| Angelica sinensis(Oliv)Diels,perennial herbs of Apiaceae,ranks first among the"Top Ten Herbal Medicine in Gansu".In recent years,however,the root rot of Angelica sinensis has been increasingly severe,which has significantly affected the quality of Angelica sinensis in Gansu Province.At present,the mainly control measure is chemical fungicides and crop rotation,but the serious agricultural residues not only polluted the soil but also decreases the control function year by year,which hinders the development of Angelica industry.Under such background,biological control in place of chemical pesticides has become a research niche for its advantages such as less environmental pollution as well as high efficiency in controlling the plant diseases.Since few reports shed lights on biological bacteria to control Angelica root rot,this study would bridge the gap.The research isolated and identified a series of pathogens from Angelica sinensis with root rot,then isolated and screened the biocontrol bacteria from rhizosphere soil,giving a systematical study of the control effect and mechanism of biocontrol bacteria.The main research results are as follows:(1)The diseased Angelica sinensis was collected from the planting area of Angelica sinensis in Weiyuan County,Gansu Province,and the Fusarium avenaceum was isolated and identified as the pathogen of root rot of Angelica sinensis.The strain SY89 and YF with strong inhibitory effect were isolated from the rhizosphere soil.The strain SY89 was identified as Bacillus tequilensis and the strain YF was identified as Paenibacillus polymyxa by molecular identification.(2)Tests in vitro showed that strains SY89 and YF could inhibit the hyphae growth of the pathogen,make hyphae expanded,multi-branched,vesicular at the end,broken and shrunk,reducing the biomass.Besides,Strains SY89 and YF could inhibit spore germination at a inhibition rate of 89.39%and 90.65%respectively.Plus,it is shown that the 10%bacteria-free filtrates had enhanced cell membrane permeability of F.avenaceum,resulting in the release of cellular contents.The control tests showed that strains SY89 and YF had better prevention effect,with the highest prevention rate of 70.45%and 68.38%,respectively.Both strains SY89and YF could produce indole-3-acetic acid(IAA)and siderophore.After the application of 10~9CFU/m L strain SY89,plant height,root length,fresh weight and dry weight increased 23.35%,56.81%,26.34%and 40.86%,respectively,compared with the control group.The indexes of10~9 CFU/m L strain YF increased 33.91%,66.57%,36.59%and 61.30%,respectively.(3)By constructing strains GFP-SY89 and GFP-YF to study their colonization in the root of Angelica sinensis,the colonization rate of both strains reached 10~3 CFU/g.The POD activity increased 209.64%and 209.03%after treating by biocontrol bacteria,respectively,and PAL activity increased 30.96%and 26.40%,respectively,suggesting that strains SY89 and YF could induce systemic resistance of Angelica sinensis,thus resisting pathogen invasion.In addition,both strains SY89 and YF could produce amylase,protease,glucanase and cellulase,which effectively inhibited the extracellular hydrolase of pathogens.Analysis by GC-MS showed that both strains SY89 and YF could produce hexahydropyrrole[1,2-a]pyrazine-1,4-dione,cyclic(L-prolinyl-l-leucamide),phthalic acid,butylhept-4-ester,cyclic(leucine-leucine)dipeptide,dibutyl phthalate 2,4-di-tert-butylphenol and other effective bacteriocidal substances.The genes of bacteriostatic substances such as iturin,surfactin,fengycin,non-ribosomal polypeptide synthetase and polyketide synthase could be amplified by PCR.In addition,strains SY89 and YF could inhibit the secretion of ENN B with rate of 77.64%and 61.93%,respectively,and degrade the toxin with rate of 76.03%and 60.69%,respectively.It proved that inhibition and degradation of toxin secretion by strain might be one of the mechanisms of pathogen control.(4)Transcriptome sequencing was used to investigate the molecular mechanism of strain SY89 in response to ENN B.A total of 926 differential genes were screened from transcriptome data mining using bioinformatics platform.The differentially expressed genes are annotated and the GO function and KEGG metabolic pathway enrichment were analyzed.It is preliminarily speculated that the oxidoreductase acyl-Co A dehydrogenase Fad E reduced the toxicity of ENN B through oxidation reaction.Hydroxymethylglutaryl-Co A lyase attenuates by degrading branched amino acids such as valine(Val)on ENN B.In addition,ENN B could increase the expression of genes related to biosynthesis of validamycin and non-ribosomal peptides of siderophore group,thus enhanced the inhibition ability of biocontrol bacteria. |
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