黄瓜青枯病内生拮抗菌株的分离及ARDRA分析

在黄瓜生长的不同阶段从根系分离内生细菌共469株。通过青枯菌平板拮抗试验,从中筛选到具明显拮抗作用的菌株59株。将内生拮抗菌纯培养物扩增近全长的16SrDNA并用限制性内切酶AluⅠ对PCR产物进行ARDRA(amplifiedrDNArestrictionanalysis)多态性分析,共得到5种不同的操作分类单元(OperationalTaxonomicUnit,OTU)。其中属于OTU1共有39株分离物,占内生拮抗菌总数的66%,为优势种群。进一步通过ERIC-PCR指纹图的方法在菌株水平上分析OTU1类群。结果表明,OTU1可分为12种不同的菌株,其中菌株HE-1和HE-2在黄瓜生长的5个不同阶段均可分离到。通过标记天然不具有利福平抗性的HE-1和HE-2菌株,获得抗利福平突变体菌株,回收检测结果表明,在栽培的不同时期,黄瓜植株根内均有HE-1和HE-2菌株的定殖。经防病效果的盆栽试验,发现HE-1和HE-2的浸种处理能有效降低黄瓜青枯病的病发率,与对照比较差异显著。因此确定HE-1和HE-2为黄瓜青枯病生物防治的优良菌株。     

焦化废水处理系统中不同培养基分离的细菌种群多样性

以焦化废水处理系统的微生物群落为对象,对3种不同培养基（YPG、LB、WW）分离细菌的能力及分离物的种群多样性组成进行了比较研究。同一悬浮污泥样品在YPG、LB和WW培养基上的活菌计数结果分别为1.6×10.6 CFU/mL、7.0×10.5 CFU/mL和98×10.5CFU/mL。从每种培养基10-4稀释度平板上共分离137株分离物。将所有分离物扩增近全长的16S rDNA并用限制性内切酶HinfI对PCR产物进行ARDRA（Amplified rDNA restriction analysis）多态性分析,共得到14种不同的操作分类单元（Operational Taxonomic Unit, OUT）。其中YPG培养基上的分离物显示了8种不同的OTUs,而WW培养基和LB培养基分离物只分别显示了6种和4种OTUs。YPGOTU1和WWOTU6所包含的菌株分别占到总分离物的30%和22.3%,为优势分离物。ERICPCR基因组指纹图分析表明,前者的34株分离物共有20种不同的指纹图类型,而后者的25株分离物只有3种。因此,就分离焦化废水处理系统中的细菌及对分离物进行种群多样性的研究而言,YPG培养基比其他两种培养基更合适。     

南京小龙山钾矿区植物根际可培养细菌的遗传多样性分析

摘要:【目的】矿区优势植物可培养细菌生物多样性研究将有助于了解植物根际细菌与矿物,植物根系相互作用及对矿物风化和土壤形成的重要影响。【方法】采用纯培养法分离南京小龙山废钾矿区野生植物野塘蒿,千金子和栽培植物甘薯根内与根周围土壤的可培养细菌, 通过16S rDNA限制性酶切多态性分析(amplified rDNA restriction analysis, ARDRA)和16S rDNA序列分析研究了可培养细菌的多样性。【结果】分离纯化到60株具不同菌落形态的可培养细菌, 在60%相似水平上可分为18个OTU. 19株代表菌株分别属于3个门, 10个科, 11个属。多数菌株属于变形菌门(α-proteobacteria, 4株, 21.1%; β-proteobacteria, 2株, 10.5%; γ-proteobacteria, 6株, 31.6%)。假单胞菌属(Pseudomonas), 泛菌属(Pantoea)和根瘤菌属(Rhizobium)为优势种群。【结论】小龙山废矿区优势植物根围具有丰富的微生物种群多样性。     

工业废水中降酚菌的分离及ARDRA多态性分析

分别将炼油废水、印染废水、造纸废水样品倍比稀释后涂布平板分离菌株,用苯酚羟化酶基因特异引物检测苯酚降解菌,共分离得到87株降酚菌。经ERIC-PCR指纹图分析,显示15种不同的类型。进一步对显示不同ERIC—PCR指纹图的15种分离物的代表菌株进行ARDRA多态性分析,结果可分为4个OTUs（Operational Taxonomic Unit,OTU）,表明实验分离得到的降酚菌至少存在4个不同的种（species）。     

