Isolation,Characteristics,Promoting Effects And16s RDNA Sequence Analysis Of Phosphorus-Solubilizing Bacteria In Rhizosphere Of Lotus Corniculatus And Trifolium Repens

The phosphorus-solubilizing bacteria from the rhizosphere of Lotus corniculatus and Trifolium repens were isolated and screened in Qiannan state using the method of phosphate-solubilizing ring. The capability of solubilizing calcium phosphate, iron phosphate, aluminum phosphate and egg yolk lecithin of the phosphorus-solubilizing bacteria strains was determined by the method of molybdenum blue. The ability of producing3-indole acetic acid (IAA) of the phosphorus-solubilizing bacteria strains was assessed by Salkowski colorimetry. The characteristics of utilizing different carbon sources was tested, and the effects of growth promoting of Trifolium repens seedlings were researched by vaccinating8dissolved phosphorus-solubilizing bacteria. Meanwhile, the effects of promoting growth characters of Lotus corniculatus and Trifolium repens from the different treatments such as phosphate-solubilizing agents, fixing bacteria agents and combination of these treatments were tested by pot experiments. Three phosphate-solubilizing bacteria were identified by16S rDNA sequence analysis. The results were obtained as follows:1. The number and distribution of phosphate-solubilizing bacteria in rhizosphere of Lotus corniculatus and Trifolium repensTwenty strains of he phosphate-solubilizing bacteria isolated from the rhizosphere of Lotus corniculatus\,and16strains from the rhizosphere of Trifolium repens The number of these phosphorus-solubilizing microorganisms in two plantspecies’rhizosphere was varied, and the distribution trend was presented as RS (root soil)> NRS (soil away from roots)> RP (rhizoplan or surface of roots)≧HP (histoplan or interior of roots).2. The isolation and phosphorus dissolving characteristics of phosphorus-solubilizing bacteriaFor different phosphorus-solubilizing strains, the sizes of the transparent rings produced on the culture mediums PKO solid agar by the source of calcium phosphate were different from those that appeared on Mengjinna solid agar in the source of egg yolk lecithin. So the values caused by the HD (transparent circle diameter) divided by the CD (colony diameter) was an imploortant index of their effectiveness.. Take the fifth day of phosphorus-solubilizing bacteria’s growth as an example, on the PKO culture mediums in the source of calcium phosphate, the values of HD/CD were between2.36and4.39. On the Mengjinna culture mediums,the values of HD/CD were between1.04and1.72. The values of HD/CD of most phosphorus solubilizing bacteria strains can reach the greatest values in their fifth growing day, and it would decrease gradually thereafter..The19phosphorus-solubilizing bacteria strains that were used had different attributesd when they dissolved calcium phosphate. The phosphorus dissolving ability was between94.35and400.49mg·L-1. There were8strains whose dissolving ability were above200.00mg·L-1, they occupied42.1%of the total. The dissolving ability of strain LC15reached400.49mg·L-1. The8phosphorus-solubilizing bacteria strains had the ability of dissolving aluminum phosphate (Al-P), iron phosphate(Fe-P) and egg yolk lecithin(EYPC). The ability to dissolving aluminum phosphate was between7.93and71.22mg·L-1, and the LC15was the highest; for the LC25, the ability of dissolving Fe-P was the highest, whose value was27.27mg·L-1. The value of dissolving ability of the8strains of phosphorus-solubilizing bacteria to the aluminum in the EYPC was between1.88and19.54mg·L-1. The dissolving ability of LC20to EYPC was the strongest. However, there was feeble dissolving ability of RW6to EYPC. Except for strains RW2and RW6, the dissolving ability of the other6strains of phosphorus-solubilizing bacteria to Ca-P、Al-P、Fe-P and EYPC sorted by volume in descending order, which showed:Ca-P> Al-P> Fe-P> EYPC.3. The ability of secreting3-indole acetic acid (IAA) of phosphate-solubilizing bacteria15strains of the19phosphate-solubilizing bacteria strains were able to secrete IAA,78.94%strains produced IAA amount in between9.17-42.39mg·L-1. Strain LC20had the strongest ability of secreting IAA, the amount of producing IAA for strain LC19was38.97mg·L-1, just below that of strain LC20.4. Carbon utilization and pH performance of phosphate-solubilizing bacteriaMost isolated phosphate-solubilizing bacteria can grow well or lush in carbon source medium in which peptone was replaced by other carbon source, including mannitol, sucrose, glucose, xylose, maltose, galactose,1/2mannitol+1/2sucrose. All strains can produce alkaline.5. The growth-promoting effects of strains(1) The effects of a single strain on white clover seeding growth8phosphate-solubilizing bacteria were inoculated in Trifolium repens seedlings, and after40days, the plant height was significantly taller than the conrol(by9.50%-111%). For the root length, it was significantly greater than the control,(except strain LC16,(by1.9% -89.12%).Both above-ground and below-ground biomass was obviously increased4.66%-242.22%and8.20%-346.17%but the biomass of strain RW2was lower than the control.(2) The growth-promoting effects of the compound strains on Lotus corniculatus and Trifolium repens in pot experimentAll treatments, including phosphorus-solubilizing bacteria combination, nitrogen fixation bacteria combination and all mixed treatment of them, promoted plant growth and above-ground biomass of Lotus corniculatus, compared with the control. Plant height, at the first-cut and the second-cut, increased significantly by20.%, and the aboveground biomass increased over100%, compared with the control. The content of total nitrogen, CP (crude protein) and total phosphorus when inoculated with phosphorus-solubilizing bacteria and nitrogen-fixation bacteria was greater. The combinational treatment was significantly higher than the control,(19.3%and61.9%respectively). The content of CF (crude fat) of treatment inoculated with phosphorus-solubilizing bacteria increased by25.1%.All the inoculated Trifolium repens seedlings, showed the growth-promoting effects on plant height, above-ground biomass, the content of total nitrogen, CP, total phosphorus and CF. When compared with the control, the growth-promoting effects on plant height and aboveground biomass of Trifolium repens inoculated with a phosphorus-solubilizing bacteria and nitrogen fixation bacteria combination were the best(10%and40%respectively), total nitrogen and CP improved over7.00%, total phosphorus levels and CF increased by19.2%and7.1%respectively, but the content of CF of Trifolium repens which was inoculated with phosphorus-solubilizing bacteria and nitrogen fixation bacteria combination was unchanged.6. The16S rDNA sequence analysis of strainsAfter PCR amplification, detection of positive clones, plasmid extraction to the genomic DNA of the strains LC15, LC20and RW18,16S rDNA sequence of three strains were analysed and compared by GenBank, the results showed that the homology of16S rDNA nucleotide sequence of strains LC15and LC20were more than99.0%, compared to multiple strains of Klebsiella sp., The homogeneousness of strain RW18was at the rate of99.8%to Leclercia adecarboxylata (Accession number was HQ242722in GenBank). Therefore, according to the primary physiological and biochemical characteristics and the position in bacterial phylogenetic tree of each strain, strains LC15and LC20were identified as Klebsiella sp., strain RW18was identified as Leclercia adecarboxylata.