16S rRNA基因高通量测序方法检测奶牛场常用干草表面微生物群落结构及多样性

【目的】随着中国奶牛业的发展,干草需求量与日俱增。作为天然牧草,干草可以成为家畜传播病原体的载体。以干草表面附着物为研究对象,了解干草中细菌群落结构以及致病菌属特征。【方法】对来自6个不同奶牛场饲草舍的干草样本,应用Illumina Mi Seq高通量测序技术测定干草表面附着物细菌的16S r RNA基因V3-V4变异区序列,分析不同干草样本细菌群落组成。【结果】干草样本中的细菌在97%的相似水平下共得到OTU个数为15 416,涵盖了29门87纲144目219科323属的细菌。微生物多样性分析表明,干草样本具有很高的细菌多样性,不同样本多样性存在差异。对干草样本菌群中丰度较高的14种病原菌属进行分析,发现相较于人工种植牧草制备的干草,天然牧草制备的干草中病原菌属丰度较高。【结论】研究解析了干草样本中微生物的多样性、丰度及主要病原菌属的特征,对奶牛场疾病防控有一定指导意义。     

青枯病与黑胫病混发烟株发病茎秆组织微生物群落结构与多样性

为了解青枯病与黑胫病混发烟株茎秆组织的微生物菌群组成,该文采用Illumina Miseq高通量测序技术研究了青枯病与黑胫病混发烟株发病茎秆和健康烟株未发病茎秆组织的真菌、细菌群落结构与多样性.结果表明:(1)发病茎秆组织中真菌群落丰富度与多样性较健康茎秆组织低,细菌群落丰富度与多样性较健康茎秆组织高.(2)健康茎秆组...     

健康与感染青枯病烟株根际土壤与茎秆细菌群落结构与多样性

【目的】了解健康烟株与感染青枯病烟株在根际土壤、茎杆发病部位、茎杆病健交界部位以及未发病茎杆的细菌群落结构与多样性。【方法】分别对土壤与茎杆样品中细菌的16S rRNA基因V3-V4区进行扩增,采用Illumina MiSeq测序技术对扩增片段进行高通量测序,然后对健康烟株与感染青枯病烟株不同部位细菌群落结构与多样性进行分析。【结果】感染青枯病烟株发病茎杆及根际土壤的细菌群落多样性高于健康烟株茎杆及其根际土壤样品,病健交界茎杆样品细菌群落多样性低于健康烟株。变形菌门(Proteobacteria)在所有样品中均为优势菌门;所有烟株根际土壤的优势菌门为拟杆菌门(Bacteroidetes)、酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)和绿弯菌门(Chloroflexi);健康烟株茎杆部位的优势菌门为蓝细菌门(Cyanobacteria);感染青枯病烟株发病茎杆和病健交界茎杆部位的优势菌门为蓝细菌门(Cyanobacteria)和厚壁菌门(Firmicutes)。所有根际土壤样品的优势菌属为劳尔氏菌属(Ralstonia)、假单胞菌属(Pseudomonas)、鞘脂单胞菌属(Sphingomonas)、黄杆菌属(Flavobacterium)和代尔夫特菌属(Delftia),而感染青枯病烟株根际土壤的劳尔氏菌属(Ralstonia)和假单胞菌属(Pseudomonas)相对丰度显著高于健康烟株根际土壤,鞘脂单胞菌属相对丰度显著低于健康烟株根际土壤。烟株茎杆的优势菌属为劳尔氏菌属和假单胞菌属等。感染青枯病烟株病健交界茎杆中劳尔氏菌属、肠杆菌属(Enterobacter)和泛菌属(Pantoea)相对丰度显著低于健康烟株样品。【结论】健康与感染青枯病烟株茎杆样品细菌群落的丰富度和多样性明显低于相应的根际土壤样品。较健康烟株而言,感染青枯病烟株根际土壤和茎杆样品细菌群落丰富度和多样性均表现出不同程度地增加,且根际土壤细菌群落结构变化较茎杆样品明显,而病健交界茎杆样品细菌群落丰富度和多样性降低。烟草青枯病为典型土传病害,其病原茄科劳尔氏菌尽管能在烟株维管束中蔓延扩增,但主要还是分布于土壤中;它的存在似乎对土壤细菌群落的影响大于茎杆样品的。该研究结果提示对于青枯病的防治不能局限于烟株本身,田间土壤也应加大防治力度。     

