喜树替代紫茎泽兰过程中根际微生物群落特征

为研究我国重要经济树种喜树根际分泌物对根际微生物群落的影响, 以及喜树在控制林业有害植物紫茎泽兰生物入侵的可行性, 采用传统培养技术和分子生物学技术PCR-单链构象多态性(SSCP)、末端限制性片段长度多态性(tRFLP)和16S rDNA文库相结合对喜树(Ca)、紫茎泽兰(Ea)和二者混栽体系(CE)根际土壤中的真核微生物和真细菌微生物群落结构进行了比较. 计数结果表明, 喜树、二者混栽以及紫茎泽兰根际中的真细菌数量依次减少, 而真核微生物数量依次增多. PCR-SSCP分析显示, 紫茎泽兰根际土壤中真核微生物条带数量(多样性)远高于喜树和混栽根际, 对部分条带克隆测序表明喜树根际中Meristolohmannia spp.为主要优势种群; tRFLP对真细菌结构分析表明, 3种根际中真细菌群落多样性丰富, 但没有差别, 对喜树根际真细菌16S rDNA文库测序比较分析, 共包含了10个已分类的门, 其中Proteobacteria 门为优势菌群, 占24.71%(其中δ-Proteobacteria占17.65%), Acidobacteria门占16.47%, Bacteroidetes门占10.59%. 另外, 色谱分析发现喜树和混栽根际土壤中分别含有较低浓度喜树碱和羟基喜树碱, 而紫茎泽兰根际二者均检测不到. 由此可见, 紫茎泽兰的扩散和蔓延依赖于特有的真核微生物群落结构模式, 并不改变真细菌群落的结构, 喜树能够通过根系分泌物改变紫茎泽兰根际真核微生物群落结构模式, 进而制约其外延. 本研究为喜树在紫茎泽兰生物替代中提供了理论基础.     

再生水补水对河道底泥细菌群落结构的影响

以北京市永定河麻峪湿地再生水补水口附近河道底泥为研究对象,应用末端限制性片段长度多态性(Terminal Restriction Fragment Length Polymorphism,T-RFLP)技术探究再生水补水口附近底泥细菌群落结构的空间差异特征以及环境因子所产生的影响,并借助典范对应分析(Canonical Correspondence Analysis,CCA)方法分析麻峪湿地底泥细菌群落结构空间差异特征的形成原因。分析结果表明:河道底泥微生物群落对再生水的净化主要发生在距补水口1200m的范围内,随着再生水与上游来水径向混合净化渐变过程,在补水口2000m处河道底泥微生物群落结构与河道再生水补水口上游趋于相似。基于样点聚类结果的群落结构多样性分析表明随着再生水净化过程的实现微生物综合多样性指数呈现下降的趋势,从均匀度指数的变化趋势看,再生水补水口具有最高均匀度指数,补水口下游1200米处由于非优势菌群所占相对丰度最低以及偶见菌群缺失,导致群落结构比较单一、群落多样性和均匀度指数都最低。CCA方面分析结果表明再生水补水口上游细菌群落与因累积效应形成的重金属有密切关系,补水口出口底泥细菌群落则主要受到总磷和总有机碳的影响较大,而再生水补水与上游来水汇水径向渐变过程可能与氨氮净化具密切关系。研究区的主要优势菌种为假单胞菌属、贪食菌属和芽孢杆菌属,是与再生水中有机碳、氮降解具有密切关系的菌属。     

水环境细菌16S rNDA限制性片段长度多型性及群落结构分析

用细菌16S核菌体RNA基因（rDNA）限制性片段长度多型性描述了水环境细菌群落结构。从环境水样中直接分离DNA,以细菌特异的引物扩增16S rDNA。构建质粒文库,随机分离重组质粒,用限制性内切酶消化获得16S rDNA基因型,用基因型的种类及频率描述特定水体生境的细菌群落结构,该方法在分析水体隐含遗传多样性、揭示污染的生物学效应和评价水环境质量等方面具有重要的应用价值。     

连作对地黄根际土壤细菌群落多样性的影响

采用末端限制性片段长度多态性(T-RFLP)技术研究地黄连作下根际土壤细菌群落的动态变化.结果表明:地黄根际土壤细菌群落的香农多样性指数、丰富度指数和相似性指数均为对照(CK)1年2年,地黄连作下细菌优势种群的比例显著下降,种植1年土壤中厚壁菌门的芽孢杆菌纲在整个细菌群落中处于主导地位,而种植2年土壤中变形菌门ε-变形菌纲处于主导地位.地黄连作使其根际土壤细菌种类大量减少,群落结构趋于简单.连作后细菌群落多样性水平的变化导致根际土壤微生物群落功能的失调,可能是引发连作障碍的原因之一.     

