A Method for Extraction and Enumeration of Teliospores of Tilletia indica, T. controversa, and T. barclayana in Soil

Abstract

A sucrose-centrifugation method was developed to extract teliospores of Tilletia indica, T. con-troversa, and T. barclayana from soil. Six soil types were artificially infested with teliospores of each of the three fungi separately to produce 102, 103, 104, or 105 teliospores per 10 g. Each 10 g of infested soil was suspended in 200 ml of water with one drop of Tween 20 and shaken for 30 s. The soil suspension was first passed through a 117-μm sieve and then through a 53-μm mesh filter, and the filtrate was collected. The filtrate was then passed through a 20-μm mesh filter, and materials caught on the mesh were washed into two 50-ml centrifuge tubes and spun for 3 min (1,200 × g). The pellet was suspended in 1.6 M sucrose solution and centrifuged for 40 s (200 × g). The supernatant was passed through a 20-μm mesh filter. The materials caught on the 20-μm mesh were collected, and the number of teliospores was determined. This procedure was initially used to extract teliospores of T. indica in soil. For extraction of teliospores of T. contro-versa and T. barclayana, 1.0 M and 1.3 M sucrose solutions, respectively, were used, and the 20-μm mesh was replaced with a 13-μm mesh filter. Teliospores of T. indica, T. controversa, T. barclayana, and T. indica-like fungus on rye grass were successfully extracted from naturally infested soils. The relationships between number of teliospores recovered from the soil and number of teliospores incorporated into the soil were Ŷ= -0.60 + 1.28(X) - 0.04(X2),Ŷ = -1.25 + 1.56(X) - 0.07(X 2), and Ŷ = -0.71 + 1.33( X) - 0.04(X2) for T. indica, T. controversa, and T. barclayana, respectively, where Ŷ = log10 of the number of teliospores recovered from soil and X = log10 of the actual number of teliospores in soil.