TECHNICAL SESSION: Mechanisms of fungal and oomycete pathogenicity
Screening method for selecting Cercospora cf. flagellaris target genes for silencing via dsRNA application
Marija Zivanovic - Department of Plant Pathology and Crop Physiology, Louisiana State University. Zhi-Yuan Chen- Department of Plant Pathology and Crop Physiology, Louisiana State Univ AgCenter
Cercospora leaf blight (CLB) of soybean (Glycine max L. Merr.) is one of the most important soybean diseases in Louisiana, primarily caused by Cercospora cf. flagellaris. C. cf. flagellaris produces a toxin, cercosporin, which is an important virulence factor. So far, there are no known CLB resistant cultivars, and substantial fungicide resistance has developed in the pathogen. Consequently, alternative methods are needed to manage CLB. One possibility is RNA interference via topical application of dsRNA, with the critical step being the selection of the target genes, whose silencing will effectively reduce fungal growth or cercosporin production. We have developed a method to test the effectiveness of candidate genes in suppressing toxin production by silencing the genes known to be involved in the production of cercosporin (CTB1, CTB8), as well as several genes recently associated with cercosporin accumulation. The desired gene fragments were cloned into a vector that enables dsRNA production upon overexpression in Escherichia coli cells. Purified dsRNA was combined with complete media broth, inoculated with macerated mycelia of C. cf. flagellaris and incubated until cercosporin was produced in the control treatments. Several of the tested dsRNAs demonstrated efficacy at various concentrations in reducing cercosporin production in culture. This method will now allow the identification of the most potent dsRNAs for potential CLB management under field conditions.