烟草种子内生细菌群落结构与多样性

【目的】为了解烟草种子内生细菌的群落结构和多样性。【方法】分别对3个品种(K326、云烟85、云烟87)烟草种子内生细菌的16S rRNA基因V3–V4区进行扩增,采用Illumina MiSeq测序技术对扩增片段进行高通量测序,并对3个品种烟草种子内生细菌群落结构和多样性进行分析。【结果】3个品种种子共获得的V3–V4区高质量序列片段128558条,Shannon指数计算为2.03–3.73,K326和云烟85内生菌多样性指数高于云烟87。3品种烟草种子内生细菌的优势门均为变形菌门Proteobacteria、放线菌门Actinobacteria、厚壁菌门Firmicute和拟杆菌门Bacteroidete。3个品种烟草种子内生细菌共有菌属共有27个,K326和云烟85的最优势菌属为假单胞菌属(Pseudomonas),云烟87的最优势菌属为大肠杆菌志贺菌属(Escherichia-shigella)。16S功能预测显示各种子中产生了丰度较高的蛋白质、核苷酸、糖类、辅酶及代谢产物合成的有益功能信息。【结论】烟草种子内生细菌多样性丰富,不同品种种子细菌群落组成基本相似,其丰度存在一定差异性。种子中存在的潜在有益细菌包括假单胞菌、类芽孢杆菌、根瘤菌、马赛菌、藤黄单胞菌、萨勒河菌、Lelliottia菌等,具有大量代谢相关的有益功能。研究结果为今后烟草种子内生菌的功能研究和利用以及种子病害生物防控提供参考信息。     

河南宝天曼不同林龄与林型森林土壤的细菌群落结构与多样性

采用高通量16S rRNA标签测序法,比较了地处北亚热带与暖温带过渡带的宝天曼自然保护区不同林龄与林分类型的土壤细菌群落结构及多样性.结果表明： 宝天曼森林土壤细菌以变形菌门(29%)、酸杆菌门(18.5%)、疣微菌门(10%)等为主,共检测到60门1209属,优势属主要有疣微菌门的DA101(6.3%)、酸杆菌门的Acidobacteria 2(5.9%)和Candidatus Solibacter(2.9%)、泉古菌门的Candidatus Nitrososphaera(2.6%)等.不同林龄和林分类型土壤分别具有特有的种属组成及高丰度和低丰度种属.林龄与林分类型都对土壤微生物群落结构影响显著,且林分类型的影响大于林龄.80年林龄的锐齿栎土壤菌群多样性在不同林龄和林分类型中均最低.pH、土壤全氮、有机碳等是不同林龄及林分类型下土壤菌群结构变化的重要影响因子.     

基于高通量测序的黄河三角洲4种人工林土壤细菌结构及多样性研究

为研究黄河三角洲不同人工林土壤细菌群落特征,应用高通量测序技术,比较分析了刺槐、榆树、白蜡、臭椿4种人工林土壤细菌结构及多样性,并结合土壤理化性质进行相关性分析。结果表明:人工林土壤中共有31门细菌;4种人工林土壤中酸杆菌门、变形菌门、放线菌门细菌以及刺槐、臭椿人工林土壤中硝化螺旋菌门细菌是土壤中的优势群落。不同人工林土壤中酸杆菌门、变形菌门、硝化螺旋菌门、芽单胞菌门、拟杆菌门、广古菌门、泉古菌门、蓝藻菌门细菌丰度差异显著。刺槐人工林土壤细菌多样性最高;白蜡人工林土壤细菌多样性最低。土壤pH、含水量、有机质含量与酸杆菌门细菌丰度呈显著负相关关系,土壤pH与变形菌门、硝化螺旋菌门、芽单胞菌门细菌丰度呈极显著正相关关系;土壤pH、有效磷含量与人工林细菌多样性呈显著正相关关系。以上研究结果表明,黄河三角洲不同人工林土壤细菌群落存在一定差异,土壤pH、含水量、有机质、有效磷含量是影响土壤细菌结构和多样性的主要土壤因素。     

Genetic diversity and population structure of Quercus fabri Hance in China revealed by genotyping‐by‐sequencing

Analysis of genetic diversity and population structure among Quercus fabri populations is essential for the conservation and utilization of Q. fabri resources. Here, the genetic diversity and structure of 158 individuals from 13 natural populations of Quercus fabri in China were analyzed using genotyping‐by‐sequencing (GBS). A total of 459,564 high‐quality single nucleotide polymorphisms (SNPs) were obtained after filtration for subsequent analysis. Genetic structure analysis revealed that these individuals can be clustered into two groups and the structure can be explained mainly by the geographic barrier, showed gene introgression from coastal to inland areas and high mountains could significantly hinder the mutual introgression of genes. Genetic diversity analysis indicated that the individual differences within groups are greater than the differences between the two groups. These results will help us better understand the genetic backgrounds of Q. fabri.     

