Functional Gene Diversiyt Of Microorganism Related The Circle Of Nitrogen Under Organic Manure Application In Red Soil

Microbial diversity plays an integral and unique role in variety of the global ecosystem; this has been proven in agricultural ecosystem by much recent research. Soil microorganism is the necessary part in global material cycle of carbon, nitrogen, and so on, thus it affects the material balance, the composition of the atmosphere and geochemistry procedure in the global scale. Soil microorganisms play a key role indegrading the animal and plant body, which directly influence the available nutrient of soil and vegetation growth. Soil microorganisms are sensitivity to the environmental change and easily indicated the change of the ecosystem functions, which had important significance to renew and protect the ecosystem. Understanding the structure and composition of soil microbial communities and their responses and apaptions to global change is critical in maintaining or restoring desirable ecosystem functions. Due to the irrational exploitation and utilization for natural resources, more and more factors impact on the diversity of soil microorganisms, such as the increasing of CO2concentration, vegetation degeneration and environmental pollution.Red soil is the typical soil resources in South China, and it is also considered as important production bases of economic forests, cash crops and cereals. However, soil degradation and utility processes, such as soil erosion, soil nutrient depletion, soil acidification, pollution, etc. exert impacts on sustainable development of agricultural economy in this area. Microorganisms are the important part of soil ecosystem, which play significant roles in material cycling promotion, ecological balance maintenance and plant nutrients transformation. For a long time, numerous researches focused on the research of soil microorganisms by traditional cultivation methods, because less than1%of microorganisms can be cultured, characterization and detection of microbial populations in natural environments present a great challenge to microbial ecologists, while molecular biology methods provide us with new perspectives to analyze the diversity, genetics and function of the microbial community.In this study, a PCR-based cloning and sequencing approach was used to investigate the molecular diversity and community structure of all bacteria in red soil by using the traditional cultural technology and analyzing the diversity of16S rDNA, which influencing the different land use patterns and different manure fertility in red soil. We also had been investigated the molecular diversity and community structure of nitrogen-fixing microbes, ammonia-oxidizing bacteria and denitrifying bacteria those connecting with the Nitrogen cycle and their key impact factors in the different manure fertility. All results from these studies are summarized as bellow.1. Extraction method of soil microbial DNA for molecular ecology studyIn environmental microbiology, molecular ecology study has been widely concerned in the world, while one of the key technologies of study uncultured microorganism in molecular level is obtain the high quality genome from environmental microbiology. It is the important basic that total extracted DNA can represent all environmental microbiology. In this study, soil microbial DNA was extracted using Zhou’s method and Raddy’s method, respectively. The crude DNA extraction was purified by gel electrophoresis dialysis method and gel midi purification kit, respectively. The result showed that the modified zhou’s method could extract DNA more efficiently and was the best useful method. Even the DNA extracted from the barren CK sample was available in amplifying its16S rDNA and functional genes fragment (such as nifH, amoA, nirK) by Polymerase Chain Reaction. Therefore, the modified Zhou’s method could be an efficient and reliable method to extract DNA from the typical barren red soils in the molecular ecology studies.2. The diversity of different amount of organic manure fertility to Soil Microbial Diversity in red soilTo understand the influence of different amount of manure fertility to soil microbial diversity, four treatments with three replicates were established in randomized plots, low manure treatment (LM), high manure treatment (HM), and high manure and lime treatment (ML). Nothing was applied in the control (CK). The organic manure was fresh pig dejecta from hoggery. the soil microbial diversity was investigated by the traditional cultural method, and DGGE molecular approach. The soil quality and microbial diversity evaluated.The diversity of microbial community has been investigated in different manure fertility treatments by the traditional cultural method and DGGE molecular approach. The quality of extracted DNA had remarkably positive correlation with the amount of CFU by statistical analysis; the diversity of microbial community had negative correlation with the amount of the organic manure adding in red soil. It was suggested that much attention should be paid and the rsourses of the microbial diversity should be prorected for long-term using organic manure in barren red soil. The structure of microbial community transformed with the different amount of the organic manure adding to red soil from the analysis of the phylogenetic tree.3. Molecular Diversity of Nitrogen-Fixing microbes in red soilIn order to understand the community structure of Nitrogen-Fixing microbes in red soil and effects of organic manure application on the structure, four nifH gene libraries were constructed (CK, LM, HM, and ML). Total150nifH gene clones were screened and grouped into21clusters of RPLP analysis. Existence of dominant patterns was observed in all libraries, which counted for over96%of clones in library HM and about56-72%in other three libraries. Representative nifH clones were sequenced and showed to be similar to nitrogenase reductase (about73-100%of similarity). These nifH clones showed very high diversity, dispersing throughout the nifH clade (alpha-, beta-and gamma Proteobacteria, Firmicutes cyanobacteri, Verrucomicrobia, and posited group). The nifH sequences of the dominant patterns in all libraries were most similar to sequences of the cyanobacteria Bradyrhizobium and Burkholderia were also important diazotrophs in low fertility soils. nifH genes in LM treatment had the highest diversity while HM had the lowest. Manure and lime treatment led to obvious community succession. High available P to total N ratio was the main reason that affected the diversity of diazotrophs in red soil. pH played important role in the community changes of cyanobacteria. Cyanobacteria diversity was positively related to the soil pH. Considering the functions of cyanobacteria in paddy soils and the climate in this test site, cyanobacteria might also play important roles under unflooded agronomy condition.4. Molecular Diversity of Ammonia-Oxidizing Bacteria in red soilIn order to understand the community structure of ammonia-oxidizing bacteria in red soil and effects of organic manure application on the structure, four amoA gene libraries were constructed (CK, LM, HM, and ML). Total206amoA gene clones were screened and grouped into20clusters of RFLP analysis. Existence of many dominant patterns was observed in all libraries, the coverage C of the four amoA gene libraries was so high (above90%) and the similarity of the four libraries was also high (above57%). Phylogenetic analysis suggested that the representative ammonia-oxidizing bacteria were only the cluster of Nitrosomonas; this was consistent with many other studies suggesting the dominance of Nitrosospira in soils. In a word, there was no remarkable effect on the community of ammonia-oxidizing bacteria adding organic manure in barren red soil.5. Molecular Diversity of Denitrifying bacteria in red soilIn order to understand the community structure of denitrifying bacteria in red soil and effects of organic manure application on the structure, four nirK gene libraries were constructed (CK, LM, HM, and ML). Total282nirK gene clones were screened and grouped into78clusters of RFLP analysis. All statistical results showed that microbial diversity of all nirK gene libraries was rich and the coverage C of all nirK gene libraries was high (above80%), four nirK gene libraries could represent the main community of denitrifying bacteria in red soil ecosystem. Existence of dominant patterns was observed in all libraries, the pairwise similarity of four libraries was about40%. The diversity of denitrifying bacteria in CK treatment was the lowest but the diversity of denitrifying bacteria in HM treatment was the highest. The diversity of denitrifying bacteria in red soil was richer with the increase of adding organic manure in to the red soil. It suggested that the organic manure must be scientifically used in red soil ecosystem to avoid so much lost of the N fertility from soil.