An optimized method for in vitro production of Verticillium dahliae microsclerotia

Verticillium dahliae causes wilt disease of many crops worldwide. Microsclerotia are the main resting structure of V. dahliae in soil and can survive for more than 10 years, serving as an important source of primary inoculum. Mass production of microsclerotia in laboratory is valuable for studying various aspects of V. dahilae, such as its biology, epidemiology and control. We failed to produce a sufficient amount of microsclerotia in vitro for one strain of our interest using several previously published methods. Therefore, we developed a new protocol for mass in vitro production of viable microsclerotia. Verticillium strains were cultured in a modified basal agar medium at pH of 11.5 under 20 A degrees C and incubated for 25 days in dark. When 16 strains were subjected to this condition, large numbers of microsclerotia were produced although varied greatly among strains, including the strain that failed to produce microsclerotia with previously published methods. Microsclerotia from 14 of the 16 strains resulted in wilt development on inoculated cotton seedlings.