蕙兰根部内生细菌多样性及季节动态变化

为了研究蕙兰（Cymbidium faberi）根部内生细菌群落结构在不同季节里的变化,于不同季节从蕙兰根部分离出内生细菌207株,采用核糖体DNA扩增片段限制性分析（ARDRA）研究了蕙兰根部内生细菌群落组成的季节动态变化。将内生细菌纯培养物扩增近全长的16SrDNA,并用ARDRA对所分离的菌株进行分型,根据酶切图谱的差异,将其分成25个ARDRA型,并选取代表性菌株进行16SrDNA序列测定。结果表明,分离出来的内生细菌分为9个属,包括芽孢杆菌属（Bacillus）、伯克氏属（Burkholderia）、类芽孢杆菌属（Paenibacillus）、假单胞属（Pseudomonas）、微杆菌属（Microbacterium）、根瘤菌属（Rhizobium）、Lysinibacillus、Cohnella和短杆菌属（Bre—vibacterium）,其中优势种群为Bacillus,次优势种群为Paenibacillus和Burkholderia。秋季分离出的内生细菌种类最多,夏季分离出的种类最少。在不同季节蕙兰内生细菌群落结构差异极显著（P〈O．001）,但在不同季节里蕙兰根部内生细菌优势种群没有差异。     

黑龙江大豆胞囊线虫胞囊可培养细菌多样性

【目的】了解黑龙江省大豆田大豆胞囊线虫胞囊可培养细菌的多样性。【方法】运用稀释平板法和16SrDNA基因序列的系统发育分析对胞囊可培养细菌多样性进行研究。【结果】用NA培养基从胞囊上分离90株具有不同菌落形态的细菌。16S rDNA序列分析结果表明:90株菌株分属于7个属22个种。46株属于变形菌门γ亚群(Gammaproteobacteria),32株属于厚壁菌门(Firmicutes),10株属于变形菌门β亚群(Betaproteobacteria),2株属于变形菌门ɑ亚群(Alphaproteobacteria)。假单胞菌属(Pseudomonas)和芽孢杆菌属(Bacillus)为优势菌属。【结果】黑龙江省大豆胞囊线虫胞囊中存在丰富的细菌物种多样性,这些细菌对大豆胞囊线虫可能具有一定的生理生态作用。     

北部湾徐闻海域红树内生细菌物种多样性及其杀线虫活性研究

为发掘红树植物内生细菌资源和寻找新型微生物杀虫剂,该研究从北部湾徐闻海域采集7种红树植物共16份样品,设计10种分离培养基,使用稀释涂布法分析红树植物内生细菌的分布特征,通过16S rRNA分子生物学方法对内生细菌进行多样性分析,并利用秀丽隐杆线虫模型,通过杀线虫活性实验测试内生细菌乙酸乙酯提取物的杀线虫活性。结果表明:(1)从16份红树植物各组织器官中获得33株内生细菌,分布于19个科23个属。其中,芽孢杆菌属(Bacillus)为优势菌属,并发现了10株潜在的新种或新属。(2)筛选到具有显著杀线虫活性的菌株IMDGX 4725和IMDGX 4744,半数致死浓度(LC₅₀)分别为61.58、100.89mg·mL^-1。研究结果证实了徐闻海域红树具有多样性丰富的内生细菌,同时部分细菌具有较强的杀线虫活性,具有发现新型微生物杀虫剂的潜力。     

大豆孢囊线虫生防菌株Myrothecium verrucaria ZW-2发酵条件优化及活性物质分析

疣孢漆斑菌(Myrothecium verrucaria)ZW-2菌株对大豆孢囊线虫具有(Heterodera glycines)明显的杀线虫活性,为了确定该菌株的最优发酵条件以及代谢产物,采用4因素3水平的正交试验进行了发酵条件优化以及代谢组学分析.结果表明:菌株ZW-2以Czapek培养基为基础培养基,最佳培养温度...     

一种基于PCR分析多样性的小鼠肠道微生物宏基因组提取方法

目的建立一种基于PCR分析分子多样性的小鼠肠道菌群宏基因组提取方法。方法比较、综合国内外小鼠肠道菌群宏基因组的提取方法后建立一种新方法,小鼠肠道内容物经丙酮洗涤,差速离心,溶菌酶、SDS裂解,CTAB处理,酚／氯仿抽提后可得到高质量的DNA,通过紫外分光光度计、琼脂糖凝胶电泳、细菌通用引物PCR和扩增核糖体限制性酶切片段分析（ARDRA）等检测该方法的实用性。结果该方法获得的小鼠肠道菌群宏基因组DNA大小在23kb左右,A260／A280在1．8—2．0,经细菌通用引物PCR后能得到适用于ARDRA的目的产物。结论该方法经济适用性较强,具备一定的应用价值。     