水葫芦根际细菌群落结构多样性分析

【目的】了解水葫芦根际细菌群落结构。【方法】运用末端限制性片段长度多态性(Terminal restriction fragment length polymorphism,T-RFLP)技术分析富营养化水体中水葫芦根际和水葫芦近、远水样的细菌群落特征及多样性,结合克隆文库技术和培养法分析根际的细菌种群类型。【结果】同一时期水葫芦根际细菌多样性(Shannon-Weiner指数H′或Simpson指数D)更高,水葫芦近水样次之,远水样最小。10月份的细菌多样性高于5月份的。通过水葫芦根际细菌的克隆文库可知变形杆菌门(Proteobacteria)是水葫芦根际细菌的主要类群,占总群体的65.1%,包括噬菌弧菌(Bacteriovorax sp.)、Dechloromonas sp.、Leptothrix sp.、红螺菌科(Rhodospirillaceae)、Rhodoferax sp.和红环菌科(Rhodocyclaceae)等。T-RFLP图谱显示159 bp为最大优势菌,247 bp为第二大优势菌,对照克隆文库及培养结果分析247 bp属于γ-Proteobacteria,159 bp为不动杆菌(Acinetobacter sp.)。【结论】水葫芦根际细菌的群落结构丰富,不同时段水葫芦根际细菌的丰度略有变化,主要类群为变形杆菌门。     

新疆低阶煤本源生物甲烷转化中微生物群落组成及变化

【目的】探究新疆低阶煤生物甲烷转化过程微生物群落组成及多样性。【方法】采用厌氧培养方法和末端限制性片段长度多态性技术(Terminal restriction fragment length polymorphism,T-RFLP)分析新疆低阶煤本源微生物对甲烷转化及有机酸含量的影响,分析新疆哈密大南湖长焰煤生物甲烷转化过程中微生物群落动态变化。【结果】研究表明长焰煤和褐煤对本源微生物产甲烷影响较小,随着低阶煤生物甲烷转化时间的延长,甲烷产量呈上升趋势,转化60 d后长焰煤甲烷产量高达10.28 m L/g,挥发性有机酸(VFA)浓度则最低;微生物多样性指数变化不明显,不同转化时间微生物主要类群为放线菌门(Actinobacteria),拟杆菌门(Bacteroidetes),厚壁菌门(Firmicutes),变形菌门(Proteobacteria);甲烷菌的群落结构相对于细菌较简单,在整个低阶煤生物转化产甲烷过程中共有古菌类群为甲烷八叠球菌属(Methanosarcina)、甲烷盐菌属(Methanohalobium)、甲烷叶菌属(Methanolobus)、甲烷食甲基菌属(Methanomethylovorans),它们是构成群落结构的基本菌群。【结论】低阶煤生物甲烷转化过程微生物群落具有丰富的多样性,且不同时期多样性有较大差异。甲烷菌群落结构相对于细菌较简单,共有类群明显。     

日粮中添加鱼油后肉牛瘤胃细菌区系的变化

目的：研究肉牛日粮中添加2%鱼油后瘤胃细菌区系的变化。方法：分别于添加鱼油前后取瘤胃液提取总DNA,根据细菌通用引物27F/1492R扩增16S rDNA基因序列,分别构建2个16S rDNA基因文库;从每个文库中各随机挑取384个克隆,对阳性克隆用HhaⅠ进行限制性片段长度多态性（RFLP）分析、聚类,取各RFLP类群中的代表克隆进行16S rDNA序列测定,构建系统发育树,研究细菌区系多样性的变化。结果：添加鱼油前、后,文库的RFLP图谱分别可以分成74和41个RFLP类群;添加鱼油前后的优势菌群均为噬纤维菌-屈挠杆菌-拟杆菌（CFB）和低（G＋C）含量的革兰阳性菌（LGCGPB）,但添加鱼油后文库中LGCGPB比例降低,而且未检测到纤维菌门细菌;有50%～60%的测定序列与GenBank数据库中的序列相似性高于97%,90%以上为未培养的细菌;添加鱼油后瘤胃细菌的多样性减少。结论：日粮添加鱼油后CFB比例增高但多样性下降,LGCGPB、纤维杆菌比例下降,这一结果为调控瘤胃发酵提供了依据。     

