本文關(guān)鍵詞：民勤退耕區(qū)次生草地土壤微生物多樣性研究及優(yōu)勢(shì)植物根際促生菌資源篩選　出處：《甘肅農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 民勤退耕區(qū) 土壤微生物多樣性 根際促生菌(PGPR) 高通量測(cè)序

【摘要】：土壤微生物是陸地生態(tài)系統(tǒng)的重要組成部分,在土壤的形成與發(fā)育、有機(jī)質(zhì)轉(zhuǎn)化、生態(tài)系統(tǒng)平衡、土壤環(huán)境凈化與生物修復(fù)等方面起著重要的作用。土壤微生物對(duì)環(huán)境的作用主要通過群落代謝功能差異來實(shí)現(xiàn)。微生物多樣性的變化能較早地反映土壤質(zhì)量的變化過程,對(duì)生態(tài)環(huán)境演變也有重要的指示作用。因此,明確不同環(huán)境中微生物群落的作用、深入了解其群落組成和結(jié)構(gòu)變化對(duì)于整個(gè)生態(tài)系統(tǒng)的研究具有重要意義。石羊河流域在我國生態(tài)安全中占有重要地位。近幾十年來,受到干旱、沙漠化及人為因素影響,該區(qū)域出現(xiàn)大面積耕地撂荒,形成草原向荒漠過渡的次生草地,生態(tài)系統(tǒng)瀕臨崩潰,已引起國家高度重視。前人從不同角度對(duì)生態(tài)修復(fù)進(jìn)行了研究,但尚缺乏有關(guān)微生物方面的研究。本研究以此為切入點(diǎn),對(duì)該區(qū)域土壤微生物多樣性及優(yōu)勢(shì)植物根際促生菌進(jìn)行研究,為改善并修復(fù)該區(qū)域退耕區(qū)次生草地生態(tài)系統(tǒng)奠定理論和實(shí)踐基礎(chǔ)。本研究以石羊河流域中下游的民勤綠洲不同退耕年限(1a、5a、10a)次生草地和從未耕種的原生地土壤及其優(yōu)勢(shì)植物為研究對(duì)象。采用時(shí)空替代法選擇四個(gè)不同年限的樣地,從不同土壤層(0~20 cm、20~40 cm)取樣,利用高通量測(cè)序技術(shù)分別對(duì)土壤細(xì)菌和真菌的多樣性進(jìn)行分析。同時(shí),選取四個(gè)樣地中的優(yōu)勢(shì)植物(鹽爪爪、駱駝蓬、梭草、鹽生草和黑果枸杞),采集植物根際(根系和土壤)樣品,通過分離、純化及篩選,獲得植物根際促生菌(PGPR),研究其促生特性,并對(duì)具有優(yōu)良促生特性的菌株進(jìn)行分子鑒定。獲得如下主要成果:1、土壤細(xì)菌多樣性研究表明:共獲得746879條優(yōu)化序列;各樣品OTU數(shù)量隨分類水平的延伸而減少,共有OTU數(shù)量達(dá)931個(gè);稀疏曲線中,退耕一年地OTU數(shù)量最多,原生地OTU數(shù)量最少。除原生地,其余年限樣品中0~20cm土壤層的OTU數(shù)量均高于20~40cm土壤層;各樣品豐度曲線跨度差異不明顯;多樣性指數(shù)計(jì)算發(fā)現(xiàn),退耕一年地(0~20cm)的多樣性指數(shù)最高,為ACE指數(shù)(2087)、Chao1指數(shù)(2087)和Shannon指數(shù)(9.17),即菌群豐富度和多樣性最高,其余各樣品豐富度和多樣性變化趨勢(shì)保持一致;PCA與聚類分析發(fā)現(xiàn),退耕五年地和十年地結(jié)構(gòu)相似性較高,而原生地與其他樣品結(jié)構(gòu)差異性較大,各年限樣品組間差異距離明顯大于組內(nèi)差異;各樣地在不同分類等級(jí)分別獲得196、616、834、1065、1094和349的菌群數(shù),且優(yōu)勢(shì)細(xì)菌種類基本相同,“門”的水平以放線菌門(Actinobacteria)、變形菌門(Proteobacteria)、綠彎菌門(Chloroflexi)、芽單胞菌門(Gemmatimonadetes)、酸桿菌門(Acidobacteria)、浮霉菌門(Planctomycetes)和擬桿門(Bacteroidetes)為主,“屬”的水平以浮霉?fàn)罹鷮?planctomycessp.)、芽孢桿菌屬(bacillussp.)、尤澤比氏菌屬(euzebyasp.)和鏈霉菌屬(streptomycessp.)