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Li J.,Gansu Agricultural University | Li J.,Sino Us Centers For Grazing Land Ecosystem Sustainability | Zhang S.,Gansu Agricultural University | Zhang S.,Sino Us Centers For Grazing Land Ecosystem Sustainability | And 4 more authors.
World Journal of Microbiology and Biotechnology

Nitrogen (N) fixing Klebsiella pneumoniae RSN19 has high inorganic phosphorus (P) solubilizing capability, but its N2-fixing capability is limited. In order to acquire a P-solubilizing mutant strain with high efficiency N-fixing capability, different microwave irradiation intensities and durations were tested on RSN19 in an attempt to produce mutants with improved N2-fixation and P-solubilization capabilities. The effect of microwave irradiation power and time were studied and the microwave mutagenesis parameters were optimized. Nitrogenase activity was tested on the mutant strains by acetylene reduction method; and their P-solubilizing capability and genetic stability were determined. The results indicated that the best conditions for microwave mutagenesis that produced better performed mutant strains were 250W, 36 s. Under these conditions a maximum positive mutation rate of 1. 66% was obtained, resulting in five genetically stable strains with promoted nitrogenase activity which was designated as RSM-219, RSM-206, RSM-224, RSM-225 and RSM-275. Subculture tests showed that RSM-219 and RSM-206 were genetically stable mutant strains with higher nitrogenase activity and phosphate solubilizing capabilities than the original strain. Both RSM-219 and RSM-206 performed better than the original strain under N-free conditions when supplied with calcium phosphate only, and produced greater increases in the biomass of alfalfa seedlings. © 2010 Springer Science+Business Media B.V. Source

Li J.,Gansu Agricultural University | Li J.,Key Laboratory of Grassland Ecosystem of Ministry of Education | Li J.,Sino Us Centers For Grazing Land Ecosystem Sustainability | Zhang S.,Gansu Agricultural University | And 10 more authors.
Journal of Biobased Materials and Bioenergy

Phosphorus (P) solubilizing and mineralizing soil microorganisms (PSMs) which effectively release P from inorganic and organic pools of total soil P have potential to improve plant yield and reduce fertilizer costs, especially for important leguminous forages like alfalfa. An evaluation program was undertaken in Gansu Province, China to select naturally occurring nitrogen (N) fixing and P solubilizing Rhizobium strains extracted from nodules collected from eight alfalfa (Medicago sativa L.) cultivars. Colony morphology, standard diagnostic tests (both physiological and biochemical) and inoculation onto alfalfa seed (cv. Longdong) were used to identify Rhizobium strains from other PSMs and compare their responses to standard non-P solubilizing (Sinorhizobium meliloti 12531) and P-solubilizing (Rhizobium. meliloti SL01) controls. From the 41 largest colonies exhibiting N-fixing capacity isolated from alfalfa nodules, only 5 PSMs were identified with capacity to solubilise from Ca 3(PO4)2 as the sole P source. Diagnostic tests showed similarity between these isolates in carbon source utilization and a failure to produce 3-ketolactose but only two isolates (L-5 and L-7) produced negative reactions to starch and citrate utilization and a positive catalase test confirming that they belong to the Rhizobium genus. L-5 and L-7 were the only isolates to produce nodules similar to the controls. L-5 and L-7 exhibited a capacity to solubilize inorganic P 1.2 to 1.9 times greater than the control strain R. meliloti SL01 under in vitro conditions. Auxin secretion was higher in L-5 resulted in higher salt tolerance, more significant nodule production and higher nitrogenase activity than L-7 and the standard inoculant S. meliloti 12531 which indicate that L-5 may provide good source material for engineering new strains with an increased capacity for P solubilization. Copyright © 2012 American Scientific Publishers. Source

Li J.,Gansu Agricultural University | Li J.,Sino Us Centers For Grazing Land Ecosystem Sustainability | Zhang S.,Gansu Agricultural University | Zhang S.,Sino Us Centers For Grazing Land Ecosystem Sustainability | And 4 more authors.
Advanced Materials Research

