Testing Seed For Tilletia indica in CaliforniaT. E. Tidwell. California Department of Food and Agriculture, Plant Pest Diagnostics Center

3294 Meadowview Road, Sacramento, CA 95832-1448

AbstractThe size-selective sieving technique developed by G. L. Peterson (USDA/ARS)(Peterson,et al., 1998) is a very useful procedure for routine testing of wheat (and other) seed todetect the presence of teliospores of Tilletia indica, the cause of Karnal bunt of wheat.The test is rapid, sensitive, requires only inexpensive equipment and materials, and issimple to teach. A 50 g sample of wheat is washed using a conventional centrifuge-wash method, but with the sdditional step of passing the rinse water through screensof different mesh sizes. This removes extraneous material that is larger and smallerthan the size range of the propagules of interest. Teliospores of T. indica range from 22µm to 47 µm, thus the sieve mesh sizes used are 20 µm and 53 µm. Size-selectivesieving sharply reduces the amount of material in a centrifuge wash that needs to beexamined with the microscope. Well over 3000 samples of wheat and other grainshave been tested for T. indica in California using the size-selective sieve technique.Samples in which teliospores are detected are also checked for the presence of buntedkernels by examining the seed using a slightly modified treated seed inspection sta-tion, commonly used for seed purity exams.

ReferencesGraham, S. 1959. The effects of various reagents, mount-ing media, and dyes on the teliospore walls of Tilletiacontroversa. Mycologia 51:477-491.

Peterson, G. L., Bonde, M. R., Schaad, N. W., and Phillips,J. G. 1998. Development and utilization of size-selectivesieving for rapid detection of teliospores of Tilletia indica,the causal agent of Karnal bunt of wheat. Phytopathology88:S131 (Potomac Division Meetings Abstract).

Materials and Methods

5. The supernatant liquid is removed and the pelletresuspended in Shears mounting medium (Graham,1959). The material is mixed and centrifuged again.

9. If teliospores are detected, the entire sample ofwheat seed is examined for bunted kernels using atreated seed inspection station.

2. The flask of wheat and water is shaken for 10min.

4. Material caught on the 20 µm sieve is washedinto a 15 ml centrifuge tube and centrifuged for 5min using a low speed clinical centrifuge.

7. The entire slide is scanned at 100X for teliosporesof T. indica. Larger pellets may require more thanone slide.

3. The wheat and wash water are poured throughnested sieves of 53 µm and 20 µm, resting on a 600ml beaker. The sieves are made of Nitex fabric meshand PVC pipe. The flask is rinsed twice and therinse water poured through the sieves as well.

1. A 50 g sample of wheat is weighed under a hood,and placed in a flask with 100 ml of water and 2drops of Tween 20.

8. Beakers and sieves are thoroughly cleaned andthen subjected to 33% bleach. This removes colorfrom any teliospores that might not be removed inthe cleaning process. Such spores can then be eas-ily distinguished from the dark teliospores of sub-sequent samples processed with the same labware.

6. The supernatant liquid is removed and the pelletis examined using a compound microscope.