PLP 6404 Epidemiology of Plant Diseases Spring 2015 LAB 2 PHASES IN THE DISEASE CYCLE: GREENHOUSE AND LAB EXERCISE

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1 PLP 6404 Epidemiology of Plant Diseases Spring 2015 LAB 2 PHASES IN THE DISEASE CYCLE: GREENHOUSE AND LAB EXERCISE "Variation in Host-Pathogen Interactions and its Effect on Epidemic Development" Purpose: Part I - To observe and quantify the results of monocyclic interactions between watermelon (Citrullus lunatus) cultivars and one isolate of Colletotrichum lagenarium causing anthracnose. Part II - To analyze the impact of differences identified in Part I on epidemics of watermelon anthracnose using a computer simulator (this will be described in a following handout). Background and General Procedure: Plant resistance provides a useful means of managing plant disease. Pathologists and breeders attempt to categorize resistance in a number of ways, e.g. differential vs. non-differential, monovs. polygenic, and rate-reducing vs. absolute. Absolute resistance is often associated with a hypersensitive response of the host to the pathogen. It is often assumed that certain groupings coincide, i.e. rate-reducing resistance is non-differential and controlled by several genes. While this correlation may exist in many instances, notable exceptions exist (Parlevliet, 1977). Watermelon anthracnose is an important disease in humid and warm climates like in Florida. Differences in host resistance exist, some cultivars exhibiting hypersensitive reactions to C. lagenarium, while others show more general resistance. In this lab you will observe the effects of different levels of resistance on four important aspects determining the rate of disease development - infection efficiency (IE), the latent period, lesion expansion and sporulation rate. Note: IE will be defined here as the proportion of viable fungal spores present in a suitable court that actually produce macroscopically visible lesions. These aspects of the infection cycle are very important in determining the overall rate of disease development. These factors are often referred to as "components of resistance". Measurement of these factors in monocyclic experiments should provide useful insight into the potential development of epidemics in the field. One obvious problem is the interpretation of these individual factors in terms of their actual effects on epidemics consisting of many infection cycles. Plant disease simulators provide us with one way of making this "quantum leap". Simulation models are constructed by identifying the mathematical relationships between various components that influence pathogen and disease development. Simulation models can be

2 constructed so that effects of environmental factors such as temperature and leaf wetness on individual phases in the infection cycle are also included. Such models can be run for many days and the output will be disease progress over time. Models are only an abstraction (and simplification) of the real processes and may not be 100% accurate. However, they can give insight in the relative importance of the various phases of the infection cycle for the overall outcome of disease development. In recent years many pathologists have managed to construct some fairly complex and extensive models of various plant diseases. The number of equations involved and the enormous amount of computation required to use them necessitates the use of computers. In this lab we will use a simulation model to analyze the effect of different infection efficiencies, latent periods and sporulation rates on epidemic development. The data you collect from the various cultivar-isolate interactions will be used in the model to generate anthracnose epidemics under a particular set of environmental conditions. We will generate disease progress curves and calculate areas under the disease progress curves (AUDPC) and final proportion of diseased tissue for comparison of disease development on the different cultivars, and determine the relative effects of different phases in the infection cycle on AUDPC and final disease levels. Specific Procedures: Part I 1. Please, form 5 groups with 4 or 5 people per group in the lab (2306 Fifield Hall). 2. Prepare inoculum ( ) Cultures of Colletotrichum orbiculare were grown on ½ strength PDA for 9-10 days. Take several petri plates (we will tell you how many) containing fungal cultures of C. orbiculare. Using a 1000 µl pipet, transfer 2.0 ml of sterile deionized water with 0.01% Tween- 20 to each plate. Gently scrape the plate using a cell scraper. Using the same pipet, carefully remove the resulting spore suspension and transfer to a clean sterile glass tube labeled original spore suspension. Be careful not to touch the agar with the pipet tip. Change the pipet tip and repeat this for each plate using an additional two ml of water. Vortex mixture so that spores are evenly dispersed in the suspension. Determine spore concentration by counting in a hemacytometer (see hemacytometer instructions below). Set up a germination test (see below). 3. Check spore viability (germination) ( , and ) On January 21, prepare inoculum as described above and determine the spore concentration with the hemacytometer. Dilute the suspension to approximately 7.0 X 10 6 spores/ml. Total volume should be 5.0 ml. Vortex the tube again before removing spores. Place sterile water and the

