| Saffron(Crocus sativus)is a perennial herhaceous plant,which belongs to the family Iridaceae,and is traditional precious Chinese herbal medicines.It has many curative effects,promoting blood circulation to remove blood stasis,cooling blood detoxification,resolving depression,anti-cancer effect and so on.The growing market demand results in the increasement of artificial cultivation year by year.In 2015,Saffron planting area exceed 6,000 mu in Zhejiang province,accounting for about 50%of the national saffron planting area.Jiande city is the biggest plant area in Zhejiang province,which Saffron planting more than 3,000 mu With the expansion of facilities cultivation and years of asexual reproduction,saffron diseases become serious year by year.Especially,Corm rot disease is the most significant that made more than 60%lose in output and the seriously decline of quality.In this study,the storage of infected corm were used as the test materials,the pathogens of corm rot disease were isolated and identified;biocontrol agents were screened out from the rhizosphere and antibacterial substances were studied.The results are as follows:1.This study investigated and collected the infected corms form Jiande city.The result showed the pathogen of corm rot disease were identified as Fusarium oxysporum ZJU-1 and Fusarium solani ZJU-2 by the morphology and molecular biology(analysis of the ITS gene and TEF-? gene).2.A strain C612 was screened out from the rhizosphere in Jiande city,and was identified as Bacillus amyloliquefaciens based on morphology and 16s rDNA/GyrB sequence analysis.The results of potting experiments showed that the biocontrol efficacy of B.amyloliquefaciens C612 against F.oxysporum ZJU-1 and F.solani ZJU-2 were 52%and 40%,respectively.When inoculated with the mixture of F.oxysporum ZJU-1 and F.solani ZJU-2,the biocontrol efficacy of strain C612 was 46%.Besides,B.amyloliquefaciens C612 could promote plant height,root length,fresh-corm weight,and average number of corms per plant.3.The stability and antibacterial spectrum of the extracellular antimicrobial crude extract of B.amyloliquefaciens C612 were determined.The results showed that the crude extract of strain C612 had strong acid and alkali resistance as well as high temperature resistance,was not sensitive to protease.It had performed broad antagonistic spectrum against all of the 8 tested strains of phytopathogenic fungi.The properties of the extract substance,were concordant to lipopeptides,so the related genes for the synthesis of lipopeptide antibiotics in B.amyloliquefacieus C612 was detected by four specific primers mycb,fenb,sfp and ituA.PCR amplification results showed that B.amyloliquefaciens C612 contained lipopeptide antibiotics synthetic genes ituA and fenB,indicating that the extracellular products of B.amyloliquefaciens C612 may contain lipopeptide antibiotics iturin A and Fengycin.Meanwhile,B.amyloliquefaciens C612 can produced protease,cellulase,chitinase,but not produced siderophore.4.According to the physical and chemical characteristics of liquid and refering to the reported extracting methods.the crude extract of active substances were isolated and purified by silica gel column chromatography and thin layer chromatography,38.4 mg of active substance was obtained finally.Using HPLC-MS and iturin A prototype to analysis the active material,the analysis results showed that active substances contained iturinA homologue C14-iturinA,C15-iturinA,C16-iturinA.This study identified the pathogenic bacteria of corm rot disease and screened out an B.amyloliquefaciens C612 that could inhibit corm rot disease from rhizosphere.On the basis of its biochemical properties,antibacterial activity and exploration on the plant biocontrol effect,it can provide technical support and theoretical basis for the development andapplication of lipopeptide antibiotics as a biological control agent. |
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