柄海鞘共附生细菌的分离培养与系统发育多样性研究

通过纯培养和16SrRNA基因序列的相似性比对,并构建系统发育树对威海海域柄海鞘共附生细菌的多样性进行了研究。用2216E培养基从柄海鞘样品中分离到78株细菌,通过形态学特征和TCBS培养基选择性筛选后,选取30株具有代表性的菌株进行了16SrRNA基因测序和基于16SrRNA基因序列的系统发育多样性分析。结果表明,30株菌株分别属于细菌域的4个系统发育类群(Actinobacteria,Bacteroidetes,Firmicutes,Gro-teobacteria)的11个科,11个属。根据16SrRNA基因序列,大多数菌株与其系统发育关系最密切的模式菌株之间存在一定的遗传差异(16SrRNA基因序列相似性为91.82%～98.93%)。其中菌株HQW7,HQYD1,HQWD4,HQE1和HQE2与相关模式菌株的16SrRNA基因最高相似度仅分别为91.82%,92.51%,92.99%,93.52%,93.87%,这5株菌可能代表新的分类单元。威海海域柄海鞘共附生细菌存在着较为丰富的物种多样性和系统发育多样性,并可能存在新的物种。     

三份南海岛礁珊瑚砂样品中可培养细菌多样性

岛礁珊瑚砂环境中存在着大量未被培养和利用的微生物资源, 微生物多样性研究是了解微生物生态功能、开发和利用微生物资源的基础。文章采用多种寡营养培养基选择性分离南海岛礁珊瑚砂中的可培养细菌, 共获得纯培养细菌菌株349株, 通过16S rRNA基因序列分析, 发现它们隶属于4门(Actinobacteria、Proteobacteria、Firmicutes和Bacteroidetes)、6纲、26目、43科、73属、134种, 可培养细菌的优势类群为放线菌门, 占所有分离菌株数量的60%; 而且还发现18个16S rRNA 基因序列相似性低于97%的潜在新种。本研究使用改良优化的寡营养培养基进行分离, 较好地显示出样品中微生物的群落组成, 且获得了大量潜在的稀有新物种资源。研究结果表明, 岛礁珊瑚砂样品的可培养细菌资源十分丰富、细菌群落所涉及的生态功能完整、潜在新种比例较高, 为后期岛礁微生物资源挖掘打下了良好的基础, 也为后期开发应用积累了丰富且稀有的菌种资源。     

俄罗斯库页岛潮间带沉积物可培养细菌的系统发育多样性

采用2216E、R2A、M1培养基,首次从俄罗斯库页岛潮间带沉积物样品中分离到82株可培养海洋细菌,经查重并菌后选取其中45株具有代表性的菌株进行了16S r RNA基因序列分析和多样性研究。结果发现,它们共分布于变形细菌门(Proteobacteria)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes)4个类群(分别占45株代表菌株的42.2%、20.0%、20.0%和17.8%),代表着6个纲、27个属、44个种;同时发现其中有8个潜在新种和1个潜在新属。本研究结果表明,俄罗斯库页岛潮间带沉积物可培养细菌的系统发育多样性非常高,大部分菌株与分离自日本和韩国近海的细菌物种在系统发育关系上高度同源,但也存在不少潜在的新物种。     

Khai岛和Pathiu岛珊瑚礁沉积物细菌多样性及细菌粗提物延缓秀丽隐杆线虫衰老活性研究*

在Khai岛和Pathiu岛珊瑚礁生长海域采集2份珊瑚礁沉积物样品, 将样品预处理后采用6种分离培养基进行分离培养, 获得活性海洋细菌菌株, 通过PCR扩增和16s rRNA基因序列测定, 分析物种多样性, 并构建系统发育树。以模式生物秀丽隐杆线虫为模型考察菌株发酵提取物延缓衰老的作用。实验结果表明, 从4种样品中分离鉴定出22株细菌, 分别属于11目13科14属。16S rRNA基因序列相似性低于98.65%的细菌有2株, 疑似为潜在新种。GXIMD014 (Vibrio galatheae)和GXIMD112 (Kocuria kristinae)的500μg·mL^-1细菌粗提物具有延缓线虫衰老的活性, 能不同程度的延长线虫寿命, 提高线虫产卵量、运动能力、热激损伤能力、抗氧化损伤能力、体内酶活性以及改善虫体表皮形态。Khai岛和Pathiu岛珊瑚礁沉积物细菌具有较高的物种多样性, 蕴藏着丰富的微生物资源, 且部分细菌菌株具有延缓衰老的生物活性。     

