Identification and characterization of Phoma tracheiphila mutants impaired in pathogenicity following Agrobacterium-mediated mutagenesis

Mal secco disease, caused by the pathogenic fungus Phoma tracheiphila, is a devastating disease of susceptible citrus species, especially lemon. To study the molecular interactions between the pathogen and its host, a method for identifying the genes involved in pathogenicity is needed. This work describes the transformation of P. tracheiphila phialoconidia by Agrobacterium tumefaciens, and the generation of mutated P. tracheiphila isolates exhibiting reduced virulence on rough lemon seedlings. A rapid, replicable, and reliable method for screening P. tracheiphila mutants to assess their virulence by using rough lemon seedlings was developed. Among 2263 transformants obtained, three were non-virulent and 43 displayed reduced virulence. In addition, one of the transformants, which exhibited virulence similar to that of the wild type, was used for in planta visualization of the fungus progression through the plant xylem. To our knowledge, this is the first report of A. tumefaciens-mediated transformation of P. tracheiphila, and subsequent screening of the transformants to identify non-virulent mutants.