As the most commonly used carrier for commercial inoculants, the development of peat has been limited because peat is a material of nonrenewable resource. Many other materials have been evaluated as alternatives to peat as carriers of rhizobia, yet seldom have been included in inoculants of phosphate dissolving rhizobia. As accessible and inexpensive carriers for rhizobial inoculants, corn stalk powder, loessal soil and vermiculite powder were used in the study to compare with peat carrier on the capacities of rhizobial solution absorption, pH value maintaining of microenvironment, viable rhizobial cells maintaining and the control of contamination. Completely randomized design and 4 replicates were used in the experiment. Twelve different compositions of selected inoculant carrier with different absorption volumes of rhizobial suspension, and were evaluated the ability of maintaining viable rhizobial cells and undesired microbes during the period of 120 days at room temperature. Thereafter, pH value, viable rhizobial cell number and undesired microbes of inoculants with selected absorption volume of rhizobial suspension that stored at 4°C and room temperature respectively, after 1a storage, were evaluated. Viable rhizobial cells in inoculants were examined after 120d and 1a storage by plate counting method, and ratio of undesirable microbes was examined by antibiotic-carrying and normal plates counting method. The result indicated that: for a period of 120days at room temperature, maximum viable rhizobial cells were found in peat, vermiculite powder, corn stalk powder and loessal soil based inoculants when the absorption volume of rhizobial suspension of inoculants were 450, 500, 1000 and 200ml/kg, respectively; viable rhizobial cell numbers were better maintained in corn stalk powder than in peat, loessal soil and vermiculite, but undesired microbes contamination was a severe problem. In the study, viable rhizobial cell numbers in loessal soil was found the highest, followed by peat, while the most serious contamination was found in peat inoculants; corn stalk powder and vermiculite could not be used as inoculant carrier because fewer viable rhizobial cells existed in these inoculants. The greatest pH change was found in peat and loessal soil based inoculants during 1a storage because of enhanced acidification caused by metabolism of phosphate dissolving rhizobia; more viable rhizobial cells were found in the 4 carriers that stored at 4°C than at room temperature after 1a storage. As carriers of phosphate dissolving Rhizobium inoculants, viable rhizobial cells of corn stalk powder after short time storage (120d) and of loessal soil after long time storage (1a) were found better than that of peat, and was also found more cost effective compared with peat, commercially. Both of the two carriers could be used as inoculant carriers at room temperature, but corn stalk powder could only be used as carrier with short shelf life. © (2011) Trans Tech Publications, Switzerland. Source

Li J.,Gansu Agricultural University | Li J.,Sino Us Centers For Grazing Land Ecosystem Sustainability | Huo P.,Gansu Agricultural University | Huo P.,Sino Us Centers For Grazing Land Ecosystem Sustainability | And 6 more authors.
Advanced Materials Research

The purpose of this study was to evaluate the effect of ampicillin as bacteriostats on the doubling time and relative survival rate of antibiotic resistant Rhizobium strains and the ratio of undesirable microbes in artificially polluted inoculants during 60 days storage, and the nodule occupancy of two bacteriostat-resistant strains were also investigated. Fully grown Yeast-mannitol liquid media culture of Rhizobium meliloti LW107 and Rhizobium sp. RSW 96 were added with ampicillin as bacteriostats at various concentrations in an artificial pollution test, and the relative survival rate of rhizobia and the counts of undesirable microbes were determined by the agar plate dilution method. The result shows that in the artificially polluted liquid inoculants, the survival rate of rhizobia was increased and the ratio of undesirable microbes declined significantly when the ampicillin was used as the bacteriostats. The growth of selected antibiotic-resistant strains was promoted at the concentration of less than 100 ug/ml and the doubling time was reduced, but inhibited and the doubling time was significantly prolonged at the concentration of more than 200 ug/ml. Inoculation tests also shows an increased nodulation competitiveness of two bacteriostat-resistant strains in five-fold dilutions of liquid inoculants containing ampicillin (at optimal concentration). According to comprehensive consideration, the optimum concentration for ampicillin as bacteriostat in Rhizobium meliloti LW107 and Rhizobium sp. RSW 96 inoculants appeared to be 100 ug/ml and 200 ug/ml, respectivly. © (2011) Trans Tech Publications, Switzerland. Source

Zhang S.-Q.,Gansu Agricultural University | Zhang S.-Q.,Sino Us Centers For Grazing Land Ecosystem Sustainability | Li J.-F.,Gansu Agricultural University | Li J.-F.,Sino Us Centers For Grazing Land Ecosystem Sustainability | And 10 more authors.
Yuanzineng Kexue Jishu/Atomic Energy Science and Technology

Various microwave powers were used to irradiate alfalfa seeds with various time to study the effect of microwave irradiation on nitrogenase activity of endogenous azotobacter and germination of seeds. Germination rate, germination speed and nitrogenase activity of pure cultures that derived from seed-carried azotobacter were tested. The results indicate that: 800 W, 20 s and 500 W, 40 s are found with highest germination rate on the 1st day, which is 122% and 88.9% times higher than the control group(P<0.01), and highest total germination rate is found within the 14-day period, which is 5.51% and 3.35% times higher than the control group, respectively. Under the two treatments, radical length on the 4th day is 29.8% and 41.9% times longer than the control group, and more sensitive nitrogenase activity is found on condition of various time than various powers. Short time treatments on condition of the two irradiation powers can increase nitrogenase activity conspicuously, and the treatments that treated more than 32 s make nitrogenase activity lower than the control group, conspicuously. Nitrogenase activity is found 104.9% times higher than the control group on condition of 24 s. Source

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