刺参消化道中蛭弧菌类的生物多样性分析

研究了刺参消化道中蛭弧菌类生物多样性。用刺参肠道内容物提取微生物总DNA,分别使用蛭弧菌类生物特异性引物Bd、Bac扩增获得16S rDNA目的片段,连接pMD19 T载体,转化到大肠埃希菌DH5α感受态细胞,经蓝白斑筛选及阳性克隆筛选,通过核糖体DNA扩增片段限制性内切酶分析(ARDAR)对阳性克隆进行分型,使用HaeⅢ和MspⅠ双酶切各60个阳性克隆,选取不同ARDAR型的阳性克隆测序,并进行生物学分析。结果显示：引物Bd、Bac的16S rDNA扩增产物分别分离获得3个和2个差异性序列,各属于一个类群,分属于蛭弧菌属(Bdellovibrio)和噬菌弧菌属(Bacteriovorax)。这些序列与数据库中相应菌株序列的最大相似性均为95.0%,这5个序列的菌株可能为潜在的新种。     

基于16S rRNA和HSP60基因序列分析粘细菌孢囊杆菌亚目形态分类的种属之间的亲缘关系

[目的]利用16S rRNA和HSP60基因分子标记分析鉴定形态分类特征不稳定的粘细菌种属.[方法]利用粘细菌的传统分离纯化方法从土壤中分离粘细菌,根据菌株的形态特征进行分类,PCR方法扩增菌株的16S rRNA和HSP60基因序列并进行系统发育关系分析.[结果]根据形态特征,分离得到的15株粘细菌菌株归入孢囊杆菌亚目(Cystobacterineae)的2个科3个属.其中11株粘细菌具有典型的所在种属的子实体结构,而菌株0085-4、0121-3、NM03和Myx9736的子实体结构发生了不同程度退化.15株粘细菌的16S rRNA基因序列的相似性在95.4%到99.5%之间.而HSP60基因序列差异较大.[结论]在属水平上,粘细菌形态分类特征和16S rRNA基因系统进化关系具有很好的一致性;在揭示粘细菌种间系统发育关系中,HSP60基因序列更为适用.     

再生水灌溉对草坪根际可培养细菌组成影响

【目的】为了了解再生水灌溉对草坪根际可培养细菌群落组成的影响,【方法】采用稀释平板法,对北京市陶然亭公园内再生水灌区及其对照自来水灌区草坪根际细菌进行了分离,并对其16S rDNA序列进行了分析。【结果】16S rDNA序列分析表明自来水样品分离得到的菌落分属于15个属的20个种,而再生水样品分离得到的菌落分属于18个属的24个种。自来水和再生水灌区草坪根际细菌主要包括变形菌门α亚群(Alphaproteobacteria,分别为9.7%和13.4%)、变形菌门β亚群(Betaproteobacteria,分别为8.1%和12.3%)、变形菌门γ亚群(Gammaproteobacteria,分别为17.9%和42.0%)、拟杆菌门(Bacteroidetes,分别为13.0%和2.9%)、厚壁菌门(Firmicutes,分别为23.6%和10.1%)和放线菌门(Actinobacteria,分别为27.6%和19.6%)其中,芽孢杆菌属(Bacillus sp.)是自来水灌区草坪根际优势菌属(23.6%),而不动杆菌属(Acinetobacter sp.)是再生水灌区根际优势菌属(17.4%)。从不同类群优势菌属看,除变形菌门γ亚群受再生水影响优势菌属在两灌区表现出一定的差异外,其余各亚群优势菌属均未受再生水影响,其中,不动杆菌属是再生水灌区变形菌门γ亚群的优势菌属(41.3%),肠杆菌属(Enterobacter sp.)是自来水灌区变形菌门γ亚群的优势菌属(45.4%)。【结论】这表明,再生水灌溉未改变细菌群落组成类型,但改变了不同类型多度分布状况。具体表现为优势种多度值增加及部分非优势种有无。再生水灌区特有机会性致病菌、植物致病菌和重金属耐性细菌的出现表明再生水灌溉中病原微生物及重金属的控制工作还有待于进一步严格。     

Rapid identification of marine bioluminescent bacteria by amplified 16S ribosomal RNA gene restriction analysis

To rapidly identify natural isolates of marine bioluminescent bacteria, we developed amplified ribosomal DNA restriction analysis (ARDRA) methods. ARDRA, which is based on the restriction patterns of 16S rRNA gene digested with five enzymes (EcoRI, DdeI, HhaI, HinfI, RsaI), clearly distinguished the 14 species of marine bioluminescent bacteria currently known, which belong to the genera Vibrio, Photobacterium, and Shewanella. When we applied ARDRA to 129 natural isolates from two cruises in Sagami Bay, Japan, 127 were grouped into six ARDRA types with distinctive restriction patterns; these isolates represented the bioluminescent species, P. angustum, P. leiognathi, P. phosphoreum, S. woodyi, V. fischeri, and V. harveyi. The other two isolates showing unexpected ARDRA patterns turned out to have 16S rRNA gene sequences similar to P. leiognathi and P. phosphoreum. Nevertheless, ARDRA provides a simple and fairly robust means for rapid identification of the natural isolates of marine bioluminescent bacteria, and is therefore useful in studying their diversity.     

