| Oilseed rape(Brassica napus L.) is one of the major oil crops in China. Sclerotinia stem rot of oilseed rape, caused by Sclerotinia sclerotiorum(Lib.) de Bary, is one of the most important diseases in the regions of rapeseed production in the world. Sclerotinia stem rot causes great economic losses in China, especially in the rapeseed production regions of the Yangtze River. The annual incidence rate of sclerotinia stem rot in this region was estimated at 15-30% on average and up to 80% in serious disease epidemic year,causing serious quality and yield reduction. Disease management through application of chemical fungicides and crop rotation for controlling sclerotinia disease have been applied in different countries for many years. However, consecutive applications of chemical fungicides not only increase production costs and lead to the adverse effects on environment, but also bring the risk of pathogens to develop resistance to chemicals.Therefore, highly efficient and environmentally friendly alternative methods to control sclerotinia stem rot of oilseed rape are desirable. Application of beneficial microbial agents,including antagonistic bacteria, fungi, actinomyces and viruses, to control plant pathogens has been considered as an eco-friendly and cost-effective component of an integrated management measures. Based on the isolation and identification of the beneficial microbial strain RSS-1, the biocontrol effects and mechanisms of Bacillus subtilis strain RSS-1 were investigated. At the same time, colonization of RSS-1 population on rapeseed leaves and in rhizospheric soil and its effects on other microorganisms in rhizospheric soil of rapeseed were studied. Preliminary purification of antifungal activity proteins and their physical and chemical properties, and antifungal spectrum of B. subtilis strain RSS-1 were studied systematically in this dissertation. The main results were as follows.1 Screening of antagonistic bacteria and their biocontrol effects on S.sclerotiorum Two-hundred and eleven bacterial strains, isolated from the rhizospheric soil of rapeseed plants, were screened for antimicrobial activitity to S. sclerotiorum using dual-culture assay. The results showed that 8 isolates, designated RSS-1, RSS-10, RSS-14,RSS-32, RSS-40, RSS-43, RSS-46 and RSS-55, exhibited antifungal activity and the strongest antimicrobial activity was exhibited by strain RSS-1 against S. sclerotiorum,showing 93.33% inhibition. Both cell suspension and cell-free filtrate treatment of RSS-1suppressed sclerotial production by 97-100%. In greenhouse experiments, all three concentrations, 1×106, 1×107 and 1×108 cfu/m L of cell suspension or cell-free culture filtrate treatment significantly reduced(P<0.05) sclerotinia stem rot severity of oilseed rape. The most effective concentration of RSS-1 was identified as 1×108 cfu/m L, which can reduce the severity by 91.1%. Both two time intervals, pretreatment with RSS-1 or treatment with RSS-1 post inoculation at 24 h, significantly reduced(P<0.05) the sclerotinia stem rot severity of rapeseed, compared with the control. B. subtilis RSS-1 was more effective in reducing disease severity when applied at 24 h before plant inoculation with S. sclerotiorum than at 24 h post plant inoculation with S. sclerotiorum.2 Identification of biocontrol bacterial strain RSS-1Bacterial stain RSS-1 was tested for various phenotypic properties including morphology, physiological and biochemical characteristics, according to the standard procedures, and confirmed by 16 S r DNA sequence using BLAST(Basic Local Alignment Search Tool). The results indicated that the strain RSS-1 was a Gram-positive, rod shaped,endo-spore forming bacteria with positive reaction for catalase activity and negative for oxidase activity. It produces amylase, hydrogen sulfide(H2S) and hydrolyzes gelatin and its growth temperature can be 10 to 45°C. Positive for V-P reaction, nitrate reduction and citrate utilization but negative for indole test and tyrosine hydrolysis. All these phenotypic characteristics are uniform with members of the genus Bacillus. In addition to the phenotypic identification, the BLAST analysis of 16 S r DNA sequence(Gen Bank accession No.: KR086418) revealed significant identity(100%) with four B. subtilis strains(Gen Bank accession No.: EU723210, KC460988, KF636527 and GU258545).Furthermore, phylogenetic tree constructed with 16 S r DNA sequences of other members of the genus Bacillus indicated that strain RSS-1 belongs to B. subtilis.3 Studies on the antagonistic mechanisms of B. subtilis strain RSS-1against S. sclerotiorum The experiments demonstrated that the B. subtilis strain RSS-1 could significantly inhibit the mycelial growth of S. sclerotiorum. Strain RSS-1 induced alterations on hyphae morphology of S. sclerotiorum, such as distortion, cytoplasm exosmosis and deformation,and inhibited sclerotial production. The activities of PPO, POD and SOD in tested rapeseed plants inoculated with S. sclerotiorum were tested and analyzed respectively in 6days. The results indicated that PPO, POD and SOD enzymes activities in leaves rose obviously compared to control, suggesting the B. subtilis strain RSS-1 could induce the rising of the activities of the 3 enzymes in the rapeseed plants. Therefore, PPO, POD and SOD all were related to the disease-resistance enzymes on oil rapeseed. Cellulose,polygalacturonases(PGs) and polymethylgalacturonases(PMG) activities were measured during the course of infection. Cellulose, PGs and PMG activities had not significant difference(P<0.01) among different treatments within 6 hours post-inoculation(hpi). The3 enzymes activity increased promptly from 12 to 96 hpi in untreated group. In contrast,although cellulose, PGs and PMG activities increased in RSS-1 treated group, the accumulation rates of the 3 enzymes activity