Study On The Antifungal Mechanism And Application Of Biocontrol Bacteria HAB-6

The strain HAB-6,isolated from Vigna unguiculata(Linn.)Walp,has antimicrobial activity.In this dissertation,identifition of strain HAB-6,antimicrobial activity,lipopeptide related-genes,the mechanisms of control plant disease and promotion plants growth of strain HAB-6 were studied.The results in the present study were summarized as below:1?Though the morphology and scanning electron microscope(SEM)observation,gram staining,and flagellum staining,the strain HAB-6 was identified as Bacillus.By the analysis of 16SrDNA sequence and specific sequences,the strain HAB-6 was further identified as Bacillus amyloliquefaciens.2?The activity of strain HAB-6 was measured against 17 plant pathogenic fungals such as Colletotrichum gloeosporioides,Colletotrichum gloeosporioides Penz,Fusarium oxysporum f.sp.cubense,by using the plate dual-culture antagonistic method.The inhibition rates were ranged from 45.48-72.20%and the rate of Colletotrichum gloeosporioides was the highest,reaching up to 72.20%.The hydrochloric acid precipitation,ammonium sulfate precipitation and organic solvent,the antifungal substances from strain HAB-6 were extracted,and their stabilities in turn were analyzed.In addition,only the n-butyl alcohol extract(5 mg/mL)had demonstrated strong antifungal activity as the diameter of its inhibition zone was 21.49 mm.The extract retained 87%antifungal activity after a 120 min exposure to UV,and 91%of that of the control after thermal treatment at 100?.However,the n-butyl alcoholhad extract no endurance extreme acid and alkali treatments.The suitable pH for the extract to remain stable was ranged from pH 5.0-9.0.3?LC-MS analysis demonstrated that strain HAB-6 could produce C14 Iturin A,C16 IturinA,C17 Iturin A or C17 Mycosubtilin,C13 Surfactin A,C15 Surfactin C.The species-unique primers designed based on the known genes of lipopeptide and genes of synthetic lipopeptide key enzyme were used to amplify the corresponding genes from the genome of strain HAB-6.The BLAST analysis showed that there were 5 lipopeptide genes,which were ituC,srfAB,ituD,fenD,yndj,and 5 genes of synthetic lipopeptide key enzyme,which were ituA,ituD,lpa-14,sfp,mycB,fenB,were cloned from the strain HAB-6 genomic DNA.But these lipopeptides extracted by ammonium sulfate precipitation were not antifungal activity,it might be that most of lipopeptides were surfactin.4?The n-butyl alcohol extract could control plant disease.The n-butyl alcohol extract inhibited the growth of C.gloeosporioides spores and hyphae,thereby effectively inhibited preventing the C.gloeosporioides infection of mango and leaves.And,the n-butyl alcohol extract could also prevent and control Oidium heveae and Alternaria solani on the tomato fruits.The anti-algal rates of different the n-butyl alcohol extract were 57.14%?73.47%?85.71%,respectively,and the EC50,7d value was 7.08 mg/L.5?The quantitative real-time PCR(qRT-PCR)investigated that the n-butyl alcohol extract could induced the induced systemic resistance in the tobacco challenged with Tobacco Moscia Virus(TMV).The expression of the hypersensitive reaction(HR)marker gene hsr203 gene was 186.9-fold increase compared with the control,when treated with the n-butyl alcohol extract upon TMV challenge during 72 h,then this value decreased.The transcriptional expression of defense-related genes PR-1b was raised by 4.4-fold in the treatment at 48 h and maintained its high level.The NPR1 following treatment was upregulated by 50.56-fold during 72 h.The n-butyl alcohol extract could induced to increase the expression of defense relative genes.6?The tomato seeds treated different concentrations of the n-butyl alcohol extract,and the n-butyl alcohol extract diluted 1500 times has the most promoting effect on the tomato seeds.The tomato seedlings was spraying with diluted 1500 times of the n-butyl alcohol extract.After 30 days the average stem length of tomato seedlings was 14.5 cm.The number of leaves,the biomass,the root lengththe and the content of chlorophyll increased 8.33%,6.89%,23.66%,21.97%,respectively,as compared with the control.The data exist significant differences at the 0.05 level.The n-butyl alcohol extract could promoted the germination of tobacco and the expression of plant growth promotion gene NtEXP2 gene was increased by qRT-PCR analysis.By spraying the n-butyl alcohol extract in the field,the growth of cowpeas were significantly better than the control group.7?Zebrafish toxicity test,The LC50 values of the n-butyl alcohol extract 17.785 mg/L at 96 hpf.The concentration more than 15.81 mg/L,the n-butyl alcohol extract can cause the zebrafish yolk cyst at 96 hpf.8?Further by vacuum pumping column method and silica gel column chromatography,gel column chromatography,reverse column chromatography separation,compound H6 was isolated from the n-butyl alcohol extract.This compound analyzed by mass spectrometry and nuclear magnetic resonance(NMR),and named 1-(3-amino-3,4-dihydronaphthalen-2-yl)ethanone.According to the comparison database,compound H6 was a new synthetic material.