Analysis Of Mutation Mechanism For Avirulence Gene Avr-pizt And Avr-pita And Polymorphism Of M. Oryzae In 2018 From Southwest Hubei Province

Rice blast is a fungal disease caused by Magnaporthe oryzae(M.oryzae)that seriously threatens rice yield.The variation of M.oryzae makes the resistant varieties lost the resistance,which leads to the decrease of annual yield and the increase of planting cost.In this experiment,the relationship between function and the sequences of avirulence gene Avr-pizt and Avr-pita of M.oryzae isolated from 2012-2016 in the southwest Hubei province were analyzed.At the same time single spores were isolated from susceptible rice stalks collected from 10 different places in same region in 2018,and classification and population composition were studied from pathogenicity identification and molecular markers,and compared with previous research data of our laboratory.The following results were obtained:1.The association analysis of the sequences and the pathogenicity to Pizt and Pita of avirulence genes Avr-pizt and Avr-pita of 489 M.oryzae from the southwest Hubei province from 2012 to 2016 was performed.The main reason for the variation of the pathogenicity was function lost of avirulence genes with deletions,substitutions and insertions in the sequence.There is an A base deletion at 10 th and 312 th of ATG downstream in Avr-pizt sequence leads to frameshift mutation.There are many mutations in the sequence of Avr-pita,including 13 different substitution,1 GTT insertion and 1 single base deletion,of which 758T> A(100%),760 del T(100%),778G> A(100%)occurred in the third intron region of Avr-pita.The protein coded by the mutated avirulence gene is unstable,which leads to the function loss of avirulence gene.2.Using the Chinese identification variety(CDVs),the 133 strains isolated from southwest Hubei province in 2018 can be divided into 7 groups and 51 physiological races,ZA is the dominant group,and A01 is the dominant physiological race.104 strains isolated in 2017 can be divided into 57 physiological races,and 133 strains isolated in 2018 can be divided into 91 physiological races by using Japanese identification variety(JDVs).133 isolates isolated in 2018 can be divided into 7genetic lineages and 133 unit types at a similar level of 50% through the pathogenicity to 40 rice identification varieties.This showed that the 40 varieties used in the experiment can completely distinguish different monospores.3.Using 21 single resistance gene identification variety(ML)to identify the pathogenicity of 133 M.Oryzae in 2018,at a similar level of 52%,there are 7 genetic lineages,132 unit types.There are the highest infection rate to ML Pikp(84.2%)and Pi3(81.2%),and the lowest infection rate to ML Piz5(22.5%),Pik(36.1%)and Pikm(36.8%).In 2017,the infection rate to ML Pikh,Pib and Pit was more than 80%,while in 2018,the infection rate dropped to below 80%.In 2018,only ML Pikp was infected by 80% strains.In two years,the infection rate to ML Pi2 and Pita was very low,and to ML Piz increased from 37.5% in 2017 to 69.3% in 2018;the infection rate to ML Pish decreased from 90.3% in 2017 to 65% in 2018.4.The 133 M.oryzae from 2018 can be divided into 7 genetic lineages and 133 unit types at a similar level of 70% based on cluster analysis of 7 avirulence genes,17 RADP,3 REMAP and 1 Rep molecular markers.At a similar level of 48%,237 M.oryzae from 2017-2018 were classified into 8 genetic lineages.Among them,the third type is dominant,accounting for 91.1% of all strains,and the other types are rare genetic lineages.The clustering results of both molecular markers and pathogenicity analysis showed no significant relationship between types of M.oryzae and sampling places.According to the amplified products of RAPD S1219,a special SCAR primer SS1219 with excellent specificity and reproducibility was design.The predominantly mating type of M.oryzae from 2018 is MAT1-2(59.4%),and there are 4 strains(3.01%)monoecious carried with MAT1-1 and MAT1-2 at the same time.There is no monoecious in M.oryzae from 2017.5.There is significantly changes in the frequency of avirulence genes of M.oryzae in different sampling places in 2018,and in different years(2017 and 2018)at the same place.There are Avr-pizt、Avr-pik、Avr-pia、Avrl-CO39、ACE1、Avr-pi9 in all 10 sampling place in 2018.6.24 rice varieties were planted in 10 different places in southwest Hubei,and the incidence of their leaf blast and neck blast were investigated between 2018 and2019.The results showed that the incidence of blast in all places in 2019 was significantly more serious than in 2018.The most serious in the 10 different places was Xuan’en,and no rice blast occurred in Xianfeng for 2 years.Among the 24 varieties tested,‘Rongyou Huazhan’ had the best resistance and it is suitable for planting in southwest Hubei.