為主;功能基因分析,各樣地共包含6大類功能基因(environmentalinformationprocessing、metabolism、cellularprocesses、geneticinformationprocessing、organismalsystemsandhumandiseases),具體功能達(dá)37種,其中,與metabolism相關(guān)的功能基因最多。2、土壤真菌多樣性研究表明:共獲得557313條優(yōu)化序列;各樣品共有otu數(shù)量達(dá)320個(gè),同一樣品otu數(shù)量在各分類水平中一樣;稀疏曲線中,退耕十年地的otu數(shù)量最多,除退耕一年地,其余年限20~40cm土壤層中otu數(shù)均高于0~20cm土壤層;退耕十年地的豐度曲線跨度最大,退耕五年地跨度最小;退耕十年地(20~40cm)的ace指數(shù)(509.57)、chao1指數(shù)(509.51)、simpson指數(shù)(0.98)和shannon指數(shù)(6.74)最高,即菌群豐富度和多樣性最高,其余各年限差異不大;pca結(jié)果發(fā)現(xiàn),退耕一年地與其他樣品組成差異性較大,且同一年限兩個(gè)樣品間差異性較小;四個(gè)年限土壤樣品聚類分析后為四類;各樣品組間差異大于組內(nèi)差異,且不同組間樣品組成差異性較小;各樣品在不同分類水平分別獲得76、154、374、545、824和1571個(gè)菌群數(shù),優(yōu)勢(shì)真菌菌種,“門”的水平以子囊菌門(ascomycota)、擔(dān)子菌門(basidiomycota)、接合菌門(zygomycota)和壺菌門(chytridiomycota)為主,其中,退耕一年地與其他樣品菌群豐度差異較大,原生地兩個(gè)土壤層中菌群組成有明顯差異,“屬”的水平主要假裸囊菌屬(pseudogymnoascussp.)、小囊菌屬(microascussp.)、支頂孢屬(acremoniumsp.)、青霉菌屬(penicilliumsp.)為主;各樣地均有特有菌種;優(yōu)勢(shì)屬關(guān)系網(wǎng)絡(luò)分析發(fā)現(xiàn),有五組優(yōu)勢(shì)屬出現(xiàn)一對(duì)一的協(xié)作關(guān)系,即rubinoboletussp.和phomasp.,aspergillussp.和micriascussp.,coniocessiasp.和solorinasp.,mortierllasp.和acremoniumsp.,chaetomiumsp.和monosporascussp.。3、根際促生菌(pgpr)進(jìn)行分離、篩選:從研究區(qū)5種優(yōu)勢(shì)植物共獲得菌株320株,其中固氮菌115株,溶解無機(jī)磷菌株107株,溶解有機(jī)磷菌株98株。pgpr菌株數(shù)量呈現(xiàn)出“根系表面根表土壤根內(nèi)”分布特征。4、根際促生菌(pgpr)促生特性研究表明:獲得具有較高固氮酶活性pgpr菌株有39株,固氮酶活性在23.13~559.68nmolc2h4·h-1ml-1之間(20株大于300.00nmolc2h4·h-1ml-1),分離自鹽生草的菌株nchp1固氮酶活性最高;溶解無機(jī)磷的菌株50株,溶磷量在19.55~574.40μg·ml-1之間(22株高于300.00μg·ml-1,占總數(shù)44%),分離自黑果枸杞的菌株ndrp2溶磷量最高;溶解有機(jī)磷的菌株37株,溶磷量含量變化較大(27.79~429.39μg·ml-1),溶磷量高于300.00μg·ml-1的菌株共10株,占總數(shù)的27.03%,分離自黑果枸杞的菌株NDRS1溶磷量最高。5、優(yōu)良根際促生菌(PGPR)分子鑒定表明:21株優(yōu)良根際促生菌(PGPR)分別屬于4個(gè)屬7個(gè)種,結(jié)合鑒定結(jié)果綜合考慮其他特性,發(fā)現(xiàn)15株具有進(jìn)一步開發(fā)潛力,即:短小芽胞桿菌(Bacillus pumilus)5株、枯草芽孢桿菌(Bacillus subtilis)6株、深紅沙雷氏菌(Serratia rubidaea)2株、暹羅芽孢桿菌(Bacillus siamensis)和蠟樣芽胞桿菌(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).

【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S812
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本文編號(hào)：1410159