Transformation-mediated chromosome loss and disruption of a gene for pisatin demethylase decrease the virulence of Nectria haematococca on pea

Isolates of the fungus Nectria haematococca pathogenic on pea are able to detoxify the phytoalexin pisatin via cytochrome P450-mediated demethylation, To examine the role of pisatin demethylating ability (Pda) in pathogenicity, mutants of N. haematococca deficient in Pda were created by transformation-mediated gene disruption (gdr). Eleven Pda(-) transformants were obtained as determined biochemically by their inability to demethylate pisatin. The transformants were of two types, Seven of the Pda transformants were gdr mutants, while the other four were Pda(-) because they had lost the 1.6-Mb chromosome which carries the gene (PDA1) fdr Pda in the recipient isolates. All of the Pda(-) mutants were more sensitive to pisatin and less virulent on pea than the Pda(+) recipient isolates. However, only the four chromosome-deletion transformants were reduced to virulence equivalent to naturally occurring Pda(-) isolates of N. haematococca. There was no evidence that the Pda(-) gdr mutants contained a cryptic PDA gene induced in planta. Lesion tissue produced by these mutants did not demethylate pisatin while that produced by the wild-type isolates did, In addition, the four chromosome-deletion transformants were as virulent on ripe tomato fruit as the wild-type isolate, These results support the hypothesis that (i) lack of Pda reduces but does not eliminate the virulence of N. haematococca on pea and (ii) an additional gene(s) required for high virulence on pea but not tomato is located on the dispensable chromosome that contains PDA1.