Genetic Diversity And Pathogenicity Of Multinucleate And Binucleate Isolates Of Rice Sheath Blight

Rice sheath blight is an important disease in rice production,which seriously affects rice yield.In this paper,multinucleate and binucleate isolates of rice sheath blight were taken as the research object.The genetic diversity was analyzed using SRAP-PCR molecular marker technology.The pathogenicity of different isolates was determined by vitro leaf inoculating,and the infection structure of different isolates was observed by scanning electron microscope and transmission electron microscope.The activities of cell wall degrading enzymes and the differences in the expression of PG genes of different isolates were investigated,providing the theoretical basic for the prevention and control for rice sheath blight.The main results were as follows:1.Genetic Diversity and Pathogenicity of Isolates causing Sheath Blight Disease of Rice in Northeast RegionIn this study,132 isolates of rice sheath blight were collected from the 20 major rice producing areas in northeast provinces.The pathogenicity and SRAP analysis were carried out among 112 multinucleate isolates(R.solani)and 20 binucleate isolates(R.oryzae sativae).The results of SRAP cluster analysis showed that the genetic similarity coefficient of the tested 112 multinucleate isolates of R.solani were 0.52 ~ 0.97,and were clustered into 15 clusters at the 78% genetic similarity level;the genetic similarity coefficient of 20 binucleate isolates of R.oryzae were 0.65 ~ 0.90,and were clustered into 7 clusters under 80% genetic similarity.There was no significant correlation between genetic structure and geographical origin.Liaoxing 1 was used to determine the pathogenicity of all isolates and the results showed that 77.27% of the isolates were highly pathogenic,8.33% of them were moderate pathogenic,14.39% of the isolates were weakly pathogenic,and the pathogenicity differentiation of R.solani was more obvious in the northeast.The result of cluster analysis and pathogenicity test showed that there was no simple correspondence between SRAP groups divided and pathogenicity identification.2.Research on the pathogenicity of multinucleate and binucleate isolates of rice sheath blightThrough the determination of cell wall degrading enzyme activity of five representative isolates with different pathogenicity,it was found that the tested multinucleate and binucleat isolates can produce five cell wall degrading enzymes,such as PG,PMG,Cx,PGTE and PMTE,and activity of PG and PMG are the highest;through optimizating enzyme-producingconditions for cell wall degrading enzymes of the binucleate isolate JLS07 of rice sheath blight,it was found that the best culture conditions were: the conducive of culture time was 10 days,the culture temperature was 28?,the initial p H value of the culture solution was 5,and the culture was still stationary,which is different from the culture conditions of multinucleate isolates culture time only.Compared with the tested multinucleate isolates and binucleate isolate,the differences in mycelial growth rate,lesion expansion time,and infection structure formation time were consistent.The ability of multinucleate isolates of rice sheath blight isolates to produce cell wall degrading enzymes differed.By comparing the pathogenicity of the tested 36 isolates of rice sheath blight pathogens and the relationship between PG and PMG enzyme activity,it was found that differences in ability of cell wall degrading enzymes produced by Rhizoctonia solani isolates,and there was a significant correlation between the ability producing cell wall degrading enzymes and its pathogenicity to rice plants(r=0.73,r=0.68),which revealed that cell wall degrading enzymes play an important role in pathogenicity and is the main pathogenic factor of rice sheath blight pathogen.Observing the infection structure of rice sheath blight after infected with rice,it was found that the main infection structure was the infection cushion,and the hypha could penetrate from the stoma and the leaf tissue.Chloroplast disintegration and thylakoid digestionafter infection with rice with multinucleate isolates,but no clear changes in the organelles after infection with rice with binucleate isolates.3.Study on the difference of PG gene expression of isolates of rice sheath blightThe target binucleate isolates PG genegene was obtained by high-throughput sequencing of the binucleate isolates JLS07,and DNAMAN was used to design primers for the target gene and the reference gene of the multinucleate and binucleate isolates.The expression of PG gene in rice was detected by quantitative real-time PCR.Tt was found that the expression of PG gene was up-regulated,and the expression of PG gene of the tested multinucleate isolates was significantly higher than that of the binucleate isolates.The PG gene expression was highest when the multinucleate isolates infected rice for 24 h,the expression level increased before 24 h,and decreased after 24 h.The binucleate isolates had the highest expression levels at 48 h after rice infected,the expression level increased before 48 h,and decreased after 48 h.