| In the agricultural production, cultivated crops are always influenced by plant diseases, caused by plant pathogens, which result in a great loss in production and economy. In the past, plant diseases were often controlled by chemical agents and pesticides, which caused environmental pollution and drug resistance of plant pathogens. For that reason, environment-friendly methods such as biological control were developed. Bacillus subtilis a ubiquitous saprophytic bacterium. It has been used as biological control strains in recently years for their endogenous spores and displayed a well stress resistance and broad-spectrum antimicrobial activity. However, environmental changes, such as temperature, humidity, pH, etc., affect the functional stability. So the effects of biological control turned to be instable. Especially in the cold region, the effect of biological control was weakened because of microbial control agents cannot colonization normally.In the previous study, a biological control strain BS5 with a suppressive effect against several phytopathogens fungi was isolated. A few aspects of research was carryed out with this strain.1.UV induction was used to breed a biological control strain which fit for low temperature named BS303, the exponential phase slope of BS5 and BS303 was calculated from the growth curve in every temperature, which showed that, the slope of BS303 was 1.6 times higher than that of BS5 in 20℃. The growth curve of BS303, which after 40 transfers on media in normal temperature, was tested in the low temperature and showed no significant differences in the growth condition.2.The activity of biological control of BS303 was tested by combined use of plate competing, bacteria-fungi interaction and greenhouse pot. In the test of plate competing, BS303 has a high activity of biological control in 20℃, for the four in five pathogenic fungi, showed a significant activity of biological control than that of BS5. Effect of biological control in normal temperature showed a few difference, there is no significance than that of biological control effect in the growth of low temperature, biological control effect of mutants grown in normal temperature showed no decrease as a whole. For the protect index of Fusarium oxysporum f. sp. Melonis, BS303 was 0.701 and BS5 was 0.667 at 28℃, there was less difference, while in 20℃, BS303 reached 0.767 compare with 0.557 of BS5, in the green house pot.3.In this study, a genetic engineering plasmid with green fluorescent protein (GFP) and antibiotics as label, was constructed in order to detect establishment ability and behaviors in the soil incubate. To insure the gene of GFP could express well, two promoters (from Bacillus cereus UW85 and Bacillus subtilis 168) was used, The efficiency of promoter was determined by the strength of fluorescence. The data of fluorescence strength showed there was little difference between the two promoters.4.Non strile soil and Citrullus lanatus was used for soil incubation to reflect the effect of biological control. Spare plate, soil fluorescence and RTm-PCR was used for reflecting the colonization trend of bacteria and fungis in soil. The result showed that, strains of biological control could well established in soil, and mutant strain was better than original strain, the phytopathogens fungi was obviously decreased when the biological control bacteria was added. Meanwhile, effect of biological control was better in mutant strain than that of original strain, which affected by Citrullus lanatus.By the four field studies described above, a biological control strain fit for the low temperature was bred. Effect of biological control in normal temperature and low temperature showed well, and the colonization dynamics in farmland was detected, which lay the foundation for follow-up apply. |
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