| Banana disease, a fusarium wilt, is a banana plant disease caused by the fungus Fusarium oxysporum f.sp. cubense. The fungus can be spread in soil and attacks the vascular bundles, which is cause destructive disease of banana plant. It had spread to all the banana-producing regions of China, such as Guangdong, Guangxi, Fujian and Hainan province. With the rapid spread and the difficult controlled of fusarium wilt disease, it has brought a huge losses to banana-growing industry. Here, we attempted to take Bacillus subtilis as a subject to control of banana disease of banana. Using the F. oxysporum f.sp.cubense race4as a target pathogen, we separated the antagonistic strains and researched on the identification, mark and colonization of relative strains. Also, the antifungal effect of the strains was studied by potted anti-efficiency and field control experiments.The Fusarium oxysporum f.sp.cubense race4as target pathogen. One hundred and twenty one antagonistic strains were isolated from soli by plate-confrontation method, and ten strains were further confirmed to have a stable effect on control pathogen. Strain of BLG-01has a strong antifungal effect (79.8%), which was confirmed by potted anti-efficiency experiment. Mycelium of Fusarium oxysporum f.sp.cubense race4can be broken and digested by the fermentation broth of BLG-01strain. Therefore, the growth of pathogen was suppressed. According to the results of morphological characteristics, physiological and biochemical characteristics and16S rDNA sequence of the BLG-01strain, it can be classified as B.subtilis.B.subtilis BLG-01R, which can resist rifamycin (200μg/ml) antibiotic, was selected by spontaneous mutant and marked from wild type strain B.subtilis BLG-01. In order to have a better tracking of BLG-01R in vivo colonization in the banana plant, B.subtilis BLG-01R was marked by GFP and we got the containing two marking genes BLG-01RK strain. The potted antagonistic efficiency of B.subtilis BLG-01RK is identical with wild type strain; both of them are more than78%.Through B.subtilis BLG-01RK, we studied the effect of wild type strain BLG-01on colonization in the soil and internal of banana plant. The number of B.subtilis BLG-01RK strains in banana rhizosphere soil will decrease with time prolonging. After cultured the B.subtilis BLG-01RK strain27days, the bacterial in the rhizosphere soil were decreased from2.8×107cfu/g to8.1×106cfu/g, which has a smaller relative decrease scope. The result indicated that the B.subtilis BLG-01R exist a stable ability of colonization. However, the strains in the bulbs always have a significantly higher amount than that in fake stem, both of them nearly has the same increase or decrease trends. Using confocal laser scanning microscope (CLSM) to observe the colonization of B.subtilis BLG-01RK in the roots of banana plant, the results confirmed that it has a higher ability to colonization on the root than on the stem internal of banana plant.To study the colonization and prevention effect in the field of wild type strain B.subtilis BLG-01by marked strain B.subtilis BLG-01RK. After inoculation of the strain46days, the higher levels of access bacteria(B.subtilis BLG-01RK) still can be isolated from banana rhizosphere soil. Nevertheless, the antagonistic efficiency in large scale field not so obviously than that in pot experiment.Taken together, through isolation, marking, colonization and field experiments, the B.subtilis BLG-01which can prevent and cure the fusarium wilt caused by F. oxysporum f.sp.cubense race4was isolated from rhizosphere soil of banana plant, and the prevention efficiency and colonization ability of B.subtilis BLG-01were identified. All the results provide a good foundation for further biological control of banana diseases. |
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