| Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense (FOC) is one of the widely distributed and devastating soil-borne diseases. Prevention and control of this disease is still a worldwide problem currently. Biological control is a developing frontier of technology and has become a hotspot of the research for its safety to human beings, animals and environment. The biocontrol-microorganism that screened by the plate confrontation assay is always difficult to colonize and secrete effective antimicrobial substances in the poor nutrition soil, because of the influence from biotic and abiotic factors in soil, which might be the reasons why the biological control is unstable and needs high cost.In present study, sixty-two of Bacillus sp. strains with different origins were evaluated in terms of the adaptability to the poor nutrition condition by simulating the soil environment. The result showed that the strain HWC-2had strong adaptability to the poor nutrition condition. This strain was identified to be Bacillus sp, and its culture conditions were optimized, then the active antimicrobial substances was isolated and identified. After that, this isolate was tagged with the GFP reporter and used to determine the impact factors during the colonization and the pathogens reduction in the soil. Then the compound bacterial fertilizer HE that contains HWC-2and biocontrol-fungus Paecilomyces lilacinus E7was obtained and its efficiency by combining with sweet potato intercrop on suppressing banana wilt disease in the field were tested. Last, the mechanisms of HE with different proportions of N, P, K nutrition to control the banana Fusarium wilt of banana were investigated in potted plant experiment. All these assays will provide detailed basic data for the comprehensive prevention and control of this disease by using biocontrol agents efficiently in the field.The main results are listed as follows:1. To evaluate the adaptability of Bacillus isolates to nutrition. The inhibition zone sizes of sixty-two Bacillus isolates with different origins to FOC were compared on different soil nutrient media including SE, SE+CN, SE+C, SE+N, SE+P, SE+K, SE+OF1and SE+OF2by the soft agar diffusion method; meanwhile, the isolates C10-1and BLG-01that have been reported were used as the control sets. With the inhibition zone as an indicator, the production of the antimicrobial substances of four Bacillus strains were promoted by the following nutrients: OF1>CN>P>C>N>OF2>K. With the colonization ability in the soil as an indicator, the inhibition rates of these Bacillus isolates in the sterilized soil inoculated with FOC were compared under two nutrition conditions (OF1, CN). The results indicated that OF1was better than CN in promoting the production of antimicrobial substrates of HWC-2stain. At days16post treatment, the inhibition rates of HWC-2to FOC under OF1was72.98%, the colonization rate of HWC-2in soil was increased by104.97%. The inhibitory effect of soil extract from that inoculated HWC-2under OF1was more significant compared with that of the single HWC-2treatment. These findings indicate that strain HWC-2has adaptability to the relatively poor nutrition. Thus, HWC-2was selected for the further research. The strain HWC-2was identified as Bacillus amyloliquefaciens by morphological, physiological and biochemical analyses as well as16S rDNA gene sequence characteristics. In addition, the optimum culture conditions for HWC-2were as follows:sugar (10g/L), urea (8g/L), inorganic salt solutions contain Mg2+(0.6g/L), Ca2+(0.4g/L), Mn2+(0.8μmol/L) and Fe2+(0.4μmol/L),200mL medium in500mL flask, pH=7,35℃,170r/min, for48hours.2. The biosynthesis genes of Surfactin, Iturin, Fengycin and Bacillomycin including sfp, ituA,fenB and bam A were identified in the genome of the strain HWC-2by PCR assays. These findings as well as the characteristics of the resistance to the acid and alkali, high temperature and the protease activity of the crude extract indicate that the strain HWC-2could produce the substances of the lipopeptide. The single factor and orthogonal optimization test on the factors that have effects on the culture of the microorganism that can produce the lipopeptide including condition of culture, mediums, initial pH, temperatures, fermentation time was explored. The results indicated the orthogonal combination2(A1B2C2D2) was the best condition for the fermentation. The lipopeptide was identified by HPLC-ESI-MS analysis, the molecular weight of the substances of the lipopeptide are appropriately1020.5,1034.6,1042.7,1056.6,1070.6,1098.4Da. Compared to the previous report, the first group of antibacterial material was predicted to be the Bacillomycin L’shomologue of