南大西洋热液区沉积物可培养细菌的多样性分析和产酶活性鉴定

为获得深海可培养微生物资源,利用2216E培养基,采用涂布平板法从南大西洋热液区沉积物中分离可培养细菌,并通过16S rRNA基因进行分类鉴定.同时测定各菌株产淀粉酶、蛋白酶、果胶酶、木聚糖酶、褐藻酸酶和脂肪酶(三丁酸甘油酯和吐温80)的能力.分离共获得216株菌株,经16S rRNA基因序列比对,共获得71株不同的菌株.其中包括变形菌纲62株、放线菌纲3株、芽孢杆菌纲2株和拟杆菌纲4株,分布于27个属,41个种.酶活检测结果显示,具有淀粉酶、蛋白酶、脂肪酶(底物为三丁酸甘油酯)和脂肪酶(底物为吐温80)的产酶细菌分别为18、14、25、32株,供测细菌中未检出产果胶酶、木聚糖酶和褐藻酸酶的菌株.这些菌株为进一步研究开发提供了深海微生物资源.     

广西茅尾海红树林土壤放线菌多样性及功能酶活性研究

研究广西茅尾海红树林自然保护区内红树林土壤中可培养放线菌的多样性,挖掘具有高产多种功能酶活性的放线菌类群。应用可培养技术和基于16S rRNA基因序列的系统发育分析研究红树林土壤中可培养放线菌的多样性;并以10种酶活底物为指示物,结合点植法和透明圈法对可培养的放线菌进行功能酶活性筛选。结果显示,从红树林土壤中共分离到444株放线菌,隶属于6目13科24属63种,其中3株放线菌为潜在新种。从63株不同种的放线菌中筛选出56株放线菌在至少1种功能酶活性检测中显示阳性,总阳性率为88.89%;其中具有2种以上功能酶活性的放线菌31株,链霉菌属、微杆菌属和短小杆菌属的产酶菌株最为丰富。综上所述,广西茅尾海红树林自然保护区内红树林土壤中可培养放线菌的种类丰富多样,潜藏着放线菌新资源,且功能酶活性显著,具有较大的挖掘潜力。     

海南红树林根系土壤中可培养细菌的多样性分析

为研究海南红树林根系土壤细菌多样性,充分发掘中国红树林特有微生物资源,作者于2012年12月份从10份采集于海南北港岛红树林根系淤泥样品中分离可培养海洋细菌,并基于16S rRNA基因序列分析的方法对样品中的细菌多样性进行研究。共分离得到122株海洋细菌,经并菌后选取其中的70株代表性菌株测序,结果发现它们分为4个类群:变形菌门(主要为γ-变形菌纲)(Proteobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)和放线菌门(Actinobacteria),分别占66%,19%,11%和4%。共代表30个属47个种,其中有9个潜在新种和2个潜在新属。本研究结果表明,北港岛红树林中存在着丰富的细菌多样性,很多类群可能是红树林特有细菌物种。     

