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| Bacillus megatherium WY4 Labeled by GFP and Its Colonization in Chinese Cabbage (Brassica chinensis) |
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Abstract Phosphate solubilizing bacteria (PSB) can increase available phosphorous in soil and improve yields in several crop species like rice (Oryza sativa), maize (Zea mays), potato (Solanum tuberosum), sunflowers (Helianthus annuus) etc. In previous studies, many phosphate solubilizing bacteria had been isolated and widely used in agricultural production. However, the colonization of phosphate solubilizing bacteria in plant rhizosphere have not been completely characterized. Phosphate solubilizing bacteria WY4 is a bacterium isolated from the soil of Brassica chinensis rhizosphere soil. WY4 is a gram-positive bacterium which is tested by Gram staining and its cell morphology is rod shape. The sequence analysis of 16S rRNA gene in the NCBI database using a BlastN analysis showed 99% similarity to Bacillus megaterium strain. To evaluate colonization in soil and Chinese cabbage rhizosphere by this microorganism, generated WY4-GFP, a green fluorescent protein (GFP) labeled derivative of WY4, and inoculated the marked strain into Chinese cabbage seedlings. Compared with the original strain, GFP-labeled had a smaller influence on the growth and phosphate solubilizing activity. While, the results demonstrated that WY4-GFP could survive in the natural soil to achieve 105 CFU/g (wet soil) at 21 days; WY4-GFP increased the plant height, root length, leaf numbers, fresh weight and dry weight of Chinese cabbage plants. Phosphorus could promote root growth of Chinese cabbage, WY4 could increase content of available phosphorus as PSB, so the root system was developed after application of WY4: The root volume that reached 1.72 mL was significantly higher than water treatment (0.38 mL). After the treatments of WY4-GFP and WY4, the fresh weight of the plant reached 17.48 and 17.52 g/plant respectively. There was no significant difference between WY4 and WY4-GFP in growth promotion. The results further indicated that WY4-GFP flourishing colonizes in Chinese cabbage plants and rhizosphere soils, colonization of WY4-GFP analysis in nature soil or rhizosphere soils showed that the population of WY4-GFP decreased gradually as the extension of time. At 0~10 d, The number of WY4-GFP decreased very fast (from 13.32~3.47×106 CFU/g), but after 20~30 d incubation in Chinese cabbage rhizosphere soils, the number of WY4-GFP were basically stabled at 104 CFU/g. Furthermore, the results indicated that Bacillus megaterium WY4 displayed good colonization ability in the rhizosphere of Chinese cabbage. Plentiful WY4-GFP colonized in different growth period (5, 10 and 20 d) of the root tips and was detected in meristernatic zone. Meanwhile, less WY4-GFP appeared to the root elongation zone, lateral root hair and hypocotyls catheter, but numerous labeled bacteria existed in intercellular spaces of Chinese cabbage roots. Taken together, these results indicated that WY4-GFP invaded Chinese cabbage through the root tips or intercellular spaces, and they could be transported into the hypocotyls catheter by the vascular bundle system of roots. Preliminary study on the mechanism of phosphorus solubilizing bacteria promoting the growth of Chinese cabbage showed the good application prospects of PSB. The results provide important information for further study on the relationship between phosphate solubilizing bacteria and plant.
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Received: 22 June 2016
Published: 07 November 2016
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