Study On Streptomyces Botrytirepellens NEAU-LD23 Against Botrytis Cinerea And Genomic Analysis

Botrytis cinerea is a fungal plant disease caused by Botrytis cinerea Pers.The strain has a wide range of infections and can infect many plants such as tomatoes,cucumbers and grapes,which are widely distributed throughout the world.Tomatoes are important economic crop in our country,and the annual yield is greatly reduced by the harm of gray mold.In the past few decades,the main method for the control of tomato gray mold is the use of fungicides.However,due to the widespread use of fungicides,not only environmental pollution,but also the chemical components remaining in tomato fruits pose a potential threat to human health,and in accordance with current conditions,the use of fungicides to control plant gray mold disease is also difficulties.Therefore,exploration of antagonistic microorganisms and their effective metabolites can be a more natural and effective alternative method for controlling plant diseases and reducing the damage caused by fungicides.In this research,we studied the isolation and physiological activities of actinomycetes in rhizosphere soil of tomato plant and screened the actinomycetes using 5 highly efficient screening media,and removing and combining replicates by observation of their morphological characteristics.Then,the pathogenic fungi of Botrytis cinerea was used as a test strain,and the antibacterial activity of the whole strain was tested in vitro.The strain with better activity was fermented and determined the antibacterial activity and stability of the fermentation broth.Combined with the tomato seedling test,the disease resistance of the obtained antagonist strain was evaluated.At the same time,through the isolation of natural products,the initial analysis of whole genomes and the identification of strains,it will provide new resources for the future biological control of tomato gray mold.In addition,this research also found and identified two new actinomycetes of the genus Lentzea and one of the genus Streptomyces,which not only provided new resources for the taxonomy of rare actinomycetes,but also enriched the microbial strain database.(1)The actinomycetes in the rhizosphere soil of tomato plant were screened by using five isolation mediums(GS?DPA?CPA?SSA and HV),and 292 actinomycetes were obtained finally.We obtained 120 actinomycetes by rearranging and combining repetitive strains.These strains were classified into the following eight genera by morphological observation and 16 S r RNA molecular level detection: Streptomyces,Micromonospora,Promicromonospora,Lentzea,Kribbella,Nonomuraea,Actinomadura and Glycomyces.(2)Antibacterial activity test was carried out on all the 120 strains isolated and purified using the pathogen of tomato gray mold as the tested fungi in vitro.Results After screening,5 strains with bacteriostatic rate above 50% were obtained,which were NEAU-LD23,NEAU-LD86,NEAU-LD90,NEAU-LD93 and NEAU-LD99.The activity of the remaining 9 pathogenic fungi was tested on the 5 strains.The strain of NEAU-LD23 had the strongest inhibitory effect on the pathogenic fungi of B.cinerea,up to 81.8%,and the best broad-spectrum antibacterial activity.The antibacterial activity and stability tests of the fermentation broth of strain NEAU-LD23 showed that the antibacterial substance had the ability to withstand high temperature and acid resistance,however,under strong alkaline condition,the activity decreased slightly.(3)The tomato seedlings inoculated with pathogenic fungi were treated with the stock solution of the fermentation broth,the 5-fold dilution solution,the 10-fold dilution solution and the 15-fold dilution solution of the strain NEAU-LD23.Compared with the CK group,the incidence rate and the disease index were significantly decreased.The highest rate dropped from 95% to 45%,and the disease index dropped from 45 to 25.(4)The genome of strain NEAU-LD23 was sequenced by single-molecule sequencing,which has a length of 8,773,497 bp after the sequence is assembled by stitching.The content of G+C mol% in genome was 71.3%.The results of genomic annotation show NEAU-LD23 has 8518 predicted protein coding genes and 69 non-coding gene.According to the COG database annotation results,527 genes are involved in the synthesis and transport of secondary metabolites,accounting for 6.2% of the total predicted genes.In the KEGG metabolic pathway annotation,the pathways of microbial metabolism in different environments,bios ynthesis of secondary metabolites,and biosynthesis of antibiotics have a higher correlation with the strain genomes.The genome of NEAU-LD23 was submitted to the anti SMASH website.The strain was found to have 61 secondary metabolite synthesis gene clusters,of which 33 genes encoding PKS and NRPS,accounting for 54.1% of the total number of predicted total gene clusters(5)By performing molecular biological analysis,morphological characteristic culture observation,physiological and biochemical characteristics comparison and chemical experiment analysis on NEAU-LD23,it was confirmed that the strain was a potentially effective strain in Streptomyces.In addition,we also identified two new actinomycetes of the genus Lentzea and one of the genus Streptomyces by the same method.