新疆醉马草内生菌群落结构

【目的】揭示醉马草不同组织内生细菌和内生真菌种群组成和分布。【方法】采用液氮研磨法分别提取醉马草种子、叶、茎、根组织总DNA,采用通用引物扩增内生细菌16S rDNA和真菌ITS,通过限制性内切酶HhaⅠ和RsaⅠ对16S rDNA PCR产物酶切,HhaeⅢ和HinfⅠ对真菌rDNA ITS PCR产物酶切得到不同的TRFs片段。TRFs经T-RFLP分析程序结合基因文库比对后,分析醉马草不同组织中内生细菌和内生真菌的群落组成及内生菌群落相似性。【结果】研究表明醉马草根部内生细菌多样性最高,而种子内生真菌多样性最为丰富。醉马草各组织内生细菌优势菌属均为Bacillus(29%以上),种子、叶、茎、根内生真菌优势菌属分别为Mycosphaerella(6.5%),Teratosphaeria(4.5%),Fragum(1.1%),Sebacina(11.3%)。聚类分析表明茎和叶内生细菌群落结构相似,种子和其他组织内生细菌群落结构相似性较远,而茎和种子内生真菌群落结构相似,叶和其他组织内生真菌群落结构相似性较远。醉马草内生菌多样性丰富且存在尚未认识的新类群。     

16S rDNA克隆文库法探索转基因香石竹对土壤细菌群落的影响

【目的】通过研究转基因香石竹对土壤细菌群落的影响,为转基因香石竹的环境安全性评价提供依据。【方法】通过构建16S rDNA克隆文库,分析种植转基因和非转基因香石竹的土壤中细菌的群落结构组成。【结果】转基因和非转基因香石竹土壤中,共有的菌群有变形菌门(Proteobacteria)、浮霉菌门(Planctomycetes)、酸杆菌门(Acidobacteria),其中α-变形菌门、β-变形菌门、浮霉菌门为优势菌群;而在放线菌门(Actinobacteria)、疣微菌门(Verrucomicrobia)及未培养菌(Uncultured bacterium clone)等菌群存在部分差异。【结论】通过16S rDNA克隆文库方法揭示了转基因香石竹的土壤中细菌多样性十分丰富,其栽培对土壤细菌群落结构影响有限。     

大庆油田油藏采出水的细菌群落结构

采用ARDRA (扩增性rDNA限制性酶切片段多态性分析)技术对大庆油田聚驱、水驱和过渡带3种油藏采出水中的细菌群落的基因组总DNA的16S rDNA克隆文库进行分析,研究了细菌群落结构.结果表明：随机挑取的596个阳性克隆可分为85个操作分类单元 (OTUs),其中聚驱、水驱和过渡带文库分别含有28、41和33个.通过对优势OTUs测序,并与GenBank进行序列比对,发现油藏采出水中的优势菌群为不动杆菌属、弓形杆菌属、厚壁菌门、假单胞菌属和硫磺单胞菌属.聚驱样品中细菌群落组成最简单,优势菌群为不动杆菌属,占库容的85%,假单胞菌属占7%;水驱样品中的优势菌群也是不动杆菌属,占库容的62%,假单胞菌属和硫磺单胞菌属各占20%和6%;过渡带文库的细菌群落的优势菌群为弓形杆菌属,占库容的50%,不动杆菌属和厚壁菌门各占19%和18%.     

Succession of bacterial community composition over two consecutive years in two aquatic systems: a natural lake and a lake-reservoir

The succession in bacterial community composition was studied over two years in the epilimnion and hypolimnion of two freshwater systems: a natural lake (Pavin Lake) and a lake-reservoir (Sep Reservoir). The bacterial community composition was determined by cloning-sequencing of 16S rRNA and by terminal restriction fragment length polymorphism. Despite large hydrogeological differences, in the Sep Reservoir and Pavin Lake the dominant bacteria were from the same taxonomic divisions, particularly Actinobacteria and Betaproteobacteria. In both ecosystems, these major bacterial divisions showed temporal fluctuations that were much less marked than those occurring at a finer phylogenetic scale. Nutrient availability and mortality factors, the nature of which differed from one lake to another, covaried with the temporal variations in the bacterial community composition at all sampling depths, whereas factors related to seasonal forces (temperature and outflow for Sep Reservoir) seemed to account only for the variation of the hypolimnion bacterial community composition. No seasonal reproducibility in temporal evolution of bacterial community from one year to the next was observed.     

