| Tomato gray mold is a destructively cosmopolitan tomato diasease caused by Botrytis cinerea.The disease can occur from area to area through human productive activities or airflow.Pathogen of tomato gray mold mainly survives in summer and winter by residue of infected plants in the soil,and it can infect the whole plant of tomato,but mainly harm the fruit,and can reduce the yield of tomato by 40%-50%,even causing total loss when the disease is very serious.Due to the lack of disease-resistant resources and few low diasease-resistant varieties,there is still a lack of disease-resistant varieties for production.As far as we know,agricultural control methods are time-consuming and labor-consuming.Meanwhile,although chemical control is effective,drug-resistant pathogens have emerged in many places in China.Biological control can be an ideal choice.Fraxinus hupehensis,an ash of the genus ashinae,is a second-class rare and protected plant,and has a medicinal value for people applying bark to Chinese medicine Cortex Fraxini in China.The buds are vulnerable to damage from disease and insect pests in the first few days of growth,but with the growth they will recover by themselves and be resistant to many diseases such as powdery mildew blight and so on.The phenomenon may indicate that there may be beneficial microorganisms in the plant,which can produce active substances resistant to disease and insect,and help it avoid the harm of diseases and insect pests.The purpose of this study was to understand the community structure of endophytic bacteria in F.hupehensis,to isolate,screen out,and identify biocontrol bacteria with strong biocontrol effect on tomato gray mold from the root of F.hupehensis,to study the biocontrol mechanisms,and to analyze the genomes.The main research results were as follows:The Next-generation sequencing results of endophytic bacteria of F.hupehensis samples collected from the garden at the west campus of Yangtze University in Jingzhou,Hubei province were as follows: 19,357 sequences were obtained from the stem of F.hupehensis,which could be divided into 291 operational taxa units(OTU)on the basis of 97% sequence similarity;16,882 sequences were obtained from the root of F.hupehensis,which were divided into 119 OTU;22,126 sequences were obtained from the leaves of F.hupehensis,which were divided into 190 OTU.There are 54 OTU of the same in the stem,leaves and roots.There was a high diversity of endophytic bacteria in the stem of F.hupehensis,and a high proportion and a low diversity of Actinophytocola in the root of F.hupehensis.A total of 125 bacterial isolates were obtained from 10 root samples collected from the garden at the west campus of Yangtze University.Twenty-five strains of them had inhibitory activity against B.cinerea during the dual culture.Among these strains,B17-1 and B17-12 A had strong inhibitory activity against B.cinerea,and the diameters of inhibition zone were 12 mm and 13 mm,respectively.B17-1 and B17-12 A had ability to produce IAA,dissolve phosphate,and produce protease and amylase.The antagonistic activity of them against 5 other pathogenic fungi such as Phytophthora infestans,Alternaria solani,Magnaporthe grisea,Fusarium oxysporum f.s.lycopersici,F.moniliforme was also determined.The results showed that the diameters of inhibition zone of them against these fungi were all above 6.95 mm.The results of detached leaf assay indicated that there was no damage on tomato leaves after treatment with strain B17-1 or B17-12 A only,and the relative efficacy of the two strains on tomato gray mold were 44.96% and 55.16%,respectively.The diameter of inhibition zone of strain B17-12 against B.cinerea was 9 mm,and the inhibition rate of volatile organic compounds(VOCs)emitted by B17-12 against B.cinerea was 80.67%.B17-12 had ability to produce IAA,dissolve phosphate,produce protease,and to form biofilm.The antagonistic activity of VOCs produced by strain B17-12 against 5 other pathogenic fungi such as P.infestans,A.solani,Rhizoctonia solani,F.oxysporum f.sp.vasinfectum,and F.oxysporum f.sp.niveum was also determined.The results showed that the inhibition rates of VOCs produced by B17-12 against these pathogenic fungi were between 75.8% and 92.4%.The VOCs produced by B17-12 reduced the fruit incidence rates of tomato early blight,tomato late blight and tomato gray mold by 78%,75% and 80% respectively,which was obviously better than the agent treatment.The culture broth of B17-12 reduced the fruit incidence of tomato early blight,tomato late blight and tomato gray mold by 62%,50% and 75% respectively.The cell suspension of strain B17-12 reduced the fruit incidence of tomato early blight,tomato late blight and tomato gray mold by 62%,55% and 82% respectively.The culture filtrate of strain B17-12 reduced the fruit morbidity of tomato early blight,tomato late blight and tomato gray mold by 57%,60% and 84% respectively.The plasmid p BBR1MCS2-Tac-EGFP was introduced into strain B17-12 by electric shock method,and the transformed strain B17-12 was obtained.The results of fluorescence microscopy and colonization recovery test showed that the B17-12 inverters could colonize in tomato roots for 29 d.Twenty-five compounds produced by strain B17-12 cultured in NA were detected by solid-phase microextraction-gas chromatography/mass spectrometry(SMPE-GC-MS).The six compounds included 2-nonanone,2-undecanone,Bis(2-ethylhexyl)phthalate,cycloclo [2.2.1]heptan-2-one,1,7,7-trimethyl-,(1s)-,2,5-cyclohexadiene-1,4-dione,2,6-bis(1,1-dimethylethyl)-and Tetradecane have been reported to have antimicrobial activities.The genome of strain B17-12 was sequenced by Illumina Hiseq×10,and its size was 6,073,173 bp after the sequences were assembled.The content of GC was 71.33%.According to the COG database annotation results,435 genes were involved in the amino acid transport and metabolism,and 378 genes were involved in transcription.In the KEEG metabolic pathway annotation,the pathways of ABC transporters,Two-component system,Biosynthesis of amino acids,and Carbon metabolism had a high correlation with the strain genomes.The genome of B17-12 was submitted to the antismash website.B17-12 was found to have 7 secondary metabolite synthesis gene clusters.The growth inhibition rates of supernatant of B17-1 against B.cinerea were 90.41% when the proportion of the supernatant in PDA medium was 40%(v/v),and at the same concentration,the growth inhibition rates of supernatant of B17-12 A and B17-12 against B.cinerea were 99.52% and 100%,respectively.According to morphological and molecular biological identification,strains B17-1 and B17-12 A both belonged to Paenibacillus sp.,while strains B17-12 were Pseudomonas koreensis.In this study,the endophytic bacteria of F.hupehensis were preliminarily analyzed by high-throughput sequencing,and it was found that the endophytic bacteria were rich in resources.To the best of our knowledge,it is the first time to isolate bacteria from the root of F.hupehensis and study their biocontrol mechanisms,and to report that the VOCs produced by a P.koreensis strain have strong control effect on tomato gray mold.B17-12 was a promising biocontrol agent for controlling tomato gray mold.At the same time,through initial analysis of the whole genome and the identification of strain B17-12,it will provide new strain resources for biological control of tomato gray mold in the future. |
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