| Phosphorus is the main nutrient element for plant growth, whereas 72% to 90% phosphate in soil is fixated by calcium, aluminum, iron and organic compounds, which makes phosphate deficiency the main limiting factor that influences the yield and quality of agricultural products. As main forage grasses that ameliorate soil situation and increase animal products, the cultivate area of alfalfa and sainfoin has exceeded 2 million hm2, which is equivalent to 3 quarters of the artificial grassland in the country, and concentrates mainly on the grain rotation area in arid and semi-arid area of northwest plateau, China. Total P content in soil increased as the application of fertilizer, but phosphate deficiency has still been performed for crops and forage grasses due to the low content of available P. P solubilizing microorganisms are capable of solving the mentioned problem, and P solubilizing rhizobium, which is also capable of fixating N and inoculation, is found excellent in promoting growth and yield of legume forage grass, which decreases the cost of agricultural production and the side effect of P application to the environment. Currently, the phosphate-dissolving Rhizobium inoculants has not been widely used in agriculture, the main reasons are as follow: 1: the low Breeding efficiency of strains, and the low comprehensive growth promoting capability of the screened strains; 2: the application of strains was limited only to host plant; 3: the serious contamination of rhizobium inoculants and onefold effect, as well as relatively short shelf life and deficient in the ability to compete for nodulation. Because of the secreting of P dissolving organic acid, the acidification of the products was found more easily, which therefore makes the death of rhizobia and the infection of the undesired bacteria; 4: the currently widely used medium peat is a kind of nonrenewable resource, the collection of which will do harm to the local environment, and the peat itself has some qualitative defects. All the above factors make the application and development of P solubilizing rhizobium inoculants been limited.According to the above problems, the study is going to focus on the following aspects: 1: optimizing for screening method of phosphate-dissolving Rhizobium and the increasing effect of mutant by a rapid intermittent microwave irradiation; 2: to apply the P dissolving and IAA secreting rhizobia as RGPR strains of rhizobacteria is on to other plants but host plants and widen its application range; 3: decrease the contamination and increase the number of viable cells as well as the nodulation competitiveness of the target rhizobium with bacteriostat 4: to use the leach liquor of green stalk powder as the liquid medium of P solubilizing Rhizobium to reduce the cost of production, and use loess soil and stalk powder to instead peat to be used as the carrier of solid inoculants, to lower down the economic and environmental cost of inoculants production. Based on the above goals, the study firstly screened out Rhizobium meliloti. L-5 and Rhizobium sp. RS-1 with P solubilizing and IAA secreting ability by isolation and breeding, and then to obtain the mutated antibiotic resistant muatnts Rhizobium meliloti LW107 and Rhizobium sp. RSW96 by microwave mutagenesis, to get greater P solubilizing and IAA secreting capability, and to get the antibiotic resistant characteristics of the 2 strains. After the verifying of genetic stability, the growth promoting capability of LW107 and RSN96 to cultivated host and other plants were observed with Ca3(PO4)2 as the only P source. During the process of anti-contamination inoculants, ampicillin was used as bacteriostat to observe the toxic performance of strains and seedlings to ampicillin with different concentrations to select the optimum concentration of bacteriostat, as well as the optimum diluted concentration in application. The optimum carbon sources were selected out for the culture of P solubilizing rhizobia to decrease cost product and increase the quality, and the feasibility of green stalk powder leach liquor with adjusted components as fermentated and plate media was also observed. Afterwards, the sterilized stalk powder, loess soil, peat and vermiculite were used as media to be selected, and finally the liquid and solid rhizobia inoculants with antibiotics were obtained. According to the test of inoculants in different periods, the stable, cheap P solubilizing inoculants with lower contamination and longer effective time was obtained, together with the important techniques in inoculants production.The main findings are listed as fellow:1. The gradual selection model of acid producing N-fixing and P solubilizing Rhizobium is capable of screening P solubilizing rhizobia with good growth promoting capability from nodules with less procedure. The selected Rhizobium meliloti L-5 and Rhizobium sp. RS-1 were found as fast growing rhizobia, and were found with greater nodulation and N fixing capability, together with P solubilizing and IAA secreting capability, and stronger saline and alkaline resistant capability, and can be used as breeding material of highly efficient P solubilizing rhizobia.2. From the single colony generated by acid secreting N-fixing microbes that isolated from nodules, the inorganic P solubilizing microbes were mainly found as Agrobacteria, Actinomycetes, Bacilli, Pseudomonas denitrificans and other N-fixing strains, but only 5 strains identified as Rhizobium strains through the inoculation test, which take up 1.78% of all N-fixing microbes, indicating lots of P solubilizing and N-fixing microbes without nodulation capability exists in plant nodules.3. To increase breeding effectiveness of the strains and to make up the weak P solubilizing deficiency of the wild species, according to the strains mutagenesis method with the existing strains resources. As a safe, clean and cheap radiation source, the conditions of microwave radiation can be controlled and changed more easily. Because of the difference of mutagenesis goals and strains, the concrete microwave radiation parameters varied from each other, while all the highest positive mutagenesis occurred at the lethality rate between 87% and 90%. Part of the obtained highly efficient strains were found genetically unstable, and some were found back mutation or a decrease in yield, at least 6 generations of genetic stability test is required.4. On condition of only inorganic P be offered, the growth of all plants were found be restrained obviously, the leaves grew retardedly, the leaf colour got darkened, and the root became thinner. The shoot height, root length and leaf area were 61-85.2%, 60.5-81.9% and 32.7-75.6% of the treatments inoculated with P solubilizing rhizobia, indicating that good growth promoting effect