Isolation of novel psychrophilic bacteria from Arctic sea ice

The phylogenetic diversity of cuhurable psychrophilic bacteria associated with sea ice from the high latitude regions of Canadian Basin and Chukchi Sea, Arctic, was investigated. A total of 34 psychropilic strains were isolated using three methods of （ Ⅰ ） dilution plating （ at 4 ℃ ）, （ Ⅱ ） bath culturing （ at - 1 ℃ ） and dilution plating, and （ Ⅲ） cold shock （ -20 ℃ for 24 h）, bath culturing and dilution plating under aerobic conditions. Sea-ice samples were exposed to -20 ℃ for 24 h that might reduce the number of common microorganisms and encourage outgrowth of psyehrophilic strains. This process might be able to be introduced to isolation psychrophilic bacteria from other environmental samples in future study. 16S rDNA nearly full-length sequence analysis revealed that psychrophilic strains felled in two phylo- genetic divisions, γ-proteobacteria （in the genera ColweUia,Marinobaeter、Shewanella,Glaciecola,Marinomortas and Pseudoalteromortas ） and Cytophaga-Flexibacter-Baeteroides （ Flavobacterlum and Psyehroflexus）. Fifteen of bacterial isolates quite likely represented novel species （16S rDNA sequence similarity below 98% ）. One of strains （BSi20002） from Canadian Basin showed 100% sequence similarity to that of Marinobacter sp. ANT8277 isolated from the Antarctic Weddell sea ice, suggesting bacteria may have a bipolar distribution at the species level.     

Sequence and phylogenetic analyses of the chloroplast 16S rRNA, tufA, and rbcL genes from Bryopsis hypnoides

Using shotgun sequencing data, the complete sequences of chloroplast 16S rRNA and tufA genes were acquired from native specimens of Bryopsis hypnoides (Qingdao, China). There are two group I introns in the 16S rRNA gene, which is structurally similar to that of Caulerpa sertularioides (Bryopsidales, Chlorophyta). The chloroplast-encoded tufA gene sequence is 1 230 bp long, very AT-rich (61.5%), and is similar to previously published 16S rRNA sequences of bryopsidinean algae. Phylogenetic analyses based on chloroplast 16S rRNA and tufA gene sequence data support previous hypotheses that the Bryopsidineae, Halimedineae, and Ostreobidineae are three distinct lineages. These results also confirmed the exclusion of Avrainvillea from the family Udoteaceae. Phylogenetic analyses inferred that the genus Bryopsis as sister to Derbesia; however, this clade lacked robust nodal support. Moreover, the phylogenetic tree inferred from rbcL GenBank sequences, combined with the geographical distributions of Bryopsis species, identified a strongly supportive clade for three differently distributed Asian Bryopsis species. The preliminary results suggesting that these organisms are of distinct regional endemism.     

Phylogenetic analysis of cultivable bacteria isolated from Arctic sea-ice

Phylogenetic analysis based on 16S rDNA of 8 strains of cultivable bacteria isolated from Arctic sea-ice was studied.The results showed that strain BJ1 belonged to genus Planococcus,which was a genus of low mole percent G C gram-positive bacteria;strain BJ6 belonged to genus Burkholderia of β-proteobacteria and the rest 6 strain all belonged to γ-proteobacteria,of which strain BJ8 was a species of Pseudoalteromonas,strain BJ2-BJ5 and BJ7 were members of genus Psychrobacter.Phylogenetic analysis also indicated that bacteria of genus Psychrobacter of the isolates formed a relatively independent phylogenetic cluster in comparison with other bacteria belonged to genus Psychrobacter.     

