有机肥施用对红壤氮素循环微生物相关基因多样性的影响
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摘要
微生物的生态多样性在各种生态系统的功能和演变中都起着综合的和独特的作用,它对持续农业系统的作用为许多近期研究所证实。土壤微生物是自然物质循环不可缺少的成员,担负着分解动植物残体,直接关系到土壤养分的有效性和植物的生长,同时微生物在土壤肥力评价和生物净化等方面也有着重要的作用。土壤微生物对环境变化很敏感,能够较早地指示生态系统功能的变化,而且对于退化生态系统的恢复和治理以及提高不同植被的生产力均有重要的理论和实践意义。
红壤广泛的存在于我国南方,该地区也是重要的粮食和经济作物产区。但近年来,水土流失、土壤生物活性下降、肥力减退及环境污染等问题已严重影响红壤地区农业经济的可持续发展。土壤微生物是土壤生态系统的重要组成部分,在土壤生态平衡、物质循环和植物养分转换等过程中起着重要的作用。长久以来,人们都是利用传统的微生物学培养方法对土壤微生物进行研究,然而环境中仅有小于1%的微生物能被研究人员在实验室条件下培养,所以在自然环境中的微生物群落结构信息及其种群的检测成为长期困扰着微生物学家和生态学家的一大难题,微生物分子生物学技术的引入为认识和了解土壤环境中微生物的多样性和功能提供了新的契机。
本研究主要通过PCR扩增、基因克隆、DGGE、RFLP和序列分析等对红壤环境中的固氮酶基因nifH、氨单加氧酶酶基因amoA、反硝化亚硝酸还原酶酶基因nirK和16S rDNA的分子多样性进行了分析,探讨了四种不同处理(对照,低有机肥,高有机肥,高有机肥加石灰改良)下红壤中参与氮素循环相关的固氮微生物、氨氧化细菌和反硝化细菌等功能微生物类群与之对应发生的群落多样性变化信息。
(1)用于分子生态学研究的土壤微生物DNA提取方法的研究
土壤微生物分子生态学研究的关键技术之一是从各种环境样品中高效的获得可以进行分子生物学研究的基因组DNA,同时高质量的提取DNA是进行后续实验的基础。提取的环境样品总DNA能否代表环境中的微生物是首要前提。本研究利用SDS高盐法(Zhou's method)和变性剂加PVPP法(Raddy's method)两种方法对红壤中微生物总DNA进行了提取,然后通过电洗脱回收和树脂柱试剂盒回收两种方法进行纯化,结果表明改良的SDS高盐法对DNA提取效率明显高于其它两种方法。PCR扩增表明,改良的SDS高盐法从贫瘠的原始红壤中可以扩增到16SrDNA片段和固氮基因nifH、硝化基因αmoA、反硝化基因nirK等功能基因。因此认为改良的SDS高盐法是一种高效、可靠的适合于中国典型红壤下微生物分子生态学研究的DNA提取方法。
(2)施加有机肥处理下红壤中微生物数量和分子多样性的变化
通过传统的培养技术和现代分子生物方法探讨了红壤在施加有机肥处理下微生物群落多样性变化,为指导红壤生态系统下科学合理的发展农业生产保护红壤中微生物资源提供重要参考。通过DGGE方法和传统培养计数方法研究了不同肥力下红壤中微生物多样性变化,统计分析发现,四种处理中DNA的提取量与微生物的计数结果成显著的正相关,但是DGGE多样性指数显示随有机肥肥力的升高微生物多样性逐渐降低。系统发育分析显示高肥和高肥改良微生物类群聚为一支,CK与底肥处理下微生物类群聚为一支。说明土壤中微生物总数量随有机肥添加量的增加而增加,微生物的种类随有机肥的增加而减少。揭示了我们在提高红壤的肥力的同时要关注红壤中微生物类群整体的变化,科学合理的施用有机肥以保护红壤中微生物多样性资源。
(3)红壤中固氮微生物的分子多样性研究
研究了向红壤中投加有机氮肥对生态系统下固氮微生物群落结构的影响,通过PCR-RFLP和测序分析对四种不同处理下微生物固氮基因(nifH)的多样性和系统发育进行了研究分析。通过RFLP分析和多样性指数计算发现,四个文库都有比较高的覆盖值(88%以上),分布在alpha-, beta-和gamma Proteobacteria, Firmicutes, cyanobacter, Verrucomicrobia和假定未知一族,代表了环境中主要的固氮微生物类群。四种文库中的nifH基因的多样性都不均匀,都存在着绝对的优势类群,种类主要以自生固氮蓝细菌为主。实验室条件下难以分离培养,主要生活在海洋中的蓝细菌是旱地红壤中固氮微生物优势种群,这为研究红壤固氮微生物提供了重要参考信息。高肥力样品中多样性指数数值在四个处理中是最低的,而低肥力样品中的多样性指数数值则在四个处理中最高。传统的计数结果显示随添加有机肥数量的增加固氮细菌数量是增加的。说明在红壤中适当投加有机肥有利于提高固氮微生物的多样性,过高投加有机肥影响了固氮微生物群落种类的多样性。科学合理的投加有机肥有利于红壤生态系统的健康平衡发展。
