Application of newly-developed dichlorvos–ammonia (DV–AM) method to direct isolation of aflatoxigenic fungi from field soils

We recently devised the dichlorvos–ammonia (DV–AM) method, a sensitive and simple visual method for detecting aflatoxigenic strains. The DV–AM method enables a prominent distinction of red (positive; aflatoxin-producing) colony from pale-yellow (negative; non-aflatoxigenic) colony via brilliant purple-red color of the backside of positive colonies. We used the DV–AM method to screen aflatoxigenic fungi from soil samples in Tsukuba, Ibaraki, Japan. We herein isolated seven positive (red) colonies and four negative (pale-yellow) colonies from one of the 100 independent samples. Chemical analyses demonstrated that these red isolates produced aflatoxins B1 and B2 without aflatoxins G1 nor G2. In contrast, 4 pale-yellow colonies with similar phenotypes to the Aspergillus flavus group were confirmed to be non-aflatoxigenic fungi. Morphological and phylogenic analyses of one positive strain (HA9-S1-1) indicated that it was A. flavus or its closely related species of Aspergillus. These aflatoxigenic strains showed differences in aflatoxin production, morphological characteristics, and sclerotia formation, suggesting that different types of aflatoxigenic strains were present in the same soil. These results demonstrated that the DV–AM method is practically useful for the direct isolation of aflatoxigenic and non-aflatoxigenic fungi from field samples.