黄河内蒙古段表层沉积物细菌多样性及群落结构类型

为掌握黄河内蒙古段表层沉积物微生物多样性、群落结构类型及其影响因素,采用高通量测序技术分析了6个采样点表层沉积物中的微生物多样性及群落结构。研究结果表明,黄河内蒙古段沉积物细菌丰度大小排序为乌拉特前旗(H3)老牛湾(H6)临河(H2)包头(H4)托县(H5)乌海(H1),微生物多样性排序为H6 H2 H5 H4 H3 H1,乌海沉积物中细菌丰度和微生物多样性都是最低的一个采样点。黄河内蒙古段表层沉积物中三大优势菌群分别为变形菌门(Proteobacteria,相对丰度32.39%)、绿弯菌门(Chloroflexi,13.25%)和拟杆菌门(Bacteroidetes,12.16%)。细菌群落丰度与环境因子之间的冗余分析结果显示,沉积物中总有机碳(TOC)、离子交换容量(CEC)和总磷(TP)、总氮(TN)等环境因子对黄河内蒙古段沉积物细菌群落分布影响较大,负相关系数分别为82.5%、80.1%、85.5%和85.2%;微生物多样性与环境理化因子相关性分析结果表明,黄河沉积物微生物多样性格局与对氮磷等营养物质的损耗有直接关系。     

Metabolic pathway of α‐ketoglutarate in Lactobacillus sanfranciscensis and Lactobacillus reuteri during sourdough fermentation

Aim: To identify metabolites of α‐ketoglutarate (α‐KG) in Lactobacillus sanfranciscensis and Lactobacillus reuteri in modified MRS and sourdough. Methods and Results: Lactobacillus sanfranciscensis and L. reuteri were grown with additional α‐KG in mMRS and in wheat sourdough. In mMRS, α‐KG was used as an electron acceptor and converted to 2‐hydroxyglutarate (2‐OHG) by both organisms. Production of 2‐OHG was identified by high performance liquid chromatography (HPLC) and confirmed by gas chromatography (GC). Crude cell extracts of L. sanfranciscensis and L. reuteri grown with or without α‐KG exhibited OHG dehydrogenase activity of 6·3 ± 0·3, 2·3 ± 0·9, 1·2 ± 0·2, and 1·1 ± 0·1 mmol l^?1 NADH (min x mg protein)^?1, respectively. The presence of phenylalanine and citrate in addition to α‐KG partially redirected the use of α‐KG from electron acceptor to amino group acceptor. In wheat sourdoughs, α‐KG was predominantly used as electron acceptor and converted to 2‐OHG. Conclusions: Lactobacillus sanfranciscensis and L. reuteri utilize α‐KG as electron acceptor. Alternative use of α‐KG as amino group acceptor occurs in the presence of abundant amino donors and citrate. Significance and Impact of the Study: The use of α‐KG as electron acceptor in heterofermentative lactobacilli impacts the formation of flavour volatiles through the transamination pathway.     

木屑添加对刺芹侧耳菌丝共生细菌多样性和群落结构的影响

细菌作为许多真菌的共生菌,能够有效地促进真菌的代谢生长,而细菌多样性及群落结构能够反映真菌的生长和利用营养物质的状况。本研究利用基于细菌16S rRNA 基因V3-V4区的高通量测序技术分析不同木屑用量对刺芹侧耳菌丝共生细菌群落结构和多样性的影响。结果表明: 5组样品共检测细菌25门52纲114目199科406属,主要的优势菌门为变形菌门(35.0%～85.9%)和厚壁菌门(6.5%～38.4%),优势菌属为不动杆菌属(14.8%～71.6%)和假单胞菌属(1.7%～22.3%)。相较于完全培养基,添加木屑能够提高刺芹侧耳菌丝中的细菌多样性,并使其中优势细菌10个门类、9个属类结构发生显著变化。刺芹侧耳菌丝在5 g木屑培养基上的菌丝生长速度最快,菌丝浓密,边缘整齐,长势优于其他几类培养基;假单胞菌属和乳酸菌属丰度及物种多样性在5 g木屑培养基上都具有一定的优势,且假单胞菌属和乳酸菌属丰度与菌丝长势具有显著的正相关性。木屑作为重要碳源之一,对刺芹侧耳生长发育及其共生细菌群落结构和多样性都有显著影响,这为进一步探索木屑及共生细菌对刺芹侧耳生长发育影响的机理研究奠定了基础。     

Long‐term nitrogen fertilization decreases bacterial diversity and favors the growth of Actinobacteria and Proteobacteria in agro‐ecosystems across the globe

