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 of an efficient inoculation method would allow many plants to be inoculated and screened for resistance. Various inoculation methods have been used including dip inoculation, pipetting the pathogen cell suspension culture into the whirl of the plant, and needle injection inoculation^8-11. With each method, the pathogen must reliably be introduced in between the plant leaves where the pathogen enters the plant through the formation of appresoria to ensure pathogen development and plant infection^12,13.

The dip inoculation method involves submerging a plant seedling into a pathogen cell suspension culture, while the pipetting method requires placing the pathogen cell suspension culture into the whirl of the plant seedling. However, there are issues with both methods. First, both methods depend on the natural movement of the pathogen from the leaf surface into the plant tissue which is highly variable. Most pathogens naturally enter the plant through stomatal openings or wounds on the plant leaf surface. However, there is significant variability in the pathogens ability to penetrate the plant leaf surface through the stomata and/or wounds on the leaf surface. Therefore, pathogen penetration cannot be controlled with either inoculation method potentially resulting in inconsistent data. Second, when screening a large number of plants, submerging the seedlings into a pathogen cell suspension culture can be time consuming and may limit the number of plants that can be screened. Conversely, the needle injection inoculation protocol described herein delivers the pathogen cell suspension culture in between the plant leaves facilitating the formation of appressoria¹⁴. The pathogen then utilizes the newly developed appressoria to enter the plant eliminating the pathogen penetration issue. Additionally, the needle injection inoculation protocol provides a range of phenotypes for maize and teosinte plants that have been inoculated with U. maydis and demonstrate good infection. The phenotypes can be used as a marker to determine the best concentration for the pathogen cell suspension culture resulting in consistent plant phenotypes within and between different experiments.

Following plant inoculation with a pathogen cell suspension culture, plants are typically screened to detect a resistant or susceptible phenotype^8-11,15. While disease rating scales have being used extensively to screen and classify plant phenotypes, rating scales differ depending on the pathogen being analyzed. Therefore, a disease rating scale protocol establishment for U. maydis and maize interactions can be utilized for similar fungal pathogens¹⁶.

The present series of protocols details needle injection inoculation with a U. maydis cell suspension culture and disease resistance reaction screening of maize, teosinte, and maize x teosinte introgression lines. The present protocols are not limited to needle injection inoculation of U. maydis into maize plants but can be utilized for relatively any fungal pathogen and plant species. Therefore, including the details of both methods in the same protocol will enable researchers to directly utilize the protocols for inoculation and screening or to manipulate the original protocols to better fit the pathogen and plant species of interest.

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 pack the soil over the seed. Planting the seed deeper or packing the soil over the seed may cause problems with seedling emergence.
4. Water the seeds into the soil. Ensure that the soil is soaked and the seeds remain under the soil after watering.
5. After watering, place plants in a growth chamber with day and night environments of 28/20 °C temperature and 14/10 hr of photoperiod, respectively and approximately 500 μmol/m² sec photosynthetically active radiations at the top of the canopy. Maintain the relative humidity during the day and night at approximately 70% and 90%, respectively.
6. Keep all plants in the same growth chamber to maintain a growth environment that is congruent across the experiment.
7. After 10 days, remove the plants from the growth chamber and inoculate the plants with the U. maydis cell suspension culture using a needle injection inoculation method. Note: Maize plants can be inoculated 7 days after planting^8-10. However, the teosinte plants are too small after 7 days. Therefore, inoculate both maize and teosinte plants 10 days after planting for consistency within the experiment (see step 2.12).

