A DIFFERENTIAL MEDIUM FOR THE ISOLATION OF Aspergillus flavus FROM COTTONSEED

Abstract

ABSTRACTAn amended Aspergilhrs Differential (BSAD) agar prepared by a modification of Bothast and Fennell's formula [Mycologia (1974) 66: 365] using botran (2,6‐dichloro‐4‐nitroaniline) facilitated the isolation and enumeration of A. flavus from cottonseeds based on the characteristic orange yellow under‐colony pigmentation after the cultures were incubated for 5 days at 28°C. Pigment production by A. flavus cultures on BSAD agar was detected by the third day of incubation. An incubation period of 5 days at 28°C is recommended for routine screening of cottonseed samples for contamination by A. flavus. At the concentrations used, botran (10 mg/liter) and streptomycin sulfate (50 mg/liter) did not interfere with the pigment production by A. flavus but decreased the numbers and colony size of other fungi and bacteria. Decrease in pH of the medium from 6.5 to 5.5, 4.5 and 3.5 resulted in decrease of the intensity of pigmentation while the sporulation of A. flavus colonies increased. When ferric citrate was omitted from ADM containing botran or was replaced with manganous sulfate, zinc sulfate or copper sulfate, orange yellow pigmentation was not produced. Kojic acid produced by strains of Aspergillus reacts with ferric citrate in the medium to produce orange‐yellow pigmentation. When esculin hydrate (6,7‐dihydrocoumarin‐6‐glucoside) was added to BSAD agar at a 1% level, deep reddish brown pigment was produced by all isolates of A. flavus and A. parasificus tested. Similarities between pigment production and nitrification by the A. flavus group of fungi was observed. Few isolates of A. oryzae produced pigmentation similar to that produced by A, flavus.