Long‐term elevated nitrogen (N) input from anthropogenic sources may cause soil acidification and decrease crop yield, yet the response of the belowground microbial community to long‐term N input alone or in combination with phosphorus (P) and potassium (K) is poorly understood. We explored the effect of long‐term N and NPK fertilization on soil bacterial diversity and community composition using meta‐analysis of a global dataset. Nitrogen fertilization decreased soil pH, and increased soil organic carbon (C) and available N contents. Bacterial taxonomic diversity was decreased by N fertilization alone, but was increased by NPK fertilization. The effect of N fertilization on bacterial diversity varied with soil texture and water management, but was independent of crop type or N application rate. Changes in bacterial diversity were positively related to both soil pH and organic C content under N fertilization alone, but only to soil organic C under NPK fertilization. Microbial biomass C decreased with decreasing bacterial diversity under long‐term N fertilization. Nitrogen fertilization increased the relative abundance of Proteobacteria and Actinobacteria, but reduced the abundance of Acidobacteria, consistent with the general life history strategy theory for bacteria. The positive correlation between N application rate and the relative abundance of Actinobacteria indicates that increased N availability favored the growth of Actinobacteria. This first global analysis of long‐term N and NPK fertilization that differentially affects bacterial diversity and community composition provides a reference for nutrient management strategies for maintaining belowground microbial diversity in agro‐ecosystems worldwide.     

Seasonal diversity and dynamics of haptophytes in the Skagerrak,Norway, explored by high‐throughput sequencing

Microalgae in the division Haptophyta play key roles in the marine ecosystem and in global biogeochemical processes. Despite their ecological importance, knowledge on seasonal dynamics, community composition and abundance at the species level is limited due to their small cell size and few morphological features visible under the light microscope. Here, we present unique data on haptophyte seasonal diversity and dynamics from two annual cycles, with the taxonomic resolution and sampling depth obtained with high‐throughput sequencing. From outer Oslofjorden, S Norway, nano‐ and picoplanktonic samples were collected monthly for 2 years, and the haptophytes targeted by amplification of RNA/cDNA with Haptophyta‐specific 18S rDNA V4 primers. We obtained 156 operational taxonomic units (OTUs), from c. 400.000 454 pyrosequencing reads, after rigorous bioinformatic filtering and clustering at 99.5%. Most OTUs represented uncultured and/or not yet 18S rDNA‐sequenced species. Haptophyte OTU richness and community composition exhibited high temporal variation and significant yearly periodicity. Richness was highest in September–October (autumn) and lowest in April–May (spring). Some taxa were detected all year, such as Chrysochromulina simplex, Emiliania huxleyi and Phaeocystis cordata, whereas most calcifying coccolithophores only appeared from summer to early winter. We also revealed the seasonal dynamics of OTUs representing putative novel classes (clades HAP‐3–5) or orders (clades D, E, F). Season, light and temperature accounted for 29% of the variation in OTU composition. Residual variation may be related to biotic factors, such as competition and viral infection. This study provides new, in‐depth knowledge on seasonal diversity and dynamics of haptophytes in North Atlantic coastal waters.     

沙埋对干旱沙区真藓结皮层细菌群落结构和多样性的影响

作为干旱沙区常见干扰之一的沙埋显著影响着生物结皮的结构和功能,但其内在的生物学机理还不清楚。利用高通量测序技术,通过对0(对照)、0.5(浅层)、2和10mm(深层)沙埋处理后的腾格里沙漠东南缘沙坡头地区真藓(Bryum argenteum)结皮层细菌群落物种组成与丰度的测定,研究了沙埋对真藓结皮层细菌群落结构和多样性的影响。结果表明:(1)共检测到沙坡头地区真藓结皮层细菌38门106纲181属,以放线菌、变形菌、蓝藻、浮霉菌、拟杆菌和酸杆菌等为主(占细菌群落的78.4%—83.0%);(2)PCA分析表明沙埋导致该地区真藓结皮层细菌群落结构组成发生明显改变。无沙埋时,真藓结皮层细菌群落中相对丰度最高的是蓝藻(18.6%),随着沙埋厚度的增加,依次变为变形菌(21.5%,沙埋厚度0.5mm)、浮霉菌(21.5%,沙埋厚度2mm)和放线菌(23.3%,沙埋厚度10mm);浅层沙埋显著增加了真藓结皮层细菌群落中光合菌、固氮菌和产菌丝体细菌等关键功能菌的丰度,但深层沙埋降低了它们的丰度;(3)沙埋显著增加了真藓结皮层细菌群落多样性(P0.05)和物种丰富度(P0.05),0.5mm沙埋后的细菌群落丰富度指数最高,2mm沙埋后的结皮层细菌群落多样性指数最高。揭示了沙埋对干旱沙区真藓结皮层细菌群落结构与多样性的影响,为深入理解沙埋对沙区生物结皮结构和生态功能影响的生物学机制提供了一定的理论依据。