Dynamic Monitoring And Avr-Pi9 Bioinformatics Analysis Of Pyricularia Oryzae Population In Liaoning Province

Rice is one of the important economic crops in China.Rice blast caused by Pyricularia oryzae is the first disease threatening rice production.Studies on physi-ological races and avirulence genes of P.oryzae and screening of germplasm re-sources for resistance to rice disease are the basis of rice disease resistance breeding.In this study,physiological races and avirulence genotypes of P.oryzae were identi-fied in various areas of Liaoning Province,avirulence gene sequencing and sequence comparison of P.oryzae in 2020 were carried out,avirulence gene Avr-Pi9 was ana-lyzed by bioinformatics,and the blast resistance of rice varieties（lines）in Liaoning Province was identified and evaluated.The results can provide a theoretical basis for the breeding of resistant rice varieties and the rational distribution of rice varieties in Liaoning Province.The main results are as follows:Physiological races of 147 strains collected from 8 regions of Liaoning Prov-ince in 2019 to 2020 were identified.The strains were divided into 6 groups and 25physiological races,among which ZA and ZB groups with strong virulence were the dominant population,and ZD and ZE groups were the weak groups.In physiological races,ZA₁ was the dominant species with a frequency of 26.53%.In terms of geo-graphical distribution,the population complexity was high in Dalian and Shenyang,with 6 groups of 12 species and 6 groups of 9 species,respectively.The population structure was simple in Anshan,with 3 groups of 6 species.Groups ZA and ZB were distributed in all regions,and the occurrence frequency of ZA group was the highest.Identification of 10 avirulence genes from 146 isolates from 2019 to 2020 re-vealed that Avr-Pi9 and ACE1 were the most frequently carried avirulence genes in P.oryzae,with the frequency of 100.00%and 99.32%,respectively.The frequencies of Avr-Pik,PWL2,Avr-Piz-t and Avr-Pita were relatively high,and Avr-Pib was the lowest avirulence gene.Avr1-CO39,Avr-Pii and Avr-Pia were not found in the province.In terms of geographical distribution,ACE1,Avr-Pi9,PWL2 and Avr-Pik were widely distributed and appeared with high frequency in Liaoning Province.The distribution of Avr-Piz-t and Avr-Pita was unstable in two years,and no Avr-Pib was detected in some areas in 19 years.Sequence alignment of avirulen genotypes of P.oryzae in Liaoning Province in2020 was carried out,and the avirulence gene Avr-Pi9 with the highest carrying rate in Liaoning Province was preliminarily analyzed by bioinformatics,subcellular lo-calization and differential expression analysis.Sequence alignment results showed that no mutations were detected in Avr-Pi9,ACE1,Avr-Pizt and Avr-Pib,and there were four alleles in Avr-Pik,among which Avr-Pik-A was first detected in Liaoning Province.There were four alleles in Avr-Pita.All PWL2-containing strains were mu-tated at 268 bp（G/A）,and the diversity of avirulence genes decreased compared with previous years.The gene structure,physical and chemical properties,hydro-philicity and hydrophobicity,and transmembrane domain of Avr-Pi9 were analyzed by bioinformatics,and the signal peptide and phosphorylation,acetylation and gly-cosylation sites of Avr-Pi9 were predicted.Differential expression analysis showed that the expression level of Avr-Pi9 was high in the early stage of P.oryzae infection,and decreased with the proliferation of P.oryzae in host tissue.Subcellular localiza-tion experiments in rice protoplasts suggested that Avr-Pi9 might be a nuclear mem-brane localization gene.There were obvious differences in the tolerance of 135 rice varieties.In 2020,28（40.00%）resistant cultivars and 42（60.00%）susceptible cultivars were identified by in vitro inoculation test.In 2021,27 resistant varieties（41.54%）and 38 suscepti-ble varieties（58.46%）were identified by indoor in vitro inoculation test.In 2020,35（50.00%）resistant cultivars（R）,17（24.29%）moderately resistant cultivars（MR）,9（12.86%）moderately susceptible cultivars（MS）and 9（12.86%）highly susceptible cultivars（HS）were identified by field natural induction test.In 2021,27 resistant cultivars（R）（41.54%）,20 moderately resistant cultivars（MR）（30.77%）,13 moder-ately susceptible cultivars（MS）（20.00%）,3 susceptible cultivars（S）（4.62%）,and 2highly susceptible cultivars（HS）（3.08%）were identified by field natural induction test.