Locality or habitat? Exploring predictors of biodiversity in Amazonia

Amazonia is an environmentally heterogeneous and biologically megadiverse region, and its biodiversity varies considerably over space. However, existing knowledge on Amazonian biodiversity and its environmental determinants stems almost exclusively from studies of macroscopic above‐ground organisms, notably vertebrates and trees. In contrast, diversity patterns of most other organisms remain elusive, although some of them, for instance microorganisms, constitute the overwhelming majority of taxa in any given location, both in terms of diversity and abundance. Here, we use DNA metabarcoding to estimate prokaryote and eukaryote diversity in environmental soil and litter samples from 39 survey plots in a longitudinal transect across Brazilian Amazonia using 16S and 18S gene sequences, respectively. We characterize richness and community composition based on operational taxonomic units (OTUs) and test their correlation with longitude and habitat. We find that prokaryote and eukaryote OTU richness and community composition differ significantly among localities and habitats, and that prokaryotes are more strongly structured by locality and habitat type than eukaryotes. Our results 1) provide a first large‐scale mapping of Amazonian soil biodiversity, suggesting that OTU richness patterns might follow substantially different patterns from those observed for macro‐organisms; and 2) indicate that locality and habitat factors interact in determining OTU richness patterns and community composition. This study shows the potential of DNA metabarcoding in unveiling Amazonia's outstanding diversity, despite the lack of complete reference sequence databases for the organisms sequenced.     

Phenotypic, genotypic and technological characterization of predominant lactic acid bacteria in Pecorino cheese from central Italy

AIMS: Identification and biotyping of lactic acid bacteria (LAB) isolated from raw-milk Pecorino cheese manufactured in the Marche region (central Italy) for selection of suitable starter cultures or adjuncts. METHODS AND RESULTS: Preliminary characterization with morphological and biochemical assays were undertaken for 112 Gram-positive and catalase-negative isolates. Unequivocal identification of the isolates was obtained through restriction analysis of the amplified 16S rRNA gene and sequencing of 360-380 bp amplicons. Fifty-nine isolates belonging to LAB species generally recognized as safe and potentially utilized as starters or flavour-producing adjuncts were preselected and tested for their acidifying, proteolitic and autolytic activities. Fifty-five of these isolates were also subject to RAPD (randomly amplified polymorphic DNA) fingerprinting and unweighted pair-group method with arithmetic averages (UPGMA) cluster analysis for the estimation of genotypic intra-species variation. As a result, in Pecorino cheese, a heterogeneous lactic acid bacteria population, which includes strains with metabolic characteristics of technological interest, was characterized. CONCLUSIONS: The polyphasic approach proposed allows the bacterial ecology of Pecorino cheese to be investigated and allows to assess the potential role of autochthonous LAB strains for the dairy industry. SIGNIFICANCE AND IMPACT OF THE STUDY: The great economic importance of Pecorino cheese encouraged a deeper knowledge of its microbiota, which is known to influence the peculiar sensory properties of this cheese, also in view of its exploitation.     

Application of Several Molecular Techniques to Study Numerically Predominant Bifidobacterium spp. and Bacteroidales Order Strains in the Feces of Healthy Children

Bifidobacteria and Bacteroides-like bacteria are strictly anaerobic nonpathogenic members of human intestinal microflora. Here we describe an analysis of the species and subspecies composition of these bacterial populations in healthy children using a combination of culture and molecular methods at two different time points. It was found that B. bifidum and B. longum are the most common dominant taxons in infants aged between 8 and 16 months. The majority of the infants carried several dominant Bifidobacterium strains belonging to different species. Examination of the dominant bifidoflora in some of these children after a 5-year period showed major shifts in both species and strain composition, but the dominant strains remained unchanged in two children. The majority of dominant Bacteroides-like isolates belonged to species B. vulgatus and B. uniformis, but members of genera Alistipes and Barnesiella were common too. In addition, a novel approach to species identification of Bacteroidales order bacteria using amplified ribosomal DNA restriction analysis (ARDRA) is described.     