广西传统发酵米粉中乳酸菌的分离鉴定

对广西传统米粉发酵液中的乳酸菌进行分离筛选,获得6株纯培养优势菌株。通过形态学鉴定及16S rDNA序列分析,结果表明其中4株乳酸菌属于戊糖片球菌(Pediococcus pentosaceus),另外2株鉴定为植物乳杆菌(Lactobacillus plantarum)。这2种乳酸菌均为对人类及动物安全的益生菌,该结果将为传统发酵米粉中有益微生物资源的挖掘和利用奠定基础。     

宜宾浓香型白酒酿造过程中可培养细菌的系统发育多样性

【目的】为较系统地了解宜宾浓香型白酒酿造过程中可培养细菌的多样性,得到一些潜在的微生物资源。【方法】采用改良的NA培养基和高氏I号培养基分离、去除冗余,测定所得细菌纯培养物的16S rRNA基因,进行系统发育分析。【结果】分离得到603株细菌,4株菌的序列与GenBank中典型菌株序列相似性低于97%,代表着潜在新类群;599株菌与GenBank中34个属、101个种的典型菌株序列相似性大于97%,其中以Bacillus为绝对优势菌(315株),Streptomyces(121株)、Lysinibacillus(35株)、Staphylococcus(45株)为次优势菌,其余各属菌株均在10株以下。而且有16个属均只检测到1株菌。【结论】宜宾浓香型白酒发酵过程中的细菌呈现出较为丰富的多样性和一定的稳定性。     

嗜盐噬菌弧菌BALOs10菌株的分离、鉴定及其裂解谱

【目的】从海水中分离得到蛭弧菌类群(Bdellovibrio-and-likeorganisms,BALOs)新型菌株,丰富BALOs的种质资源。【方法】从中国深圳大亚湾取回海水样品后,使用本实验室分离得到的Vibrio alginolyticus LF TCBS 15作为宿主,通过海水双层平板法分离得到BALOs菌株,通过光学显微镜及透射电镜观察菌体形态,对16S rDNA序列进行系统发育分析,完成分子鉴定。采用双层平板滤纸片法分析NaCl浓度、pH及温度对菌株BALOs10生长的影响并测定菌株BALOs10对16株细菌的裂解效果。【结果】成功分离出一株以Vibrio alginolyticus LF TCBS 15为宿主的BALOs菌株BALOs10。噬菌斑呈圆形、透明且边缘光滑整齐,菌体为弧状,极生单鞭毛,菌体大小(0.21–0.44)μm×(1.25–1.87)μm。菌株最佳生长温度、NaCl浓度和pH范围分别为35–37°C、2%–3%(W/V)和7–8。菌株BALOs10可以裂解9株不同种的受试菌,占总试验菌株数(16株)的56.3%,主要是海杆菌属和弧菌属;菌株BALOs10的16S rDNA与最相近的典型菌株Halobacteriovorax marinus SJ的相似性只有92.14%,可能是一个全新的物种,将其命名为Halobacteriovorax sp. BALOs10。【结论】本文研究发现了Halobacteriovorax属(嗜盐噬菌弧菌属)的一个新型菌株,丰富了BALOs种质资源,为后续的应用及理论研究奠定物质基础。     

一株抑制黄曲霉毒素的深海微杆菌的分离与鉴定

从南大西洋深海海水中分离到一株放线菌菌株R104,该菌株的发酵无细胞上清液对黄曲霉毒素合成的抑制率高达96.2%,经16S rDNA序列分析,初步将该菌株鉴定为微杆菌（Microbacterium）,这是首次报道深海来源的微杆菌具有抑制黄曲霉毒素合成的能力。     

金川镍矿可培养细菌的多样性及耐镍菌株筛选

目的:了解金川镍矿可培养细菌的多样性和分离筛选有较高镍耐受性的菌株.方法:2007年8月从金川镍矿采集土壤样品,经过在TSA固体培养基上稀释涂布和划线分离得到55株纯培养菌株,对分离的所有菌株均提取基因组DNA,利用通用引物27F和1492R扩增得到16S rDNA的序列并测序,同时利用含不同浓度镍离子的MH培养基对所分离的菌株做了MIC分析.结果:55株菌属于11个属14个分类单元(16S rDNA序列相似性在97%以上为同一个分类单元),其中2株属于变形菌门γ亚群(Gammaproteobaaeria)(3.6%)、3株属于变形菌门α亚群(Alphaproteobacteria)(5.5%)、12株属于厚壁菌门(Firmicutes)(21.8%)、38 株属于放线菌门(Actinobacteria)(69.1%),优势菌是节杆菌属(60%),其中菌株C4、D3、D7的MIC最高达到20mmol/L.结论:金川镍矿有着较丰富的细菌多样性,菌株C4、D3、D7有潜在的应用价值.     