2. Needle Injection Inoculation

1. Do all work in a laminar flow hood. Remove U. maydis glycerol stocks from freezer storage. Use a sterile loop and streak glycerol stocks of U. maydis wild-type strains ½ (mating type a1b1) and 2/9 (mating type a2b2, near isogenic to ½) on to potato dextrose agar (PDA) plates. Maintain strains separately.
2. Place PDA plates streaked with U. maydis in a 30 °C incubator for two days. If using a different biotrophic pathogen use the appropriate strain, media and growth conditions. Monitor the growth of the pathogen over the two day period to ensure that the U. maydis strain is growing well.
3. Remove the PDA plates from the incubator after two days. The plates should have good pathogen growth and contain single colonies (Figure 2A). It is important to obtain single colonies. If single colonies are not present restreak the plates at a lower concentration.
4. Do all of the work in a laminar flow hood. Use a sterile toothpick to select a single colony for each strain from the PDA plates. Place the toothpick containing a single colony into a 3 ml potato dextrose broth (PDB). It is advised to have 2-3 cultures.
5. Place the 3 ml PDB cultures into a 30 °C incubator/shaker for two days at 200 rpm. Monitor the growth of the culture over the two day period to ensure growth of the culture. The culture should appear very cloudy.
6. Remove the liquid cultures from the incubator/shaker and measure the concentration at OD₆₀₀ to ensure that the cells were grown to an OD of 1.0 (~1 x 10⁷ cells/ml)¹⁷.
7. Bring the U. maydis cell suspension cultures to a final concentration of 1 x 10⁶ cells/ml, using water in a final 30 ml culture volume. This concentration consistently results in good infection of the plants with the pathogen cell suspension culture.¹⁷

Note: Various cell suspension concentrations should be tested when using different pathogen strains to determine the appropriate cell titer needed for inoculation^18,19. The given final concentration for the cell suspension culture can be used as a starting point for tittering. The appropriate concentration of the pathogen cell suspension culture should be verified by visualizing the plant phenotypes with good infection (Figures 3A-E).

8. Mix equal volumes of the two U. maydis strains prior to inoculation. If using one pathogen strain proceed to step 2.9. Prepare fresh U. maydis cell suspension cultures for each inoculation experiment and discard cell suspension cultures after two days.
9. For the experimental needle injection inoculation, fill a 3 ml syringe with the U. maydis cell suspension culture by drawing the cell suspension culture into the syringe.
10. For the control needle injection inoculation, fill a 3 ml syringe with water¹⁷. Use the same procedure for the experimental needle injection inoculation.
11. Attach a 0.457 mm x 1.3 cm hypodermic needle to the end of each 3 ml syringe. The selected needle size will deliver the cell suspension culture in between the plant leaves with minimal damage to the plant tissue.
12. Remove the experimental and control plants from the growth chamber 10 days after planting in preparation for needle injection inoculations (Figure 2B) (see step 1.7).
13. Carefully insert the hypodermic needle containing the U. maydis cell suspension culture into the stem of an experimental plant at a 90° angle just above the soil line. Insert the needle until it is in the middle of the stem. Do not push the needle through the stem (Figure 2C).
14. Inject the experimental plant with about 100 μl of the U. maydis cell suspension culture^18,19. This will vary slightly depending on the height of the seedling. The cell suspension culture will push through the stem and move into the whirl of the plant. The cell suspension culture will be visible in the whirl of the plant. Continue injecting 100 μl of the cell suspension culture into each individual plant until the 3 ml syringe is empty.
15. After the injection, carefully remove the needle from the plant stem. Remove the needle from the now empty 3 ml syringe and fill with water. Attach the needle back to the syringe and push the water through the needle to remove any plant tissue that may be caught in the needle tip.
16. Repeat steps 2.9-2.15 for each experimental plant. Follow the same protocol for the control plants by injecting water.
17. Place the inoculated experimental and control plants back into the growth chamber. Water the plants daily by wetting the soil not the plant tissue.
18. Check the plants daily to detect pathogen development and plant resistance reactions.

3. Resistance Reaction Screening

1. Score and record the resistance reactions for each plant 7, 10, 14, and 21 days post inoculation (dpi) using a 1 to 5 resistance reaction rating scale. Disease severity increases as the numerical values on the rating scale increases (Table 2). A 1C (Leaf chlorosis), 1A (Leaf anthocyanin production), or 2 (small leaf galls) resistance reaction indicates resistance. A 3 (stem galls), 4 (basal gall), or 5 (plant death) resistance reaction indicates susceptibility (Figures 3A-E and Table 2)^18,19.
2. Score both experimental and control plants and record resistance reaction ratings.
3. Compare the resistance reactions of the experimental and control plants. Select experimental plants with a 1C, 1A, or 2 resistance reaction rating. These plants are considered to be resistant to U. maydis^18,19.
4. Repeat the entire experiment to verify the plant phenotypes.

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 s...

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

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