3 appropriate volume of your original spore suspension in the tube labeled Diluted spore suspension and gently vortex. Place 10 µl of the diluted spore suspension in the center of the marked area on a plate of water agar and spread over marked area only with sterile yellow plastic loop. 25 C for 24 hours (put in refrigerator). Check germination on and [It may be too difficult to see germination on , so, preferably on ]. Cut out the marked area with inoculated agar, place on an object glass, cover it with a cover slip and determine the percent spore germination. A spore is considered germinated when the length of the germ tube is equal to or longer than the width of the spore. Calculate the percentage of viable spores. 4. Go to the greenhouse ( ) Prepare inoculum and set up spore germination test. Take to the greenhouse: 1) test tube rack, 2) tube of diluted spore suspension, 3) tube of sterile DI water with 0.01% Tween 20, 4) cardboard template, 5) a marker 6) 200 ul pipet, 7) box of yellow tips, and 8) cell scraper. Place all items in the small plastic box that has been provided for transport. 5. Inoculate plants ( ) Seeds from three different varieties of watermelon (Citrullus lanatus) were planted on 12/01/14 and watered daily. The varieties are: Georgia Rattlesnake (GAR), Sweet Favorite (SWF), and Jubilee (JBL). Three seeds per pot were planted for each variety and germination was close to 100%. As the seedlings emerged, plants were thinned to one plant per pot when the first true leaves appeared. Osmocote fertilizer was applied on 01/09/15 at the rate of 1 teaspoon per pot. Plants have been watered for you prior to inoculation. Immediately after inoculation, the plants will be placed in boxes with lids to prevent dehydration of the inoculation droplets and maintain high relative humidity. There are 5 boxes; each group will have one box; this will be a block in the experiment. There will be 6 inoculated plants and 6 control plants per box (left or right, randomized) and 2 pots of each variety in each group of inoculated and control plants per box (the varieties are randomized). Block1 GAR SWF SWF GAR SWF JBL GAR JBL JBL GAR JBL SWF Block2

4 Block3 Block4 Block5 Prior to beginning the inoculation procedure, wet the paper towels that are in the bottom of the large plastic boxes. Inoculation procedure: Using a 3 cm X 3 cm cardboard square as a template, draw a square on two leaves per plant, with a marker, WHILE THE PLANTS ARE STILL OUTSIDE THE BOXES. For each group, two plants of each variety (six plants total) will be inoculated with the spore suspension. Also - two plants of each variety (six plants total) will be inoculated using only sterile water and Tween 20. These are your control plants. Therefore, each group will inoculate a total of twelve (12) plants. To inoculate plants, hold plant leaf securely in a horizontal position, supported by the palm of your hand. Using the pipet place 100 µl of spore suspension or the control DI water % Tween in the center of the marked area. Using the cell spreader, gently spread the spore suspension evenly over the entire marked area. Inoculate all the controls first, using only the DI water and then inoculate plants using the spore suspension. Gently mix the spore suspension each time before removing inoculum. Place labels in the pots!! Place each of the plants into the boxes immediately after its inoculation to prevent drying of the leaves! See diagram above to determine how to place the plants in the boxes. 6. Open boxes and removal of plants ( ) Leave plants in plastic boxes for 24 hours, then remove the lid from the box (on Saturday!); each group of students is responsible for their own box. Two days later (on Monday), remove the plants from the box and place them on the greenhouse bench. Water plants daily, and observe daily for symptom development.

