| To2050, the world’s population will mount to9billion.It arises a problem that how we can support such a large population, and it is of great significane to make sustainable use of the soil we live on. With the increasing contradictions between population and resources and environment, soil degradation, including soil acidification and depletion, has become a major global problem. Soil microorganism is the the most important and active biotic factor in soil structure, nutrition and fertility. It has always been the hotspot for study of sustainable utilization of soil to change the soil fetility through its effect on soil microbial ecology.This experiment is focused on soil microbial ecology.Traditional analysis methods and molecular biology methods are combined to study the ecological influence of beneficial microorganisms to provide reference for evaluation of beneficial bacterial manure and theoretical basis for soil sustainable utilization study. In this study, kudzu, alfalfa, aciduric kudzu, and rhizobium meliloti were chosen as experimental subjects. Growth of aciduric kudzu, and rhizobium meliloti with acid stress as well as its effect on rhizosphere micro-ecology was studied. Alkaline substances produced by aciduric kudzu and rhizobium meliloti under acidic conditions and impact of bacterial manure on physical and chemical properties of rhizosphere soil of highland barley were also discussed. Through research, the following results have been obtained:(1) The sequence of the kudzu and rhizobium meliloti strain16s rDNA is1345-1380bp. Kudzu rhizobium068,389,390,021and Rhizobium constitute a branch, the similarity of various species is respectively99.35%(Rhizobium lusitanum),98.46%(Rhizobium),98.24%(Rhizobium), and98.54%(Rhizobium). Alfalfa rhizobium3,4,5,8and Sinorhizobium constitute a branch, and has high homology with Sinorhizobium meliloti with the similarity of98.83%,99.20%,98.83%and99.20%respectively.(2) The measurement of growth curve of the kudzu rhizobium and alfalfa rhizobium with different acidity showed that acidity significantly reduces the growth rate of rhizobium, increases its growth lag phase and delays the logarithmic phase and stable phase. Kudzu rhizobium can grow in the liquid culture medium with the pH4.6and alfalfa rhizobium can grow in the liquid culture medium with the pH5.0, which indicates with low acidity aciduric alfalfa rhizobium will still be able to grow rapidly after lag phase with range tolerance of acid.(3) Acidity remarkably reduces the deformation rate of root hair of kudzu and alfalfa. With the decreasing of pH, deformation rate of root hair gradually reduces. Under the neutral condition (pH7.0) and acidic conditon (pH4.8) root hair deformation rate can be improved by rhizobium inoculation. Acid stress greatly reduces the root aciticity of kudzu and alfalfa. Their root activity gradually reduces with the decreasing of pH. Rhizobium inoculation can enhance their root activity. Under neutral and acidic conditions inoculation of acidic kudzu and alfalfa rhizobium can result in nodule of kudzu and alfalfa. But under neutral condition the nodule number is significantly higher than the conditon with pH4.8. Under acidic condition, the delay of nodules for kudzu and alfalfa will arise. Inoculation of acidic kudzu rhizobium and alfalfa rhizobium can improve the pH of nutrient solution. There is remarkable influence of of kudzu and alfalfa rhizobium inoculation on raw weight of kudzu and alfalfa plant, raw weight of overground part, raw weight of root, plant height, raw weight of nodules and total nitrogen content, which shows that under acidic conditon, environmental pH is the main factor affecting the yeild and quality of kudzu and alfalfa, efficient inoculation of aciduric kudzu and alfalfa rhizobium, slection of good breed of alfalfa plants can reduce the negative impact of pH on alfalfa growth to a certain extent.(4) Aciduric kudzu rhizobium and alfalfa rhizobium inoculation improve the content of bacterium, fungus, actinomycetes and rhizobium in rhizosphere soil and it is better than the treament without inoculation. After inoculation, urease, acid phosphatase and catalase activity have improved notly in rhizosphere soil. The microbial biomass carbon, nitrogen and soil respiration intensity have also been improved. Inoculation of aciduric kudzu and alfalfa rhizobium increases the absolute content of bacterial PLFA, the absolute content of Gram-negative bacterium PLFA, as well as that of Gram-positive bacterium PLFA, fungal PLFA and actinomycetes PLFA. Aciduric alfalfa rhizobium has improved the soil nutrient in acidic soil and soil fertility.(5) Thin layer chromatograph (TLC) and vitro chromogenic reaction indicate that the bacterial strain of kudzu068and bacterial strain of alfalfa5can produce alkaline substances during cultivation process. Low concentration of rhizobium can enhance the seed germination and root growth, whereas high concentration is the just opposite. (6) Chemical analysis, PCR-DGGE and DNA sequencing methods were used to investigate effects of bacterial manurevarying in application rate and applied at diffentent time on soil physicochemical properties and microbial community diversity in the rhizosphere of highland barley in Tibet. Results show that application of bacterial manure significantly increased total N, total P, total K, organic matter, alkalytic N, available P and readily available K in the soil, for instance, by13.32%,28.42%,16.20%,9.81%,21.36%,39.35%and30.48%respectively over that in CK when750ml hm-2of bacterial manure was applied before sowing; and by7.25%,29.35%,18.04%,12.86%,15.90%,43.27%and53.99%, respectively, when2250ml hm-2of bacterial manure was applied at the jointing stage of the crop. DGGE analysis demonstrates that DGGE atlases of microorganisms in the soils the same in application method, were all quite similar regardless of application rate. Cluster analysis with UPGMA sorted the DGGE atlases into two clusters. Shannon indices suggest that soil microbial diversity in soils applied bacterial manure increased first and decreased gradually afterwards. Application of750ml hm-2of bacterial manure prior to sowing brought about the highest soil microbial diversity, while application of2250ml hm-2at the jointing stage did. Moreover, the former was higher than the latter in nutrient releasing and Shannon index as well. DNA sequencing shows that microbial populations were widely distributed and dominated with bacteria of Actinobacteria class, and a few uncultured strains in the soils applied with bacterial manure, regardless of application rate. Canonical correspondence analysis (CCA) reveals that the distribution of DGGE fingerprints was strongly related to physical and chemical properties of the soil. AN (Alkalytic N), TP and TN were the main environmental factors influencing structure of the microbial community. All the findings demonstrate that application of bacterial manure significantly improves physicochemical properties of the soil and microbial community diversity of the rhizosphere soil of highland barley. |
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