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。     

一株海水氨氧化细菌的系统发育分析

应用PCR技术扩增16S rRNA基因和amoA(ammonia monooxygenase subunit A)的基因片段,并测定其序列,对一株源于海水养殖水体的高效氨氧化细菌(ammonia-oxidizing bacteria,AOB)进行了系统发育分析。结果表明,经PCR扩增得到了1 098 bp的16S rRNA基因片段和491 bp的amoA基因片段,将其序列用NCBI-Blast软件在GenBank数据库中进行同源性检索后发现,该菌株的16S rRNA基因序列和amoA基因序列分别与亚硝化单胞菌Nitrosomonas sp.NS20的相对应基因片段相似性分别为98.4%和96.7%。在此基础上构建了系统发育树,表明用该菌株的16S rRNA基因片段和amoA基因片段构建的系统发育树均与亚硝化单胞菌属类聚在一起,结合该菌株形态和生理生化特性,鉴定该株氨氧化细菌属亚硝化单胞菌。     

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)。     

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     

蛤蜊科3种贝类16S rRNA基因片段及ITS2核苷酸序列分析

利用PCR技术分别扩增连云港及启东沿海蛤蜊科的西施舌(Coelomactra antiquata)、中国蛤蜊(Mactrachinensis)和四角蛤蜊(Mactra veneriformis)3种双壳贝的16S rRNA基因片段和ITS2核苷酸序列,测序后用DNA star软件分析了核苷酸差异。结果显示:三种贝类16S rRNA基因片段长度相同,均为306bp(去除引物),核苷酸存在多态性,共有45个变异位点,54个核苷酸发生了变异,全部为碱基置换。西施舌与中国蛤蜊此片段核苷酸的同源性为88.9%,与四角蛤蜊的同源性为88.6%,中国蛤蜊与四角蛤蜊的同源性为90.6%。三种蛤蜊ITS2序列分别为390 bp(西施舌)4、41 bp(四角蛤蜊)和466 bp(中国蛤蜊),存在长度多态性,ITS2核苷酸差异分析结果显示,西施舌与中国蛤蜊的同源性为70.9%-71.1%,西施舌与四角蛤蜊的为70.5%-71.0%,中国蛤蜊与四角蛤蜊的同源性为88.1%-88.8%。ITS2序列分析结果与16S rRNA基因片段分析结果一致,2种分子分析法均显示中国蛤蜊与四角蛤蜊的亲缘关系近。     

环境16S rDNA和16S rcDNA序列分析湖光岩玛珥湖的浮游细菌组成

通过提取环境总16S rDNA和总16S rRNA分别构建DNA克隆文库和反转录cDNA文库,并进行克隆测序和序列分析,探讨湖光岩玛珥湖浮游细菌和浮游活性细菌的遗传多样性。结果表明:构建两个水层的4个克隆文库,即1 m和10 m水层总菌的16S rDNA文库和总活性菌的16S rcDNA文库,共获得413条序列,分属15门、32目、52科;1 m水层的rDNA文库与rcDNA文库中Verrucomicrobia类群所占比例最大(36.8%和32.1%),其次为Alphaproteobacteri(a 15.1%和16%)和Betaproteobacteri(a 17.9%和16%);在10 m水层的2个文库中,Alphaproteobacteria类群均占绝对优势,分别为63.5%(rDNA)和43.8%(rcDNA),其次为Verrucomicrobia(11.5%和14.3%),其他细菌类群包括Acidobacteria、Chloroflexi、Firmicutes、待定门OD1、Planctomycetes、Spirochetes和Deltaproteobacteria,比例在0.8%~3.8%之间;在科的水平上,SAR11为湖光岩玛珥湖最为丰富的类群,占总克隆数的26.9%,其中在10 m水层的比例高达51.5%。序列同源性分析表明,绝大多数序列(89.8%)来源于新种,其中19.6%的序列可能来自于新属,69%的序列可能来自于全新的科。湖光岩玛珥湖浮游细菌群落结构较为独特,有大量的浮游细菌种类尚未获得相应的纯培养物,蕴含着丰富而新颖的微生物资源。     

海洋短小芽孢杆菌ZH1-6菌株的鉴定及生物学特性

从新鲜近江牡蛎中分离到一株拮抗细菌,命名为ZH1-6,该菌对金黄色葡萄球菌、乙型副伤寒沙门氏菌及大肠杆菌等多种病原菌有较强的抑制作用。通过形态学观察、常规生理生化指标测定、16SrDNA序列测定和同源性分析,鉴定该菌株为短小芽孢杆菌。研究表明:ZH1-6能利用多种碳、氮源,其生长温度范围为10-50℃,生长pH范围为pH3-10,最适初始pH为6.0。     

基于分子生物学的大庆油田聚驱后内源微生物资源评价

为提高内源微生物驱采收率,调查大庆油田聚驱后区块的油藏微生物,并采集油水样,分析内源微生物的总数、类型和分布规律.研究聚驱后油藏中的细菌和古菌,利用PCR-DGGE指纹图谱、16S rDNA克隆文库的序列的生物进化树(确立微生物种类)和油藏微生物群落的16S rDNA多态性,得到内源微生物群落的16S rRNA基因分布...     

