民勤退耕区次生草地土壤微生物多样性研究及优势植物根际促生菌资源筛选

发布时间：2018-01-11 15:35

  本文关键词：民勤退耕区次生草地土壤微生物多样性研究及优势植物根际促生菌资源筛选　出处：《甘肃农业大学》2017年博士论文　论文类型：学位论文

  更多相关文章： 民勤退耕区 土壤微生物多样性 根际促生菌(PGPR) 高通量测序

【摘要】：土壤微生物是陆地生态系统的重要组成部分,在土壤的形成与发育、有机质转化、生态系统平衡、土壤环境净化与生物修复等方面起着重要的作用。土壤微生物对环境的作用主要通过群落代谢功能差异来实现。微生物多样性的变化能较早地反映土壤质量的变化过程,对生态环境演变也有重要的指示作用。因此,明确不同环境中微生物群落的作用、深入了解其群落组成和结构变化对于整个生态系统的研究具有重要意义。石羊河流域在我国生态安全中占有重要地位。近几十年来,受到干旱、沙漠化及人为因素影响,该区域出现大面积耕地撂荒,形成草原向荒漠过渡的次生草地,生态系统濒临崩溃,已引起国家高度重视。前人从不同角度对生态修复进行了研究,但尚缺乏有关微生物方面的研究。本研究以此为切入点,对该区域土壤微生物多样性及优势植物根际促生菌进行研究,为改善并修复该区域退耕区次生草地生态系统奠定理论和实践基础。本研究以石羊河流域中下游的民勤绿洲不同退耕年限(1a、5a、10a)次生草地和从未耕种的原生地土壤及其优势植物为研究对象。采用时空替代法选择四个不同年限的样地,从不同土壤层(0~20 cm、20~40 cm)取样,利用高通量测序技术分别对土壤细菌和真菌的多样性进行分析。同时,选取四个样地中的优势植物(盐爪爪、骆驼蓬、梭草、盐生草和黑果枸杞),采集植物根际(根系和土壤)样品,通过分离、纯化及筛选,获得植物根际促生菌(PGPR),研究其促生特性,并对具有优良促生特性的菌株进行分子鉴定。获得如下主要成果:1、土壤细菌多样性研究表明:共获得746879条优化序列;各样品OTU数量随分类水平的延伸而减少,共有OTU数量达931个;稀疏曲线中,退耕一年地OTU数量最多,原生地OTU数量最少。除原生地,其余年限样品中0~20cm土壤层的OTU数量均高于20~40cm土壤层;各样品丰度曲线跨度差异不明显;多样性指数计算发现,退耕一年地(0~20cm)的多样性指数最高,为ACE指数(2087)、Chao1指数(2087)和Shannon指数(9.17),即菌群丰富度和多样性最高,其余各样品丰富度和多样性变化趋势保持一致;PCA与聚类分析发现,退耕五年地和十年地结构相似性较高,而原生地与其他样品结构差异性较大,各年限样品组间差异距离明显大于组内差异;各样地在不同分类等级分别获得196、616、834、1065、1094和349的菌群数,且优势细菌种类基本相同,“门”的水平以放线菌门(Actinobacteria)、变形菌门(Proteobacteria)、绿弯菌门(Chloroflexi)、芽单胞菌门(Gemmatimonadetes)、酸杆菌门(Acidobacteria)、浮霉菌门(Planctomycetes)和拟杆门(Bacteroidetes)为主,“属”的水平以浮霉状菌属(planctomycessp.)、芽孢杆菌属(bacillussp.)、尤泽比氏菌属(euzebyasp.)和链霉菌属(streptomycessp.)为主;功能基因分析,各样地共包含6大类功能基因(environmentalinformationprocessing、metabolism、cellularprocesses、geneticinformationprocessing、organismalsystemsandhumandiseases),具体功能达37种,其中,与metabolism相关的功能基因最多。2、土壤真菌多样性研究表明:共获得557313条优化序列;各样品共有otu数量达320个,同一样品otu数量在各分类水平中一样;稀疏曲线中,退耕十年地的otu数量最多,除退耕一年地,其余年限20~40cm土壤层中otu数均高于0~20cm土壤层;退耕十年地的丰度曲线跨度最大,退耕五年地跨度最小;退耕十年地(20~40cm)的ace指数(509.57)、chao1指数(509.51)、simpson指数(0.98)和shannon指数(6.74)最高,即菌群丰富度和多样性最高,其余各年限差异不大;pca结果发现,退耕一年地与其他样品组成差异性较大,且同一年限两个样品间差异性较小;四个年限土壤样品聚类分析后为四类;各样品组间差异大于组内差异,且不同组间样品组成差异性较小;各样品在不同分类水平分别获得76、154、374、545、824和1571个菌群数,优势真菌菌种,“门”的水平以子囊菌门(ascomycota)、担子菌门(basidiomycota)、接合菌门(zygomycota)和壶菌门(chytridiomycota)为主,其中,退耕一年地与其他样品菌群丰度差异较大,原生地两个土壤层中菌群组成有明显差异,“属”的水平主要假裸囊菌属(pseudogymnoascussp.)、小囊菌属(microascussp.)、支顶孢属(acremoniumsp.)、青霉菌属(penicilliumsp.)为主;各样地均有特有菌种;优势属关系网络分析发现,有五组优势属出现一对一的协作关系,即rubinoboletussp.和phomasp.,aspergillussp.和micriascussp.,coniocessiasp.和solorinasp.,mortierllasp.和acremoniumsp.,chaetomiumsp.和monosporascussp.。3、根际促生菌(pgpr)进行分离、筛选:从研究区5种优势植物共获得菌株320株,其中固氮菌115株,溶解无机磷菌株107株,溶解有机磷菌株98株。pgpr菌株数量呈现出“根系表面根表土壤根内”分布特征。4、根际促生菌(pgpr)促生特性研究表明:获得具有较高固氮酶活性pgpr菌株有39株,固氮酶活性在23.13~559.68nmolc2h4·h-1ml-1之间(20株大于300.00nmolc2h4·h-1ml-1),分离自盐生草的菌株nchp1固氮酶活性最高;溶解无机磷的菌株50株,溶磷量在19.55~574.40μg·ml-1之间(22株高于300.00μg·ml-1,占总数44%),分离自黑果枸杞的菌株ndrp2溶磷量最高;溶解有机磷的菌株37株,溶磷量含量变化较大(27.79~429.39μg·ml-1),溶磷量高于300.00μg·ml-1的菌株共10株,占总数的27.03%,分离自黑果枸杞的菌株NDRS1溶磷量最高。5、优良根际促生菌(PGPR)分子鉴定表明:21株优良根际促生菌(PGPR)分别属于4个属7个种,结合鉴定结果综合考虑其他特性,发现15株具有进一步开发潜力,即:短小芽胞杆菌(Bacillus pumilus)5株、枯草芽孢杆菌(Bacillus subtilis)6株、深红沙雷氏菌(Serratia rubidaea)2株、暹罗芽孢杆菌(Bacillus siamensis)和蜡样芽胞杆菌(Bacillus cereus)各1株。
[Abstract]:Soil microorganism is an important component of the terrestrial ecosystem, the formation and development of soil, organic matter transformation, ecological balance, soil environmental pollution plays an important role in bioremediation and so on. Effects of soil microbes on the environment mainly through community metabolic differences to achieve. Changes of microbial diversity. Can reflect the soil quality and the evolution of the ecological environment has important implications. Therefore, specific microbial communities in different environments, in-depth understanding of the community composition and structure change is of great significance