Study On The Inhibitory Mechanism Of Muscodor ZJLQ024 On Pyricularia Oryzae And Mutants

Rice blast is one of the most serious rice diseases caused by Pyricularia oryzae.As a result,the rice in the world is reduced by about 10% per year and seriously threatens the safety of food production and economic development.At present,the control measures of Pyricularia oryzae mainly include: chemical control and biological control.Because of the loss of resistant varieties and chemical pesticide residues,Biocontrol is considered to be the most safe,effective control method.As an endophytic fungus of Cladosporaceae,Muscodor can produce complex volatile organic compounds(VOCs),including acids,ketones,esters,alkanes,alcohols,nitrosamines and terpenoids.These compounds have strong inhibitory effect on pathogenic fungi.Through the construction of the mutant library of Pyricularia oryzae.and hi TAIL-PCR technology,it was predicted that the two key genes MGG-09531 and MGG-00779,might play a role in the inhibition of Pyricularia oryzae,but the mechanism of inhibition was not clear.Based on this,we knocked out MGG-09531 and MGG-00779 genes in Pyricularia oryzae to study the role of these two genes in the growth and development of Pyricularia oryzae,and further studied the growth inhibition effect.We studied the effects of Muscodor ZJLQ024 on mycelial morphology,growth,conidia germination and appressorium formation of Pyricularia oryzae and its mutants,combined with transcriptome sequencing we explored the mechanism of Muscodor ZJLQ024 inhibiting Pyricularia oryzae.The main test results are as follows:1.Biological function analysis of MGG-09531 and MGG-00779 in Pyricularia oryzae.MGG-09531 and MGG-00779 knockout mutants were successfully constructed by homologous recombination,which were named mutant ΔMo09531 and ΔMo00779respectively.According to the phenotypic analysis of mutant ΔMo09531,The results showed that:(1)compared with wild type Guy11,the mycelial branches of ΔMo09531mutants were increased,the color was light brown,and the growth of mycelium slowed down;(2)the spore produced by ΔMo09531 mutant was normal,but the germination of conidia was delayed,and the formation rate of appressorium decreased and pathogenicity was lost;(3)the resistance of ΔMo09531 mutant to salt stress and cell wall stress was decreased.It was suggested that MGG-09531 was involved in the process of the growth of Pyricularia oryzae,the development of appressorium,pathogenicity and response to environmental stress.Phenotypic analysis of mutant ΔMo00779,the results showed that:(1)compared with wild Guy11,the colony center of ΔMo00779 mutant was prone,the color was light white,and the growth of mycelium was slightly slower.(2)The conidia of ΔMo00779 mutant decreased significantly,but the morphology of appressorium was normal,the pathogenicity and maturation of appressorium were not affected.(3)The tolerance of ΔMo00779 mutant to salt stress and cell wall stress was not significantly different from that of wild type Guy11.It was suggested that MGG-00779 gene was involved in the sporulation related process of Pyricularia oryzae.2.Study on the inhibition of Muscodor on Pyricularia oryzae and mutants.Six different strains of Muscodor were compared with wild-type Guy11 and mutant strains respectively ΔMo09531 and ΔMo00779.The results showed that:(1)different strains of Muscodor had different antibacterial ability,the Muscodor strain with strong antibacterial ability is WT,followed by ZJLQ024.(2)Under the confrontation of ZJLQ024,the wild strain Guy11 hyphae were severely shrunk and dried,and The mycelial shrinkage of ΔMo09531 and ΔMo00779 was lighter than that of the wild type,the mutant ΔMo09531and ΔMo00779 had some full hyphae.(3)Compared with the wild-type Guy11,the mutantΔMo09531 and ΔMo00779 was more sensitive to ZJLQ024.The mycelial growth inhibition rate of the two mutants decreased,which indicated that the two mutants were more resistant to VOCs produced by ZJLQ024.(4)The filtrate of ZJLQ024 made the spores of wild-type Guy11 unable to form normal appressorium,and the mutants ΔMo09531 and ΔMo00779had a small amount of appressorium formation.These results indicate that the genes MGG-09531 and MGG-00779 affect the inhibitory effect of ZJLQ024 on the rice blast fungus,and gene knockout can enhance the resistance of the rice blast fungus to the inhibitory effect of strain ZJLQ024.3.Transcriptional analysis of the inhibitory effect of Muscodor ZJLQ024 on Pyric ularia oryzae.In this study,1386 differentially expressed genes(DEGs)were identified,including 616 up-regulated genes and 770 down-regulated genes,by sequencing and comparing the transcriptome of Pyricularia oryzae under confrontation and non confrontation culture conditions(control)of ZJLQ024.Through go functional annotation and enrichment analysis,it was found that the gene functions of DEGs were mainly enriched in the composition and structure of ribosome,the development of mycelium,the development process of single organism,the metabolism process of single organism and the composition of cell membrane.Through KEGG pathway enrichment analysis,16 of them were found to be significantly enriched,mainly related to ribosome,amino acid synthesis and fatty acid metabolism.In ribosome pathway,40 DEGs were identified,39 of which were down regulated.the DEGs of 60 S ribosomal protein S4-A,60 S ribosomal protein L26 and 40 S ribosomal protein S6 B,40S ribosomal protein S14 were significantly down regulated,which inhibited the synthesis of proteins.The expression of α-1,3-glucan synthase AGS2,chitin deacetylase and α-1,6-mannotransferase related to cell wall were down regulated,which affected the synthesis of glucan,chitin and mannan in cell wall.the process of amino acid metabolism,25 DEGs were identified,22 of which were down regulated.The expression of serine hydroxymethyl transferase,branched amino acidtransaminase,ornithine aminoformyltransferase,etc,were significantly down-regulated,which affected the biosynthesis of serine,branched amino acid,ornithine,etc.The results of transcriptional group showed that the VOCs produced in Muscodor ZJLQ024 inhibit the hypha growth of Pyricularia oryzae by affecting the cell wall synthesis,amino acid metabolism and destroying the structure of cell membrane and ribosomes.