Fusarium circinatum, the causal agent of pine pitch canker, is one of the most destructive pathogens of Pinus species worldwide. Infections by this pathogen result in serious mortality of seedlings due to root and root collar disease, and growth reduction in trees due to canker formation and dieback. Although much is known about the population biology, genetics, and genomics of F. circinatum, relatively little is known regarding the molecular basis of pathogenicity in F. circinatum. In this study, a protoplast-based transformation using CRISPR-Cas9-mediated genome editing was utilized to functionally characterize a putative pathogenicity gene in three different strains of the fungus. In silico analyses suggested the gene likely encodes a small secreted protein, and all isolates in which it was deleted displayed significantly reduced vegetative growth and asexual spore production compared to the wild-type isolates. In pathogenicity tests, lesions induced by the deletion mutants on detached Pinus patula branches were significantly shorter than those produced by the wild-types. The putative pathogenicity gene was named Pgs reflecting its role in pathogenicity, growth, and sporulation. Future research will seek to explore the molecular mechanisms underlying the mutant phenotypes observed. Overall, this study represents a significant advance in F. circinatum research as the development and application of a Cas9-mediated gene deletion process opens new avenues for functional gene characterization underlying many of the pathogen's biological traits.
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