Screening And Identification Of Biocontrol Agents Against Phytophthora Melonis And Preliminary Analysis Of Its Antimicrobial Substance

Cucumber phytophthora blight caused by Phytophthora melonis is an important soil-borne disease in the production of cucumber, which cause the growth, yield and quality decline of cucumber in production.Without effective chemical control methods and ecological safety problems are inevitable. Screening, using of beneficial microorganisms and their metabolic products to control Cucumber phytophthora blight is effective measures to implement the green plant protection. For screening effective antagonistic agents to Phytophthora melonis, some strains were isolated from collected soil with gradient dilution method and 2 strains showed obvious antagonistic effects to Phytophthora melonis, furthermore, To take the cultural characters, physio-biochemical tests and molecular biological identification. Then optimization of fermentation conditions and preliminary analysis antimicrobial active substances for 1 strain. The main research and results are as follows:(1) For screening effective antagonistic agents to Phytophthora melonis,269 strains were isolated from collected soil in Hunan and Beijing with gradient dilution method.7 strains showed obvious antagonistic effects to Phytophthora melonis by using agar plate dual culture, and strain X54 and strain P3 were finally screened out because their diluted 5 times filtration both showed the inhibition rate of up to 70%. In addition, the stain X54 showed different inhibition activity against many kinds of pathogenic such as Colletotrichum capsici, Phytophthora parasitica var. nicotianae, Rhizoctonia solani, Gibberella zeae, Colletotrichum gloeosporioides, Fusarium oxysporum and so on.(2) Research on X54 and P3 strains cultural characters, physio-biochemical tests, PCR from genome DNA amplification of 16SrRNA sequences and combined with bioinformatics methods of analysis. It pointed out that strain X54 was Streptomyces virginiae, strain P3 was Bacillus licheniformis. The accessions No. of them in GenBank were KM078926 and KJ872538.(3) The optimization of fermentation conditions of antagonistic strain X54 were studied, the optimal medium carbon and nitrogen were determined first:maltose 20.0g/L, peptone 3.0g/L.Then through the one variable method and response surface experiments indicated that the best seed age was 37h, fermentation temperature 28℃, rotational speed on 230r/min, 8%inoculation amount, broth content 50ml, optimization pH7.0 and fermentation times 5d.(4)The stability, secretory type, grade-precipitation with ammonium sulfate and organic solvent extraction of antagonistic substance produced by X54 were studied. The results showed that active substances with high genetic stability, light stability, and high temperature, it may be non-protein substances. Poor stability in the pH environment, and in the compound purification of late to maintain the pH of 7.0. It is not sensitive to ultraviolet light, the inhibition is relatively stable. Through the substance secretion, antibacterial substances belong to the extracellular secretion. The result of grade-precipitation with ammonium sulfate indicated that the active substance could be non-protein substances. By extraction of different polar organic reagents, we found that it can be soluble in polar methanol and insoluble in chloroform. According to the principle of "similarity and intermiscibility", we can infer the antibacterial substances have certain polarity.