Screening Actinomycetes Anti Phytophthora And Studying Its Bioactive Substance

In recent years, biological control using antagonistic microorganisms has gradually become a major trend on plant disease control. Actinomycetes, a class of microorganisms which can produce the most of active antibiotics substances, has great potential and broad prospects for plant disease control. In order to find effective antagonistic actinomycetes strain for biological control of phytophthora pathogens, we isolated sactinomycetes strain L24 from a tobacco,tfield in Liuyang of Hunan and carried a series research based on it. The main results are as follow:1. Isolation and Screening of actinomycetes:Antagonistic actinomycetes strains L24 was isolated by dilution separation from soil samples collected from a Tobacco fields in Liuyang of Hunan, and it showed significant antifungal activity to Phytophthora according to the confront culture in the first round of screening and inhibition test of fermentation filtrate in vitro.5 times dilution fermentation filtrates showed significant antagonistic effect against the Phytophthora, for example the inhibition rate of mycelium reached 97.3%,86.5%,95.4% and 100% respectively, against theP.melonis, P.capsici, P.nicotianae and P. infestans.2. Identification of Actinomycetes strain L24:According to morphological, cultural characteristics, physiological and biochemical characteristics and 16S rDNA analyze results, actinomycetes strain L24 was phylogenetic classified and identified as Streptomyces humidus preliminarily3. Optimization fermentation medium of Actinomycetes strain L24:The fermentation conditions regarding the carbon source species, nitrogen source species and C/N ratio were optimized using the unit factor variable method on the basis of the Gauserime agar medium. The optimal formula was as follows:rice flour 40g/L, yeast flour 1.35g/L, K2HPO40.5 g/L, NaCl 0.5 g/L, KNO31 g/L, MgSO4·7H2O 0.5 g/L,FeSO40.01 g/L.4. The stability of the fermentation broth was studied under different condition as temperature, pH, ultraviolet. The results showed that the fermentation broth was stable under the condition of acidic, UV and temperature and was unstable under alkaline conditions.5. Separation and collection of antagonistic substances:The antagonistic substances of L24 were confirmed to be primarily included within the cells when treated with ultrasonic and methanol. In addition, we have collected 400 ml concentrated liquid.6. The polarity of antagonistic substances:Four organic reagents, petroleum ether, chloroform, ethyl acetate and n-butanol were selected according to their increasing polarity to determine the polarity of the fermentation broth. The results showed that n-butanol is suitable for extraction of antagonistic substances which means it is high polarity substances.7. Biological control test of L24 strain cucumber phytophthora blight:the control efficiency of the fermentation filtrates to cucumber blight was studied by in-vivo and in vitro with no wound inoculation method by mycelium blocks. The inhibition ratio was 37.5% and 22.2% respectively. It revealed that antagonistic substances produced by L24 could inhibit phytophthora invasion and disease progression.8. Preliminary study of the function mechanism of active ingredients produced by actinomycetes strain L24 on Phytophthora:Compared with control the mycelium morphological changes were observed on P. melonis treated with fermentation filtrates, including hyphae fracture and deformity.