscopy without requiring expensive equipment.

Can this technique be applied to other plant species?

Yes, further studies may explore its applicability to various plant-fungal interactions across different pathosystems.

What is the importance of pectin in plant-pathogen interactions?

Pectin is crucial for maintaining cell wall integrity and can influence the outcome of pathogen invasion and plant defense mechanisms.

How does the double staining method enhance visualization?

Double staining with cotton blue and ruthenium red allows clear differentiation of fungal structures and plant tissue under light microscopy.

What are some observed effects on plant cells during fungal infection?

The protocol reveals cell wall breakdown, loss of integrity in interaction zones, and presence of reproductive fungal structures in the plant tissue.

Este protocolo describe un método microscópico para detectar la pectina en la interacción café-hongo.

Este protocolo es significativo ya que permite identificar la interacción planta-hongo. Además de colorear la pectina, también demuestra defectos en el polímero de la pared celular del patógeno y permite la identificación de estructuras de hongos. Esta técnica simple y sin complicaciones se puede realizar utilizando microscopía de luz y no requiere ningún equipo costoso, como el microscopio electrónico de barrido.

Además, es una técnica rápida que obtiene una excelente distinción de estructuras vegetales para estudios histopatológicos. La técnica identifica la interacción planta-hongo. De ahí que colabore en el diagnóstico de las enfermedades causadas por hongos biotróficos y necrotróficos como la roya del café y la cercosporiosis.

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