卵形鲳鲹结节病病原的分离与鉴定

从湛江港、阳江闸坡海水网箱养殖的患结节病卵形鲳鲹(Trachinotus ovatus)中分离一株细菌,回归感染结果证明该菌是引起卵形鲳鲹结节病的病原,对卵形鲳鲹半致死浓度为45 g-1。该菌株呈革兰阳性,好氧,具有弱抗酸性,菌体呈短杆状或细长分枝状,过氧化氢酶阳性,氧化酶阴性,还原硝酸盐,不水解酪素、黄嘌呤、酪氨酸、淀粉、明胶,以柠檬酸盐为唯一碳源生长。对其16S rDNA序列作Blast分析,构建系统进化树。结果表明,该菌株与诺卡氏菌属的菌株亲缘关系最近,且与鰤鱼诺卡氏菌(Nocardia seriolea JCM 3360T)的16S rDNA序列同源性高达99%。综合患病鱼的症状及病原菌形态、生理生化特性及16S rDNA序列的结果,该菌被鉴定为鰤鱼诺卡氏菌(N.seriolea)。     

养殖场底泥中芽孢杆菌属细菌的生态学研究

对近海养殖场底泥细菌的研究 ,发现养殖池底泥中芽孢菌为优势菌群。应用形态学、生理生化和 16SrDNA序列测定 ,对芽孢杆菌属细菌做进一步分类的结果表明 ,该属细菌在底泥中的主要种群为蜡状芽孢杆菌、巨大芽孢杆菌、短小芽孢杆菌、坚强芽孢杆菌、Bacillusbaekryungensis等。同时 ,还分析了这些芽孢杆菌属细菌在底泥元素的地球化学循环中的作用 ,认为这类细菌应属于陆生微生物 ,在养殖环境中代谢有机化合物产生氨、亚硝酸盐和H2 S ,对养殖环境产生不利的作用和影响     

红树林共附生微生物HSL36的鉴定及其主要代谢产物GC/MS分析

从湛江红树林的根部中分离筛选一株潜在益生菌,命名HSL36,通过形态观察,并进行生理生化特性分析和16S rDNA序列分析后,鉴定为戊糖乳杆菌(Lactobacillus pentosus)。采用气-质联用技术(GC-MS)对菌株HSL36胞外代谢产物进行成分分析,菌株HSL36发酵液二氯甲烷萃取物的主要化学成分为邻苯二甲酸二异丁酯、邻苯二甲酸二丁酯、2,6-二叔丁基-4-甲基环己醇、柏木脑、碘代十六烷和2个未确定化合物。     

Construction of Porphyra yezoensis Pure Line from Protoplasts and Its 18S rDNA Sequence Determination

1　Introduction　PorphyraisthemainobjectforalgafarmingandplaysaveryimportantroleinChinesemarineindus tries.Recently ,therehasbeenagreatlossinPor phyracultivationduetothedegenerationoftheculti var,sothereisanincreasingdemandforgoodPor phyracultivars .Theprerequisiteofthetraditionalbreed ingandbioengineeringresearchofPorphyraistheconstructionofpurelines .Traditionally ,theclassifi cationofPorphyrawasaccordingtotheirmorphologi calcharacteristics .However ,mostmorphologicalfea turesofPorphyraar…     

具有多种胞外酶的对虾肠道黏附菌的分离和鉴定

用对虾饲料培养基从健康凡纳滨对虾肠道分离出500株黏附细菌,以产淀粉酶、脂肪酶和蛋白酶能力为指标,筛选出产该3种消化酶的细菌90株,占总菌株的18%.对其中生长较快的69株进行16S rDNA 基因测序,确定其分类地位.结果显示,69株菌分别属于不动杆菌属(Acinetobacter)、芽孢杆菌属(Bacillus)、葡萄球菌属(Staphylococcus)、假交替单胞菌属(Pseudoalteromonas)、气单胞菌属(Aeromonas)、嗜盐单胞菌属(Halomonas)、利斯顿氏菌属(Listonella)、莫拉氏菌属(Moraxella)等,其中数量最多是芽胞杆菌属,占鉴定细菌总数的53.62%,数量最少是气单胞菌属和嗜盐单胞菌属,均占鉴定细菌总数的2.90%.表明对虾肠道黏附菌群中具有较多能分泌多种消化酶的细菌,可进一步开发为促进对虾消化功能的益生菌     