海洋动物肠道中抗氧化活性乳酸菌的分离及多样性分析

【目的】分析海洋动物肠道中抗氧化活性乳酸菌的多样性,并对活性菌株胞外与胞内代谢物清除1,1-二苯基-2-三硝基苯肼(DPPH)自由基能力进行测定。【方法】采用溶钙圈分离培养技术和DPPH自由基清除法,从湛江海域6种海洋动物肠道中分离活性乳酸;通过构建16S rDNA基因序列系统发育树,分析其多样性;利用DPPH自由基清除法,测定其抗氧化活性。【结果】共分离得到16株抗氧化活性乳酸菌,分属于细菌域厚壁菌门(Firmicutes)的4个科(Leuconostocaceae,Streptococcaceae,Lactobacillaceae,Enterococcaceae)、6个属(Weissella,Pediococcus,Leuconostoc,Lactococcus,Lactobacillus,Enterococcus),其中除菌株zy-3、LY-87、ZH-54的16S r DNA基因序列与数据库EzBioCloud中的模式菌相似性100%外,其余13株活性菌株与模式菌存在一定的遗传差异(97.42%～99.93%),菌株zy-I与模式菌株Weissella cibaria(KACC11862T)的相似性仅为97.42%,推测为Weissella cibaria属中的潜在新种;16株活性菌株胞外代谢物对DPPH的抗氧化活性均大于胞内代谢物,且5株乳酸菌株(zy-4、zy-s6、YM-49、ZH-53、ZH-54)的胞外的DPPH清除能力均在45%以上,高于其它11株菌株。【结论】湛江海域动物肠道中存在较为丰富的抗氧化活性乳酸菌,并蕴藏潜在乳酸菌新种。     

钦州湾牡蛎线粒体16S rRNA和COⅠ基因片段的序列变异分析

利用线粒体16S rRNA基因和线粒体细胞色素氧化酶亚基Ⅰ(cytochrome oxidaseⅠ,COⅠ)基因片段初步研究钦州湾牡蛎(Ostrea)的物种组成。以特异引物进行PCR扩增,对扩增产物进行纯化、测序,分析表明,16S rRNA基因部分长度为413bp,COⅠ基因部分长度为535bp,2种牡蛎序列的碱基组成均显示出较高的A+T比例:16S rRNA基因59.8%;COⅠ基因60.5%。对位排序比较表明,16S rRNA片段种内个体间变异较小,存在7个变异位点,4种单倍型,其中包括5个转换位点突变,2个颠换位点突变;COⅠ片段有30个碱基存在变异,7种单倍型,其中包括16个转换位点突变,14个颠换位点突变。运用MEGA4软件计算出不同个体间的遗传距离,并构建了NJ和UPGMA系统树。香港牡蛎(Crassostrea hongkongensis)16S rRNA和COⅠ序列与白肉牡蛎的遗传距离均为0.000,有明巨牡蛎(Crassostrea ariakensis)16S rRNA和COⅠ序列和红肉牡蛎(red meat ostrea)的遗传距离分别为0.000和0.011,白肉牡蛎16S rRNA和COⅠ序列和红肉牡蛎之间的遗传距离分别为0.035和0.146。结果表明,钦州湾牡蛎分为2个不同的种,线粒体16S rRNA和COⅠ基因在种间存在明显的多态性,证实了16S rRNA和COⅠ基因序列适用于牡蛎的系统学分析。     

钦州湾牡蛎线粒体16SrRNA和COI基因片段的序列变异分析

利用线粒体16SrRNA基因和线粒体细胞色素氧化酶亚基Ⅰ（cvtocllrome oxidase Ⅰ，COI）基因片段初步研究钦州湾牡蛎（Ostxea）的物种组成。以特异引物进行PCR扩增，对扩增产物进行纯化、测序，分析表明，16SrRNA基因部分长度为413bp，COI基因部分长度为535bp，2种牡蛎序列的碱基组成均显示出较高的A＋T比例：16SrRNA基因598％；COI基因605％。对位排序比较表明，16SrRNA片段种内个体间变异较小，存在7个变异位点，4种单倍型，其中包括5个转换位点突变，2个颠换位点突变；COI片段有30个碱基存在变异，7种单倍型，其中包括16个转换位点突变，14个颠换位点突变。运用MEGA4软件计算出不同个体间的遗传距离，并构建了NJ和UPGMA系统树。香港牡蛎（Crassostrea hongkongensis）16SrRNA和COI序列与白肉牡蛎的遗传距离均为0000，有明巨牡蛎（Crassostrea ariakensis）16SrRNA和COI序列和红肉牡蛎（redmeatostxea）的遗传距离分别为0000和0011，白肉牡蛎16SrRNA和COI序列和红肉牡蛎之间的遗传距离分别为0035和0146。结果表明，钦州湾牡蛎分为2个不同的种，线粒体16SrRNA和COI基因在种间存在明显的多态性，证实了16SrRNA和COI基因序列适用于牡蛎的系统学分析。     

Antifouling activities of marine bacteria associated with sponge (Sigmadocia sp.)

