The Screening And Identification Of A Antagonistic Bacteria Against Botrytis Cinerea

The strawberry gray mould that caused by botrytis cinerea is one of the most devastating diseases occurred in most strawberry production areas worldwide. The low temperature and high humidity always cause heavier infection with the strawberry yield and quality being decreased. The pathogen mainly infected the strawberry fruits by forming a gray mould layer, then make the leaves appear tawny disease spots. In the actual production, procymidone, carbendazim and diethofencarb are the commonly used chemical agents to control gray mould. However, the long term usage of chemicals could result in such problems as pollution of chemical fertilizer and drug resistant, and it is difficult to prevent the chemical resistant mutant effectively. So it is urgent to look for a new way to prevent and, control the strawberry mould. The biological control technology is an ideal substitute for chemical pesticide to control the strawberry grey mould for its eco-friendly, lower pesticide residue and hard to develop resistance. Screening and application of antagonistic bacteria against plant pathogens has become a subject of general interest. Therefore this study firstly screened and isolated an effectively antagonistic bacterium from strawberry field, then carried out the morphologic observation, physiological determination and 16S rDNA sequence analysis of the antagonistic bacterium, finally we optimize the components of its culture medium and condition. The main research results are as follows:1) The obtaining of a bacterium antagonize botrytis cinereaA total of 426 bacteria were isolated from soil, strawberry stem and the fruit surface by means of dilution plate count, in which 100 strains were from soil,126 from the stem surface and 200 from the fruit surface. The plate confrontation method was carried out to detect the antagonistic bacterium. The results showed that four bacteria named ZY13, ZY34, ZY86, ZY98 had the antagonism effect to gray mould. Among them, the ZY86 isolated from soil, not only had the best anti-effect to botrytis cinerea, but also inhibit a variety of pathogenic fungi such as rhizoctonia solani, magnaporthe oryzae, colletotrichum capsici, phytophythora capsici and sclerotinia scleotiorum, can be regarded as a new microbiological resource for development of biopesticide.2) The identification of the antagonism strain of ZY86The gram-negative ZY86 was in a rod shape, no spores and no flagellum, which can grow rapidly on PDA medium with the colonies of round, swell and white. It grew slowly on GSYH medium with the colonial morphology as follows:irregular, sticky, swell, milk white, smooth in surface and opaque. The bacterial colony is opaque, smooth in surface as well as neat in edge when grew on PDA and NA medium. The results of physiological and biochemical tests for ZY86 were as follows:oxidase negative, catalase positive, citrate utilization positive, methyl red test negative. The amylolysis, gelatin liquefaction, nitrate reduction, V-P and urease test were all negative. Most of the physiological and biochemical indexes were the same between ZY86 and the type strain ATCC17909, and a few differences indicated the different sources of the two strains. After the PCR amplification of the 16S rDNA on ZY86 genome, a 1431bp DNA fragmentation was obtained. The sequencing result showed that the ZY86 belonged to the acinetobacter sp., with the closest relatives to the acinetobacter johnsonii (ATCC 17909). All the results above indicated the ZY86 belonged to acinetobacter johnsonii.3) The optimization of fermented medium for ZY86The fermentation test was carried out by single-factor method and orthogonal method with 7 kinds of carbon sources,5 kinds of nitrogen sources and 5 kinds of inorganic salts. The basic medium composition were as follows:peptone 1.0%, yeast extract powder 1.5%, NaCl 1.0%, KH2PO40.15%,MgSO4.7H2O 0.15%. According to the activity of antagonism, the carbon sources were arranged as follows:dextrin> soluble starch> maltose> saccharose> glucose> corn flour; the nitrogen sources were arranged as follows: (NH4)2SO4>peptone> NH4Cl>urea> soybean meal; the inorganic salts were arranged as follows:MgSO4>CaCl2>MnSO4>GuSO4>FeSO4. The orthogonal test results showed that based on the basal medium, the dosage of 0.8% dextrin,0.8%(NH4)2SO4 and 0.15%MgSO4 could reach to an optimum fermentation effect, the maximum inhibitory rates of the fermentation products on botrytis cinerea was 94.03%.In conclusion, the antagonistic bacterium strain ZY86 can be developed as the biopesticide for its wide antibiotic spectrum and effectively antagonism capacity.