浙江镇海潮间带沉积物中可培养微生物初步研究

针对浙江镇海潮间带沉积物样 品,采用纯培养法分离培养海洋微生物,并基于16S rRNA基因序列,开展系统发育学研究,分析沉积物细菌群落结构及多样性。分离获得细菌39株,16S rRNA基因序列分析表明,这些菌株分别属于厚壁菌门Firmicutes(51. 3%)、变形杆菌门Proteobacteria(30.8%)、拟杆菌门Bacteroidetes(15. 4%)和放线菌门Actinobacteria(2. 6%)4个类群。厚壁菌门和变形杆菌门的菌株主要归属于芽孢杆菌纲Bacilli和a-变形杆菌纲Alphaproteobacteria,它们可能在近海海洋生态系统的元素地球化学循环中发挥了重要作用。此外,部分菌株16S rRNA基因序列与已报道物种的相似性较低,可能代表了新的分类单元。研究丰富了浙江近海微生物种质资源,并拓展了对浙江近海可培养细菌多样性的认识,为今后开发利用海洋微生物资源积累了资料。     

红鳍东方鲀(Takifugu rubrips)病原杀对虾弧菌的主要生物学特性研究

采用表观分类学及分子生物学方法,对从发病的养殖红鳍东方()中分离的细菌进行了主要生物学特性研究.主要包括形态特征、理化特性等较系统的表观分类学指征鉴定;同时对代表菌株进行了16S rRNA基因的分子鉴定,测定了16S rRNA基因序列、分析了相关细菌相应序列的同源性、构建了系统发生树;结果表明,分离菌为弧茵属(Vibrio Pacini 1954)的杀对虾弧菌(Vibrio penaeicidasp.nov.Ishimaru et al 1995).代表菌株(Hc051105-6株)16s rRNA基因序列长度(不包括引物结合区)为1443bp(GenBank登录号:EU073023),代表菌株(HC051105-8株)16S rRNA基因序列长度(不包括 引物结合区)为1450bp(GenBank登录号:Eu073024).择代表菌株做对健康红鳍东方鲀的人工感染试验,认为分离鉴定的杀对虾弧菌在被检红鳍东方鱼电病例具有一定的病原学意义.药敏试验结果显示,对供试37种抗菌药物中,在一些药物中存在耐药性的菌株间差异.     

北极海洋沉积物中可培养细菌及其多样性分析

利用Zobell 2216E培养基和涂布平板法对北极海洋沉积物中可培养细菌进行分离纯化,并利用16SrRNA基因进行分子鉴定与系统发育分析。根据菌落形态学特征,从59个站点的沉积物样品中共分离纯化获得570株细菌;基于16SrRNA基因的分子鉴定与系统发育分析表明,分离到的可培养细菌分别属于细菌域的4个门,5个纲,12个目,23个科,47个属,102个种,其中γ-Proteobactria占绝大多数;有14株菌株与模式菌株的16SrRNA基因序列相似性小于97%,为6个潜在的新种。北极海域的海洋沉积物中存在着丰富的微生物种质资源,为开发新型生物活性物质和特殊功能基因打下了基础。     

Isolation, distribution and evaluation of cytotoxic and antioxidant activity of cultivable actinobacteria from the Oman Sea sediments

Screening bioactive natural products from bacteria is a determinative step in the drug discovery programs. The present study aim to isolate actinobacteria from the Oman Sea sediments for determining the effects of different culture media and treatments on the yield of the isolation process, and measure the DPPH radical scavenging and Artemia cytotoxic activity of culture extracts of the actinobacterial isolates. A total of 290 actinobacterial isolates were collected from 14 sediment samples. Heat treatment(40.68%) and M_4 medium(29.31%) exhibited the maximum isolation rates of actinobacteria. Streptomyces isolates were dominantly distributed in all of the investigated stations according to 16 S rRNA gene sequencing. The distribution pattern of Streptomyces followed a depth-dependent frequency trend, whereas the members of rare genera including Micromonospora, Nocardia Actinoplanes, Nocardiopsis, Saccharopolyspora and Crossiella were distributed in deeper stations. Approximately,25% of the examined isolates could scavenge 90% of 10~(–4) mol/L DPPH solutions at 1 250 μg/mL final concentration of their ethyl acetate culture extracts. Furthermore, the most potent extracts could scavenge DPPH radicals with IC50 ranges from 356.8 to 566.4 μg/mL. Brine shrimp cytotoxicity tests showed that 38.88% of the examined culture extracts exhibited LC_(50) lower than 1 000 μg/mL against the Artemia cells. Moreover, the most potent culture extracts exhibited LC_(50) range from 335.4 to 534.4 μg/mL. Phylogenetic analysis by 16 S rRNA gene sequence revealed that the OS 005, OS 263 and OS 157 closely related to Streptomyces djakartensis, Streptomyces olivaceus and Nocardiopsis dassonvillei respectively. These results suggested the widespread distribution of the antioxidant and cytotoxic producing actinobacteria in the Oman Sea sediments, which could be considered as promising candidates for the discovery of microbial bioactive compounds.     