The present study aimed at assessing the antifouling activity of bacteria associated with marine sponges. A total of eight bacterial strains were isolated from the surface of sponge Sigmadocia sp., of them, SS02, SS05 and SS06 showed inhibitory activity against biofilm-forming bacteria. The extracts of these 3 strains considerably affected the extracellular polymeric substance producing ability and adhesion of biofilm-forming bacterial strains. In addition to disc diffusion assay, microalgal settlement assay was carried out with the extracts mixed with polyurethane wood polish and coated onto stainless steel coupons. The extract of strain SS05 showed strong microalgal settlement inhibitory activity. Strain SS05 was identified as Bacillus cereus based on its 16S rRNA gene. Metabolites of the bacterial strains associated with marine invertebrates promise to be developed into environment-friendly antifouling agents.     

Identification and characterization of pathogen to bacterial septicaemia in cultured turbot, Scophthalmus maximus

Bacteria-infected turbots Scophthalmus maximus with septicaemia were examined between 2001 and 2004 in aspects of the conditions of disease occurrence, clinical syndromes and pathological changes. The phenotypic information of pathogenic bacteria was studied, including morphology, physiological and biochemical characteristics, and the mol% G C of the DNA. In addition, representative strains (S010623-1, LH031120-1) were selected for molecular identification by partial 16S rRNA gene sequencing. The results show that the isolates (LH031120-1 to LH031120-6, HT040308-1 to HT040308-6, HT040620-1 to HT040620-6) from three farms were identified as Edwardsiella tarda. The isolates (S010610-1 to S010610-10, S010623-1 to S010623-20) from one farm were identified as Listonella anguillarum. We conducted studies on the pathogenicity of isolates by artificial infection, and revealed all infected groups in morbidity and mortality. The septicaemia infected turbot showed a syndrome similar to that of the naturally infected fish. Antibiotic sensitivity showed that of 37 antimicrobial agents, E. tarda was sensitive to 27 agents, and L. anguillarum was sensitive to 21 agents.     

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。     

Polyphasic examination on Merismopedia tenuissima CHAB 7021 from Ganjiang River,China revealed the polyphyly of the genus Merismopedia(Cyanobacteria)

Species in the cyanobacterial genus Merismopedia are present in freshwaters at different trophic levels, with some species even as the components of cyanobacterial blooms. However, species diversity in this genus was not fully verified by molecular investigation and polyphasic taxonomic studies. In this study, Merismopedia-like strain tenuissima CHAB 7021 was isolated from Ganjiang River in Jiangxi Province, China, and polyphasic characterization of this strain was performed by morphological observation, ultrastructural examination, chemical detection of pigments and phylogenetic analysis based on 16 S rRNA gene sequences. Morphological identification of the strain was supported by the ultrastructural features, as the tiny species Merismopedia tenuissima Lemmermann. The phylogeny based on 16 S rRNA gene sequences revealed at least three clades formed by the strains of Merismopedia. The three M. tenuissima strains including M. tenuissima CHAB 7021 was gathered in clade III with distant relationship to the clade I formed by the six Merismopedia strains including the type species M. punctata, and such a genetic distance may propose Merismopedia tenuissima to separate from Merismopedia genus. However intermixture relationship in between strains of M. punctate and M. glauca in the phylogenetic tree still complicated the taxonomic status in the genus Merismopedia. The process for taxonomic revision in the Merismopedia genus still await for examination and further information on more strains of type species M. punctata.     

Gel microbead cultivation with a subenrichment procedure can yield better bacterial cultivability from a seawater sample than standard plating method

A gel microbead (GMD) cultivation method was employed to cultivate microorganisms from an amphioxus breeding zone in Qingdao, P. R. China. The culture results were compared with those by standard plating method. In the GMD-based method, the microcolony-forming GMDs were sorted by fluorescence-activated cell sorting (FACS). To further get pure cultures, a subsequent enrichment culture and a streaking purification procedure were conducted on marine R2A medium. Eighty bacterial strains isolated by the GMD-based method were randomly selected for sequencing. These isolates belonged to Alphaproteobacteria (33%), Gammaproteobacteria (44%), Bacteroidetes (11%), Actinobacteria (5%), Firmicutes (5%), Epsilonproteobacteria (1%), and Verrucomicrobia (1%), the last two groups being usually difficult to culture. The 16S rRNA gene sequences revealed a diverse community with 91.1%-100% of the bacterial rRNAs similarities. Thirteen strains were sharing 16S rRNA gene sequence which was less than 97% similar to any other rRNA genes currently deposited in TYP16S database. Seventy isolates derived from the standard plating method fell into 4 different taxonomic groups: Alphaproteobacteria (9%), Gammaproteobacteria (81%), Bacteroidetes (7%) and Firmicutes (3%) with a 16S rRNA gene sequence similarities between 95.8%-100%, in which only 3 strains were sharing 16S rRNA gene sequence of less than 97%. The results indicated that the GMD-based method with subenrichment culture yielded more taxonomic groups and more novel microbial strains, including members of previously rarely cultured groups, when compared with the standard plating method, and that this method markedly improved the bacterial cultivability.