were found of P solubilizing rhizobia with IAA secreting capability to both host and other plants, and the P deficiency stress can be solved effectively, the assimilation of N and P, and the growth and biomass of the plants increased. The functional process of P solubilizing rhizobia was: firstly dissolve inorganic P to abate the P deficiency stress of plants, and to offer the plants a relatively balanced nutrient condition, simultaneously, to infect the host plant and generate nodules for N fixation. To other plants, P solubilizing rhizobia were found resemble the characteristics of rhizospheric P solubilizing bacteria, but the growth promoting effect on host plants was found better than on other plants.5. On condition of low P content, normal rhizobia were found capable of infecting host plants and nodulation to fixate nitrogen, but on condition of P deficiency, the diameter, fresh weight and nitrogenase activity of nodules were found decreased conspicuously. On condition of low P content, the growth and the accumulation of biomass of plants inoculated with 12531 rhizobia were found performed better than un-inoculated and non fertilized plants (CK1), but the leaf area was only 65.9%, 79% and 67.4% of the treatments inoculated with LW107. RSW96 and applied with 1/4 P solubilizing rhizobia (CK2), respectively. and also poorer than treatments applied with chemical fertilizer. The dry weight of plants inoculated with 12531 strain was found increased by 35.93%, compared with CK1, and only 73.1% and 85.29% of the treatments inoculated with LW107 and RSW96, and also found 17.92% lower than the treatment applied with chemical fertilizer.6. As bacteriostat with low toxity, ampicillin was found with stimulatory effect of low dose and toxic effect of high dose to plants. 200mg/L ampicillin could cause the decrease of nodule number, and to made the waning and lighten of nodules, simultaneously, the nitrogenase activity of nodules decreased to 1/5 or lower to that of the normal nodules. Ampicillin with low concentration has not depression effect on nodulation and nitrogenase activity, and could even the number of nodulation. Because of the difference between species, the reaction of alfalfa and sainfoin to ampicillin was found different. 7. Among the solid inoculants with different materials as the medium, after 120d of storage, the number of viable cells increased as the increase of the absorption of bacterial liquid. Because of the loose and porous structure, green stalk powder could absorb the bacterial liquid that is 2.7 times of its weight, and the powder also contains mass of carbon source, nucleic acid, soluble sugar and almost all the mineral elements that is required of the growth of the microorganism, which will result in the fast proliferation of bacteria, within the 120d of storage, the effective bacteria content of which were found 155.48% and 138.44% times of the numbers found in peat, which was found of great potential in short time inoculant production. But after 1a storage, because of the accumulation of acidic metabolite generated by P solubilizing rhizobia, the pH value was found decreased significantly, the number of undesired bacteria increased, and the nodule numbers decreased sharply. According to the comparison of inoculants after 1a storage, the effective bacteria numbers of the two strains in inoculants with loess soil as the medium was found significantly higher than inoculants with other materials as media(P<0.05), followed by peat. Both the 2 media reached the national standard for inoculant, and could be used as inoculant carrier. Because of the low cost and the accessibility, and the high viable cells as well as the low undesired bacteria number, and the stable characteristic, loess soil was found with good potential.8. Both the carbon source and P in medium prepared with corn stalk leach liquor (CSE) could be used by bacteria. When replenished with glucose (with adjusted pH value) (CSE-2, the ratio of corn stalk powder and distilled water was 1:50), the CSE was found good to the fermentation of LW107, the fermentation speed was increased and the cost also decreased, and was found better than YMA medium; to CSE-3 medium (the ratio of corn stalk powder and distilled water was 1:100), when added with glucose and agar, the obtained solid medium was found with good rhizobia culture effect, equivalent to or better than standard YMA medium, and could be used as cheap medium for plate culture of rhizobia.9. The decrease of temperature or the addition of bacteriostat could increase the number of effective bacteria, decrease the number of undesired bacteria and maintain the pH stability in inoculants. Temperature influences most for the number of viable cells and the undesirable microbes in 1a stored inoculants, bacteriostats were secondly. Viable cell number could be maintained and the number of undesired bacteria will be controlled under the condition of addition of bacteriostat and at low temperature simultaneously, the most active metabolism and organic acid accumulation was found at room temperature, and the pH was found relatively lower, while for inoculants stored at low temperature or added with bacteriostat was found relatively stable. The pH order of the inoculants after 1a storage was: inoculants stored at low temperature and with bacteriostat > inoculants stored at room temperature and with bacteriostat > inoculants stored at low temperature > inoculants stored at room temperature.10.The study synthesizes the screening, mutagenesis and the further utilization, and use double antibiotic and inorganic P solubilizing as marked feature of the strains, and the finally obtained strains could be spiked and detected by observing P solubilizing circle and by plates that contain certain chemical compounds, all the above make the test procedure of target and undesired bacteria more concisely, and also make the production of antibiotic inoculation as well as the nodulation competitiveness of target bacteria more achievable.11.According to the determination of nitrogenase activity by ethyne reduction method, because of the lack of oxygen or the protection of low oxygen conditions to nitrogenase, the nitrogenase activity of pure cultured material was found far lower than the induced plant nodules, but positive correlation was found between the nitrogenase activity of the pure culture material and the nitrogenase activity of nodules induced by the same original strain, accordingly, the same result was found in mutant strains derived from Rhizobium meliloti L-5 and sainfoin Rhizobium sp. RS-1. This phenomenon is worth of further study and exploration, and might be a new way for the fast breeding of highly efficient nitrogen fixation mutant strains.
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