Biocontrol Agents Screening Of Potato Late Blight And Its Biocontrol Mechanism

Potato late blight（PLB）is one of the top ten oomycete diseases in the world,which significantly reduces the yield and quality of potato tubers,its pathogen is Phytophthora infestans.Biological control mainly uses microorganisms and their metabolites to control plant diseases and it is an environmentally friendly measure for sustainable development.At present,there is a lack of biological control agents（BCAs）for PLB.It is urgent to find and screen suitable antagonistic microorganisms and metabolites for the PLB biocontrol.To screen the ideal BCAs,the membership function method was used to comprehensively evaluate several antagonistic actinomycetes isolated from potato producing areas,and two Actinomycetes with high biocontrol potential were selected for follow-up research.The main research results are as follows:（1）Biocontrol of PLB by Streptomyces violacens A10 and its metabolites:Actinomycete A10 isolated from potato rhizosphere soil was identified as a new isolate of S.violacens by polyphasic taxonomy.The results in vitro showed that S.violacens A10 significantly inhibited the hyphae growth and sporangium germination of P.infestans,damaged the hyphae ultrastructure,and reduced the expression of effector genes.Genome analysis of S.violacens A10 predicted the possibility that the A10 strain produced cytoprotective agents,siderophores,volatile organic compounds,and other secondary metabolites.The presence of these predicted secondary metabolites was confirmed using metabolic analysis.This study also found that a volatile sulfide methionol（3-Methylthio-1-propanol,3-MP）produced by S.violacens A10 can significantly inhibit the hyphae growth of P.infestans.Fumigation or direct contact treatment were both effective.（2）Identification and genomic analysis of a new species of Streptomyces sp.A2-16:Actinomycete A2-16 isolated from potato root zone soil was identified as a new species of Streptomyces by polyphasic taxonomy.The anti SMASH analysis showed that Streptomyces sp.A2-16 had 28 secondary metabolite synthesis gene clusters,including5 polyketide synthase（PKS）,5 terpenoids,and 4 siderophore gene clusters.Transcriptional and metabolic analysis of confrontation culture between Streptomyces sp.A2-16 and P.infestans:Streptomyces sp.A2-16 significantly affected the metabolite secretion of P.infestans,so that the differentially expressed genes of P.infestans were significantly enriched in tryptophan metabolism and the ABC transporter pathway,and the differentially expressed genes enriched in these two pathways were significantly downregulated,suggesting that Streptomyces sp.A2-16 can regulate the growth and pathogenicity of P.infestans by inhibiting tryptophan biosynthesis and ABC transporter.（3）Isolation and identification of anti-oomycete active substances of Streptomyces sp.A2-16 and its biological mechanism:1)Based on metabonomics:the volatile and nonvolatile metabolites produced by Streptomyces sp.A2-16 was identified by metabolomics analysis.It was found that the high abundance metabolites（penicillic acid,rufloxacin,n-octanoic acid,2-heptanol and Phenylethyl alcohol）had significant anti-oomycete activity against P.infestans.Among them,the inhibitory activities of 2-heptanol and n-octanoic acid against P.infestans were reported for the first time.2)Based on the activity tracking method:the main active substances of P.infestans were separated by semi preparative liquid chromatography,and three pure compounds（A2-9,A2-8,A2-6）were obtained.After multi spectral analysis such as mass spectrometry and nuclear magnetic resonance spectroscopy,the three compounds were all compounds of the manumycin family.A2-9 was the known compound nisamycin.A2-8 and A2-6 had low similarity with the known manumycin family compounds,which were speculated to be new compounds,and their chemical structures have not been identified.Further,the inhibitory activities of the three manumycin family compounds against P.infestans were detected,and the EC₅₀ values were calculated to be 1.29,0.79 and 7.67 mg/L,respectively.At the same concentration,the anti-oomycete activity of the three manumycin family compounds was much higher than that of agricultural antibiotics validamycin,which showed great biocontrol potential.The potential new manumycin family compound A2-6 controlled P.infestans by destroying the main infection morphological structure and reducing the expression of pathogenic related genes.The above results showed that Streptomyces sp.A2-16 secreted many types of metabolites（including sulfide,enzyme,antibiotic,organic acid,etc.）to biocontrol P.infestans.（4）Field experiment results of Streptomyces A10 and A2-16:in the two-year field experiment,the inhibitory effect of Streptomyces A10 and A2-16 on PLB was only limited to the early and middle stages.S.violacens A10 could increase the weight of potato tubers during harvest,and the results were consistent in the two-year field experiment.Although the application of Streptomyces sp.A2-16 alone had no significant effect on the weight of potato tubers,the weight of potato tubers increased significantly after Streptomyces sp.A2-16 combined with chemical pesticide reduction application,suggesting the great biological control potential of Streptomyces sp.A2-16in the combined chemical pesticide reduction application.The above results laid a foundation for Streptomyces A10 and A2-16 as BCAs to control PLB.At the same time,the genomic analysis of Streptomyces sp.A2-16provided information for the study of gene function and metabolite biosynthesis pathway of Streptomyces sp.A2-16.The research results of Streptomyces A10 and A2-16 can not only provide a theoretical and experimental basis for the PLB biocontrol,but also provide an important reference for the other plant diseases biocontrol.