Antagonize The Breeding Of Actinomycetes Of Zhanhua Winter Jujube Black Spot Disease

Winter jujube is a late-maturing and fresh-eating variety of Ziziphus jujube,of which Zhanhua Winter jujube is an excellent representative.With a thin skin,thick and brittleness meat,Zhanhua Winter jujube is benefical to the health of cardiovascular.However,as a kind of widely distributed diseases all over the world,Black Spot has a huge influence upon the quality of Winter jujube;its provence and control needs to be paid attention during the process of plants cultivation,fruit growth and maturity.Nowadays,the prevention and control of Zhanhua Winter Jujube Black Spot mainly depends on chemical pesticides,which has a great impact on the environment,may enhance the drug resistance of pathogenic bacteria and is harmful to human health.However,biological pesticide is safety,has the small ecological environment pollution,and has a wide raw material sources.Besides it does not easily develop drug resistance of pathogenic bacteria.Therefore it's an urgent problem to develop the biological pesticides.This study is aimed at screening an antagonistic actinomycetes of Winter Jujube Black Spot from the soil of Zhanhua Winter Jujube cultivated area,thus providing the new strain and accumulated theoretical basis for exploitation biological control of Winter Jujube Black Spot and bio-pesticide development.The methods and results of reasearch turned out as follow:1.The initial and secondary screenings were determined by agar block method and Oxford cup method respectively.And then 2 strains of actinomycetes were obtained which have inhibition effect against black spot.According to the results of morphological observation,physiological and biochemical identification,and 16 S rDNA sequence analysis,N19 was identified as Streptomyces moderatus with an inhibitory cycle of 13.50 mm in diameter and the GenBank accession number as KX149095;and F5 was identified as Streptomyces variabilis with an inhibitory cycle of 13 mm in diameter mm and the GenBank accession number KX149094.2.The original strain of N19 was treated by twice UV mutagenesis,the best dose for UV was the concentration of 105/mL of the bacteria liquid exposed to 20 W UV lamp for 150 s and120 s that 30 cm away from super clean bench.The results showed that strain UV23 was obtained with an inhibitory cycle of 13.50 mm,which increased by 22% than the original strain.3.Then fermentation conditions of the strain UV23 was optimized through the response surface methodology.Box-Behnken experimental design and Design Expert 8.0.6 software were adopted to analyzed data,then we got the regression model equation and ascertained the best combination of ingredients and conditions: A:0.428;B:0.405;C:-0.719;D:0.194;E:-0.233;F:0.227,that is: 24.28 g/L of corn meal;24.05 g/L of soybean meal;0.36 g/L of KH2PO4;thevolume of liquid is 147.76 mL/500 mL;pH is 7.12;inoculation amount is 445.40 ?L/100 m L.The significance discriminate analysis showed that carbon source,nitrogen source,the volume of liquid and inoculation amount are the factors that have a significant impact on the antibacterial activity of strain UV23.Under the optimal cultivate conditions,the diameter of inhibition zone of strain UV23 is 20.50 mm,which has been increased 24.47% than the original one.4.At last,this study researched the stability of the supernatant of fermentation broth by transparent circle method.The result showed that the bacteriostatic efficacy of fermentation broth was stable when pH was at 4~8,but in the strong acid and alkaline system,it would be volatile.When the temperature was at 4?,the bacteriostatic efficacy of fermentation broth basically remained the same during a week;however,when it was at-20?,it would remained the same at the first 3 days and then decreased gradually.The fermentation broth has the light stability,thermal instability and ultraviolet irradiation stability.According to the research of polarity of supernatant of fermentation broth,we could concluded that this antibacterial substance can't solube in nopolar organic matter and it is yellowish-orange strong polar material.