for the study of the ecological system. The Shiyang River Basin occupies an important position in the ecological security of our country in recent decades. Come, by drought, desertification and human factors, the large area of arable land fallow, the formation of secondary grassland steppe to desert ecosystem is on the verge of transition. Collapse, has aroused national attention. Previous researches from different angles on the ecological restoration were studied, but there is still a lack of research related to microbe. This research as a starting point, the area of soil microbial diversity and dominant plant growth promoting rhizosphere of endophytic bacteria, to improve and repair the area of returning farmland to grassland ecosystem area secondary lay the theoretical and practical basis. Based on the lower reaches of the Shiyang River Basin in the Minqin oasis in different vegetation restoration years (1a, 5A, 10a) of plant secondary grassland and cultivated land soil and never native advantage as the research object. Using space-time substitution method to choose four different time plots from different soil layers (0~20 cm, 20~40, CM) sampling, using high-throughput sequencing respectively on the diversity of soil bacteria and fungi were analyzed. At the same time, dominant species in four plots were selected in (Kalidium, harmaline, shuttle grass, grass and salt Lycium ruthenicum), rhizosphere plants (roots and soil) samples, through the separation, purification and screening for plant growth promoting rhizobacteria (PGPR), to study the growth characteristics and the growth characteristics of the strains with excellent molecular identification. The main results are as follows: 1. Soil bacteria diversity research shows that a total of 746879 optimized sequence; extension number of each sample OTU with the classification level decreased, OTU total number of 931; sparse curve, the number of returning a year at most OTU, native to the number of at least OTU. In addition to the original, the number of OTU years 0~20cm soil layer in the sample the soil layer was higher than that of 20~40cm; the sample abundance curve span difference is not obvious; calculate diversity index for one year (0~20cm) the highest diversity index, ACE index (2087), Chao1 index (2087) and Shannon index (9.17), namely the flora richness and diversity is the highest, the rest The samples of richness and diversity variation trend is consistent; PCA with cluster analysis found that returning for five years and ten years of structural similarity is high, and the original and other samples structuredifferences, the age difference between groups was significantly greater than the sample distance between groups of 19661683410651094 and 349; the number of bacteria in different classification, and the dominant species of bacteria are basically the same, "door" level to actinobacteria (Actinobacteria) (Proteobacteria), Proteobacteria, Chloroflexi (Chloroflexi), gemmatimonadetes (Gemmatimonadetes), acidobacteria (Acidobacteria), planctomycetes (Planctomycetes) and the door bar (Bacteroidetes), belonging to the level of Planctomyces (planctomycessp.), Bacillus sp. (bacillussp.), Jo Ze Be S (euzebyasp.) and Streptomyces (streptomycessp.); gene function Analysis of a total of 6 major categories including functional genes (environmentalinformationprocessing, metabolism, cellularprocesses, geneticinformationprocessing, organismalsystemsandhumandiseases), the specific function of 37 kinds, among them, the function of genes associated with metabolism in the.2, the research of soil fungal diversity showed that we obtained 557313 samples of each optimization sequence; OTU