Appendix: Filter Wash Test for Bunt, Smut, and Other Pathogens

This test is for detecting the presence of spores of bunts and smuts, conidia of imperfect fungi, and oospores, either as contaminants or on asymptomatic seed.

Place a 10- or 20-g seed sample in 100 ml of water plus 2-5 drops of Tween 20 and agitate on a shaker for 30 min at 250-300 rpm (if Tween is not available, one drop liquid laundry detergent can be used). Filter the wash water through 50- and 20- µm sieves. Large spores and debris will be caught on the 50-µm sieve, and Tilletia indica teliospores, with an average diameter of 25-40 µm, will remain on the 20 µm sieve.

Rinse sieve with a water gun, catching the overflow in a beaker. Then pass the water through Whatman #1 filter paper placed in a Buchner funnel that is inserted into a flask attached to a vacuum pump. Whatman #1 filter paper will trap all structures smaller than 20 µm.

After filtering the water, moisten the filter with a 3% KOH solution, and examine under a stereo microscope for the presence of spores. Mark any suspicious structures and confirm under a compound microscope.

Top