A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Summary

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described. The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

Abstract

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately $1.0 billion annually in the U.S.¹ Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut². However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually^3-5. Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants.

Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.

Introduction

Fungal diseases of plants represent one of the most eminent threats to agriculture. The need to develop crops with improved disease resistance is increasing due to the food needs of a growing world population. Plant pathogens naturally infect crop plants in the field causing diseases that negatively impact crop yield⁶. It has been shown that identifying and utilizing resistant plants can improve resistance and decrease yield loss. Resistant cultivars have been identified in many plant species including maize, wheat, rice, and sorghum by inoculating the plants with a plant pathogen and selecting for resistant lines⁷. Therefore, development and use....

Protocol

1. Growth of Plant Material

1. Select plant lines for inoculation and screening. Two maize lines, five teosinte lines, and forty maize x teosinte lines with uncharacterized resistance to U. maydis were used for this work (Table 1).
2. Plant seeds for experimental (U. maydis injection) and control (water injection) needle injection inoculation experiments. Do this for each plant line.
3. Plant four seeds (replicates) for each plant line in small flats by pushing the seeds about ½ inch into the soil with finger and covering with soil lightly (Figures 1A and 1B). Do not ....

Results

A successful needle injection inoculation can be determined by visualizing the phenotype of the plants inoculated with U. maydis (experimental). The majority of the experimental plants were susceptible to U. maydis infection. The susceptible plants showed very severe disease development demonstrated by stem and basal gall formation with black teliospores (Figures 3D and 3E, Table 2). Several plants were dead after inoculation due to the severity of the .......

Discussion

In this study the needle injection inoculation method used to deliver a strain of U. maydis into the stem of 700 maize and teosinte plants was successful. Additionally, a revised disease resistance rating scale was used to screen the plants and detect pathogen development. As a result of using both methods, plant lines that are resistant to U. maydis were identified among 700 maize and teosinte plants that can now be combined and tested in breeding programs for improved disease resistance.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Seed for plants			Collected from original crosses
Growth chamber	Conviron	PGR14 REACH-IN
Planting flats	Hummert International	14-3385-2
Soil (3 parts pine bark; 1 part peat moss with perlite)	Hummert International	10-1059-2
Laminar flow hood	Lab Conoco	70875372
Glycerol stock of pathogen (U. maydis) or fungal pathogen of interest			Stocks were grown from original culture
Sterile loop	Fisher Scientific	S17356A
Potato dextrose agar (PDA) plates	Fisher Scientific	R454311
Incubator set to 30 °C	Fisher Scientific	11-690-650F
Sterile toothpicks	Walmart		Purchased from Walmart and sterilized by autoclave
Potato dextrose broth (PDB)	Fisher Scientific	ICN1008617
Incubator-shaker set to 30 °C	New Brunswick	14-278-179
Spectrophotometer	Fisher Scientific	4001000
U. maydis cell suspension culture (1 x 106 cells/ml)			Grown from glycerol stock as described in the methods
3 ml Syringes	Becton Dickinson	309606
.457 mm x 1.3 cm Hypodermic needles	Kendall Brands	8881250321