Molecular analysis of the bacterial community in a continental high-temperature and water-flooded petroleum reservoir

Water from a continental high-temperature, long-term water-flooded petroleum reservoir in Huabei Oilfield in China was analysed for its bacterial community and diversity. The bacteria were characterized by their 16S rRNA genes. A 16S rRNA gene clone library was constructed from the community DNA, and using restriction fragment length polymorphism analysis, 337 randomly selected clones were clustered with 74 operational taxonomic units. Sequencing and phylogenetic analyses showed that the screened clones were affiliated with Gammaproteobacteria (85.7%), Thermotogales (6.8%), Epsilonproteobacteria (2.4%), low-G+C Gram-positive (2.1%), high-G+C Gram-positive, Betaproteobacteria and Nitrospira (each <1.0%). Thermopilic bacteria were found in the high-temperature water from the flooded petroleum reservoir, as well as mesophilic bacteria such as Pseudomonas-like clones. The mesophilic bacteria were probably introduced into the reservoir as it was being exploited. This work provides significant information on the structure of bacterial communities in high-temperature, long-term water-flooded petroleum reservoirs.     

Characterization of the bacterial community associated with the surface and mucus layer of whiting (Merlangius merlangus)

The bacterial community inhabiting the mucus layer and surface of whiting was examined to determine whether the bacteria present are a reflection of the surrounding water or an indigenous bacterial flora is present. The outer mucus, mouth mucus and gut of four whiting harvested from a site in the Irish Sea and the surrounding water were examined by terminal restriction fragment length polymorphism (tRFLP) analysis of the 16S rRNA gene and clone library construction. The water community was the most diverse, with only a small number of shared water-mucus phylotypes present. The bacterial flora associated with the outer mucus layer were more diverse than that of the mouth mucus and gut. All three mucus layers were characterized by the presence of a dominant phylotype, identified as clone wom-1, highly similar to Photobacterium iliopiscarium. In addition to other Photobacterium phylotypes, members of the CFB and Clostridia groups were also detected. Subsequently, whiting from 11 different sites along the east and south coast of Ireland were compared by tRFLP analysis. Strikingly, the mucus layer of whiting at all sites was characterized by the presence and dominance of a TRF corresponding to the clone wom-1 which was virtually absent from the water column.     

长春花内生细菌多样性与柑橘黄龙病菌的相关性

【目的】分析感柑橘黄龙病长春花植株与健康长春花植株不同部位内生细菌菌群结构变化,为柑橘黄龙病菌与长春花内生细菌的相关性研究提供理论基础。【方法】本研究利用兼性厌氧可培养技术、16S rDNA限制性片段长度多态性分析(Restriction fragment length polymorphism,RFLP)以及16S rDNA序列分析相结合的方法。【结果】分别从感病和健康长春花叶、茎、根的组织中分离获得67株内生细菌,与GenBank中29种细菌的相似性达到97%-100%。其中短小杆菌属(Curtobacterium sp.)、欧文氏菌属(Erwinia sp.)、蜡样芽胞杆菌(Bacillus cereus)为感病长春花内生细菌的优势菌群,鞘胺醇单胞菌属(Brevundimonas sp.)、芽胞杆菌属(Bacillus sp.)为健康长春花内生细菌的优势菌群;马胃葡萄球菌(Staphylococcus equorum)为两者的共同优势菌群。通过RFLP方法分析,感病株得到16个、健株得到23个操作分类单元(Operational TaxonomicUnits,OTUs),感病植株中除柑橘黄龙病菌Candidatus Liberibacter asiaticus外,还有丰富的CandidatusLiberibacter sp.存在。【结论】感病与健康长春花植株中均含有丰富的内生细菌,黄龙病菌的存在改变了长春花原有内生细菌的菌群结构,且菌群多样性下降。可见长春花内生细菌在一定程度上受到柑橘黄龙病菌的抑制。     

Comparison of DNA‐ and RNA‐based bacterial community structures in soil exposed to 2,4‐dichlorophenol

Aims: To examine the effect of the pollutant 2,4‐dichlorophenol on DNA‐ and RNA‐based bacterial communities in soil. Methods and Results: Soil was exposed to 100 mg kg^?1 of 2,4‐dichlorophenol (2,4‐DCP), and degradation was monitored over 35 days. DNA and RNA were coextracted, and terminal restriction fragment length polymorphism (T‐RFLP) was used to report changes in bacterial communities in response to the presence of the chlorophenol. The phylogenetic composition of the soil during degradation was determined by creating a clone library of amplified 16S rRNA sequences from both DNA and reverse‐transcribed RNA from exposed soil. Resulting clones were sequenced, and putative identities were assigned. Conclusions: A significant difference between active (RNA‐based) and total (DNA‐based) bacterial community structure was observed for both T‐RFLP and phylogenetic analyses in response to 2,4‐DCP, with more pronounced changes seen in RNA‐based communities. Phylogenetic analysis indicated the dominance of Proteobacteria in both profiles. Significance and Impact of the Study: This study describes the response of soil bacterial communities to the addition of the xenobiotic compound 2,4‐DCP, and highlights the importance of including RNA‐based 16S rRNA analysis to complement any molecular study in a perturbed soil.     

