Population Dynamic Monitoring Of Pyricularia Oryzae In Liaoning Province And Preliminary Study On The Function Of Avirulent Gene Avr-pi9

Rice blast caused by Pyricularia oryzae is one of the most widely distributed and harmful rice diseases in the world.In recent years,the occurrence of rice blast in Liaoning region of China has also shown an increasing trend year by year,which has become one of the main obstacles limiting the high and stable yield of rice in this region.The population structure of P.oryzae is complex,the rate of variation is fast,and the pathogenicity differentiation is serious,which leads to the rapid formation of dominance of the population that can overcome host disease resistance.Most resistant varieties lose resistance after 3-5years of field promotion.This study monitored the dynamic changes of physiological races and avirulence genes of P.oryzae,identification of blast resistance and blast resistance genes of tested rice varieties,and preliminary study on the function of avirulent gene Avr-pi9.The results are as follows:The population dynamics of the races of P.oryzae in Liaoning province in 2017-2018: In this study,the total of 151 strains of P.oryzae isolated from different rice regions in Liaoning province were identified by physiological races using seven rice identification varieties.The tested strains were divided into 6 groups and 41 physiological races,among which the dominant populations were ZA,ZB and ZF,accounting for 42.36%,31.76% and 12.58%respectively;The frequency of ZC,ZD and ZE was low,5.96%,3.97% and 3.31%,respectively;No ZG population was detected;The dominant races were ZA1,ZB1 and ZF1,accounting for 19.87%,13.91% and 12.58%,respectively.In terms of geographical distribution,the population composition of physiological races of P.oryzae in Tieling city,Shenyang city,Dandong city,Fushun and Panjin city is complex,while that in Dalian and Yingkou city is relatively single.The dynamics of physiological small populations fluctuate continuously within a certain range,but the overall change trend is basically consistent with the last decade.The dominant populations have always been ZA and ZB populations.Avirulence gene identification of P.oryzae in Liaoning province in 2017-2018: In this study,specific primers were designed according to the cloned CDS sequences of 10 avirulence genes(PWL2,Avr1-CO39,Avr-Pita,ACE1,Avr-Piz-t,Avr-Pia,Avr-Pii,Avr-Pik,Avr-Pi9 and Avr-Pib),and 151 strains of P.oryzae were tested by PCR.Sequencing analysis of PCR products of 76 strains of P.oryzae in 2018.The avirulent gene Avr-Pi9 carries the highest frequency,accounting for 100.0%;The carrier rates of Avr-Pita,Avr Piz-t,Avr-pik,PWL2 and ACE1 were higher,and the carrier frequencies were 90.07%,94.04%,93.38%,92.72% and 98.68%,respectively.The frequency of avirulence gene Avr-pib was relatively low,only 27.81%.Avirulent genes Avr-Pii,Avr1-CO39 and Avr-Pia were not detected.Geographically,the frequencies of different avirulence genes carried in the same region are basically the same,and the frequencies of the same avirulence gene carried in different regions are different.The avirulence genes ACE1,Avrpiz-t,Avr-pib and Avr-pi9 of P.oryzae were relatively stable,and no other genotypes were detected;Avr-pik,Avr-Pita and PWL2 are prone to mutation,Avr-pik detected 4 genotypes: Avr-pik-B,Avr-pik-C,Avr-pik-D,Avr-pik-F,There are different mistranslations of amino acids at 46(H/N),47(P/A),48(G/D),67(A/D)and 78(M/I),respectively;The total of 6 genotypes were detected in Avr-Pita,except for synonymous mutations at 312 bp(G/T),and different amino acid misinterpretations occurred at 82(K/S),83(T/C),104(K/N),136(G/E),174(V/I),192(Y/C)and 207(K/R),respectively;PWL2 detected a mutation(G/A)at 268 bp,resulting in a change in the amino acid from aspartate(D)to asparagine(N)genotype.The rice blast resistant identification of the tested rice varieties(lines)and identification of blast resistance genes: In this study,the total of 139 rice varieties tested in Liaoning province were identified for blast resistance by indoor inoculation identification and field natural induction identification.The blast resistance genes of 62 tested rice varieties were identified by PCR detection.Among the 139 rice accessions,there were 4 high resistant materials(3.10%),85 resistant materials(65.89%),19 medium resistant materials(14.73%),11 medium sensitive materials(8.53%),4 susceptible materials(3.10%)and 6 high sensitive materials(4.65%).Identification of blast resistance genes showed that: The blast resistance genes Pi21,Pia,Pid2,Pid3,Pil,Ptr were most frequently carried in the tested rice in Liaoning,Jilin and Heilongjiang provinces,with the carrier rate exceeding 90.00%.Pikm has the highest carrying rate of 100.0% in Liaoning province and the lowest carrying rate of50.00% in Jilin province.The carrying rates of Pikh in Liaoning,Jilin and Heilongjiang provinces were 80.95%,47.37% and 63.64%,respectively.Pita carrier rate was higher than70.00% in Liaoning,Jilin and Heilongjiang provinces.Pi5 was absent in Liaoning,Jilin and Heilongjiang provinces,with a low carrier rate of 22.00%.Pi9 is absent in Liaoning province,the detection rate is only 9.52%;Pib was deleted in Heilongjiang province,with a carrier rate of 27.27%;Most of the tested rice carried multiple blast resistance genes,and no rice variety carrying a single blast resistance gene was detected.Preliminary study on the function of avirulent gene Avr-pi9 of P.oryzae : In this study,avirulent gene Avr-pi9 and four disease resistance protein of the disease-resistant gene Pi9 were cloned(NBS2-Pi9、NBS4-Pi9、NBS5-Pi9、NBS6-Pi9),and subcellular localization and yeast two-hybrid validation of Avr-pi9 were performed by constructing vectors.The results show that: The avirulent gene Avr-pi9 is a gene expressed in both cell membrane and nucleus,and has no obvious direct interaction with the four disease-resistant proteins of Pi9..