Disease Resistance Induced By Nonpathogenic Mutants Of Magnaporthe Oryzae In Rice

Rice blast is one of the most important fungal diseases on rice.The research and management of rice blast have always attracted the attention of scholars in the world.Up to now,many great advances have been made in the field of revealing the pathogenic mechanism of Magnaporthe oryzae at the molecular level.However,it has not been systematically studied by using nonpathogenic mutants of M.oryzae to induce rice disease resistance.The exploration in this respect can provide research materials and data for the study of green prevention and control of rice blast,which is of great significance.In this study,based on the screening and identification of the nonpathogenic T-DNA insertion mutants of M.oryzae,the resistance to rice blast induced by four nonpathogenic mutants was assayeded and the activity of defense-related enzymes and the expression of pathogenesis-related genes were further determined.The main results are as follows:1.23 nonpathogenic T-DNA insertional mutants were obtained.5707 T-DNA insert transformants of M.oryzae were obtained by ATMT method.Among them,23 nonpathogenic mutants were identified through three rounds of barley cut-leaf pathogenicity assays.After a series of phenotypic analysis,we found that 6nonpathogenic mutants were unable to produce conidia and 7 nonpathogenic mutants could produce conidia but lost the ability of forming appressoria on hydrophobic surface,and 10 reduced or nonpathogenic mutants that could produce conidia and form appressoria as well.By the method of TAIL-PCR,Hi TAIL-PCR or NI-PCR,7 T-DNA tagged genes were identified as follows:MGG＿06637(D919),MGG＿17198(D1308),MGG＿04296(D2902),MGG＿07619(D3519),MGG＿04677(D4241),MGG＿05883(D4979)and MGG＿02696(D5338).The biological function the above T-DNA tagged genes and the T-DNA insertional sites of the other mutants need to be further studied.2.Treatment with nonpathogenic mutants of M.oryzae can improve resistance to rice blast.The nonpathogenic mutant strains D4979 and D3519 obtained in this study and the gene deletion mutantsΔmst12 andΔznf1 of M.oryza were selected as the inductive strains to spray-inoculate rice seedlings(variety Co39)with conidia suspension(concentration 5×10~4/m L),respectively.For the inductive strains,the mutant D4979andΔmst12 can form appressorium on the hydrophobic surface,while D3519 andΔznf1lose the ability to form appressorium.The wild-type strain Guy11 was used for challenge inoculation two days after induction.After 5 days,disease incidence and disease index were evaluated and counted.The results showed that the rice blast disease of induction treated groups was significantly less than that of the untreated group,which the disease indexes decreased 33.83%to 40.48%.However,there was no significant difference of disease occurrence between the inductive mutants with or without the ability to form appressorium.The above research results show that the nonpathogenic mutants of M.oryzae could induce rice blast resistance.3.A preliminary study on the mechanism of induced defense response activated by the nonpathogenic mutants of M.oryzae in rice.Rice seedings(Co39)were sprayed with the spore suspensions of the inductive strains D4979,Δmst12,D3519 andΔznf1(the same as above).It was found that,the activities of superoxide dismutase(SOD)and peroxidase(POD),both are antioxidant defense enzyme,increased significantly one day after inoculation.The activities of polyphenol oxidase(PPO)increased significantly by the third day post-inoculation.Similarly,the activity of phenylalanine ammonia-lyase(PAL)also increased significantly,reaching a peak by the third day,which was significantly higher than the control groups.In summary,nonpathogenic mutants of M.oryzae could significantly induce the enzyme activities of SOD,POD,PPO and PAL in rice leaves.SOD and POD respond more rapidly to induction than PPO and PAL.The activity changes of these enzymes may be related to the defense response of rice plants against the infection of M.oryzae.The fluorescence quantitative RT-PCR method was used to determine the expression of PR genes after the inoculation of the induced strain in rice leaves.The results showed that the expression levels of defense genes PR1b,PR4,PR5,and JIOs PR10 were up-regulated,while the expression levels of Cht-1 and CHS were not affected.The expression level of PR1b and PR4 were significantly higher than CK on the 7th day,while the expression levels of PR5 and JIOs PR10 reached to the peak by the first day post-inoculation.Taken together,the above preliminary research results indicate that nonpathogenic mutants of M.oryzae can induce defense response and thus improve resistance against pathogens in rice.The improvement of blast resistance may be related to the increase activities of SOD,POD,PPO and PAL in rice leaves,and the up-regulation of the expression levels of pathogenesis-relatred genes PR1b,PR4,PR5 and JIOs PR10.Our study confirmed that the use of induced resistance by treatment with nonpathogenic mutants of M.oryzae is efficient and has a good prospect on green prevention and control of rice blast.