Species identification of clinically important Aeromonas spp. by restriction fragment length polymorphism of 16S rDNA

AIMS: The aim of the study was to characterize 16S rDNA of Aeromonas spp. to rapidly identify clinically important species of these bacteria. METHODS AND RESULTS: Sequence analysis of published 16S rDNA for unique restriction sites revealed prospect of species identification. Extraction of genomic DNA followed by amplification and step-by-step restriction endonuclease digestion of 16S rDNA was able to identify Aeromonas spp. of medical significance. Validation of the method was performed by subjecting 53 Aeromonas strains of multiple origin to similar treatment. Results of the study were in agreement with corresponding species of the isolates. CONCLUSIONS: The method developed offers an easily interpretable tool for the identification of Aeromonas spp. of clinical relevance. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed methodology should facilitate routine laboratory diagnosis of Aeromonas spp. from clinical cases to species level.     

16S rDNA clone library-based bacterial phylogenetic diversity associated with three South China Sea sponges

Culture-independent molecular techniques, 16S rDNA clone library alongside RFLP and phylogenetic analysis, were applied to investigate the bacterial diversity associated with three South China Sea sponges, Stelletta tenui, Halichondria rugosa and Dysidea avara. A wide bacterial diversity was detected according to total genomic DNA-based 16S rDNA clone library, abundant clones with low identify with sequences retrieved from database were found as well as uncultured sponge symbionts. The phylogenetic analysis shows that the bacterial community structure of Stelletta tenui is similar to that of Halichondria rugosa comprising gamma-Proteobacteria and Firmicutes. Whereas, alpha-Proteobacteria, gamma-Protebacteria, Bacteroidetes and uncultured sponge symbionts were found in sponge Dysidea avara, suggesting that Dysidea avara has the highest bacteria diversity among these sponges. A specific sponge–microbe association is suggested based on the difference of bacterial diversity among these three sponges from the same geography location and the observed sponge species-specific bacteria.     

Characteristics of the microbial community in rhizosphere of Camptotheca acuminata cultured with exotic invasive plant Eupatorium adenophorum

The traditional culture-dependent plate counting and culture-independent small-subunit-ribosomal RNA gene-targeted molecular techniques, Single-Strand Conformation Polymorphism (SSCP) and ter-minal Restriction Fragment Length Polymorphism (tRFLP) combined with 16S rDNA clone library were adopted to investigate the impacts of secretion from Camptotheca acuminata (abbreviated to Ca) roots on the quantities and structure of eukaryotic microbes and bacteria in the rhizosphere, and the possi-bility that Ca controls exotic invasive plant Eupatorium adenophorum (Ea). The counting results indi-cated that the number of bacteria increased in turn in rhizospheres of Ea, Ca-Ea mixed culture and Ca, while that of eukaryotic microbes decreased. PCR-SSCP profiles showed eukaryotic microbial bands (corresponding to biodiversity) in rhizosphere of Ea were more complex than those of Ca and CE. Meristolohmannia sp., Termitomyces sp. and Rhodophyllus sp. were the dominant populations in the rhizosphere of Ca. Bacterial terminal restriction fragments (TRFs) profiles showed no difference among three kinds of rhizospheres, and the sequences of the 16S rDNA clone library from Ca rhizospheres were distributed in 10 known phyla, in which phylum Proteobacteria were the absolute dominant group and accounted for 24.71% of the cloned sequences (δ-Proteobacteria accounted for up to 17.65%), and phyla Acidobacteria and Bacteroidetes accounted for 16.47% and 10.59% of the cloned sequences, respectively. In addition, high performance liquid chromatography detected a trace amount of camp-tothecin and hydroxycamptothecin in the rhizospheric soil of Ca and CE, but examined neither camp-tothecin nor hydroxycamptothecin in rhizospheric soil of Ea. Therefore, invasion and diffusion of Ea evidently depended on distinguishing the eukaryotic community structure, but not on that of the bac-terial pattern. Ca was able to alter the eukaryotic community structure of invasive Ea by secreting camptothecin and hydroxycamptothecin into rhizospheres, and may benefit the control of overspread of Ea. This study provided theoretical evidence for rhizospheric microbial aspects on substituting Ca for Ea.