水产养殖用芽孢杆菌制剂的检测

通过平板菌落计数法检测编号为GD1、GD2、GD3、GD4、GD5、JS、JX、SD1和SD2等9种水产养殖用芽孢杆菌制剂的有效活菌数,结合形态、生理生化和16S rRNA基因序列分析对各制剂中分离的产芽孢细菌代表菌株进行鉴定。结果显示:除GD1、GD2和GD5制剂未标明芽孢数量外,其余制剂检测到的芽孢数量均低于或远低于标注数量;经鉴定,各制剂中最优势类型,GD2、GD3、GD5、SD1和SD2制剂为地衣芽孢杆菌(Bacillus licheniformis),GD4、JS和JX制剂为解淀粉芽孢杆菌(B.amyloliquefaciens),GD1制剂为枯草芽孢杆菌(B.subtilis)。除GD4、GD5和SD1制剂未标明具体菌种以及GD2的标注菌种与检测结果不一致外,其他制剂的标注菌种与被鉴定的优势类型芽孢杆菌基本一致。     

PCR-DGGE analysis of intestinal bacteria and effect of Bacillus spp.on intestinal microbial diversity in kuruma shrimp(Marsupenaeus japonicus)

In this study,the intestinal microbiota of kuruma shrimp(Marsupenaeus japonicus) was examined by molecular analysis of the 16S rDNA to identify the dominant intestinal bacteria and to investigate the effects of Bacillus spp.on intestinal microbial diversity.Samples of the intestines of kuruma shrimp fed normal feed and Bacillus spp.amended feed.PCR and denaturing gradient gel electrophoresis(DGGE) analyses were then performed on DNA extracted directly from the guts.Population fingerprints of the predominant organisms were generated by DGGE analysis of the universal V3 16S rDNA amplicons,and distinct bands in the gels were sequenced.The results suggested that the gut of kuruma shrimp was dominated by Vibrio sp.and uncultured gamma proteobacterium.Overall,the results of this study suggest that PCR-DGGE is a possible method of studying the intestinal microbial diversity of shrimp.     

Construction of Porphyra yezoensis Pure Line from Protoplasts and Its 18S rDNA Sequence Determination

The wild Porphyra yezoensis collected from the Qingdao coast was used to prepare protoplasts by enzyme digestion. The pure line was constructed by cultivating the protoplasts. The 18S rDNA of the P. yezoensis pure line was cloned and sequenced. Sequence analysis was executed for this sequence and other 22 sequences retrieved from GenBank. A phylogenetic tree was constructed using the neighbor-joining method. The results revealed a high diversity of 18S rDNA sequences in genus Porphyra and the considerable variation of 18S rDNA sequences in different strains of the same species P. yezoensis and P. tenera. Significant difference of 18S rDNA sequence was observed between P. yezoensis from Qingdao, China, and the two strains of P. yezoensis from Japan, but the three strains of P. yezoensis formed a stable clade in the phylogenetic tree. These results indicate the possibility of interspecies and intraspecies discrimination of Porphyra using the 18S rDNA sequences.     

PCR-DGGE analysis of intestinal bacteria and effect of Bacillus spp. on intestinal microbial diversity in kuruma shrimp (Marsupenaeus japonicus)

In this study, the intestinal microbiota of kuruma shrimp (Marsupenaeus japonicus) was examined by molecular analysis of the 16S rDNA to identify the dominant intestinal bacteria and to investigate the effects of Bacillus spp. on intestinal microbial diversity. Samples of the intestines of kuruma shrimp fed normal feed and Bacillus spp. amended feed. PCR and denaturing gradient gel electrophoresis (DGGE) analyses were then performed on DNA extracted directly from the guts. Population fingerprints of the predominant organisms were generated by DGGE analysis of the universal V3 16S rDNA amplicons, and distinct bands in the gels were sequenced. The results suggested that the gut of kuruma shrimp was dominated by Vibrio sp. and uncultured gamma proteobacterium. Overall, the results of this study suggest that PCR-DGGE is a possible method of studying the intestinal microbial diversity of shrimp.     