工业废水中降酚菌的分离及ARDRA多态性分析*

分别将炼油废水、印染废水、造纸废水样品倍比稀释后涂布平板分离菌株,用苯酚羟化酶基因特异引物检测苯酚降解菌,共分离得到87株降酚菌。经ER IC-PCR指纹图分析,显示15种不同的类型。进一步对显示不同ER IC-PCR指纹图的15种分离物的代表菌株进行ARDRA多态性分析,结果可分为4个OTUs(Operational Taxonom ic Un it,OUT),表明实验分离得到的降酚菌至少存在4个不同的种(species)。     

白色念珠菌体外诱导氟康唑耐药性的实验研究（简报）

随着广谱抗生素的普遍应用以及免疫缺陷人群的增加,机会性致病菌念珠菌感染日益增多。深部念珠菌感染已经成为重症患者死亡的重要原因,白色念珠菌(C．albicans)是其中的主要致病菌。低毒广谱的唑类抗真菌药氟康唑是既能治疗严重真菌感染又不会产生明显副作用的少数抗真菌药之一,它的广泛使用取得良好治疗效果,但也导致菌株耐药率增加而使临床治疗失败。近10年来,在治疗     

藏北地区传统发酵乳中乳杆菌的多样性分析

摘要：【目的】针对藏北地区的乳杆菌来源及种属,对其多态性进行研究。【方法】采用ERIC-PCR技术和NTSYS-pc2.1软件对从藏北地区牧民家庭制作的发酵乳制品中分离出的77株乳杆菌进行多样性分析。【结果】ERIC-PCR扩增出的条带清晰,重复性好,多态性高。聚类分析表明,在0.73的水平上,77株乳杆菌共分为4大类群：干酪乳杆菌群A1、发酵乳杆菌群A2,瑞士乳杆菌群A3和植物乳杆菌群A4。其中,干酪乳杆菌群A1和发酵乳杆菌群A2分别占供试乳杆菌的35.06%和61.04%,为优势菌群。进一步对优势菌群     

Molecular typing of native Bacillus thuringiensis isolates from diverse habitats in India using REP-PCR and ERIC-PCR analysis

Bacillus thuringiensis is a bacterium of great agronomic and scientific interest. The subspecies of this bacterium colonize and kill a large variety of host insects and even nematodes, but each strain does so with a high degree of specificity. Therefore molecular typing and diversity analysis of B. thuringiensis has enormous importance for discrimination of strains isolated from different sources. In this study, 113 native B. thuringiensis isolates collected from diverse habitats and locations in India and 27 B. thuringiensis type strains obtained from the Bacillus Genetic Stock Centre (BGSC), Ohio State University, USA and used as reference, were analyzed for molecular typing. Genotypic data of 140 B. thuringiensis isolates and type strains was generated by using REP-PCR and ERIC-PCR primers and unweighted pair group method with arithmetic mean (UPGMA) analysis using NTSYSpc2.2 and grouped into 4 main clusters. All the groups have isolates from diverse origins. No group was found to represent any specific origin or location. The observed patterns of REP-PCR and ERIC-PCR pattern were discriminatory enough to reveal differences in the B. thuringiensis isolates and reference strains. The resolution power and marker index of the ERIC-PCR (RP 9.39, MI 6.34) was found to be higher than that of the REP-PCR (RP 6.20, MI 4.48). The REP-PCR and ERIC-PCR markers have been found to be useful for discrimination of B. thuringiensis isolates and reference strains. ERIC-PCR was the more informative of the two techniques. This study showed that the B. thuringiensis isolates collected from diverse habitats in India had a high degree of genetic diversity.     

Molecular fingerprinting of Salmonella enterica subsp. enterica Typhimurium and Salmonella enterica subsp. enterica derby isolated from tropical seafood in South India

