Screening And Identification Of Biocontrol Streptomyces(HS1) And Its Control Mechanism On Poplar Canker

Poplar canker is a major disease in China,which is widely distributed and seriously infested,and caused a great threat to the healthy development of poplar plantation industry in China.In this study,Against Botryosphaeria dothidea,the main pathogen of poplar canker,the potential biocontrol Streptomyces strains were studied.Whole genome sequencing and16S r DNA sequence analysis were used to clarify its taxonomic status;the fermentation conditions and fermentation technology of 5-50l large-scale equipment were explored;and the antibacterial mechanism,control effect and induced resistance of the fermentation broth were studied.The main results are as follows:（1）About 104 strains of Streptomyces were isolated from 18 soil samples collected from different forest stands in Changbai Mountain National Nature Reserve of Jilin Province.And13 strains with antagonistic effect against B.dothidea were screened out.Among them,strain HS1 had the best antibacterial effect and a wide antibacterial spectrum.The strain HS1 was identified belong Streptomyces by traditional classification and identification methods.The strain had the highest homology with Streptomyces scopuliridis in16S r DNA sequence.The phylogenetic tree constructed with 243 known Streptomyces strains showed that HS1 had no unique sister branch.Based on the existing database,the taxonomic status of HS1 could not be determined at the species level,and there was a possibility of new species.（2）The genome of strain HS1 accords with the basic characteristics of Streptomyces genome.4970 genes were annotated in GO database;3459 genes were enriched in 213KEGG metabolic pathways,among which more genes were involved in regulation of secondary metabolism and antibiotic biosynthesis;5525 genes were successfully annotated in COG database,among which 302 genes were related to secondary metabolite synthesis;6842genes were in NR;The results of cazy database annotation showed that there were 119carbohydrate related modules（CBMS）,22 carbohydrate esterase（CES）,117 glycoside hydrolase（GHS）,91 glycosyltransferase（GTS）,2 polysaccharide lyase（PLS）and 6oxidoreductase（AAS）genes.There are 38 gene clusters in the genome of Biocontrol Strain HS1,most of which belong to unknown gene clusters.It has the potential to synthesize a variety of secondary metabolites and gene elements,and has great biological control potential.（3）Through the optimization of fermentation conditions,the optimal fermentation medium formula was obtained as follows:soluble starch 80 g,peanut cake powder 35 g,potassium nitrate 2 g,calcium carbonate 4 g,sodium chloride 4 g,water 1000 ml,pH 7.0.The fermentation conditions were as follows:shaking speed 150 R/min,fermentation temperature 28℃,inoculum 6%,strain age 4 D,fermentation time 4 D,pH 7.0.Parameters of secondary fermentation system:set speed of 150 R/min in 5L seed tank,fermentation temperature of 28℃,ventilation of 3.0 L/min,initial pH of 7.0,fermentation time of 4 d.The fermentation process of 50L fermentor:rotation speed 250 R/min,temperature 28℃,ventilation 10.0 L/min,fermentation time 4 D,pH 7.0.Results of physical and chemical properties of fermentation broth:the fermentation broth has good light stability,the best bacteriostatic effect at pH 7.0,a wide range of storage temperature,long-term storage should be below 4℃.（4）The fermentation broth could obviously inhibit the growth of mycelium,and had effects on mycelium morphology,growth rate and growth amount.The increase of electrical conductivity caused by the leakage of the contents indicated that the fermentation broth could increase the permeability of the cell membrane;the accumulation of malondialdehyde in the cells indicated that the fermentation broth could cause the serious lipid peroxidation of the cell membrane;therefore,it could be determined that the action site of the fermentation broth on the pathogen was the cell membrane.The activity of hexokinase（HK）,Phosphofructokinase（PFK）and pyruvate kinase（PK）increased after fermentation broth treatment,which accelerated the glucose metabolism of the pathogen,resulting in the decrease of total glucose content and the increase of total pyruvate content,resulting in the excessive glycolysis pathway in the cell;meanwhile,the activity of Ca²⁺Mg²⁺-ATPase increased,which seriously affected the nutrients and energy accumulation in the mycelium Tired.It can accelerate the physiological metabolism of the pathogen and accelerate the aging and death of the pathogen.（5）The fermentation liquid of strain HS1 had good preventive and therapeutic effects on poplar canker.It can also induce poplar disease resistance,the content of phenolic substances,flavonoid substances and lignin in poplar leaves treated with fermentation broth increased in varying degrees;phenylalanine ammonia lyase（PAL）activity increased,which can effectively induce the accumulation and synthesis of phenolic antibacterial substances,enhance the ability of plants to resist the invasion of pathogens;the activity of chitinase increased,which can be degraded The formation of cell wall hydrolase can effectively inhibit the proliferation and diffusion of the pathogen after invasion;the increase of salicylic acid content in poplar leaves can effectively induce the synthesis of a variety of antibacterial substances and improve the disease resistance of poplar.In conclusion,a strain of Streptomyces with obvious antagonistic effect on poplar canker was obtained,and its taxonomic status,fermentation technology,antibacterial mechanism,forest control effect and induced resistance were systematically studied.The fermentation broth of strain HS1 can effectively control the occurrence and harm of poplar canker,and has a promising development and application prospect.