Molecular typing of Pasteurella pneumotropica isolated from rodents by amplified 16S ribosomal DNA restriction analysis and pulsed-field gel electrophoresis

A total of 52 isolates of Pasteurella pneumotropica obtained from rodents were examined for their genetic heterogeneity. On the basis of DNA restriction analysis, including amplified 16S ribosomal DNA restriction analysis (ARDRA) and pulsed-field gel electrophoresis (PFGE), differences were identified among the isolates. ARDRA typing with Hae III revealed 4 different banding patterns of the P. pneumotropica isolates. Eighty-two percent of the 23 isolates identified as a-1 were derived from mice, whereas all the isolates identified as a-3 were derived from rats. Most of the isolates, which showed hemolytic activity on blood agar, obtained from mice and rats, were identified as a-2 and a-4, respectively. By restriction analysis of genomic DNA, Apa I and Not I digestion differentiated 9 variants and an undiscriminating group. However, no close relation with regard to the phenotypic characteristics was observed among the variants. The isolates identified as a-2 and a-4 could not be distinguished by PFGE analysis. DNA restriction analysis revealed that the genetic diversity of the P. pneumotropica isolates was more complex than the phenotypic characteristics among the species, and that at least the P. pneumotropica isolates were clearly differentiated into 4 groups by ARDRA typing with Hae III.     

Effect of restriction endonucleases on assessment of biodiversity of cultivable polar marine planktonic bacteria by amplified ribosomal DNA restriction analysis

To choose a suitable restriction endonuclease for quick assessment of bacterial diversity in polar environments by ARDRA, we investigated the effect of restriction enzymes on ARDRA patterns of cultivable marine planktonic bacteria isolated from polar region. Thirty-three isolates were analyzed by ARDRA using five enzymes (HinfI, HaeIII, AluI, and the mix AfaI/MspI), respectively, resulting in different groups, each group corresponding to a particular genotype. A comparison of the ARDRA patterns was carried out, and phylogenetic position of all thirty-three bacteria was obtained by 16S rDNA sequencing. Consistent with phylogenetic analysis, ARDRA pattern comparison revealed that AluI, being sensitive and reliable enough to generate species-specific patterns, was a suitable restriction enzyme used for evaluating bacterial diversity, suggesting a combination of ARDRA with AluI and 16S rDNA sequencing can provide a simple, fast and reliable means for bacterial identification and diversity assessment in polar environments.     

不同风化程度钾长石表面矿物分解细菌的筛选及遗传多样性

【目的】不同风化程度钾长石表面矿物分解细菌生物多样性研究将有助于了解矿物生物风化、生物成矿和土壤形成的演化规律和机理。【方法】采用纯培养法自南平钾矿区高、中、低风化度钾长石以及矿区土壤样品中分离矿物分解细菌,通过摇瓶释硅实验比较不同菌株分解矿物能力,采用16SrDNA限制性酶切多态性分析(Amplified rDNA Restriction Analysis,ARDRA)研究了供试菌株的遗传多样性。【结果】分离筛选到35株生长良好的矿物分解细菌,与对照相比,接菌处理发酵液中有效硅增加了101～206%;所有供试菌株可分为11个OTU,分别属于5个门,6个科,7个属。多数菌株(74%)属于γ-变形杆菌纲(γ-Proteobacteria)。泛菌属(Pantoea),沙雷氏菌属(Serratia),假单胞菌属(Pseudomonas)为优势种群。【结论】南平钾矿区矿物分解细菌具有丰富的微生物种群多样性,且γ-变形杆菌纲(γ-Proteobacteria)细菌在钾长石风化过程中可能起了重要的作用。     

温度对厚指海绵Pachychalina sp.体内真细菌的影响

摘要：温度是影响微生物多样性的重要因素之一。【方法】本研究采用16S rDNAs扩增产物酶切片段多态性（amplified rDNA restriction analysis , ARDRA）和16S rDNAs序列分析方法,对生长在16℃和30℃条件下厚指海绵Pachychalina sp.体内真细菌（eubacteria）的多样性进行了研究,【目的】探讨温度对海绵体内细菌的影响。【结果】根据ARDRA 聚类分析,16℃条件下海绵体内100个真细菌的16S rDNAs克隆片断被分成34个类群,而30℃条件下,海绵体内100个细菌的16S rDNAs克隆片断则被分为32个类群,说明在不同温度条件下,海绵体内真细菌的组成与群落结构有所变化,但研究发现温度没有明显改变海绵体内真细菌总的群落结构。【结果】根据厚指海绵Pachychalina sp.体内真细菌的16S rDNAs序列分析结果发现：在16℃和30℃条件下,海绵体内的真细菌均属于α、γ、δ-变形菌、硫细菌、硫还原菌和烃类分解菌,此外还有少数放线菌。16℃条件下海绵体内的优势菌为γ-变形菌,而且体内的硫细菌和硫还原菌主要是耐寒细菌,而30℃条件下海绵体内的优势菌为α-变形菌。该研究还发现：同一ARDRA类型的克隆序列分析表明在同一ARDRA群内各组分间彼此关系比较密切。