Rice blast disease is one of the most destructive fungal disease of rice worldwide. The avirulence (AVR) genes of Magnaporthe oryzae are recognized by the cognate resistance (R) genes of rice and trigger race-specific resistance. The variation in AVR is one of the major drivers of new races. Detecting the variation in the AVR gene in isolates from a population of Magnaporthe oryzae collected from rice production fields will aid in evaluating the effectiveness of R genes in rice production areas. The Pik gene contains 5 R alleles (Pik, Pikh, Pikp, Pikm and Piks) corresponding to the AVR alleles (AVR-Pik/kh/kp/km/ks) of M. oryzae. The Pik gene specifically recognizes and prevents infections by isolates of M. oryzae that contain AVR-Pik. The molecular variation in AVR-Pik alleles of M. oryzae and Pik alleles of rice remains unclear. We studied the possible evolutionary pathways of AVR-Pik alleles by analyzing their DNA sequence variation and assaying their avirulence to the cognate Pik alleles of resistance genes under field conditions in China. The results of PCR products from genomic DNA showed that 278 of the 366 isolates of M. oryzae collected from Yunnan Province, China, carried AVR-Pik alleles. Among the isolates from six regions of Yunnan, 66.7–90.3% carried AVR-Pik alleles. Moreover, 10 AVR-Pik haplotypes encoding five novel AVR-Pik variants were identified among 201 isolates. The AVR-Pik alleles evolved to virulent from avirulent forms via stepwise base substitution. These findings demonstrate that AVR-Pik alleles are under positive selection and that mutations are responsible for defeating race-specific resistant Pik alleles in nature. We demonstrated the polymorphism and distribution of AVR-Pik alleles in Yunnan Province, China. By pathogenicity assays used to detect the function of the different haplotypes of AVR-Pik, for the first time, we showed the avoidance and stepwise evolution of AVR-Pik alleles in rice production areas of Yunnan. The functional AVR-Pik possesses diversified sequence structures and is under positive selection in nature.

The product of the avirulence (Avr) gene of Magnaporthe oryzae can be detected by the product of the corresponding resistance (R) gene of rice and activates immunity to rice mediated by the R gene. The high degree of variability of M. oryzae isolates in pathogenicity makes the control of rice blast difficult. That resistance of the R gene in rice has been lost has been ascribed to the instability of the Avr gene in M. oryzae. Further study on the variation of the Avr genes in M. oryze field isolates may yield valuable information on the durable and effective deployment of R genes in rice production areas. AvrPiz-t and Piz-t are a pair of valuable genes in the Rice-Magnaporthe pathosystem. AvrPiz-t is detectable by Piz-t and determines the effectiveness of Piz-t. To effectively deploy the R gene Piz-t, the distribution, variation, and evolution of the corresponding Avr gene AvrPiz-t were found among 312 M. oryzae isolates collected from Yunnan rice production areas of China. PCR amplification and pathogenicity assays of AvrPiz-t showed that 202 isolates (64.7%) held AvrPiz-t alleles and were avirulent to IRBLzt-T (holding Piz-t). There were 42.3–83.3% avirulent isolates containing AvrPiz-t among seven regions in Yunnan Province. Meanwhile, 11 haplotypes of AvrPiz-t encoding three novel AvrPiz-t variants were identified among 100 isolates. A 198 bps insertion homologous to solo-LTR of the retrotransposon inago2 in the promoter region of AvrPiz-t in one isolate and a frameshift mutation of CDS in another isolate were identified among 100 isolates, and those two isolates had evolved to virulent from avirulent. Synonymous mutation and non-AUG-initiated N-terminal extensions keeps the AvrPiz-t gene avirulence function in M. oryzae field isolates in Yunnan. A haplotype network showed that H3 was an ancestral haplotype. Structure variance for absence (28.2%) or partial fragment loss (71.8%) of AvrPiz-t was found among 39 virulent isolates and may cause the AvrPiz-t avirulence function to be lost. Overall, AvrPiz-t evolved to virulent from avirulent forms via point mutation, retrotransposon, shift mutation, and structure variance under field conditions.

Due to the harmful nature of the rice blast fungus, Magnaporthe oryzae, it is beneficial to characterize field isolates to help aid in the deployment of resistance (R) genes in rice. In the present study, 252 field isolates of M. oryzae, collected from rice fields of Yunnan Province in China, were assessed for mating type, DNA fingerprinting, and disease reactions to differential rice lines. In total, 94 isolates (37.3%) were MAT1-1 and 158 (62.7%) were MAT1-2 based on polymerase chain reaction assays, and some of them were verified with the tester isolates. All MAT1-1 and MAT1-2 isolates were virulent to some of the International Rice Research Institute–Japan International Research Center for Agricultural Sciences monogenic lines harboring 22 major resistance genes as differential varieties. Three simple-sequence repeat markers were used to examine genetic diversity in all isolates. The existence of regional patterns of genetic diversity, sexual reproduction potential, and pathogenicity suggests that M. oryzae populations have been independently asexually adapted in rice fields during crop cultivation.

The rice blast disease (caused by Magnaporthe oryzae) is a devastating disease in China. Understanding the molecular mechanisms of interaction for the cognate avirulence (AVR) gene with host resistance (R) genes, as well as their genetic evolution is essential for sustainable rice production. In the present study, we conducted a high-throughput nucleotide sequence polymorphism analysis of the AVR-Pi9 gene that was amplified from the rice-growing regions of the Yunnan Province in China. We detected the presence of seven novel haplotypes from 326 rice samples. In addition, the sequences of AVR-Pi9 were also obtained from two non-rice hosts, Eleusine coracana and Eleusine indica. The sequence analysis revealed the insertions and deletions in the coding and non-coding regions of the gene. The pathogenicity experiments of these haplotypes on previously characterized monogenic lines showed that the newly identified haplotypes are virulent in nature. The breakdown of resistance was attributed to the development of new haplotypes. Our results suggest that the mutation in the AVR-Pi9 gene is an alarming situation in the Yunnan province and thus needs attention.

Rice blast is a very serious disease caused by Magnaporthe oryzae, which threatens rice production and food supply throughout the world. The avirulence (AVR) genes of rice blast are perceived by the corresponding rice blast resistance (R) genes and prompt specific resistance. A mutation in AVR is a major force for new virulence. Exploring mutations in AVR among M. oryzae isolates from rice production fields could aid assessment of the efficacy and durability of R genes. We studied the probable molecular-evolutionary patterns of AVR-Pib alleles by assaying their DNA-sequence diversification and examining their avirulence to the corresponding Pib resistance gene under natural conditions in the extremely genetically diverse of rice resources of Yunnan, China. PCRs detected results from M. oryzae genomic DNA and revealed that 162 out of 366 isolates collected from Yunnan Province contained AVR-Pib alleles. Among them, 36.1–73.3% isolates from six different rice production areas of Yunnan contained AVR-Pib alleles. Furthermore, 36 (28.6%) out of 126 isolates had a transposable element (TE) insertion in AVR-Pib, which resulted in altered virulence. The TE insertion was identified in isolates from rice rather than from Musa nana Lour. Twelve AVR-Pib haplotypes encoding three novel AVR-Pib variants were identified among the remaining 90 isolates. AVR-Pib alleles evolved to virulent forms from avirulent forms by base substitution and TE insertion of Pot2 and Pot3 in the 5′ untranslated region of AVR-Pib. These findings support the hypothesis that functional AVR-Pib possesses varied sequence structures and can escape surveillance by hosts via multiple variation manners.