5 7. Observations Monday Jan-26 Check plants for symptoms; write down for each plant if there are symptoms Wednesday Jan-28 Check plants for symptoms; count lesions per plant; estimate size of lesions. Friday Jan-30 Estimate size of lesions again, place the plants in the boxes, rewet paper towels in the bottom of the boxes and close them to induce sporulation. Monday Feb-2 Check plants for sporulation with hand lens; remove spores with sticky tape and count in the lab under the microscope If there is no sporulation yet, continue to check the following days. 8. Calculations: Infection Efficiency: number of lesions per marked area divided by the number of VIABLE spores applied (first calculate number of viable spores based on the germination%) Lesion expansion: While scoring your plants, select from each cultivar-isolate combination four leaflets that have a low number of infections (preferably three or less). Observe and measure lesion size on these leaflets by recording lesion diameter both parallel and perpendicular to the mid-vein. Repeat your measurements two days later. Sporulation: Count the number of spores per inoculated area for each cultivar. Calculate the number of spores produced per number of spores applied during inoculation. Latent period: determine the period between inoculation and the start of sporulation for each plant. There may be variation in the time from inoculation to sporulation. The latent period ends when 50% of the plants of a certain cultivar is sporulating. 9. Reports Prepare group reports of about 3 pages with a brief introduction (250 words), materials and methods, including observations and statistical analysis (400 words), Results (300 words), Discussion (250 words), References (about 5, only from refereed journals, no websites). Graphs and tables can be added, are not part of the page limit. Letter size not smaller than Times New Roman 11. Points to Address and Consider: 1) With the data you collect and your information on the initial concentration of inoculum, compute IE for each cultivar-isolate interaction. 2) Can you speculate about the severity of epidemics on the 3 cultivars? 3) Which of the four components of the infection cycle might have most effect on epidemic development? 4) What can you say about the resistance present in each of the three cultivars?

6 Use of the Hemacytometer 1. Clean hemacytometer and cover slip using alcohol and kimwipes. Handle the coverslip gently as it is very fragile. DO NOT DISCARD COVERSLIPs. Hemacytometer coverslips are expensive and used multiple times. 2. When these are dry, place coverslip on hemacytometer. 3. Vortex the spore suspension. 4. Using the 10 ul eppendorf, pipette, take 10 ul of spore suspension. Insert the pipet tip into the grove between the hemacytometer and the coverslip at the edge of the chamber and slowly eject the cell suspension under the coverslip. 5. Let the cell suspension be drawn under the coverslip by capillarity action. Add liquid until the sliver area is covered. Do not overfill or under fill the chamber and examine closely to be sure that there are no air bubbles under the coverslip. (Figure 1) 6. You may want to practice with water before using the spore suspension. In between each use, rinse hemacytometer with deioinized water, then clean with alcohol. 7. Remix the tube of cells. Refill the pipet with cells and fill the second chamber. 8. Pick up the hemaycytometer, holding it level. Place it on the microscope stage. Allow a few minutes for the spores to stop moving settle in one place. Then turn the objective to 10X. 9. Count the spores in A,B,C, and D (Figure 2) and take the average Count the cells in the other chamber of the hemacytometer and repeat the calculation. Take the average of the two numbers to determine the concentration of your spore suspension 10. To calculate the number of spores /ml.: The size of each section ( A for example) = 1 mm x 1 mm (Figure 2). The height of the liquid between the hemacytometer and the covergalss is 0.1 mm. Use this information to calculate the number of spores /ml. 11. When counting is complete, rinse the hemacytometer and coverslip with deionized water and then clean with ethanol and kimwipes. 12. Adjust the concentration of spores to 7.0 X 10 6 spores/ml.

7 Figure 1: Correctly and incorrectly filled hemacytometer = 1.0 mm Figure 2: The photograph above on left is a hemacytometer. Each silver coated section contains a grid that is pictured on the right.