(4)红壤中硝化细菌的分子多样性研究
研究了向贫瘠红壤中投加有机氮肥对红壤生态系统下硝化微生物群落结构的影响,通过PCR-RFLP和测序分析对四种不同处理下氨氧化细菌中氨单加氧酶基因(amoA)的多样性和系统发育进行了研究分析。通过RFLP分析和多样性指数计算发现,四个文库的覆盖值都很高(90%以上),可以代表了环境中绝大部分的氨氧化细菌。四种文库中的amoA基因的多样性均匀性不高,但文库之间都存在着共同的主要类群,主要以亚硝化螺菌为主。四个文库群落之间相似性比较高(57%以上)。MPN计数结果显示随投加有机肥量的增加氨氧化细菌数量是逐渐增加的。因此说明向贫瘠的红壤中施加有机肥和改良红壤有利于增加氨氧化细菌数量,对氨氧化细菌群落种类多样性的影响不明显。这也与红壤环境下氨氧化细菌自身种类少有一定的关系。
(5)红壤中反硝化细菌的分子多样性研究
研究了向贫瘠红壤中投加有机氮肥对红壤生态系统下反硝化微生物群落结构的影响,通过PCR-RFLP和测序分析对四种不同处理下微生物亚硝酸还原酶基因(nirK)的多样性和系统发育进行了研究分析。通过RFLP分析和多样性指数计算发现,四个文库都有比较高的覆盖值(80%以上),代表了环境中主要的反硝化微生物类群。四种文库中的nirK基因的多样性均匀性较高,而且也存在着主要类群的反硝化细菌,主要Alcaligenes faecalis细菌为主。四文库之间的相似性在40%左右。原始对照样品中多样性指数数值是四个处理中最低,而高肥力样品中的多样性指数数值则是四个处理中最高的。MPN计数结果显示随有机肥投加量的增加反硝化微生物数量成递增的趋势。说明在红壤中反硝化微生物的多样性是随投加有机肥量的增加而提高。揭示了要科学控制有机肥的投加量,减少土壤中有机氮肥的流失。
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 study
In 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 soil
To 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 soil
In 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 soil
In 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 soil
In 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.
红壤广泛的存在于我国南方,该地区也是重要的粮食和经济作物产区。但近年来,水土流失、土壤生物活性下降、肥力减退及环境污染等问题已严重影响红壤地区农业经济的可持续发展。土壤微生物是土壤生态系统的重要组成部分,在土壤生态平衡、物质循环和植物养分转换等过程中起着重要的作用。长久以来,人们都是利用传统的微生物学培养方法对土壤微生物进行研究,然而环境中仅有小于1%的微生物能被研究人员在实验室条件下培养,所以在自然环境中的微生物群落结构信息及其种群的检测成为长期困扰着微生物学家和生态学家的一大难题,微生物分子生物学技术的引入为认识和了解土壤环境中微生物的多样性和功能提供了新的契机。
本研究主要通过PCR扩增、基因克隆、DGGE、RFLP和序列分析等对红壤环境中的固氮酶基因nifH、氨单加氧酶酶基因amoA、反硝化亚硝酸还原酶酶基因nirK和16S rDNA的分子多样性进行了分析,探讨了四种不同处理(对照,低有机肥,高有机肥,高有机肥加石灰改良)下红壤中参与氮素循环相关的固氮微生物、氨氧化细菌和反硝化细菌等功能微生物类群与之对应发生的群落多样性变化信息。
(1)用于分子生态学研究的土壤微生物DNA提取方法的研究
土壤微生物分子生态学研究的关键技术之一是从各种环境样品中高效的获得可以进行分子生物学研究的基因组DNA,同时高质量的提取DNA是进行后续实验的基础。提取的环境样品总DNA能否代表环境中的微生物是首要前提。本研究利用SDS高盐法(Zhou's method)和变性剂加PVPP法(Raddy's method)两种方法对红壤中微生物总DNA进行了提取,然后通过电洗脱回收和树脂柱试剂盒回收两种方法进行纯化,结果表明改良的SDS高盐法对DNA提取效率明显高于其它两种方法。PCR扩增表明,改良的SDS高盐法从贫瘠的原始红壤中可以扩增到16SrDNA片段和固氮基因nifH、硝化基因αmoA、反硝化基因nirK等功能基因。因此认为改良的SDS高盐法是一种高效、可靠的适合于中国典型红壤下微生物分子生态学研究的DNA提取方法。
(2)施加有机肥处理下红壤中微生物数量和分子多样性的变化