Salmonella enterica subsp. enterica Typhimurium and Salmonella enterica subsp. enterica Derby strains isolated from different seafood were genotyped by PCR-ribotyping and ERIC-PCR assays. This study has ascertained the genetic relatedness among serovars prevalent in tropical seafood. PCR-ribotyping exhibited genetic variation in both Salmonella serovars, and ribotype profile (II) was most predominant, which was observed in 10/18 of Salmonella enterica subsp. enterica Typhimurium and 7/17 Salmonella enterica subsp. enterica Derby isolates. Cluster analysis of ERIC-PCR for Salmonella enterica subsp. enterica Typhimurium strains exhibited nine different banding patterns and four strains showed >95% genetic homology within the cluster pairs. ERIC-PCR produced more genetic variations in Salmonella enterica subsp. enterica Typhimurium; nevertheless, both methods were found to be comparable for Salmonella enterica subsp. enterica Derby isolates. Discrimination index of PCR-ribotyping for Salmonella enterica subsp. enterica Typhimurium isolates was obtained at 0.674 and index value 0.714 was observed for Salmonella enterica subsp. enterica Derby strains. Molecular fingerprinting investigation highlighted the hypothesis of diverse routes of Salmonella contamination in seafood as multiple clones of Salmonella enterica subsp. enterica Typhimurium and Salmonella enterica subsp. enterica Derby were detected in same or different seafood throughout the study period.     

Functional characterization of antagonistic fluorescent pseudomonads associated with rhizospheric soil of rice (Oryza sativa L.)

Antagonistic fluorescent pseudomonads isolated from rhizospheric soil of rice were characterized by 16S rRNA amplicon and fatty acid methyl ester (FAME) analyses. Antagonistic isolates were grown in the fermentation media, and production of antibiotics was confirmed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Production of fungal cell-wall-degrading enzymes such as protease, cellulase, pectinase, and chitinase was determined. Dendrogram based on the major and differentiating fatty acids resulted into 5 clusters, viz., cluster I (P. pseudoalcaligenes group), cluster II (P. plecoglossicida group), cluster III (P. fluorescens group), cluster IV (P. aeruginosa group), and cluster V (P. putida group). Characteristic presence of high relative proportions of cyclopropane (17:0 CYCLO w7c) was observed in antagonistic bacteria. Data revealed biodiversity among antagonistic fluorescent pseudomonads associated with the rice rhizosphere. Results presented in this study will help to identify the antagonistic isolates and to determine their mechanisms that mediate antagonism against fungal pathogens of rice.     

Low recovery frequency of <Emphasis Type="Italic">Gluconacetobacter diazotrophicus</Emphasis> from plants and associated mealybugs in Cuban sugarcane fields

This study was aimed to isolate and identify the N₂-fixing bacterium Gluconacetobacter diazotrophicus from 11 sugarcane varieties, grown under field conditions in four Cuban provinces, and from their associated mealybugs Saccharicoccus sacchari. Identification was based on morphological and biochemical tests and PCR-amplification of 16S rRNA genes using species-specific primers. From all sugarcane varieties and numerous mealybug colonies sampled, G. diazotrophicus isolates were recovered from inside sugarcane stems of only three varieties, and one from S. sacchari colony. These four isolates showed acetylene reduction activity in nitrogen-free media and contained nifH genes which were PCR-amplified using specific primers. ERIC-PCR fingerprinting was used to compare the Cuban G. diazotrophicus isolates with type and reference strains of N₂-fixing Gluconacetobacteria. The very low frequency of G. diazotrophicus isolates recovered is probably related with the high doses of nitrogen fertilizers applied to the sugarcane in the Cuban fields for almost 30 years. Some genetic differences, using ERIC-PCR, were detected among G. diazotrophicus strains, which could be related with its source.     

Analysis of Antarctic proteobacteria by PCR fingerprinting and screening for antimicrobial secondary metabolites

Fifty-seven proteobacterium species were successfully isolated from soils of Barrientos Island of the Antarctic using 11 different isolation media. Analysis of 16S rDNA sequencing of these isolates showed that they belonged to eight different genera, namely Bradyrhizobium, Sphingomonas, Methylobacterium, Caulobacter, Paracoccus, Ralstonia, Rhizobium, and Staphylococcus. All isolates were studied for capability of producing antimicrobial and antifungal secondary metabolites using high-throughput screening models. Approximately 23 (13/57) and 2% (1/57) of isolates inhibited growth of Candida albicans ATCC 10231(T) and Staphylococcus aureus ATCC 51650(T), respectively. These results indicated that proteobacterium species isolates from Antarctic could serve as potential source of useful bioactive metabolites. Enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting produced nine clusters and 13 single isolates, with a high D value of 0.9248. RAPD fingerprinting produced six clusters and 13 single isolates, with a relatively low D value of 0.7776. ERIC-PCR analysis proved to have better discrimination capability than RAPD analysis and generated better clustering for all proteobacterium species isolates. We conclude that ERIC-PCR is a robust, reliable and rapid molecular typing method for discriminating different genera of proteobacteria.