Screening, Identification Of Antagonistic Actinomyces Against Valsa Mali In Apple And Optimization Of The Fermentation Condition

Apple canker caused by Valsa mali Miyable et Yamada is an important fungal disease, Which discovered in 1916 in china. It has happened more and more seriously in south of Liaoning area in 1948-1949 and1960 and has already caused great economic losses. Along with the appearance of environmental pollution caused by chemical pesticide and 3R including Resistance, Residue, Resurgence, Utilizing natural antagonistic microorganisms is a potential safe tool for crop protection and offers a promising alternative. The most aim of this experiment is to isolate and screen the antagonistic actinomyces from the soil , Which collected from zhongshan city, guangdong province, In the test, we make further research on the identifing, optimizing of the fermention condition of the antagonistic actinomyces. The main content and results are as follows:1 In order to decrease the common actinomyces, The soil was treated by thermotherapy and chemotherapy method. In this study, Two hundred and ninty-three strains of actinomyces were isolated with dilution isolation method. The quantities of the soil unsettled, thermotherapy and chemotherapy treatment is hundred and fourteen, twenty-two, fifty-seven respectively. The experimental data indicates that the quantities of actinomyces isolated from treated soil decreased obviously, At the same time, The rare actinomyces may be increase.2 Using confronted culture method we had separated sixty-one actinomyces antagonistic to Valsa mali. In which inhibition rate of fourteen strains are above 50%, 4.78% of total. Using cylinder plate method we discovered one actinomyces of the strain Z-6 in the following selection , The results showed that this strain inhibited Valsa mali growing obviously, Inhibition zone account for 32.87 mm. In order to inditify the effection of the strain Z-6, We did experiment in further, In the mensuration of excised twigs Z-6 also showed evident effection. The inhibition rate is 32.9%.3 According to the cultural, morphological characteristics, such as spore and spore chains, as well as physiological, biochemical characteristics and inhibition spectrum test, The strain Z-6 cultured using filling-in bits culture was identified as the type of Aureus, belonging to the genus of Streptomyces. At the same, In the following test of antagonistic spectrum of the strain Z-6, The strain could not only inhibit the growth of the Valsa Mali, but also effect other sixteen plant pathogenic fungi, especially on Gaeumannomyces graminis, Botrytis cinerea, and Pyricularia grisea, Which Inhibition rate are abve 70%.4 The test on confronted culture showed that the pathogen grow slowly and the red substance appear around, It indicate the strain product or excrete some substance inhibite the pathogen growing. Through obserbation under the microscope we know that there were a lot of vesicles like prayer beads appearing in the apex and middle of the mycelia of both pathogens, dilate, deforaiedand formed empty vesicles. a series of diluted culture supernatant of the strain Z-6 from zero to one thousand time had been tested and the results showed that the hypha can hardly grow at zero time, one thousand time was completely lose inhibition effect, In conclusion, The inhibition mechanism is Producting anti-fungal substance to restrain the pathogen nutural growing.5 In order to increase the production of the fungicidal substance, the liquid media composition was optimized and the fermentation condition was primarily studied. The optimum fermentation medium of the strain Z-6 was composed of com powder 30.0g/L, soybean cake powder 27.7 g/L, glucose 35.5 g/L, starch 10.0 g/L, CaCO₃ 3.0 g/L, NH₄Cl 3.0g/L. The optimum fermentation condition was the initial pH of medium is 6.0, fermentation tempertarure 28°C, inoculum size is four (Φ5 mm), liquid volue 50 mL(in 250 mL flask), strain culture time 120 h, 210 r/min for 108 h.