Plant Growth-Promoting Rhizobacteria Of Mulberry Rhizosphere

The rhizosphere soils were obtained by three point sampling method from mulberry soils with different fertilities in silkworm and mulberry scientific research base of Shandong Agricultural University, and the mulberry variety was Husang 32. Rhizosphere microbes were isolated through selective medium, quantity analysis of rhizosphere bacteria, fungi and actinomyces that in fertile and infertile soils was conducted. Rhizosphere azotobacter, phosphobacteria, potassium-bacteria were cultivated by plate cultivation method with selective medium. The repetitive-element PCR genomic fingerprinting (rep-PCR) were used to fingerprint pure DNA extracted from mulberry rhizosphere bacteria. The diversity of Growth-Promoting Rhizobacteria was analyzed by the Shannom-Wiener index, richness index, evenness index and domination index. Rhizosphere azotobacter, phosphobacteria, potassium-bacteria were identified by their morphological properties, physiological and biochemical characteristics and sequence analysis of 16S rDNA. Nitrogenase activity of nitrogen-fixing bacteria was measured by acetylene reduction assay (ARA) with gas chromatography (GC). Phosphate-solubilizing ability of phosphobacteria was compared by measuring circle size of lecithin plate and phosphorite plate. Potassium-releasing ability of potassium-bacteria was measured by flame spectrophotometer. The main results were as follows.In the different soil fertilities, the quantities of rhizosphere total bacteria, azotobacter, phosphobacteria, potassium-bacteria and actinomyces in fertile soils were larger than that in infertile soils, and the quantity of fungi in fertile soils was lower than that in infertile soils. The quantities of Ammoniation bacteria, Cellulose-decomposing bacteria, Nitrifiers, Anti-Sulfurizing bacteria in fertile soils were more than that in infertile soils, and the quantities of denitrifiers and sulfurizing bacteria in fertile soils were lower than that in infertile soils. Under the same soil fertility, the quantity of the phosphobacteria was the largest, that of potassium-bacteria the second, and that of azotobacter the least.Based on the rep-PCR fingerprints cluster analysis of rhizosphere bacteria, rhizosphere bacteria in fertile and infertile soils were divided into 71 and 33 clusters at the dissimilarity of 0.2 with rep-PCR fingerprints respectively. The PGPR in fertile soils were divided into 33 clusters, including 10 clusters of azotobacter, 14 clusters of phosphobacteria, 14 clusters of potassium-bacteria. The PGPR in infertile soils were divided into 28 clusters, including 11 clusters of azotobacter, 13 clusters of phosphobacteria, 10 clusters of potassium-bacteria. 18 azotobacter, 18 phosphobacteria and 23 potassium-bacteria were obtained by rep-PCR fingerprints cluster analysis.The diversity index, richness index and evenness index of rhizosphere bacteria, phosphobacteria and potassium-bacteria in fertile soils were larger than that in infertile soils, but the domination index was lower than that in fertile soils. The diversity index, evenness index and domination index of azotobacter were the lower in fertile soil, but the richness index was the larger. These results showed that the soil fertility had impacts on bacteria distribution, and the fertile soils provided suitable growth micro-environments for most bacterial genera, implied by larger number of bacterial genera and lower domination index. In infertile soils, relatively few bacteria genera could colonize due to the lack of nutrition, leading to a higher domination index.The identification results of rhizosphere azotobacter, phosphobacteria, potassium-bacteria showed that Variovorax sp.were the majority of azotobacter, and Pseudomonas sp. were the majority of phosphobacteria and potassium-bacteria. Their 16S rDNA sequences had been registered at Genbank.The comparison results of nitrogenase activity of azotobacter showed that the activities of PA16 and PA2 were the highest, and their activities were 388.369nmolC₂H₄/hmL and 178.60nmolC₂H₄/hmL respectively. The strains PYP2, PYP4, PYP6, FYP2 had high organic phosphate solubilizing capacity. The strains FWP7 and PWP4 had high inorganic phosphate solubilizing capacity. The strains FWP1,FWP9,FWP11,PWP6,FYP6 had both organic phosphate solubilizing capacity and inorganic phosphate solubilizing capacity. The Potassium-releasing activities of FK2 and FK3 were higher than that of others.