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Laboratory Exercise Instructor Notes
Following the disease progression of an ectotrophic root-infecting fungus

1. Perithecia production requires a minimum incubation period of 6 weeks. Assays may be set up weeks in advance to ensure students will observe all steps of the G. graminis disease cycle in a timely manner. See Note #6 below.
2. Depending on lab size and supplies available, students may work individually, in pairs or small groups. Adjust the number of test tubes and wheat seeds needed accordingly. The wheat cultivar Norm and the creeping bentgrass cultivar Penneagle have always been used to establish these assays at Ohio State; however, all cultivars of wheat and creeping bentgrass are susceptible to take-all and should work for this assay.
3. Isolates recently collected from infected plant material work best. G. graminis var. avenae was chosen for this exercise because it will infect both wheat and creeping bentgrass. All isolates tested with this method are maintained on 1/5 strength PDA. It is unknown what impact, if any, full strength media would have on cultures and this procedure.
4. Depending on environmental conditions and seed age, this time may vary. Test the timing of 10-12 seeds under normal lab conditions to determine when to perform this step. Seedlings should not become rooted into the filter paper or the paper towels because it then becomes difficult to inoculate the tubes.
5. Petri dishes with small discs of filter paper or paper towels can be used in place of the plastic bag. This may be a preferred method as 8-12 seeds can be placed in one dish, and the dishes can be distributed to groups or tables of students.
6. Besides maintaining plant growth, exposure to light is a requirement for the formation of perithecia by G. graminis.
7. The cotton plug should be tight enough to block air flow and potential contaminants from entering the test tube but loose enough to permit the wheat plant to continue to grow.
8. Test tubes with creeping bentgrass do not need to be covered with parafilm until 28 days after set-up and only if perithecia are desired. Perithecial formation begins as small spherical mycelial balls, that form perithecial necks during maturation. Depending on isolates, perithecial initials may be observed as early as 28 days with mature perithecia by 35-42 days.
9. It is normal to see the water agar disappear over time as the water is utilized by the plants. The decrease in media will be more noticeable in the wheat tubes. Additional water agar (cooled-until a lumpy gel) can be added to wheat tubes if necessary, but this may delay maturation of perithecia.

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