| Soybean blight caused by Phytophthora sojae is one of the most important diseases, bringing serious damage to soybean production every year. This dissert action focused on biological control of the disease. Based on theisolation, identification, antimicrobial spectrum test and the optimization of fermentation conditions of Bacillus pumilus strain BS-4, the inhibition activity and biocontrol effets of BS-4 and its metabolites against P. sojae were investigated. The main results were as follows:1. Isolation, identification and physiological and biochemical properties of antagonistic bacteria against P. sojaeIn the study, an antagonistic bacteria strain BS-4 against P. sojae strongly was isolated from the healthy soil of soybean field in Huaiyuan, Anhui. Extracted total DNA of BS-4 strain by CTAB method, the molecular identification of the strain was conducted by analyzing the 16 S r DNA sequence and testing the colony morphology, physiological and biochemical characteristics. The effects of different temperature, p H, incubation time and different carbon and nitrogen sources on the growth of BS-4 strain were also tested. The results showed that the 16 S r DNA fragments sequence of BS-4 strain was 1416 bp in length, which has a 89% homology with the Bacillus pumilus strain Y58(Accession No. EF203211.1) published in Gen Bank and the phylogenetic tree based on 16 S r DNA sequence and the physiological and biochemical properties showed that strain BS-4 belonged to Bacillus genus. In initial stage the colony of strain BS-4 was circular, yellowish, and translucent, with smooth surface on LB plate. After 36 h, the colony was changed to a dark yellow, and adiaphanous, with uneven surface and wrinkles and irregular margins. The growth of strain BS-4 was affected by temperature, p H, cultural time and different carbon and nitrogen source. The optimum growth temperature was 37℃ and it could grow in the p H range from 7 to 9 with the best p H of 7.0. The growth period of strain BS-4 was about 72 h, with growth curve of “S” type. Maltose and NH4 Cl were the best carbon and nitrogen source of strain BS-4, respectively.2. Test of antibacterial spectrum of strain BS-4The inhibition activity of BS-4 and its metabolite against 15 fungal pathogens and 8 Phytophthora pathogens were tested, respectively. It was concluded that there was a obvious difference between the inhibition of BS-4 to the different pathogens. The strain BS-4 has a strong inhibition to Sclerotinia sclerotiorum and Botrytis cinerea, with the inhibition rates aboved 70%. Magnaporthe oryzae, Fusarium avenaceum, F. culmorum, Gaeumannomyces graminis var. tritici, Bipolaris maydis and F. moniliforme were inhibited in a moderately level, with inhibition ratio between 41.67 and 50.89%. The inhibition of BS-4 against Colletotrichum gloeosporioides, F. nivale, F. oxysporum and Verticillium dahliae were mild and has hardly effect to F. solani and Rhizoctonia solani almostly. The antagonistic effects of strain BS-4 to the pathogens in Phytophthora were all more than 50% except P. capsic. The inhibition activities of metabolites produced by BS-4 strains to different pathogens were also different. S. sclerotiorum, B. cinerea, R.cerealis, G. graminis var. tritici, R. solani were all could not grow on the plate when the metabolite was presented and the inhibition activity to F. moniliforme and F. culmorum were reduced more than 50% compared with the confront culture. Significantly, there was no significant change between the metabolite and confront culture to Phytophthora pathogens.3. Optimization of fermentation conditions of strain BS-4The effects of different carbon and nitrogen sources and inorganic salts to the biological activity of production of BS-4 were tested, respectively. The result indicated that D-fructose, D-xylose, maltose, D-glucose, sucrose, D-sorbitol, D-mannitol and lactose could be used as carbon sources by BS-4, and maltose was the best carbon source for the antifungal production. The inhibition rate of the metabolite to P. sojae was 70.03% when maltose was used as carbon source. The inhibitory action of the fermenting liquor from the medium containing NH4 Cl has no significant differences compared with the medium containing tryptone, and the inhibitory rate were 86.14% and 85.76%, respectively. It is indicated that KH2PO4 was an important inorganic salt to the BS-4. Optimal proportion of the four nutrient media components was tested using L16(44)factors and levels of the orthogonal test, and the result showed that the optimal proportion of maltose, NH4 Cl, KH2PO4 and tryptone was 