The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Summary

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

Abstract

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

Introduction

Rice blast, caused by M. grisea, is one of the most serious diseases for rice varieties worldwide^1,2. The process by which M. grisea infects host plants includes a conidia production and surface attachment, a conidia germination and appressorium formation, a formation of the penetration peg and infectious hypha differentiation, and a disease spread³. All these stages are common in many other plant pathogenic fungi, and, indeed, a blockade of any single stage prevents the infection of host plants. Owing to its economic importance and genetic tractability, M. grisea<....

Protocol

1. Spray Inoculation with a Suspension of M. grisea Conidia

1. Fungal culture for M.grisea
   1. Prepare the oatmeal tomato agar (OTA) culture medium for fungal strains.
   2. Weigh 30 - 50 g of oatmeal, add this to 800 mL of distilled/deionized water (ddH₂O) and boil the mixture for 30 min in the electric pot.
   3. Filter the boiled oatmeal juice into the beaker through a piece of gauze.
   4. Add 150 mL of tomato juice and 20 g of agar to the filtrate in the beaker and add ddH₂O up to 1,000 mL.
2. Preparation of experimental materials
   ....

Results

The entire workflow for the technique is shown in Figure 1. The plant infection assays were performed on 14-day-old susceptible rice seedlings (O. sativa cv CO-39) or susceptible 7-day-old barley leaves (H. vulgare cv Golden Promise)^7,8,9. To test for an infection on the rice leaves, a conidial suspension (1.0 x 10⁵ spores/mL) of the .......

Discussion

Plant disease resistance genes play an essential role in preventing infections by pathogens, including fungal pathogens^1,12. Rice blast has been used as a model to understand the nature of pathogen population structures and to identify plant resistance genes⁴. Therefore, it is necessary to examine the disease resistance genotype and avirulence genotypes of the main varieties of agricultural plants on a large scale to identify disease-resis.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Filter paper	GE Healthcare brand(Sweden)	10311387
50-mL tube	CORNING(Amercia)	430290
Centrifuge	Eppendorf(Amercia)	5804R
Culture dish	Thermofisher(Amercia)	150326
0.5-5 mL pipette	Eppendorf	4920000105
100-1000uL pipette	Eppendorf	4920000083
Vacuum pump	Leybold	D25B
Dissection needle	FST	26000-35
Incubator	MEMMERT	PYX313
Inoculation ring	Greiner Bio One	731175