Screening And Identification Of A Bacterium Against Phytophthora Sojae And The Preliminary Analysis Of Its Bioactive Substances

Phytophthora sojae causes destructive disease of soybean in the world. Oomycetes, including Phytophthora, are evolutionary very distant from fungi and fall within the kingdom Stramenopiles. Common fungicide applied in fungi can not be used for controlling the diseases caused by oomycetes. Screening Bacteriums against Phytophthora sojae will help researches to find new targets of chemical control and provide good ideas for designing new control strategies.Five hundred and twenty three isolates were collected from rhizosphere soil of growing soybean in three different area of Heilongjiang province. Fifty seven bacterial isolates, twenty two actinomycetes isolates and five fungi isolates showed antagonistic activities against P. sojae in vitro. The proportion of antagonistic isolates was16.1%in all collected isolates. One bacterial isolate showed the strongest antagonistic activities to P. sojae, and it was named as strain B048. Based on the analysis of morphological and16S rDNA gene sequences, it was identified as Bacillus pumilus.The isolate B048showed strongly inhibited the mycelial growth of P. sojae in vitro, the inhibitory rate was97.5%, and the width of clean zone still was wider than20.0mm until21days after coculture. In pot experiment, isolate B048strongly suppressed the Phytophthora blight of soybean, and the efficacy rate were100%at seven days after treatment. Isolate B048successfully colonized in the root of soybean, maintained the population density in the root of soybean up to5.70×105cfu·g-1(root weight) until60days after inoculation.The fermented filtrates of B048from optimum culture conditions were centrifuged for20min at8000rpm,4℃and then filtered through0.45μm filter membrane before analysis. After that, fermentation supernatant was precipitated with (NH4)2SO4solution at70%saturation degree. Aseptic crude extractions were dialyzed from precipitation solution, which still have antagonistic ability against Phytophthora sojae. The antagonistic substance stability with different treatments of temperature, pH, ultraviolet radiation, proteases were obtained by further study as follows:Inhibition diameter was89.3%control after crude antagonistic protein of B048was treated by100℃,1h,it means crude antagonistic protein can be resistant to high temperature, but lost the antagonistic activity completely after the treatment with121℃,20min. Crude antagonist protein of B048should be at neutral pH for antagonistic ability was the highest, regardless that the antagonistic protein has antagonistic activity at pH4to pH10, a wide range of pH. The antagonistic substance was not sensitive to Ultraviolet. Inhibition diameter was95.7%of control after exposed for3h with distance20cm under UV light which power was30w. The antagonistic substance was sensitive to proteinase E and proteinase K, whereas insensitive to Pepsin.