Stripe rust, caused by Puccinia striiformis f. sp. tritici, is a devastating disease of wheat (Triticum aestivum) in the United States. The fungal pathogen can rapidly evolve, producing new virulent races infecting previously resistant cultivars and genotypes adapting to different environments. The objective of this study was to investigate the long-term population dynamics of P. striiformis f. sp. tritici in the United States. Through genotyping 1,083 isolates taken from 1968 to 2009, using 14 simple sequence repeat (SSR) markers and 92 secreted protein single nucleotide polymorphism (SP-SNP) markers, 614 and 945 genotypes were detected, respectively. In general, the two types of markers produced consistent genetic relationships among the P. striiformis f. sp. tritici populations over the 40-year period. The prior-to-2000 and the 2000-to-2009 populations were significantly different, with the latter showing higher genotypic diversity and higher heterozygosity than the earlier populations. Clustering analyses using genotypes of either SSR or SP-SNP markers revealed three molecular groups (MGs), MG1, MG2, and MG3. The prior-to-2000 and the 2000-to-2009 groups both had evidence of MG1 and MG2; however, MG3 was only found in the 2000-to-2009 population. Some of the isolates in the period of 2000 to 2009 formed individual clusters, suggesting exotic incursions. Other isolates of the same period were clustered with prior-to-2000 isolates, indicating that they were developed from the previously established populations. The data suggest the coexistence of newly introduced populations alongside established populations in the United States. Twenty SP-SNP markers were significantly associated to individual avirulence genes. These results are useful for developing more accurate monitoring systems and provide guidance for disease management.

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