Development of an innovative in vitro method for mass production of Verticillium dahliae microsclerotia

The soil-borne fungal plant pathogen Verticillium dahliae can infect more than 300 plant species including important economic crops, causing great economic loses. V. dahliae can persist and survive more than 14 years in the soil by resistance structures, known as microsclerotia, which constitute the primary inoculum in the field. In vitro mass production of microsclerotia is essential for performing many pathological assays. Nevertheless to harvest the microsclerotia is not an easy task and several protocols have been described although none of them is completely satisfying for different reasons. here we present a new protocol that is reproducible, robust, simple and fast allows to overcome the difficulties for obtaining massive amounts of microsclerotia. In summary, we developed a new culture medium that we called Pluronic Potato Medium (PPM) because it is essentially potato dextrose media with the hydrogel, Pluronic F127 as a solidifying agent. The microsclerotia collected in form PPM were infectious in tomato plants were they were able to reproduce the disease and we recovered and quantitated V. dahliae in infected plants.