number 320 the number of samples, the same as OTU in each classification level; sparse curve, the number of OTU to ten years at most, except to a year, the remaining life of 20~ 40cm soil layer in OTU were higher than that of 0~20cm soil layer; the abundance curve of the maximum span of ten years to five years, to the minimum span; for ten years (20~40cm) of the ace index (509.57), Chao1 index (509.51), Simpson index (0.98) and Shannon index (6.74) is the highest, flora richness and the highest diversity of the rest The age difference is small; the results of PCA showed that farmland a year with other sample differences and two samples of the same age difference among the smaller four years; analysis of soil samples clustered into four categories; each sample is greater than the differences between groups differences within groups and between different groups of sample composition differences small; each sample was obtained in 76154374545824 and 1571 the number of bacteria in different level of classification, the advantage of fungi, "door" level to Ascomycota, Basidiomycota (Ascomycota) (Basidiomycota) (Zygomycota), Zygomycota and Chytridiomycota (Chytridiomycota), which, to a year with the other samples of bacteria abundance differences, two native soil microflora have obvious difference, "is" false level gymnoascus (pseudogymnoascussp.), microascus (microascussp.), supporting genus (acremoniumsp.), Penicillium (P Enicilliumsp.); all kinds of special strains; network analysis found that there are five genera, dominant genera group cooperative one to one relationship, namely rubinoboletussp. and phomasp., aspergillussp. and micriascussp., coniocessiasp. and solorinasp., mortierllasp. and acremoniumsp., chaetomiumsp. and monosporascussp..3, promotingrhizobacteria (PGPR) were isolated and screened: 5 dominant species from the study area received a total of 320 strains, including 115 strains of nitrogen fixing bacteria, 107 strains of dissolved inorganic phosphorus, the amount of dissolved organic phosphorus of 98 strains of.Pgpr isolates showing a.4 distribution of the root surface in root surface soil in root, promotingrhizobacteria (PGPR) growth characteristics research shows that get high nitrogenase activity of strain PGPR 39 strains, nitrogenase activity between 23.13~559.68nmolc2h4 and h-1ml-1 (20 strains of more than 300.00nmolc2h4 - h-1ml-1), strain NC from salt grass HP1 highest nitrogenase activities; 50 strains of dissolved inorganic phosphorus, dissolved phosphorus content between 19.55~574.40 g and ml-1 (22 strains was higher than that of 300 g ml-1, accounting for 44% of the total), isolates ndrp2 from Lycium ruthenicum soluble phosphorus was the highest; 37 strains of dissolved organic phosphorus, dissolved phosphorus content changes high content (27.79~429.39 g ML-1), soluble phosphorus content is higher than 300 g and ml-1 10 strains, accounting for 27.03% of the total isolates of NDRS1 from Lycium ruthenicum soluble phosphorus was highest in.5, excellent promotingrhizobacteria (PGPR) molecular identification showed that 21 strains of fine root promoting students bacteria (PGPR) respectively belong to 4 genera and 7 species, with the identification results considering other characteristics, found 15 strains with further development potential, namely: Bacillus pumilus (Bacillus pumilus) and 5 strains of Bacillus subtilis (Bacillus subtilis) 6 strains of Serratia rubida (Serratia rubidaea) 2 strains in Siam bacillus (Bacillus siamensis) and wax 1 strains of Bacillus cereus (Bacillus cereus).

【学位授予单位】：甘肃农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S812
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本文编号：1410159