Molecular diversity of Enteromorpha from the coast of Yantai: a dual-marker assessment

We collected nine Enteromorpha specimens from the coast of Yantai and evaluated their diversity based on analyses of their ITS （internal transcribed spacer） and 5S rDNA NTS （non-transcribed spacer） sequences. The ITS sequences showed slight nucleotide divergences between Enteromorpha linza and Enteromorpha prolifera. In contrast, multiple highly variable regions were found in the ITS region of Enteromorpha flexuosa. In general, there were more variable sites in the NTS region than in the ITS region in the three species. The variations in 5S rDNA NTS sequences indicated that the molecular diversity of Enteromorpha from the coast of Yantai is very high. However, a phylogenetic tree constructed using 5S rDNA NTS sequence data indicated that genetic differences were not directly related to geographical distribution.     

Design of Vibrio 16S rRNA Gene Specific Primers and Their Application in the Analysis of Seawater Vibrio Community

The pathogenic species of genus Vibrio cause vibriosis, one of the most prevalent diseases of maricultured animals and seafood consumers. Monitoring their kinetics in the chain of seafood production, processing and consumption is of great importance for food and mariculture safety. In order to enrich Vibrio-representing 16S ribosomal RNA gene （rDNA） fragments and identify these bacteria further real-timely and synchronously among bacterial flora in the chain, a pair of primers that selectively amplify Vibrio 16S rDNA fragments were designed with their specificities and coverage testified in the analysis of seawater Vibrio community. The specificities and coverage of two primers, VF169 and VR744, were determined theoretically among bacterial 16S rDNAs available in GenBank by using BLAST program and practically by amplifying Vibrio 16S rDNA fragments from seawater DNA. More than 88.3% of sequences in GenBank, which showed identical matches with VR744, belong to Vibrio genus. A total of 33 clones were randomly selected and sequenced. All of the sequences showed their highest similarities to and clustered around those of diverse known Vibrio species. The primers designed are capable of retrieving a wide range of Vibrio 16S rDNA fragments specifically among bacterial flora in seawater, the most important natural environment of seafood cultivation.     

Lytic Characteristics and Identification of Two Alga-lysing Bacterial Strains

Lytic Characteristics and Identification of Two Alga-lysing Bacterial StrainsBeaulieu, S. E., M. R. Sengco, and D. M. Anderson, 2005. Using clay to control harmful algal blooms: deposition and resuspension of clay/algal flocs. Harmful Algae . (4): 123-138…     

3种柔鱼线粒体基因与核核糖体基因片段序列比较分析

为准确检测柔鱼（Ommαstrephesbartram川、茎柔鱼（ Dosidicus gigas）与阿根廷滑柔鱼（ IIIex argentinus ） 的种间遗传差异,对线粒体16SrRNA、细胞色素b（Cytb）与编码核糖体大亚基的基因（28SrDNA）片段序列进 行测定。经比对获得同源片段序列的长度分别为444、430、464坤,其中16SrRNA与28SrDNA基因片段上分别存在3处和47处碱基插入/缺失。核昔酸组成分析表明;3种柔鱼在3个基因片段上的核音酸组成差异不显著, 在线粒体2个基因片段上的A＋T含量（16SrRNA;69.90%、72.01%、74.66%; Cytb; 63.61%、68.91%、71.65% ） 均明显高于C＋C含量（16SrRNA;30.10%、27.99%、25.34%; Cytb; 36.39%、31.09%、28.35% ）,而在28SrDNA 基因片段上的A＋T含量（37.16%、36.74%、38.29% ）明显低于C＋C含量（62.84%、63.26%、61.71 %）0 3种柔鱼 在28SrDNA基因片段上检测到的核昔酸替代率最低,为6.68%,而蛋白质编码基因Cytb核昔酸替代率最高,为 20.93%,核营酸替代均发生在密码子第3位点上,而且未引起氨基酸替代。基于邻接法、最大简约法与最大似然 法重建的系统树显示,柔鱼与茎柔鱼的亲缘关系较近。根据C严b基因片段序列分析,柔鱼与茎柔鱼和阿根廷滑柔鱼的分歧时间分别为653-790万a和765 - 925万a,种间分化事件发生在中新世至上新世间。     

