Identification Of Actinomycetes PY-1 Against Plasmopara Viticola And Structure Determination Of Antimicrobial Substances

Grapevine downy mildew, caused by pathogen Plasmopara viticola (Berk. & Curtis) Berl.& de Toni, is a serious fungal disease for viticulture. It is considered to be the most destructive and widely distributed disease and a major cause of crop loss in grape production system. Nowdays, application of chemical fungicides are the major measure for grapevine downy mildew control. However, a large number, long-term and single continuous use of chemical fungicides could cause 3R problems. Biocontrol for P.viticola is attracted a wide spread attention for environmentally friendly, safety to people, animals and sustainable control effect. In this paper, some actinomycetes with inhibitory activity on P. viticola are screened from soil, and its inhibition mechanism would be researched. The study would provide technique support for biocontrol agent development and application.1. Isolation and identification of actinomycetes against P. viticola. Fifty-three rhizosphere soil samples were collected from Liaoning, Yunnan and Tibet vineyard. And total of 151 actinomycetes were isolated. By using Plasmopara capsici as indicator fungi for the first screening, and P. viticola as indicator fungi for the rescreening,28 actinomycetes were founded which could both against P. capsici and P. viticola. Among these strains, strain PY-1 showed strong inhibition activity against P. viticola, which control efficiency was 91.11%. Antagonistic strain PY-1 was identified by morphological feature, chemical classification and molecular identification. Strain PY-1 was identified as Streptomyces atratus. Inhibitory activity test in vitro showed that PY-1 strains showed varying degrees of inhibition against many pathogenic fungis, such as Botrytis cinerea, Bipolaris sorokiniana^ Phytophthora infestans?Fusarium oxysporum?Colletotrichum orbiculare?Trichothecium roseurn?Rhizoctonia solani?Curvularia caryopsida and Fusarium graminearum.2. The optimization of the best fermentation condition of PY-1 strain. The fermentation mediums and fermentation conditions of strain PY-1 were optimized by single factor experiment and orthogonal analysis, respectively. The optimized mediums component for this biological control agent was 5% corn flour,0.5% glucose,0.5% peptone,0.5% NH4Cl and 0.05% NaCl. The optimal fermentation parametes are liquid medium volume 90 mL/250mL, inoculation volum 5%, initial pH7.0, temperature 28?, rotated speed of shaker 180r/min and culture time 5 days, which has the strogest antagonistic activity against P.viticola. After optimization, the ability of producing antagonistic compounds of PY-1 strain had been significantly enhanced, and the inhibitory rate increased 8.35%.3. The antibiotic substance was extracellular secondary products of PY-1 strain. The active antagonistic compounds fermented by PY-1 strain were stable when the temperature is below 65?, exposure to 40W fluorescent lamp, pH value 6-10 and proteinase K. After exposure to sunlight for 72h, the antagonistic activity began to decline, and strong acids or alkali ferment filtrate also affected antagonistic activity. There was no significant change in antagonistic activity after being incubrated at 4? for 6 months. But at room temperature, the antagonistic activity began to decline after 4 months; Metal ions was significantly affected the antagonistic activity.4. The sporangium and sporangiophore of P. viticola are wrinkled, wizened and fracture after dealing with extracts of PY-1 strain metabolite. PY-1 strain metabolite included chitinase, protease, siderophore, ACC deaminase, HCN, IAA, exclud cellulase. These metabolites were related to the biological control mechanism of competitivet, growth-promotingt and induced resistance. In two years of field experiment, the fermentation solution of PY-1 strain has great control effect on grape downy mildow. The metaphase control effect on grapevine downy mildew is up to 90%, and the end control effect could be above 86%. It is slightly lower than 52.5% Equation-pro WG with 2000-fold, but significantly higher than 58% Metalaxyl-mancozeb with 1000-fold. When the fermentation solution of PY-1 strain is diluted for 700-fold, the control effect is equal to 58% Metalaxyl-mancozeb 1000-fold. And the most important is that PY-1 strain is safe for grapevine.5. The antibiotic substance were purification and structure identification of PY-1 strain. The crude extract of PY-1 metabolite was further purified by dichloromethane, silica gel column chromatography, Sephadex LH-20, thin-layer chromatography and HPLC methods. Tow antagonistic pure compounds PY1-7-1 and PY1-7-2 were isolated. The biocontrol effect of the different diluted concentrations (10-2 mg·mL-1,10-4 mg·mL-1,10-6 mg·mL-1) of PY1-7-1 and PY1-7-2 on grapevine downy mildew were 92.59%,86.30%?64.81% and 97.04%?91.85%?84.07%. The structure of antagonistic compounds was identified by ESI-MS,1H NMR,13C NMR and spectra analysis. PY1-7-1 and PY1-7-2 were identified as 5-Acetoxycycloheximide and Cycloheximide, respectively.