An improved ethanol medium for efficient recovery and estimation of Verticillium dahliae populations in soil

Abstract

The population of microsclerotia of Verticillium dahliae in soil affects the severity of wilt diseases caused by the fungus. The number of microsclerotia (MS) in soil is usually estimated by colonies recovered from plating of soil samples in various forms on solid media, and this value can be useful in making decisions on disease management. Most protocols, however, lack efficiency in making accurate estimates of V. dahliae populations in soil. In this research, it was found that most of the regular ingredients used in the isolation media were inhibitors of MS germination and their subsequent growth. An improved medium, Ethanol Potassium Amoxicillin Agar (EPAA), which is Ethanol Agar amended with potassium salts and amoxicillin, was able to recover 98% of the added MS population from non-sterile soil samples with high precision (SE = 1.37%). The medium was highly specific, allowing only minor growth of the other microorganisms. Distinct V. dahliae colonies were obtained within seven days of incubation at 20 °C.