Pyrenophora tritici-repentis was described first as a pathogen of wheat (tan spot) in Japan in the 1920s, but, since then, no reports on P. tritici-repentis race structure or its effectors in Japan have been published. In this study, 10 single-spore isolates of P. tritici-repentis were collected from bread wheat in Japan. These isolates were evaluated for virulence on four differential wheat genotypes and tested for the presence/absence of the effector-encoding genes, ToxA and ToxB, in multiplex PCR assays. These isolates were identified as ToxA producers, of which eight were designated as race 2 (ToxA producers) and two were classified as race 1 (ToxA and ToxC producers) based on their virulence patterns. Sequence analysis of the ToxA amplicons from these 10 isolates indicated the presence of a novel ToxA haplotype (denoted PtrA2). A comparative sequence analysis and resequencing of ToxA from reference P. tritici-repentis isolates showed that all previously published ToxA haplotypes in P. tritici-repentis were identical, and are hence denoted PtrA1 in this study. A total of 163 PtrToxA sequences from global origins were already deposited in GenBank and were confirmed identical to PtrA1. Sequence variation in PtrA1 and PtrA2 open reading frames were found at three positions: one synonymous mutation at position 412 (C/G) and two nonsynonymous mutations at positions 342 and 362 that alter amino acid sequence. These mutations did not seem to affect the necrosis development on a ToxA-sensitive wheat genotype when rated for symptoms 5 to 7 days after inoculation. This is the first report correctly confirming the presence of an additional novel ToxA haplotype in P. tritici-repentis for which we have predicted its isoform and updated the ToxA haplotype evolutionary network.
Keywords: ToxA haplotypes; necrotrophic effector; race structure; tan spot.