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Hayslett, Maya C. 2017. Investigating Resistance in a Population with Rapid-cycling Brassica species and Albugo candida. The Plant Health Instructor. 10.1094/PHI-T-2017-0711-01

Maya C. Hayslett, Paul H. Williams, Douglas I. Rouse, and Victoria Kartanos, Department of Plant Pathology, University of Wisconsin – Madison

Instructor Notes

INSTRUCTOR'S NOTE #1

Discussion of learning goals

The basis of host resistance is an important topic in plant pathology and we focus on this concept in our introductory plant pathology course with several lectures and lab exercises. Based on the exercises we were using in class previously and a review of lab exercises available from previous APS lab manual publications (Baudwin 1988, Boothroyd et al., 1962), many lab exercises for concepts of resistance focus on monogenic, complete resistance of one to a few plant genotypes to a single pathotype. When there is only a comparison between two genotypes, one with resistance and one without, students may be left with the impression that neither the “background” genotypes of the plants nor the environment influence degree of disease expression (which we call the interaction phenotype (IP)). When students can observe populations of genetically diverse plants with and without major resistance genes, they have the opportunity to grasp the complexity of the IP through examination of their own data. We wanted to draw students’ attention to the fact that variation in IP occurs that is not explained by a single major gene even in systems where such a gene is clearly present. 

Within the species Brassica rapa there is a major dominant resistance gene to pathotype 2 of Albugo candida. We use a population of B. rapa where approximately 70% of the plants happen to contain this gene. We want students to see the variation due to this gene and also (and especially) the variation from plant to plant due to minor unknown factors perhaps attributable to the differing genotypes of each plant or alternatively very local effects of environment. We use a population of B. juncea which contains no major gene resistance to A. candida as a control comparison. In that case there will be variation in susceptibility from plant to plant with no obviously highly resistant plants unless there is an escape from the inoculation procedure. When this happens it makes a good discussion point. Notice that although all plants are susceptible in the B. juncea population, there is still variation in the interaction between pathogen and host using our rating scale. This is most easily discussed as variation in the interaction phenotype rather than as variation in the susceptibility phenotype.

We ask the students to interpret their data by first recognizing that the two populations of plants (B. rapa population versus B. juncea population) differ in a fundamental way as it is clear that many plants in the B. rapa population have a very high level of resistance. We can group almost all of them into one of two categories. We ask them whether they think there is variation beyond that due to a single dominant gene and why or why not. This leads to a discussion of the fact that there are probably a number of genes that influence the IP. Also it may be possible that the environmental conditions are a factor even though in our experiment we kept the environment as uniform as possible.

The lab activity described here allows students to see a diversity of interaction phenotypes based on a genetically diverse population of hosts.

INSTRUCTOR'S NOTE #2

Preparation of materials

This protocol was adapted from Crucifer Genetics Cooperative Resource Book (Williams 1985).

Timing:

• 5 days before class: Plant seeds
• 2 days before class: Thin to one plant per cell and prune apical meristem
• 1 day before class: Prune apical meristem
• 1 hour before class: Start inoculum​ incubating

When class starts: Check for zoospores every half hour. Inoculate as soon as zoospores are released. Zoospores should be released between 1 and 3 h after starting incubation.  If your lab is shorter than 2 h, you may want to start incubating more than 1 h before class.  Zoospores will continue to swim for 30 min when the temperature is lowered (see “Inoculum” below for details).

After inoculation: Place in moist chamber and incubate

• 6 to 12 h after class: Return plants to normal growing conditions
• 2 days after class: Fertilize and prune meristem
• 7 days after class: Rate for disease
• 8 days after class: Collect sporangia from infected leaves (for future use)

Seed and pathogen

Seed is available from the Rapid-Cycling Brassica Collection (https://rcbc.wisc.edu/). For the B. rapa seed we use 1-033 Sta B. rapa and for the B. juncea we used 4-001 Bpo B. juncea. A. candida pathotype 2 is available from Maya Hayslett at the University of Wisconsin – Madison. An APHIS import permit is required for movement of plants pathogens. A. candida is a common pathogen and it may be possible to collect some locally though it may be a different pathotype. Each time the experiment is run, the resulting sporangia can be collected from infected leaves for future use. Break open pustules with a dissecting needle and tap or shake leaves to release sporangia. Sporangia can then be collected, transferred to microcentrifuge tube, and stored at -20°C. Sporangia remain viable for at least another year if kept at this temperature.

Growing plants

We grew seventy to one hundred plants per lab section using the plant growth system designed by Paul Williams and described below. You may use this or another system to grow a large number of small plants in an organized way so that students can rate disease for each individual plant. Seeds are planted 5 days before inoculation. Fertilize (1 g of Peters in 2 liters water) at planting and 5 to 7 days later. At both 3 and 4 days after planting, use fine forceps to prune the apical meristem from the center of the growing cotyledons to enlarge the cotyledons.

Plant growth system

The plant growth systems are easy to make and can be reused. See Figure 5 for photographs of each step. A plastic tub of approximately 12.5”(W) × 7.5” (D) × 4.5” (H) is the base. The base is fitted with a piece of corrugated plastic (commonly used for yard signs) about halfway in. This is held up with wire (we used coat hangers) inserted through the base. Capillary cloth (available at garden stores) is used to create a self-watering system and seed starter trays are used to grow the plants in individual containers. For more detailed instructions contact Maya Hayslett at the University of Wisconsin – Madison.

Inoculum

In a 1.5-ml microcentrifuge tube, place a small amount of sporangia, approximately 10 µl in volume, and fill with DI water. Place in an ice bath at 20°C. After 30 min, place a drop of inoculum on a slide and look for zoospores. If zoospores are present, inoculate immediately or cool ice bath to 10°C. If zoospores are not present check again every 30 min until you see swimming zoospores. Zoospores are usually present within 3 h of starting incubation. Once zoospores are released, you can lower the temperature of the ice bath to 10°C and the zoospores will continue to swim for approximately 30 min.

Incubation after inoculation

Cover plants with another plastic tub lined with wet paper towels to create a moist chamber. After inoculation, plants should be incubated in the dark at 20°C for 6 to 12 h. Moist chambers are then taken off and plants returned to normal growing conditions.

INSTRUCTOR'S NOTE #3

Explanation of the expected results:

B. juncea

This population has no known resistance to this pathotype and so should be universally susceptible to the pathotype. The rating scores are typically mostly 9s with a few 7s and an occasional 5.

B. rapa

About 70% of the population has the single resistance gene and shows complete resistance. 30% of the population does not have the gene and shows variation in susceptibility. Individuals rated for a high amount of disease development may have no resistance. Individuals rated for low to medium amounts of disease development likely have some type of incomplete, multigene resistance. The variability is likely related to the host genetics and not the pathogen genetics because the same pathogen was applied to all the plants. The hosts however, are genetically variable. Variation due to environment is also possible but less likely as the environment is very similar for all plants.

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