通过传统的培养技术和现代分子生物方法探讨了红壤在施加有机肥处理下微生物群落多样性变化,为指导红壤生态系统下科学合理的发展农业生产保护红壤中微生物资源提供重要参考。通过DGGE方法和传统培养计数方法研究了不同肥力下红壤中微生物多样性变化,统计分析发现,四种处理中DNA的提取量与微生物的计数结果成显著的正相关,但是DGGE多样性指数显示随有机肥肥力的升高微生物多样性逐渐降低。系统发育分析显示高肥和高肥改良微生物类群聚为一支,CK与底肥处理下微生物类群聚为一支。说明土壤中微生物总数量随有机肥添加量的增加而增加,微生物的种类随有机肥的增加而减少。揭示了我们在提高红壤的肥力的同时要关注红壤中微生物类群整体的变化,科学合理的施用有机肥以保护红壤中微生物多样性资源。
(3)红壤中固氮微生物的分子多样性研究
研究了向红壤中投加有机氮肥对生态系统下固氮微生物群落结构的影响,通过PCR-RFLP和测序分析对四种不同处理下微生物固氮基因(nifH)的多样性和系统发育进行了研究分析。通过RFLP分析和多样性指数计算发现,四个文库都有比较高的覆盖值(88%以上),分布在alpha-, beta-和gamma Proteobacteria, Firmicutes, cyanobacter, Verrucomicrobia和假定未知一族,代表了环境中主要的固氮微生物类群。四种文库中的nifH基因的多样性都不均匀,都存在着绝对的优势类群,种类主要以自生固氮蓝细菌为主。实验室条件下难以分离培养,主要生活在海洋中的蓝细菌是旱地红壤中固氮微生物优势种群,这为研究红壤固氮微生物提供了重要参考信息。高肥力样品中多样性指数数值在四个处理中是最低的,而低肥力样品中的多样性指数数值则在四个处理中最高。传统的计数结果显示随添加有机肥数量的增加固氮细菌数量是增加的。说明在红壤中适当投加有机肥有利于提高固氮微生物的多样性,过高投加有机肥影响了固氮微生物群落种类的多样性。科学合理的投加有机肥有利于红壤生态系统的健康平衡发展。
(4)红壤中硝化细菌的分子多样性研究
研究了向贫瘠红壤中投加有机氮肥对红壤生态系统下硝化微生物群落结构的影响,通过PCR-RFLP和测序分析对四种不同处理下氨氧化细菌中氨单加氧酶基因(amoA)的多样性和系统发育进行了研究分析。通过RFLP分析和多样性指数计算发现,四个文库的覆盖值都很高(90%以上),可以代表了环境中绝大部分的氨氧化细菌。四种文库中的amoA基因的多样性均匀性不高,但文库之间都存在着共同的主要类群,主要以亚硝化螺菌为主。四个文库群落之间相似性比较高(57%以上)。MPN计数结果显示随投加有机肥量的增加氨氧化细菌数量是逐渐增加的。因此说明向贫瘠的红壤中施加有机肥和改良红壤有利于增加氨氧化细菌数量,对氨氧化细菌群落种类多样性的影响不明显。这也与红壤环境下氨氧化细菌自身种类少有一定的关系。
(5)红壤中反硝化细菌的分子多样性研究
研究了向贫瘠红壤中投加有机氮肥对红壤生态系统下反硝化微生物群落结构的影响,通过PCR-RFLP和测序分析对四种不同处理下微生物亚硝酸还原酶基因(nirK)的多样性和系统发育进行了研究分析。通过RFLP分析和多样性指数计算发现,四个文库都有比较高的覆盖值(80%以上),代表了环境中主要的反硝化微生物类群。四种文库中的nirK基因的多样性均匀性较高,而且也存在着主要类群的反硝化细菌,主要Alcaligenes faecalis细菌为主。四文库之间的相似性在40%左右。原始对照样品中多样性指数数值是四个处理中最低,而高肥力样品中的多样性指数数值则是四个处理中最高的。MPN计数结果显示随有机肥投加量的增加反硝化微生物数量成递增的趋势。说明在红壤中反硝化微生物的多样性是随投加有机肥量的增加而提高。揭示了要科学控制有机肥的投加量,减少土壤中有机氮肥的流失。
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 study
In 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 soil
To 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 soil
In 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 soil
In 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 soil
In 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.
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