| The rice blast,caused by Magnaporthe oryzae,has resulted in drastically reduction in rice production.The population structure of M.oryzae is complex,and the avirulence gene of M.oryzae is closely related to the blast resistance of rice.The co-evolution of the two resulted in the loss of resistance of resistant varieties 3-5 years after planting.In this experiment,susceptible rice stalks were collected from 9different regions in southwestern Hubei from 2019 to 2020,and monospores were isolated,and their colony morphology was recorded.The pathogenicity and molecular markers were used to classify and study of the population structure.The results were compared analyze with the data from previous years.At the same time,the sequence analysis of the avirulence genes Avr-Pia,PWL2 and Avr-Pi54 of the M.oryzae was carried out.The results are as follows:1.From 2019 to 2020,177 and 276 M.oryzae monospores were isolated from rice straws collected from 9 different regions in southwestern Hubei（the same 99varieties were planted in each region）,and there were 107 and 158 isolates with pathogenicity respectively.1)According to the colony morphology,the population of M.oryzae in 2019 can be divided into 10 morphologically types,and there is one more type in 2020 than2019.Compared with the previous data,there were more morphological distinctions in the past two years.This may be indicated that the population structure of M.oryzae in southwestern Hubei was more complex,and the diversity of colonies increased in recent 2 years.2)Using 7 Chinese identification varieties（CIV）,the 107 isolates in 2019 were divided into 7 groups and 40 physiological races.ZA and ZC were the dominant groups,and C15was the dominant physiological race.The 158 isolates in 2020 were divided into 7 groups and 42 physiological races,ZC was the dominant group,and C15was the dominant physiological race.3)Using 21 single resistance gene identification varieties（SRGIV）to identify the pathogenicity,107 isolates in 2019 were divided into 12 types and 106 haplotypes at a similar level of 49%,and the resistance genes with the highest infection rates were Pia（81.0%）and Pikp（64.5%）,and the lowest were Pish（19.1%）,Piks（30.0%）and Pikm（32.7%）.The 158 isolates in 2020 were divided into 15 types and 156haplotypes at a similar level of 44%,the resistance genes with the highest infection rates being Pia（86.1%）and Piz（57.6%）,and the lowest infection rates were Pib（18.4%）and Pi2（19.0%）).Comparing the susceptibility rates of the 265 isolates in2019 and 2020 to 21 SRGIVs,the susceptibility rates to SRGIV containing Pi2 and Pikh resistance genes were lower than 40%.The susceptibility rates to the SRGIV containing Pikp,Pii,Pizt,and Pib were decrease between 2019 and 2020,and the other SRGIV showed no significant change.4)Using 39 rice varieties for pathogenicity identification,the isolates in 2019were divided into 7 groups and 106 haplotypes at a similar level of 44%;the isolates in 2020 were divided into 16 groups with 158 haplotypes at a similar level of 46%.Principal coordinate analysis showed no significant correlation between pathogenicitiy and regions.5)Avirulent gene ACE1,Avr-Pia,Avr-Pi9,Avr-Pik,Avr-Pita,PWL2,Avr-Pizt can be detected in the 9 different regions of southwestern Hubei in 2019-2020,and Avr-Pia only appears in Baiyang,Xuanen and Laifeng,while Avr1-CO39 undetected.The frequencies of avirulent genes ACE1,AVR1-CO39,AVR-Pia,and AVR-Pi9 were relatively stable in the past two years,but the frequency of AVR-Pik and AVR-Pita had increased significantly.The frequency of avirulent genes in different sampling regions was significantly different,this indicating that there was obvious geographical differences in various rice planting areas in southwestern Hubei.6)The results about the mating type of M.oryzae showed that the mainly mating type of the isolates was MAT1-2 in the past two years.In 2019,131 isolates（74.01%）are MAT1-2,and one isolates（0.56%）is hermaphrodite which MAT1-1 and MAT1-2appeared at the same time,and 5 isolates（2.82%）did not have MAT1-1 and MAT1-2.In 2020,219 isolates（79.35%）are mating type MAT1-2,19 isolates（6.88%）are MAT1-1 and MAT1-2 at the same time,and 6 isolates（2.17%）did not have MAT1-1and MAT1-2.7)The diversity of M.oryzae was analyzed using molecular markers.At the similar level of 60%,the 107 isolates in 2019 can be divided into 8 groups,at the similar level of 60%,the 158 isolates in 2020 can be be divided into 7 groups.The results with cluster analysis of the isolates in two years showed that with a similarity of 60%,the 265 isolates could be divided into 8 groups.Comparing the clustering results of pathogenicity identification and molecular clustering,it was found that there was no significant correlation between them,and the PCo A results were consistent with the clustering analysis.8)The number of alleles（Na）,the number of effective alleles（Ne）,and the Nei gene diversity index（H）of the 9 different regional populations of M.oryzae in southwestern Hubei were 1.3917-1.9822,1.2611-1.5875,0.0812-0.3438.67.39%of the genetic differentiation in the population resulting from the difference isolates within the population,while 32.61%of the genetic differentiation was from the population and the population.This indicated that the genetic variation in southwestern Hubei is rich.The gene flow（Nm）was 1.0334,which indicated that there was less gene exchange among different groups in the southwest of Hubei.The genetic diversity（Hs）of 0.3400 indicated that the genetic diversity of M.oryzae was mainly derived from individuals.2.PCR amplification and sequencing of the avirulent genes PWL2 and Avr-Pi54were carried out,and two types of mutations,base substitution and deletion,were detected in PWL2,but no mutation was detected in Avr-Pi54.The Avr-Pia promoter and CDS sequences were amplified and sequenced,and combined with the results of the pathogenicity identification of single-resistance gene rice varieties,it was found that there was no mutation in the CDS region,and a base mutation G/T occurred at the 109 position upstream of the start codon.This site was not on the predicted transcription start site and cis-acting element,which needs further study. |
