Agrobacterium tumefaciens-mediated transformation (ATMT) system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformation efficiency of up to 60 transformants per 10(6) conidia was achieved by this system. The integration of the hph gene into the fungal genome was verified by polymerase chain reaction (PCR) amplification and sequencing. These transformants tested were also shown to be mitotically stable. Southern blot analysis of 14 randomly selected transformants showed that the hph gene was randomly integrated as single copy into the fungal genome of P. digitatum. Thus, we conclude that ATMT of P. digitatum could be used as an alternatively practical genetic tool for conducting insertional mutagenesis in P. digitatum to study functional genomics.

Penicillium digitatum, causing green mold, is the most important postharvest pathogen of citrus fruits worldwide. Fungicide imazalil has been used in control of this mold for more than three decades. In the presence of imazalil pressure, imazalil resistance strain has risen worldwide. To explore the potential role of multidrug resistance (MDR) in imazalil resistance, a membrane efflux transporter Penicillium digitatum major facilitator surperfamily1 (PdMfs1) was cloned, and its functions in imazalil resistance and pathogenicity were analyzed. PdMfs1 has an open reading frame (ORF) of 1,876 bp and 3 introns of 55, 49 and 71 bp, respectively. It encodes a protein of 566 amino acids that shares a high degree of similarity with members of the drug: H + antiporter efflux family of the major facilitator surperfamily (MFS) transporters of other fungi. Expression of PdMfs1 was up-regulated by treatment with imazalil and other fungicdes in both imazalil-sensitive and-resistant P. digitatum. Disruption of PdMfs1 gene rendered P. digitatum more sensitive to imazalil and other DMI fungicides, and the imazalil-resistance could be rescued by reintroducing the wild-type PdMfs1 gene into the PdMfs1 disruption mutant (PdMfs1). Overexpression of PdMfs1 rendered P. digitatum more resistant to imazalil. These results indicate that PdMfs1 is a multidrug transporter of P. digitatum that could pump imazalil out of cells, thus contributing resistance to imazalil partially. Pathogenicity analysis showed that the disease on the citrus fruits inoculated with the PdMfs1 developed much slower than that induced by the parental strain PdW03, suggesting that PdMfs1 also plays a role on the virulence of P. digitatum.