| Grape downy mildew, caused by Plasmopara viticola, is one of the most severe fungal diseases in grape production. This disease occurs widespread and causes serious harm. Chemical pesticides are always the main measures for preventing it. However, due to the large number of long-term continuous use of chemical pesticides, causing3R problem (3R problem means resistance, residue and resurgent). Further more, the pesticide residues can also cause environmental pollution and declined the quality of products. The chief preoccupation is to find a safe and effective way to control the diseases. Nowadays, biological control has already become a hot topic in plant diseases. This method is the greatest potential way to control plant diseases, and it has the advantages of cost-effective and environmentally friendly. This study focuses on the bacteria isolated from grape leaves, and screens the effective antagonistic strain against P. viticola from them. Further more, carry out of the fermentation conditions and field efficacy evaluation; the antagonistic mechanisms against P. viticola in depth. The detailed results of the study are shown in followings.1. Screening and identification of biocontrol bacteria against P. viticola.303bacterial strains are isolated from the leaves of the nine grape growing areas in Liaoning province. SY286strain exhibits highest inhibitory activity against P. viticola through the test of control efficiency in vitro. The control efficiency is93.18%. In addition, this strain also shows high inhibitory activity against other pathogens such as Phytophthora capsici; Botrytis cinerea; Coniella diplodiella; Phytophthora infestans; Trichothecium roseurn; Botryosphaeria berengeriana and so on. Above all, this strain as biocontrol research has some potential applications.By physiological and biochemical tests of SY286strain, combined with molecular biology information to determine its taxonomic status. The strain is gram negative and rod-shaped. The biochemistry and biochemical features of SY286strain are as following:catalase reaction, oxidase reaction, amylohydrolysis test and nitrate reduction test are positive; gelatin liquefaction test, esculin hydrolysis test and indole test are negative. The16S rDNA sequence of SY286strain is1385bp. It is compared with known sequences from GenBank shows that its homology is99%with Ochrobactrum sp. Above all, SY286strain is identified as Ochrobactrum sp.2. The optimization of fermentation condition of SY286strainThrough single factor and orthogonal experiments, find the most suitable fermentation conditions for SY286strain to produce antibacterial substances. According to the antagonistic activity of zymotic fluid and its grown, the fermentation medium is modified and optimized medium is composed of glucose10g·L-1, beef extract7.5g·L-1, KNO35g·L-1, NaCl2.5g·L-1. The optimal fermentation parametes are90mL culture in250mL flask,5%inoculation volume,150r·min-1,27℃, pH7and shake culture for7h, which has the strongest antagonistic activity against P. viticola. After optimization of culture conditions, the ability of producing antibacterial substances of the SY286strain has significantly enhanced, inhibitory rate increased6.98%.3. The antibacterial mechanism and field efficacy of SY286strain The metabolite of SY286strain has strong inhibitory activity against P. viticola. The sporangium and sporangiophore of pathogen are wrinkled, wizened and fracture after dealing with extracts of metabolite from SY286strain. The bacteria can produce some inhibiting activity substances such as cellulase, proteinase and siderophores. In two years of field test, the fermentation solution of SY286strain has great control effect on grape downy mildew. Seven days after spraying the fermentation liquid, the control effect on grape downy mildew is up to90%, ten days can be above80%. It is slightly lower than52.5%Equation WG with2000-fold, but significantly higher than58%Metalaxyl-mancozeb with1000-fold. When the fermentation solution of SY286strain is diluted for400times sprayed7-10days, the control effect is quite to58%Metalaxyl-mancozeb1000times, and it is about70%. And most important, this strain is safe for grapevine.4. The analysis of crude antimicrobial protein from SY286strainThe inhibiting antibacterial substances of SY286strain are extracellular secondary metabolites. The activity protein salts out from saturation of60%ammonium sulfate precipitation have the strongest inhibitory activity to P. viticola. The antifungal rate is up to81.98%. Temperature, pH and ultraviolet have certain influence on activity of antimicrobial protein. The antimicrobial protein shows stability under condition of lower than50℃and pH4-10. Proteinase has a great impact on inhibiting activity substances. Proteinase K, trypsase, trypsase and nematolyt can suppress its activity.5. Cellulase gene cloning and expression of SY286strainThe primers are designed according to the known cellulase gene. The related gene segment of cellulose is amplified by PCR. The specific primers are designed according this related gene segment. The upstream and downstream sequence are amplified by Tail PCR, and the open reading frame of cellulase gene is obtained from jointing upstream sequence, downstream sequence and that related gene segment. The cellulase gene is named SY286gene. The open reading frame of SY286gene is1494bp. Initiation codon and termination codon are ATG and TAG respectively. The protein which codes by SY286gene contains497amino acids. The molecular weight of protein is about55.04kDa, and isoelectric point is8.12. Sodium dodecyl sulfate polyacrylamide gel electrophoresis is used for detecting whether the recombinant plasmid is expressed in E.coli BL21(DE3). The result shows that recombinant plasmid is expressed successfully in the condition of15℃,22℃,30℃,37℃under ITPG concentration is0.5mM. The result of scanning electron microscope shows that expression product can inhibit hyphae and sporangium of P. viticola. Their surface is smooth in control group. However, the phenomenon of fracture and wrinkle of P. viticola is appeared after being processed by expression product. The phenomenon is the same as the hyphae and sporangium are treated by crude metabolite and cellulase. |
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