玉米和大豆根内生细菌多样性及促生细菌鉴定评价

发布时间：2017-12-27 18:34

本文关键词：玉米和大豆根内生细菌多样性及促生细菌鉴定评价　出处：《东北农业大学》2016年博士论文　论文类型：学位论文

【摘要】：内生细菌是植物微生态系统的重要组成部分,在不同植物器官内分布数量存在差异,其中,根内生细菌数量远远超过其它部位,具有寄主植物多样性和种属多样性特征,且群落结构受环境因素影响。本研究以中国科学院东北地理与农业生态研究所水土保持监测研究站在黑龙江省海伦市光荣村建立的玉米(Zea mays cv.,新垦5号)和大豆(Glycine max cv.,东生1号)轮作的长期定位试验地为平台,研究对象为不同施肥方式与不同侵蚀条件下大豆和玉米根内生细菌,采用Locked Nucleic Acid(LNA)oligonucleotide-PCR Clamping(LNAPCR)和454高通量测序方法,比较黑土区大豆和玉米根内生细菌群落结构差异;探讨在表层土壤剥离条件下,施肥方式对根内生细菌多样性和群落结构的影响,揭示作物种类、土壤侵蚀和施肥管理与根内生细菌的关系,并建立了一种基于LNA-PCR和454高通量测序技术研究内生细菌多样性的新方法,同时采用传统可培养手段筛选植物内生促生细菌,研究其促生效果,为促生菌种资源开发提供材料。主要研究结果如下:(1)采用LNA寡核苷酸引物对玉米和大豆根内生细菌DNA进行PCR扩增,以未加LNA寡核苷酸为对照,454高通量测序结果显示,采用LNA扩增的植物样品中测得的基因序列基本上来源于内生细菌,而在玉米和大豆中,未采用LNA的样品测序结果中来源于植物细胞器的序列所占比例分别达97.4%和67.8%;并且,采用LNA扩增的样品比未加LNA扩增的样品具有较高的OTU数量、ACE、Chao1和Shannon指数及较低的Simpson指数,表明采用LNA扩增的样品内生细菌群落结构多样性较高,改进了植物内生细菌多样性研究方法。(2)采用LNA-PCR和454高通量测序方法比较大豆和玉米根内生细菌多样性差异,研究发现假单胞菌属(Pseudomonas)、慢生根瘤菌属(Bradyrhizobium)和黄杆菌属(Flavobacterium)是大豆内生细菌的主要成员,而链霉菌属(Streptomyces)和Niastella是玉米内生细菌的主要成员,结果中还发现一些数目较少和不可培养的内生细菌,进一步说明大豆和玉米根内生细菌群落结构多样性丰富。(3)采用LNA-PCR和454高通量测序方法分析土壤表层剥离30cm和施用有机肥的玉米苗期根内生细菌群落结构,并以表层土未剥离和单施化肥处理为对照。结果共获得37820条16S r RNA有效序列,主要分布在4个门,35个纲,214个属和782个OTU,其中,变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和拟杆菌门(Bacteroidetes)为优势菌门,但不同样品中分布比例存在差异。内生细菌群落α多样性指数分析发现,土壤侵蚀降低了玉米苗期根内生细菌群落多样性,但在表层黑土剥离和未剥离条件下,增施有机肥均可以增加根内生细菌群落多样性,这种作用以表层土剥离30 cm处理表现得更为突出。(4)采用传统梯度稀释平板分离法测定不同土壤剥离、施肥方式及采样时期下大豆和玉米根部可培养内生细菌数量,结果表明,1/10 NA 30°C培养7 d后,不同样品根内生细菌数量不同,在0.79×104~(-1)9.95×104 CFU g~(-1)·fresh·weight之间,并发现在大豆和玉米根内,7月10日样品可培养内生细菌数量均明显高于6月22日和8月24日样品,大部分剥离样品内生细菌数量小于相同施肥条件下的未剥离样品。(5)采用可培养方法分别从大豆和玉米根部获得119株和277株内生细菌,发现其中39.6%的内生细菌具有分泌IAA能力,分泌能力在1-23 mg·L~(-1)之间,对其中分泌IAA能力大于10 mg·L~(-1)的14株内生细菌进行16S r RNA基因鉴定和潜在促生能力评价。16S r RNA基因鉴定结果证实14株内生细菌分别属于嗜冷芽孢杆菌属(Psychrobacillus)、微杆菌属(Microbacterium)、赖氨酸芽孢杆菌属(Lysinibacillus)和芽孢杆菌属(Bacillus),盆栽回接试验证实大部分菌株具有促生能力但促生效果不同,其中,菌株C4和C9(分别属于Microbacterium和Lysinibacillus)对大豆和小麦的促生效果较好。(6)采用平板对峙培养法,从获得的396株内生细菌中筛选出12株具有拮抗作用的细菌,对大豆立枯丝核菌(Rhizoctonia solani)表现出明显拮抗作用,抑菌圈半径在1.4 cm-3.5 cm之间。这12株内生细菌对黄瓜尖孢镰刀菌(Fusarium oxysporum f.sp.cucumerinum)和番茄枯萎病菌(Fusarium oxgsporum f.sp.lycopersici)均具有拮抗作用,16S rRNA基因鉴定结果证实12株内生拮抗细菌分别属于芽孢杆菌属(Bacillus)、类芽孢杆菌属(Paenibacillus)和假单胞菌属(Pseudomonas)。选取5株进行盆栽回接试验,结果表明其均对大豆根腐病具有一定的防治效果,同时能够促进大豆幼苗生长。(7)研究发现分泌IAA内生细菌C9可能为一株潜在新种细菌,故采用多相分类学方法对其进行分类地位确定。16S r RNA基因序列分析显示菌株C9~T属于赖氨酸芽孢杆菌属(Lysinibacillus),与Lysinibacillus chungkukjangi NBRC 108948~T(序列相似性98.1%)和Lysinibacillus sinduriensis DSM 27595~T(序列相似性98.0%)的16S r RNA基因序列相似性最高,与Lysinibacillus sp.其它典型菌株相似性均低于98%。通过neighbour-joining、maximum-parsimony和maximum-likelihood 3种方法将菌株C9~T与Lysinibacillus sp.的典型菌株构建系统发育树,发现3种方法得到的拓扑结构基本一致,菌株C9~T与Lysinibacillus sp.的各成员聚在一起。通过形态学特征、生理生化特征、细胞化学组分分析及分子学鉴定确定菌株C9~T属于Lysinibacillus sp.的一个新种,命名为Lysinibacillus endophyticus,典型菌株为C9~T,分别在中国普通微生物菌种保藏中心(CGMCC 1.15291~T)和德国微生物和细胞培养物保藏中心(DSM 100506~T)进行保藏。