were slower than untreated group. The results showed that bacterial strain RSS-1 significantly suppressed(P<0.01) the PGs and PMG activities during the course of infection. Oxalic acid accumulation was also detected during the course of infection. Significant difference(P<0.05) of oxalic acid accumulation among different treatments were detected at 12 hpi. In untreated group, the concentration of oxalate increased from 9.38 mg/g FW at 12 hpi to 12.03 mg/g FW at 96 hpi. However, in treated group, the concentration of oxalate increased from 8.41 mg/g FW at 12 hpi to 9.43mg/g FW at 96 hpi. The results indicated that B. subtilis strain RSS-1 significantly inhibited(P<0.05) the accumulation of oxalic acid during S. sclerotiorum infection.4 Investigation of the colonization of B. subtilis strain RSS-1 on rapeseed leaves and in rhizospheric soil and its influence on the microbial diversity around rapeseed plants Double-resistant mutant(streptomycin and rifampicin) of B. subtilis strain RSS-1 was tested for ability to survive on rapeseed leaves and in rhizospheric soil under greenhouse conditions. The B. subtilis strain RSS-1 population on rapeseed leaves significantly decreased over time under greenhouse conditions. During the first 6 days, the RSS-1population size significantly(P<0.05) decreased, and was reduced by 3.45 log units at day6. The RSS-1 was not detected on rapeseed leaves at 10 days after being applied on leaves.Whereas, the RSS-1 population size significantly(P<0.05) increased in rhizospheric soil during the first 4 days after being bacterized on leaves. At day 4, the population size in rhizospheric soil had no significant(P<0.05) changes until day 10. Then, the population size in rhizospheric soil was declined at day 10, and was reduced by 2.00 log units at day20, and then maintained no significant(P<0.05) variation until day 30. Dynamic of strain RSS-1 population in rhizospheric soil with the treatment by irrigating root was also tested.The RSS-1 population size significantly(P<0.05) increased in rhizospheric soil during the first 4 days after being bacterized in rhizospheric soil. At day 4, the RSS-1 population size in rhizospheric soil was increased by 0.75 log units, and then leveled off until day 30. Through traditional training methods, we analysed the quantity changes for bacteria, fungi and actinomycetes on rapeseed plants and in soil samples by isolating and counting the plants and rhizosphere microbial populations. The resuits showed that in 10 days after application of the B. subtilis strain RSS-1, the number of bacteria showed no significant change. However, the number of bacteria was obviously higher than the control group treated by sterile water when 20 days after application of biocontrol strain RSS-1. The number of fungi and actinomyces showed no significant change compared to the control group with different treatments.5 Optimization of the fermentation conditions for production of antibiotic substances by B. subtilis strain RSS-1The optimal nitrogen source, carbon source and salt for the production of antifungal substances by B. subtilis strain RSS-1 were tested. The medium composition and fermentation conditions were optimized by single factor and orthogonal experimental designs. The results showed that the optimal carbon source, nitrogen source and salt were cornflour, tryptone and Mg SO4·7H2O, respectively. The composition of the medium was2.0%(W/V) cornflour, 1.5%(W/V) tryptone and 0.05%(W/V) Mg SO4·7H2O. The optimal fermentation conditions were the combination of initial p H 6.0, 0.1% inoculum volume percentage and shaking flask volume at 25 m L/100 m L, 180 rpm for 5 d under 28 °C. The inhibition activity of B. subtilis strain RSS-1 cell-free culture filtrate had a significant rise after fermentation condition optimization.6 Test of antifungal spectrum of B. subtilis strain RSS-1The antifungal spectrum of B. subtilis strain RSS-1 was tested using the method of agar-plate dual culture. The results showed that strain RSS-1 could inhibit the mycelial growth of 41 isolates among 17 genera of plant pathogens. The inhibitory effect of strain RSS-1 against Botrytis genera were extremely strong, with the inhibitory effects of 90-96%.The B. subtilis strain RSS-1 could also strongly inhibit the mycelial growth of Trichothecium, Colletotrichum, Pyricularia, Sphaceloma, Phomopsis, Phytophthora,Gaeumannomyces and Botryosphaeria. Howeber, the B. subtilis strain RSS-1 showed relatively weak antagonistic activities to Pythium, Gibberella and partial members of Fusarium and Rhizoctonia genera, but the average mycelial growth inhibitory rate still reached more than 28%. This work indicated that B. subtilis strain RSS-1 has a broad antifungal spectrum.7 Isolation and initial purification of antifungal proteins from B. subtilis strain RSS-1 and its physical and chemical characteristics The crude antifungal protein was initial purified from cell-free culture filtrate of B.subtilis strain RSS-1 by the method of ammonium sulfate precipitation. The results showed that the highest inhibitory effect of antifungal protein on mycelial growth of S.sclerotiorum was treated with ammonium sulfate saturation of 20%. Meanwhile, the effects of heat, p H, UV and proteinases on inhibiting activity of antifungal protein were studied. The result indicated that it was stable in various p H and temperature and resistant to proteinases and UV. The antifungal activity was retained 71.3% after at 100 °C for 60 min. The antifungal protein is active in the range of p H 3-10, the antifungal activity has no significant difference in the range of p H 5-8. The antifungal protein is insensitive to UV and 3 kind of proteinases, the inhibition rate on mycelial growth of S. sclerotiorum was retained 88.3% under UV for 180 min. |
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