the lipopeptide antibiotic substances, the second group of antibacterial material was predicted to be the Iturin A’s homologue of the lipopeptide antibiotic substances, the third group of antibacterial material was predicted to be Bacillomycin F which belongs to the lipopeptide antibiotic substances.3. The strain HWC-2was marked with GFP and tetracycline resistance by the way of chemical transformation. Comparing the effects of soil texture, temperature, moisture and nutrients and crop on the inhibition effects of the HWC-2-GFP on FOC and its colonization ability. The most suitable soil for inhibition and colonization was loam soil, followed by sand and clay; the most suitable soil temperature successively was37℃,30℃,23℃and16℃. The optimum soil relative humidity was20%, followed by25%,15%and30%, and the most suitable crop was sweet potato crop, followed by peanuts and corn. Adding the nutrition OF1promoted the inhibition of HWC-2-GFP to FOC and its colonization, the nutrition P, N and K.4. By simulating the soil environment,16formulas that were composed of B. amyloliquefaciens HWC-2, Paecilomyces lilacinus E7, Trichoderma viride H06and nutrient substance OF1were screened. The best formula was HWC-2+E7+OF1and it could reduce70%of FOC (log (cfu/g)) in the soil and was named as HE. When the OF1humidity was controlled fewer than5%in the temperature of room temperature,15℃,25℃and35℃, the shelf life of HWC-2and E7could be extended. The number of living bacteria was over20million within12months. The number of viable microorganisms meets the standard of composite microbial fertilizer.5. A randomized block experiment was conducted to test the effects of a single bacterial fertilizer and the compound bacterial fertilizer with sweet potato intercropping or not on the growth of banana plants, disease severity index (DSI) of Fusarium wilt, the quantity of FOC, other culturable microbial population and the total soil respiration rate in banana rhizophere. The results showed that, after transplanting for120days, the growth of banana and disease-control efficiencies were increased by dealing with single bacterial fertilizer H or single bacterial fertilizer E or the compound bacterial fertilizer HE in both intercropping and monocropping model compared to the control treatment in the monocropping model; the combination of the intercropping with compound bacterial fertilizer HE showed the most efficient to Fusarium wilt and the banana height, stem thickness, leaf width, fresh weight of aerial and underground part and control efficiency were increased. Moreover, the number of FOC in banana rhizosphere at different depth was decreased significantly; with applications of H, E and HE in the intercropping model, the DSI was decreased by47.06%-72.22%, the number of culturable fungi, bacteria and actinomyces in rhizosphere was increased significantly by1.10-6.59,16.08-39.00and2.63-3.32times respectively, the number of FOC was decreased significantly by97.12%-99.81%, total soil respiration rate of the rhizosphere was increased by46.64%-74.53%. The DSI positively correlated with the number of FOC at a significant level, while the DSI negatively correlated to the number of culturable fungi, bacteria and the total soil respiration rate at a significant level.6. The mechanisms that the HE compound bacterial fertilizer with different proportion of N, P, K nutrition employed in controlling Fusarium wilt of banana were investigated in potted experiment. The results showed that the applications of combinations of the compound bacterial fertilizer HE and additional different nutrients had certain effects on controlling Fusarium wilt of banana. Among these treatments, the treatment of HE+P had best disease-controlling effect, then followed by HE+N, HE, HE+K treatments respectively. At days120post transplanting, DSI was decreased by50.34%-72.15%, the log cfu/g of FOC was decreased significantly by27.18%-55.85%, the number of culturable fungi, bacteria and actinomyces in banana rhizosphere was increased significantly by2.28-49.27,5.89-241.57and1.87-4.69times, respectively. The total soil respiration rate of the banana rhizosphere was increased by8.20%-127.95%. The HPLC-ESI-MS analysis at the days30and days60post inoculation showed that HWC-2can secrete Iturin in the banana rhizosphere, and the mass fractions with crude quantitative analysis are7.55%-50.54%and4.84%-50.03%. The DSI had a positive correlation with the number of FOC at a significant level, while it negatively correlated the number of the culturable fungi, bacteria as well as the culturable actinomyces and the total soil respiration rate at a significant level. |
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