八门湾红树林土壤放线菌多样性及抗病原菌活性分析

红树林土壤中蕴藏着丰富的放线菌资源,是新菌种的重要来源。为获得更多新的放线菌资源以用于开发新的生物活性化合物,了解放线菌的多样性是非常重要和必要的。研究采用扩增子高通量测序和传统培养法对八门湾红树林土壤放线菌进行了非培养和培养水平多样性研究,同时对其抗病原菌活性进行了分析。对于非培养水平多样性研究,与细菌通用引物相比,利用放线菌相关引物可以提高放线菌丰度的检测水平,其百分比含量提高2.47倍;可以检测到放线菌门更多的目、科和属;对于在目水平上的放线菌类群组成来说,Acidimicrobiales、Corynebacteriales、Gaiellales、Kineosporiales、Solirubrobacterales是优势类群,但是在用两对不同引物得到的结果中,其百分含量差异较大。放线菌相关引物更适合环境样品中放线菌多样性的分析。对于培养水平多样性研究,分离到256株放线菌,属于7个目、9个科、14个属, Shannon-Wiener多样性指数为1.32, Streptomyces(42.58%)和Micromonospora(42.19%)是优势属;与模式菌株的相似性小于98.5...     

Phylogenetic diversity of bacteria associated with the marine sponge Agelas robusta from South China Sea

It is well known that marine sponges harbor large numbers of bacteria, some of which have been proved to be sponge-specific. The diversity of bacteria in marine sponges distributed along the coast of South China Sea has been previously studied but that of bacteria in sponges inhabiting the open sea has been rarely investigated. In this report, the diversity of bacteria associated with the marine sponge Agelas robusta from a remote coral reef in the South China Sea was documented employing 16S rDNA library construction, amplified ribosomal DNA restriction analysis (ARDRA) and phylogenetic analysis. A total of 32 phylotypes were finally categorized in nine phyla including Cyanobacteria, Proteobacteria, Chloroflexi, Firmicutes, Actinobacteria, Acidobacteria, Planctomycetes, Bacteroidetes and Gemmatimonadetes. The dominant phylum was Proteobacteria whereas the dominant genus was Synechococcus in Cyanobacteria. Some spongespecific bacteria were also found in the bacteria population, but the proportion (5 OTUs/32 OTUs) was much lower than other sponges. This study reveals the phylogenetic diversity of bacteria in A. robusta and confirms the presence of some sponge-specific bacteria in the South China Sea. Understanding the diversity of sponge-associated bacteria in China assists to exploit the bacteria resources for biotechnology.     

海绵共附生微生物基因多态性的RAPD-PCR分析

采用传统分离手段对中国南海海域的细薄星芒海绵（Stelletta tenui（Lindgren,1897）1、皱皮软海绵（Halichondria rugosa（Ridley＆Dendy））、澳大利亚厚皮海绵（Craniella australiensis（Carter））、贪婪倔海绵（Dysidea avara（Schmidt））4种海绵体内的微生物进行了分离培养,随机挑选64株芽孢杆菌属细菌（每种海绵16株）进行了RAPD-PCR基因多态性分析。研究表明,一些海绵微生物是可以通过传统分离手段得到的,来自同一或者不同海绵的微生物均具有丰富的基因多态性。     

