Accurate virulence test method for Cochliobolus heterostrophus wild-type and mutant strains in the post-genomic era

Cochliobolus heterostrophus is an agriculturally important and emerging model pathogen for studying the signalling hierarchy’s role during maize colonization. In particular, G-protein and MAPK-linked pathways play a major role during pathogenesis. Although gene disruption studies are an efficient method for identifying the role of these cascades, differentiating between the mutant strains’ virulence ability may become an intricate task. For example, in C. heterostrophus, mutants in a G-protein α subunit gene, cga1, are defective in mating and appressorium formation, but unlike mutants in homologous genes in other fungal pathogens, cga1 mutants remained highly virulent to corn under some host physiological conditions. Here, we used the cga1 strain as a model for developing an in vivo sensitive and accurate pathogenicity assay. A detailed and well-controlled analysis of wild-type (WT) and cga1 pathogenic behavior revealed that detached leaves are significantly more vulnerable to the disease than intact ones. In intact leaves, cga1 mutants were less infective of maize under most conditions. This difference was maximized when the first seedling leaf was chosen for inoculation and when the infected leaves, with spores or mycelia fragments droplets, were incubated for a period of four days. This optimal condition set enabled us to classify the C. heterostrophus G-protein signalling mutants (deficient in α, β or both subunits) in order of decreasing virulence: WT > cga1 > cgb1 > cga1 cgb1. The method presented here proves to be accurate and sensitive enough to identify even slight variations in virulence. Moreover, it could be modified for use in the studies of other foliar phytoparasitic fungi.