| Potato late blight caused by Phytophthora infestans ( Mont. ) de Bary is one of the most important diseases restricting potato production in the potato-cultivated areas all over the world. In this study, we found that Bacillus subtilis strain EB-28 had potential as a biocontrol agent in the control of potato late blight.B. subtilis strain EB-28 was an endophytic bacterial strain isolated from tomato stem and showed strong antagonism against Botrytis cinerea Pers. In this study, EB-28 was found to express antibiosis activities against P. infestans in vitro. The inhibitory activity of the bacterial suspension and the filtrate of B. subtilis EB-28 against P. infestans were tested at different stages (the release of zoospores and the germination of sporangia), in vitro. The results showed that the filtrate of strain EB-28 strongly inhibited the release of zoospores from sporangia (inhibition rate: 91.26%). But the filtrate of strain EB-28 almost had no effect on the inhibitory activity of the germination of sporangia.The growth of EB-28 and the inhibition activity of EB-28 filtrate on zoospore release of P. infestans were evaluated under different cultural conditions. In rye broth, cultured under 30℃, 180 rpm, in 48 h, the bacterium number could reach 108 cfu/mL, and the inhibition activity of EB-28 filtrate on zoospore release of P. infestans got the highest(90.30%). The results showed that the optimum culture condition was under 30℃and at pH 7 for 48 h.Control effects on potato late blight(PLB) of the bacterial suspension and the filtrate of strain EB-28 were tested on detached-leaf trials and potted plant trials. The results showed that control efficacy of the bacterial suspension on potted plant trials could be 74.22%, which was significant higher than that on detached-leaf trials (55.68%). While control efficacy of the filtrate of strain EB-28 were 61.80% on potted plant trials and 60.54% on detached-leaf trials.The survival ability of B. subtilis EB-28 on potato leaves were investigated. In 3 days after inoculation, the number of bacterium could reach up to 108 cfu/g in fresh leaf, and after 15 days, the number of bacterium was maintained 103 cfu/g in fresh leaf. As the change trend of the number of bacterium, the control effects on potato late blight(PLB) of the bacterium expressed same trend.The filtrated fermentation liquid of strain EB-28 was precipitated in grades with the ammonium sulfate to draw the antifungal proteins, and characters of the crude protein obtained with ammonium sulfate at 30%-80% saturation were studied. The antifungal activity of the crude protein extracts at 40℃treated 20 min was no significant difference with control, and decreased at 60, 80 and 100℃, but still showed certain antifungal activity after heat treatment at 100℃for 20 min. The inhibitory activity of the crude protein extracts was variable in the range of pH value from 5 to 11, and lost full antifungal activity when pH value was 4. The crude protein extracts kept almost full antifungal activity after treated with trypsin, pepsin, protease k, some organic solvents and ultraviolet. Results of SDS-PAGE showed that B. subtilis EB-28 secreted abundance of proteins.The crude protein extracts were eluted through the DEAE52 cellulose column with stepwise elution and six peaks were found, only the fourth fraction (fraction IV) had antifungal activity. After the fraction IV were pooled and eluted in the Sephadex G-75 column, only one peak was obtained. And the only peaks showed only one protein band in the SDS-PAGE,with 24.3 kDa molecular weight compared with standard proteins.
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