Macrofauna associated to Mycale microsigmatosa (Porifera, Demospongiae) in Rio de Janeiro State, SE Brazil

The macrofauna (endo- and epi-biotic) associated to the sponge Mycale (Carmia) microsigmatosa Arndt, 1927 was studied at three sites in Rio de Janeiro State, Brazil (Arraial do Cabo, Niterói, and Rio de Janeiro). A total of 2235 individuals (over 1 mm long) of 75 invertebrate species were found associated to 19 specimens of the sponge. The most abundant and diverse taxa were the crustaceans (83%, 31 spp.), polychetes (10%, 18 spp.), and molluscs (3.7%, 15 spp.). Cnidarians, platyhelminthes, ascidians, echinoderms, pycnogonids, bryozoans, and sponges were also represented. Amphipod crustaceans were the dominant group, comprising 61% of all individuals collected. Species richness and abundance of associated fauna were highly correlated with sponge volume, but diversity and evenness were not. The site of collection influenced the species composition of the fauna associated to M. microsigmatosa but did not change significantly its diversity, abundance, richness, and dominance patterns of higher taxa. Pregnant females and juvenile stages of 29% of the species associated, including crustaceans, molluscs, echinoderms, and pycnogonids were frequently found inside M. microsigmatosa. Although many of these organisms do occur and reproduce in other habitats outside the sponge as well, M. microsigmatosa is also important for their reproduction and survivorship, thus contributing for the maintenance of biodiversity in Southwestern Atlantic sublittoral rocky shores.     

Sponge epibionts on ecosystem-engineering ascidians: The case of Microcosmus sabatieri

The study of epibionts on habitat engineering ascidians is of increasing interest because changes in the population structure of the latter may affect associated communities, especially in the case of commercially exploited species. The solitary ascidian Microcosmus sabatieri lives on rocky cliffs in the Eastern Mediterranean and is harvested in certain Aegean areas. Its hard, wrinkled tunic is usually fouled by various epibionts both sessile and motile. Sponges are an important component of this complex and their biomass may be higher than that of the ascidian itself, strongly affecting diversity and abundance of the motile epifauna. The aim of this study was to examine in detail the structure of the epibiotic sponge assemblage on ascidians collected from their main fishing grounds in the South Aegean Sea. A rich (41 species) and taxonomically diverse sponge assemblage was found, while only eight species contributed 80% of the total sponge cover. Most of the epibiotic sponges commonly grow on the surrounding sublittoral cliffs. The encrusting sponge growth form prevailed in cover of the ascidian tunic, while two massive species dominated in terms of frequency of appearance and abundance. Ascidian dimensions, weight and volume were significantly correlated with sponge diversity, abundance and cover area, thus structuring the epibiotic sponge assemblage. Spatial patterns in sponge cover were not clear, but a general declining NW to SE trend in sponge richness, abundance and cover appeared in accordance with previous records. Sponge distribution on the ascidian tunic presented a clear pattern related with characteristic features of the ascidian: the posterior zone supported the richest and most expansive sponge fauna. The ecosystem-engineering process performed by the ascidian is enhanced by the diverse epibiotic sponge assemblage, thus further increasing habitat complexity in this space-limited, temperate, sublittoral, rocky environment.     

Five new species of lichomolgid copepods associated with ascidians from Korea,with proposal of two new genera (Crustacea,Copepoda, Lichomolgidae)

Five new species of the family Lichomolgidae associated with solitary ascidians are described from the east coast of Korea. Two new genera are proposed: Protomolgus n. gen. to incorpotate Protomolgus duplex n. sp. and P. singularis n. sp., and Dontimolgus n. gen. to incorporate Dontimolgus brevicaudatus n. sp. Protomolgus n. gen. characteristically has a four-segmented female maxilliped and a bipartite mandible. Dontimolgus n. gen. possesses a large, tooth-like process on the first maxillary segment and three spines on the third exopodal segment of leg 3. Other two new species described are Lichomolgus infirmus n. sp. and L. pectinatus n. sp.