[Abstract]:Endophytic bacteria are an important part of micro ecosystem of plants, the number of differences, distribution in different plant organs in which the number of root endophytic bacteria far more than other parts, with host plant diversity and species diversity, community structure and environmental factors influence. In this study, China Academy of Sciences, Northeast Institute of geography and agricultural ecology study on Soil and water conservation monitoring station was established in the village of Helen glorious city of Heilongjiang province maize (Zea mays cv., the newly reclaimed 5) and soybean (Glycine Max cv., Dongsheng No. 1) long-term experiment for the rotation platform, the research object for different fertilization and under different erosion conditions of soybean and maize root endophytic bacteria, using Locked Nucleic Acid (LNA) oligonucleotide-PCR Clamping (LNAPCR) and 454 high throughput sequencing method, comparison of black soil area of soybean and maize root endophytic bacteria community structure difference; on the surface soil peeling conditions, fertilization effect on bacterial diversity and on the community structure of the root, to reveal the relationship between soil erosion and crop species, fertilization management and root endophytic bacteria, and the establishment of a research and LNA-PCR 454 high-throughput sequencing technology based on the diversity of Endophytic Bacteria The new method, at the same time, uses the traditional culture method to screen the endophytic bacteria of plant, study the effect of promoting the growth, and provide materials for the development of the resources for the growth of the bacteria. The main results are as follows: (1) using LNA DNA oligonucleotide primers for PCR amplification of endophytic bacteria in the roots of corn and soybeans, by not adding LNA oligonucleotide as control, 454 high throughput sequencing results showed that the gene sequence of LNA was measured in plant samples basically comes from endophytic bacteria in maize and in the soybean sequence without using the sequencing results of LNA samples in plant organelles accounted for 97.4% and 67.8% respectively; and amplified by LNA samples is higher than that without LNA amplification of the sample number of OTU, ACE, Chao1 and Shannon index and lower Simpson index showed that amplified by LNA samples of endophytic bacterial diversity is higher, improved the bacterial diversity of Endophytic method. (2) LNA-PCR and 454 using high-throughput sequencing methods of soybean and maize root endophytic bacterial diversity differences, the study found that Pseudomonas sp. (Pseudomonas), Bradyrhizobium sp. (Bradyrhizobium) and yellow (Flavobacterium) is the main member of Endophytic Bacteria of soybean, and Streptomyces (Streptomyces) and Niastella is the main member of Endophytic Bacteria in maize, also found some small number and non culturable endophytic bacteria results in further description of soybean and maize root endophytic bacteria community structure diversity. (3) LNA-PCR and 454 high flux sequencing method were used to analyze the endophytic bacterial community structure of maize root exfoliated 30cm and organic manure during the seedling stage. Results of the 37820 16S R RNA sequences were obtained, mainly distributed in 4 phyla, 35 classes, 214 genera and 782 OTU, among them, Proteobacteria (Proteobacteria), Firmicutes (Firmicutes), actinobacteria (Actinobacteria) and