Resources Exploitation Of Biocontrol Strains In Rhizosphere Microorganisms Of Lycium Barbarum In Ningxia

Root rot has caused serious economic losses to Lycium barbarum in Ningxia,and rational control methods are important to reduce the losses.Biological control has attracted more and more attention because of its low pollution,low cost,and long-lasting effect.Screening of indigenous microorganisms with good antibacterial effect from L.barbarum rhizosphere soil is the foundation of developing biological control agents.In this study,the rhizosphere soil of healthy L.barbarum cv.Ningqi-1 and Ningqi-5 was used as materials for isolation of microbes.Firstly,rhizosphere microorganisms were isolated using dilution coating method and classified after purification.On this basis,the primary screening was carried out according to the plate confrontation experiment,and then the secondary screening was carried out based on the mycelium growth inhibition experiment using the culture solution.The biocontrol effect of antagonistic strains was explored by pot experiment,and the influence of antagonistic strains on the microbial community structure in the rhizosphere and root surface of barbarum was simultaneously investigated.Furthermore,the possible bacteriostatic substances were detected using metabonomics and genomic technology,aiming at providing efficient microbial resources for the development of biological control agents for root rot of L.barbarum.The main results are as follows:1.In total,50 strains of fungi belonging to 18 genera were isolated from the rhizosphere soil of L.healthy plants.67 strains of bacteria belonged to 10 genera and 61 strains of Actinomycetes belonged to 4 genera.There is little difference in the species of strains isolated from different soil samples.Fusarium ranged the highest in fungi,Bacillus ranged the highest in bacteria,and Streptomyces ranged the highest in Actinomycetes.2.Taking four main pathogenic fungi of L.barbarum root rot(Rhizoctonia solani,Fusarium oxysporum,Fusarium redolens and Fusarium solani)as test pathogens,23 strains of antagonistic bacteria and 11 strains of antagonistic bacteria with obvious inhibitory effects were screened through plate confrontation experiment and mycelium growth inhibition experiment.These antagonistic strains can produce extracellular antibacterial substances with different degrees of thermal stability,among which the extracellular antibacterial substances of Bacillus TB01 and Streptomyces TA16 have better thermal stability.3.The biocontrol effects of strains TB01 and TA16 were verified by pot experiments and the soil microecology was studied.It was found that Bacillus TB01 and Streptomyces TA16 had certain control effects on L.barbarum root rot caused by F.oxysporum.Strain TB01 can significantly reduce the relative abundance of pathogens in rhizosphere and root surface,and improve the richness and diversity of fungal and bacterial communities in L.barbarum root Although strain TA16 can not reduce the relative abundance of pathogens in rhizosphere and root surface,the richness and diversity of fungal and bacterial communities in rhizosphere were increased significantly.4.Based on genome sequencing,it is predicted that strain TB01 has 12 secondary metabolite synthesis gene clusters,producing surfactin,butirosin A/butirosin B,macrolactin H,bacillaene,fengycin,difficidin,bacillibactin and bacilysin.Through LC-MS analysis,the potential antibacterial substances in Bacillus sp.TB01 were detected,among which surfactant is supposed to be the key antibacterial substance.5.By genome sequencing,it was predicted that there were 24 gene clusters related to the synthesis of secondary metabolites in strain TA16,of which 20 gene clusters had clear information.Metabonomics analysis showed that staurosporine and ectoine were consistent with the prediction results of secondary metabolites gene clusters.To sum up,178 strains of rhizosphere microorganisms were isolated from the rhizosphere soil of healthy plants of two L.barbarum varieties,including 50 strains of fungi,67 strains of bacteria,and 61 strains of Actinomycetes,and 23 strains of antagonistic bacteria and 12 strains of antagonistic Actinomycetes were screened.The biocontrol effects of antagonistic strains TB01 and TA16 and the corresponding positive effects on the microbial community in the roots of L.barbarum were further confirmed by pot experiments.Finally,the possible antagonistic substances produced by Bacillus sp.TB01 and Streptomyces sp.TA16 were predicted by combining genomics and metabonomics techniques.