Identification of new pisatin demethylase genes (PDA5 and PDA7) in Nectria haematococca and non-Mendelian segregation of pisatin demethylating ability and virulence on pea due to loss of chromosomal elements

Previous studies have shown that high virulence on pea in Nectria haematococca Mating Population VI is linked to the ability to detoxify the pea phytoalexin, pisatin, via demethylation (Pda). To test this linkage further, a highly virulent Pda(+) isolate (34-18) was used as the recurrent parent in backcrosses to Pda(-) isolates, but most of the progeny were low in virulence on pea, and tetrad analysis gave conflicting ratios for the genetic control of Pda. Southern analysis of 34-18 and progeny showed that 34-18 carries a gene similar to PDA1 (PDA1-2), two new PDA genes, PDA5 and PDA7, and that all three genes can be lost during meiosis. Southern analysis of electrophoretic karyotypes showed that PDA1-2 is on a 1.5-Mb dispensable chromosome in 34-18 and that PDA5 and PDA7 are on a 4.9-Mb chromosome in 34-18 but are found on variably sized chromosomes in progeny. Loss of PDA5 or PDA7 in progeny was not generally associated with morphological phenotypes, except in progeny from some crosses between PDA5 parents. Loss of PDA5 was associated with growth abnormalities in these crosses, suggesting that in some genetic backgrounds at least a portion of the PDA5/PDA7 chromosome is essential for normal growth. All highly virulent progeny had PDA1-2 or a combination of PDA5 and PDA7 while isolates that lacked the three genes were low in virulence, supporting the hypothesis that Pda, or genes linked to PDA genes, are necessary for virulence on pea. However, low virulence isolates with PDA genes were also identified, suggesting that there are pathogenicity genes that can segregate independently of PDA genes.