Bacteroidetes (Bacteroidetes) for advantage from gate, but there are differences in the distribution proportion in different samples. Endophytic bacteria community diversity index analysis showed that soil erosion reduced maize seedling root endophytic bacterial community diversity, but in black topsoil stripping and non stripping conditions, organic fertilizer can increase the root endophytic bacterial community diversity, the role in topsoil stripping of 30 cm more prominent. (4) using the traditional gradient dilution plate separation method for the determination of different soil stripping, fertilization methods and sampling period of soybean and maize root number, endophytic bacteria culture results showed that 1/10 NA 30 C after 7 d culture, different samples of root endophytic bacteria in different quantity, 0.79 * 9.95 * 104 104~ (-1) CFU g~ (-1) between fresh and weight, found in soybean and maize roots in July 10th, number of training samples can be endophytic bacteria were significantly higher than that in June 22nd and August 24th samples, most stripped samples of endophytic bacteria quantity is less than the same fertilization conditions without stripping samples. (5) the culture methods respectively from soybean and maize roots obtained 119 strains and 277 strains of endophytic bacteria, found that 39.6% endophytic bacteria can secrete IAA, Mg and L~ in 1-23 secretion (-1), the ability of secreting IAA more than 10 mg - L~ (-1) were evaluated by 16S r identification of RNA gene and potential growth promoting ability of endophytic bacteria in 14 strains. 16S r confirmed that RNA gene identification results of 14 strains of endophytic bacteria were psychrotrophic bacillus (Psychrobacillus), Microbacterium sp. (Microbacterium), lysine (Lysinibacillus) and Bacillus bacillus (Bacillus), pot inoculation experiment confirmed that most strains with growth promoting ability but the growth promoting effect of different. Among them, the strains of C4 and C9 (belong to Microbacterium and Lysinibacillus) of soybean and wheat growth promoting effect. (6) using plate confrontation culture, 12 antagonistic bacteria were screened from 396 endophytic bacteria obtained, showing obvious antagonism against Rhizoctonia solani, and the radius of inhibition zone is between 1.4 cm-3.5 cm. The 12 strains of endophytic bacteria on Cucumber Fusarium oxysporum (Fusarium oxysporum f.sp.cucumerinum) and Fusarium oxysporum (Fusarium oxgsporum f.sp.lycopersici) has antagonistic effect, 16S rRNA confirmed that the gene identification results of 12 strains of endophytic bacteria belonging to the genus Bacillus (Bacillus), Bacillus (Paenibacillus) and Pseudomonas (Pseudomonas). 5 plants were selected for pot grafting test, the results showed that all of them had certain control effect on soybean root rot disease, and could promote the growth of soybean seedlings. (7) the study found that the secretion of IAA endophytic bacteria C9 may be a potential new species of bacteria, so the classification status of the bacteria was determined by multiphase taxonomy. 16S R
【学位授予单位】：东北农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S513;S565.1

【相似文献】