4:2:3:1, and the fermenting liquor has a spectacularly inhibition to P. sojae, which the mycelial growth was inhibited above 71.42%. The fermentation conditions were also tested, including the temperature, p H, inoculum volume, shaking speed and fermented time. The result displayed that the best antifungal activity of fermentation production was promising under the conditions of inoculation for 2% inoculum volume, fermentation for 72 h, sharking for 150 rpm and p H 7.0 at 30℃.4. Test of physiological and biochemical properties and inhibition effect of BS-4 metabolite on P. sojaeThe physiological and biochemical characteristics of the fermentation liquid of the strain BS-4 and the biological activity were tested. The results showed that EC50 value of the metabolites against P. sojae was 75.5μL/m L, and the metabolites displayed a higher antifungal activity in a range of p H 7.0 to p H 9.0 and excellent thermal stability at a temperature 121°C for 30 min. The metabolites was moderately resistant to hydrolysis by trypsin, proteinase K and pepsin, after which its relative activities were 66.35%、64.89% and 64.86%, respectively. The mycelial fresh weight and dry weight of P. sojae were reduced seriously, after being treated by the metabolites, while the germination of the zoospore and elongation of germ tube were also inhibited. In addition, the size and numbers of oospore were changed after treatment with the fermentation.5. Effects of BS-4 on the infection of P. sojae and the dynamics of enzyme activities related to the resistance insoybean plants treated by BS-4In order to clarify the effects of strain BS-4 and its metabolite against the zoospore and mycelium infected in vitro and in vivo, the infection of zoospores and mycelium in the leaves and soybean plant after treatment with BS-4 and its metabolite were investigated and the activities of PAL, POD and PPO which were related with the induced resistance were also tested, respectively. The results indicated that the zoospores could not infect the leaves which were made pretreatment and post-treatment with strain BS-4. The mycelium could not infect the pre-treated leaves also, but made a mild infection on the post-treated leaves, in which the soybean plants could resisted the infection of the mycelium after treatment with the strain BS-4, displaying only a small and non-expansible spot at the inoculation point. Meantime, the plants in control groups were infected and diedat 6d after inoculation with the mycelium or zoospores of P. sojae. The results suggested that the strain BS-4 could prevent and inhibit the infection of P. sojae on soybean plants.The experimental results also indicated that enzymes activities related to the resistance were changed, the activities of PAL, POD and PPO were raised and reached a peak after challenge vaccinated with P. sojae, then the enzymes activities were reduced, but there was a small increase at 6 d after being inoculated. It was also found that the PAL and POD activities in plants treated with BS-4 alone were significantly increased and lasted for 4 d, while PPO activity was not increased significantly. These results indicated that the induced resistance was one of the mechanisms of strain BS-4 to control P. sojae.6. The colonization of biocontrol strain BS-4 in soybean plant and effect on the microbial community in soilThe colonization of strain BS-4 in soybean leaves and rhizosphere and the influences on soil microbial population were investigated. The results show that the strain BS-4 could colonized in soybean leaves in a shorter period, which was only be maintained for 11 d; at 7 d after inoculation, the number of colonies isolated on the leaves was only 8.0 ′ 105 cfu / g and at 11 dafter being inoculated, strain BS-4 almost could not be detected. But the strain BS-4 has good ability to survive in the soil. At 1d after being inoculated, the number of colonies appeared a peak, then gradually reduced, at 5d, after being inoculated, the number of colony was reached to astable level which numbers was maintained in 107 orders of magnitude. The fungal and bacterial colonies in soil were affected by strain BS-4. The number of fungi in the early stage of inoculation was reduced, but it was increased at10 dafter being inoculated. Meanwhile, at the beginning of inoculation, the number of bacteria has a sharp rise, but in the late inoculation it tended to be stable. In addition, the colonization of strain BS-4 had a mild effect on the actinomycetes which numbers of colonies did not appear significant change. |
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