| Industrial hemp(Cannabis sativa L)has a long history of cultivation and is an important economic crop.Its fiber is an important textile raw material.The fruit is both oil and medicinal,so it is widely used in textile,oil extraction,health care,building materials and other fields.In recent years,industrial cannabis extract CBD(cannabidiol)has attracted much attention because of its good effects and broad medicinal prospects for nervous system diseases such as anxiety,insomnia,pain relief and epilepsy.Therefore,the industrial cannabis industry has ushered in vigorous development,and the national industrial cannabis planting area has rapidly exceeded100 acres.Sclerotium rolfsii Sacc is a fungal soil-borne disease caused by Sclerotium rolfsii Sacc,which can seriously affect the growth and development of hemp,resulting in a significant decline in the yield and quality of fiber and hemp seed,resulting in the death of the whole plant and a large area of production reduction.The use of beneficial microorganisms to inhibit soil-borne diseases is a hot topic in recent years.In order to further explore the microbial resources of biocontrol strains of cannabis southern blight and develop corresponding green prevention and control technologies,this study used industrial cannabis as the source of separation,and carried out the isolation and purification of cannabis endophytic bacteria,plate confrontation,morphological and molecular identification,condition optimization and omics analysis.The main results are as follows :1.21 endophytic bacteria and 25 endophytic fungi were screened by plate confrontation method.Among them,4 endophytic bacteria had more than 70 %antagonistic effect on Sclerotium rolfsii,while strain 024 A had the strongest inhibitory effect on Sclerotium rolfsii,with a relative inhibition rate of 80.1 %.Based on the morphological,molecular biological,physiological and biochemical characteristics of the strain,the strain 024 A was identified as Bacillus velezensis.2.Antagonistic bacteria 024 A has the ability to secrete cellulase,protease and amylase,has siderophore activity,but can not secrete chitinase,nitrogen fixation.In addition,the strain has a weak ability to dissolve phosphate,and the ability to dissolve potassium is relatively weak.The bacterial solution has strong biofilm formation ability,and the increase of bacterial concentration will lead to the increase of biofilm formation ability and then decrease.3.The results of antimicrobial spectrum showed that strain 024 A had certain inhibitory effect on various plant diseases.Among them,the relative inhibitory rate of the strain against Sclerotium rolfsii Sacc,Phytophthora capsici,Colletotrichum gloeosporioides and F.oxysporum f.sp.melonis was between 69.4 % and 84.7 %.The inhibition zone radius of Ralstonia solanacearμm,Pectobacterium carotovorum subsp.carotovorum,Erwinia carotovora,Xanthomonas campestris pv.campestris(Xcc)and Xanthomonas oryzae could reach 5.56-13.52 mm.In vitro leaf experiments showed that strain 024 A had a strong inhibitory effect on the infection of Sclerotium cannabinum.The lesion area of leaves treated with strain 024 A was slightly smaller than that treated with the fungicide difenoconazole,indicating that the control effect of strain 024 A was similar to that of the heterocyclic fungicide difenoconazole.The results of pot experiment showed that strain 024 A also had strong control effect on cannabis southern blight under greenhouse conditions,and its control effect reached 32.24 %.4.The optimal carbon source and nitrogen source of fermentation medium were explored by single factor experiment,and then the experimental scheme was designed by Design-Expert software.Taking the optimal carbon source(A),optimal nitrogen source(B),fermentation temperature(C)and fermentation time(D)obtained in the early stage as the influencing factors,and the bacteriostatic rate(Y)of strain 024 A against Sclerotium rolfsii as the response value,the response surface test of 4 factors and 3 levels was carried out.The fermentation conditions for the maximum bacteriostatic rate of 024 A fermentation filtrate were as follows : yeast powder concentration 14.12 g / L,glucose concentration 11.84 g / L,fermentation temperature31.56 °C,fermentation time 17.91 h,and the predicted bacteriostatic rate was up to89.27 %.According to the regression equation,the optimal culture conditions of 024 A were simulated,and three repeated experiments were carried out.The bacteriostatic rates were 87.00 %,86.00 % and 90.00 %,respectively,with an average of 87.66 %,which was close to the predicted value of 89.27 %.The reliability of the model was confirmed,and the bacteriostatic rate of the optimized 024 A was increased by nearly10 % compared with the plate confrontation bacteriostatic rate of 80.10 % when the medium was not optimized.5.The whole genome sequence of strain 024 A predicted that the number of coding genes was 4433,the total length of coding region accounted for 89.82 % of the whole genome,and the total length of all coding genes was 3,719,877 bp.After comparative analysis,GO annotation,COG annotation,KEGG annotation and NR annotation were performed on the genome,and it was found that the genes involved in energy metabolism,cell division and differentiation,and enzymatic reactions in the genome accounted for the highest proportion.In addition,the gene clusters encoding secondary metabolites in the genome of strain 024 AA were predicted.A total of 13 secondary metabolite synthesis gene clusters were predicted,including fengycin,pantazolicin,and lanthipeptide synthesized through the non-ribosomal pathway(NRPS).The polyketide synthase(PKS)pathway includes butirosin A,macrolactin H,surfactin,and mersacidin.Lanthipeptid and difficidin are produced by PKS-NRPS hybrid pathway;mersacidin and bacilycin synthesized by other ways.6.Metabolomic analysis of the supernatant of strain 024 A showed that : According to the biological function,the metabolic active substances produced by the biological process of strain 024 A contained four antibiotics,namely aminoglycoside gentamicin C,polyketides and non-ribosomal peptide erythromycin,hypericin and geldanamycin,quinolone nalidixic acid and other types of mitomycin,cycloheximide and 2-oxazolidinone.In addition,there are arachidic acid,leukotriene E4,marquinoline and other substances.The pesticide-related metabolites of strain 024 A include Indole-3-acetic acid and Oxytocin S,as well as 2-biphenyl and endosulfan.Phytochemical compounds include growth-related substances such as gibberellin A44,gibberellin A37,and indole-3-acetic acid,as well as dihydrocapsaicin jasmonate,chelidonine,and lauryl alcohol oxide.The lipids produced by strain 024 A contain n-leucine,cucurbitacin B,glutaric acid and other substances. |
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