| As everyone knows, China is a large potato producing country in the world, according to "China Statistical Yearbook" statistics in 2008, the potato planting area in China has reached more than 580 million hectares, and the cultivated area is the largest in the world. The potato late blight is caused by Phytophthora infestans (Mont.) de Bary, and it is one of the most serious diseases which has threated potato production.This disease has caused huge economic losses every year. At present, the control measures of potato Late Blight are mainly chemical control, physical control and variety improvement, but the long-term large-scale application of chemical pesticides has caused environmental pollution, pesticide residues, and also lead to drug-resistant strains and cross-resistance appear. So people are forced to look for new control measures constantly. Therefore, biological control method has become a research hotspot in recent years.Alternaria alternata is one of the antagonistic fungus strains which has a strong antagonistic effects on P. infestans. The fermentation conditions of A. alternata against P. infestans were investigated in this experiment based on former experiment results, and then antimicrobial substances in fermentation liquids were reserched in this experiment. The aim of this study is to provide a theoretical basis and practical basis for the application of the antimicrobial substances of A. alternata against potato late blight.First,in this experiment the fermentation conditions of A. alternata were optimized with a single factor design using a rubbing method or filter paper method in stationary culture status. The results showed that the fermentation liquid at temperature 28℃, the natural pH, seed age 8 d, broth content 80mL/250mL, inoculation amount 6% and darkness for culture 8d had the strongest inhibitory effect on P. infestans. And the inhibitory rate reache 88%, which increased by 18% in comparison with the condition that before optimization. It indicates that there is a great potential for using fermentation substances to control potato late blight.Second, on the base of the fermentation optimization, the physical and chemical properties of fermentation broth were researched. The results indicated that the antifungal rate against P. infestans was 71.4% under optimal fermentation broth concentration (0.95mg/mL), when the fermentation broth concentration was diluted to 0.48mg/mL, the antifungal rate to P. infestans still had 66.2%; the fermentation broth had good thermal stability, under 121℃high-pressure sterilization its inhibitory rate still had 80%; fermentation broth had a certain acid-base stability, but the antifungal activity was completely disappeared under the condition of strong acid (pH2); fermented liquid was not sensitive to ultraviolet light within 12h; fermentation broth had a good storage stability, it was suitable for long-term storage at low temperature (4℃) ; when the fermentation broth was treated with denaturant phenol-chloroform, its antimicrobial activity was almost lost completely.Finally, a preliminary study about the effective antimicrobial substances in fermentation liquid was carried on. After determined the anti-bacterial substance as mainly kinds of proteins by ammonium sulfate fractionation, then through ammonium sulfate precipitation, the best saturation of ammonium sulfate precipitation was identifed. And then it was carried on a further verification to prove the anti-bacterial substances that was mainly protein, usinga secondary sediment method and protease K, and denaturant phenol-chloroform. The antifungal proteins and fermentation broth basically had the same physicochemical properties .They had both good thermal stability andlight stability. The antifungal activity of the proteins was 68% when the antifungal proteins’concentrationwas 3.5mg/mL, compared with the fermentation broth, the antifungal activity decreased a little. The antifungal protein was the kinds of small molecule substance, and its molecular size was estimated to be about 41 kDa, according to SDS- PAGE. |
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