Identification of two Skeletonema costatum-like diatoms by fluorescence in situ hybridization

A harmful algae bloom (HAB) is a dense aggregation of algae in a marine or aquatic environment that can result in significant environmental problems. To forecast the occurrence of HAB, development of a rapid and precise detection method is urgently required. In this study, two Skeletonema costatum-like diatoms (SK-1 and SK-2), were identified morphologically under a light microscope, and detected using fluorescent in situ hybridization (FISH). Strain SK-1 was isolated from a frequently HAB affected area of the East China Sea, and strain SK-2 from an aquatic farm in Qingdao, China. Fluorescent DNA probes were designed that were complementary to the ITS sequence (including 5.8S rDNA) of strain SK-1. After hybridization, strong green fluorescence was observed in cells of strain SK-1 under an epifluorescence microscope; however, no such fluorescence was observed with strain SK-2, which indicates that probes hybridized only the DNA of the target strain, SK-1, in species-specific manner, and that the two strains do not belong to a same species. This finding was confirmed by ITS sequence analysis. The FISH technique used in this study was sensitive, simple, and rapid, and is a promising tool for detecting target HAB species in natural environments.     

一株分离自红树林沙雷氏菌的鉴定及杀虫活性测定

【目的】为开发用于害虫生物防治的细菌杀虫剂,对分离自红树林的一株沙雷氏菌进行鉴定并初步测试其杀虫活性。【方法】采用形态特征比较、细菌理化特性测定和基于16S rDNA序列构建系统发育树对从湛江红树林根际分离的菌株ZJ9进行综合鉴定,采用喂毒法和注射法进行杀虫活性测定。【结果】该菌为黏质沙雷氏菌Serratia marcescens,对草地贪夜蛾3龄幼虫7d校正死亡率为80.21%,且与苏云金芽孢杆菌和隐地杆菌无显著差异,菌体及菌液对大蜡螟老熟幼虫血腔注射24 h死亡率均达100%。【结论】菌株ZJ9为黏质沙雷氏菌,对草地贪夜蛾具有很好杀虫活性,可用作害虫的生物防治资源。     

Integrating a DNA barcoding project with an ecological survey: a case study on temperate intertidal polychaete communities in Qingdao, China

In this study, we integrated a DNA barcoding project with an ecological survey on intertidal polychaete communities and investigated the utility of CO1 gene sequence as a DNA barcode for the classification of the intertidal polychaetes. Using 16S rDNA as a complementary marker and combining morphological and ecological characterization, some of dominant and common polychaete species from Chinese coasts were assessed for their taxonomic status. We obtained 22 haplotype gene sequences of 13 taxa, including 10 CO1 sequences and 12 16S rDNA sequences. Based on intra- and inter-specific distances, we built phylogenetic trees using the neighbor-joining method. Our study suggested that the mitochondrial CO1 gene was a valid DNA barcoding marker for species identification in polychaetes, but other genes, such as 16S rDNA, could be used as a complementary genetic marker. For more accurate species identification and effective testing of species hypothesis, DNA barcoding should be incorporated with morphological, ecological, biogeographical, and phylogenetic information. The application of DNA barcoding and molecular identification in the ecological survey on the intertidal polychaete communities demonstrated the feasibility of integrating DNA taxonomy and ecology.     

Sequence analysis of the ITS region and 5.8S rDNA of <Emphasis Type="Italic">Porphyra haitanensis</Emphasis>

The sequences of the ITS(internal transcribed spacer) and 5.8S rDNA of three cultivated strains of Porphyra haitanensis thalli(NB,PT and ST) were amplified,sequenced and analyzed.In addition,the phylogenic relationships of the sequences identified in this study with those of other Porphyra retrieved from GenBank were evaluated.The results are as follows:the sequences of the ITS and 5.8S rDNA were essentially identical among the three strains.The sequences of ITS1 were 331 bp to 334 bp,while those of the 5.8...