| Planting watermelon has a long history and is an important gardening industry of the world. With watermelon planting area increases year by year, especially in the continuous watermelon-cropping systems, the soil-borne disease of watermelon becomes more and more severe. Fusarium wilt of watermelon is one of the most serious and destructive diseases worldwide. Therefore, it is extremely urgent to seek safe and effective methods to control Fusarium wilt of watermelon. However, the conventional chemical control has brought environmental pollution and microbial resistance to drug. Effects of rotation and intercropping on disease control are limited while many difficulties exist in breeding resistant varieties in practice. Biocontrol has been proven to be pollution-free and environment-friendly alternative to the tranditional methods in control of soil-borne disease and has potential significance in maintaining ecological balance and developping sustainable agricultural practices. The present study was aimed to isolate highly pathogenic Fusarium oxysporum pathogens from field watermelon suffered from Fusarium wilt disease, to isolate antagonistic bacteria against the screened the pathogenic fungi; and to prepare bio-organic fertilizers that could be used in biological control of Fusarium wilt of watermelon by fermentation of the screened antagonists with organic fertilizers. The results of this reseach were listed as follows.1.Thirty-four suspected pathogens were isolated from the roots, stems and leaves of the diseased watermelon plants in field. Re-inoculation of watermelon plants with six strains of obvious morphological variations showed that strains LD and LB could cause wilt symptom with100%and72%of disease incidence, respectively,14days after inoculation while strain XA had no wilt symptom. Morphological observation and28S D1/D2sequence analysis showed that strains LD and LB were Fusarium oxysporum and strain XA was Fusarium moniliformis.2. A greenhouse pot experiment with a paddy soil, which was never planted with watermelon before the experiment, showed that inoculation of soil with non-pathogenic strain XA did not cause any wilt symptom. Microscopic observation and culture showed that XA could successfully colonize roots of the watermelon. In contrast, inoculation of soil with pathogenic strain LD led to100%death of watermelon. The pathogenic numbers of F.oxysporum in crown, roots, rhizosphere soil and bulk soil of watermelon reached to1.58×104CFU/g,1.77×104CFU/g,4.85×104CFU/g and3.45×104CFU/g, respectively. As compared with the LD treatment, dual inoculation of soil with strain XA and LD decreased the pathogenic numbers of F.oxysporum in crown, roots, rhizosphere soil and bulk soil of watermelon by63.3%,66.1%,3.3%and24.4%, respectively, and gained57.8%of control efficiency, suggesting non-pathogenic strain XA effectively inhibited pathogenic strain LD to infect watermelon plants.3. A total of172bacterial strains with antagonistic activity against pathogenic F.oxysporum were isolated from four soils in field. The plate-confrontation analysis showed that13strains had more than60%of inhibition rate against the pathogen and strains Cy5and CR38performed best. Antibiotic spectrum experiments showed that strains Cy5and CR38were able to inhibit the growth of other plant pathogens such as F. oxysporum f.sp.cubense, F. oxysporum f.sp.melonis, Verticillium dahliae and P. parasitica var. nicotiana, but strain Cy5performed better than strain CR38. Physiological and biochemical characterization and16Sr DNA gene sequence analysis showed that strain Cy5belonged to Paenibacillus jamilae (Genbank number JQ323092).PCR amplification and sequencing analysis showed that β-1,3-1,4-glucanase gene from strain Cy5had99%of homological similarity with that (Genbank No. AY164457) of Paenibacillus polymyxa. Additionally, strain Cy5also was able to produce several secondary metabolites beneficial to plant growth.4. Two seasons of greenhouse pot experiments with heavily diseased soil showed that bio-organic fertilizers could improve soil microflora, increase disease control efficincy and promote plant growth. Control efficiencies of bio-organic fertilizers against Fusarium wilt of watermelon in the two experiments were different. For examples, the control efficiencies of bio-organic fertilizers made of strain Cy5and strain CH8were75.0%and62.5%, respectively, in the first experiment, whereas those were37.5%and31.2%, respectively, in the second experiment. However, as compared with the common organic fertilizer that was not fortified with any antagonist, bio-organic fertilizers significantly increased the above-ground biomass, increased the numbers of bactiria and Bacillus species, and decreased the numbers of fungi and pathogenic F. oxysporum in the rhizosphere soil of watermelon. 5. A greenhouse pot experiment with non-diseased soil showed that bio-organic fertilizers were able to promote plant growth. At the flowering stage (30days after transplantation), plant height and dry weight of the plants in these treatments using bio-organic fertilizers increased significantly in the range of36.5%to40.3%and38%to70%, respectively, as compared with the common organic fertilizer treatment. Total length and total surface area of roots in these bio-organic fertilizer treatments were increased by47.1%to77.6%and26.3%to57.7%, respectively, as compared with the common organic fertilizer treatment.In conclusion, this study gained a strong pathogenic F.oxysporum strain and a non-pathogenc congeneric strain, which inhibited the infection of plants by the pathogen, from the plants of field watermelon.We also gained several potencial biocontrol agents, of which bio-organic fertilizers were made. Application of these bio-organic fertilizers significantly controled Fusarium wilt of watermelon, promoted watermelon growth and improved microbial flora in rhizosphere soils of watermelon. Further study should be focused on the field performance of these bio-organic fertilizers and related factors that affect the performance. |
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