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Analysis of Genetic and Molecular Identity Among Field Isolates of the Rice Blast Fungus with an International Differential System, Rep-PCR, and DNA Sequencing

The Pi-ta gene deployed in southern U.S. rice germplasm is effective in preventing the infection by strains of Magnaporthe oryzae isolates that carry the avirulence (AVR) gene AVR-Pita1. In the present study, 169 isolates from rice (Oryza sativa) cultivars, with and without Pi-ta, were analyzed for their genetic identity using an international differential system, repetitive element-based polymerase chain reaction (Rep-PCR), and sequence analysis of PCR products of AVR-Pita1. These isolates belong to the races IA1, IB1, IB17, IC1, and IC17 of M. oryzae. These isolates were further classified into 15 distinct groups by Rep-PCR. There was a predominant group within each race. Pathogenicity assays on ‘Katy’ (Pi-ta) and ‘M202’ (pi-ta) rice determined that IC1 was virulent to Katy and M202; IB17, IC17, and most of IA1 and IB1 were avirulent to Katy and virulent to M202, suggesting that the Pi-ta gene in Katy is responsible for preventing infection by these isolates. Consistently, AVR-Pita1 was not amplified from 28 virulent isolates. One AVR-Pita1 allele was amplified by AVR-Pita1-specific primers in 78 avirulent isolates. Interestingly, different AVR-Pita1 alleles were found in each of the 12 avirulent isolates, as determined by DNA sequencing. Sequence analysis of 90 PCR products revealed 10 AVR-Pita1 haplotypes, 4 of which were new. In total, 12 amino acid changes were identified in the new variants when compared with the first described AVR-Pita sequence (AF207841). The finding of isolates with altered AVR-Pita1 from rice cultivars with and without Pi-ta suggests that these virulent isolates were adapted to the field environments in the southern United States. Further research will be needed to verify this prediction.