Morphological and molecular characterization within 26 strains of the genus Cylindrospermum (Nostocaceae,Cyanobacteria), with descriptions of three new species

Twenty‐six strains morphologically identified as Cylindrospermum as well as the closely related taxon Cronbergia siamensis were examined microscopically as well as phylogenetically using sequence data for the 16S rRNA gene and the 16S‐23S internal transcribed spacer (ITS) region. Phylogenetic analysis of the 16S rRNA revealed three distinct clades. The clade we designate as Cylindrospermum sensu stricto contained all five of the foundational species, C. maius, C. stagnale, C. licheniforme, C. muscicola, and C. catenatum. In addition to these taxa, three species new to science in this clade were described: C. badium, C. moravicum, and C. pellucidum. Our evidence indicated that Cronbergia is a later synonym of Cylindrospermum. The phylogenetic position of Cylindrospermum within the Nostocaceae was not clearly resolved in our analyses. Cylindrospermum is unusual among cyanobacterial genera in that the morphological diversity appears to be more evident than sequence divergence. Taxa were clearly separable using morphology, but had very high percent similarity among ribosomal sequences. Given the high diversity we noted in this study, we conclude that there is likely much more diversity remaining to be described in this genus.     

Bacterial microbiota assemblage in Aedes albopictus mosquitoes and its impacts on larval development

Interactions between bacterial microbiota and mosquitoes play an important role in mosquitoes’ capacity to transmit pathogens. However, microbiota assemblages within mosquitoes and the impact of microbiota in environments on mosquito development and survival remain unclear. This study examined microbiota assemblages and the effects of aquatic environment microbiota on the larval development of the Aedes albopictus mosquito, an important dengue virus vector. Life table studies have found that reducing bacterial load in natural aquatic habitats through water filtering and treatment with antibiotics significantly reduced the larva‐to‐adult emergence rate. This finding was consistent in two types of larval habitats examined—discarded tires and flowerpots, suggesting that bacteria play a crucial role in larval development. Pyrosequencing of the bacterial 16S rRNA gene was used to determine the diversity of bacterial communities in larval habitats and the resulting numbers of mosquitoes under both laboratory and field conditions. The microbiota profiling identified common shared bacteria among samples from different years; further studies are needed to determine whether these bacteria represent a core microbiota. The highest microbiota diversity was found in aquatic habitats, followed by mosquito larvae, and the lowest in adult mosquitoes. Mosquito larvae ingested their bacterial microbiota and nutrients from aquatic habitats of high microbiota diversity. Taken together, the results support the observation that Ae. albopictus larvae are able to utilize diverse bacteria from aquatic habitats and that live bacteria from aquatic habitats play an important role in larval mosquito development and survival. These findings provide new insights into bacteria's role in mosquito larval ecology.     

Morphology and Phylogenetic Position of an Unusual Stentor polymorphus (Ciliophora: Heterotrichea) Without Symbiotic Algae

We investigated the live morphology, infraciliature, and small subunit rRNA gene sequences of an unusual population of Stentor polymorphus without symbiotic algae that was isolated from the southeastern region of Brazil. The morphological and molecular data confirmed the identity of this strain as S. polymorphus. The Brazilian S. polymorphus organism is 850–2,000 μm in length in vivo and has colorless cortical granules, a moniliform macronucleus with 6–12 nodules, somatic ciliature composed of 50–60 kineties, a single contractile vacuole located to the left of the cytostome, and a conspicuous oral pouch, and it does not build a lorica. Based on the phylogenetic analyses, the Brazilian S. polymorphus was located within a cluster consisting of four other S. polymorphus sequences, with high support values using both the Bayesian inference and maximum likelihood algorithms. Our study presents the first report of a S. polymorphus population without its symbionts under natural conditions. On the basis of our findings, we propose that the presence or absence of symbiotic algae should not be used as a taxonomic character for the identification of Stentor species.     

一株纤维素降解细菌的筛选、鉴定及产酶条件分析

目的筛选高活性的纤维素降解细菌,并进行初步鉴定和产纤维素酶条件分析。方法采集吉首旗帜山松树林的土壤样品,通过富集培养和刚果红平板染色法筛选分离纤维素降解细菌;通过形态观察、生理生化特性检测和基于16S rRNA基因序列的系统发育分析对分离的菌株进行初步鉴定。利用单因素实验对产纤维素酶条件进行优化。结果分离获得1株高活性纤维素降解细菌JDM11,初步鉴定其为Bacillus velezensis;菌株JMD11产纤维素酶最佳培养温度、最适初始pH和培养时间分别为28℃、7.0～7.5和32h,在该条件下其滤纸酶(FPase)和羧甲基纤维素酶(CMCase)活力分别为260.32U/ml和651.75U/ml。结论菌株JDM11是1株高活性纤维素降解的Bacillus velezensis。     

津巴布韦烟叶中淀粉酶和蛋白酶产生菌的分离及鉴定

目的:从津巴布韦烟叶中分离产蛋白酶菌和产淀粉酶能力最高的菌株,并对其进行鉴定。方法:采用淀粉富集培养基和酪蛋白富集培养基分别分离津巴布韦烟叶中的产淀粉酶和产蛋白酶菌株,通过生理生化实验和16SrRNA序列分析鉴定分离的菌株。结果:产蛋白酶菌株菌体不透明、表面有褶皱,蛋白酶酶活为52.10±0.13 U/mL;产淀粉酶菌株菌体表面呈黏状,淀粉酶酶活为3.69±0.07 U/mL;产蛋白酶与产淀粉酶的2株菌均与枯草芽孢杆菌的16S rRNA序列有100%的相似性,结合生理生化指标初步鉴定为枯草芽孢杆菌。结论:获得的2株菌在降解烟叶的蛋白质和淀粉过程中可能起重要作用。     

人体肠道微生物多样性和功能研究进展

人体肠道中庞大而复杂的微生物群落对人体自身代谢表型有深远的影响.肠道微生物群落在亚种或菌株水平上表现出极大的多样性.利用微生物分子生态学、元基因组学和代谢组学研究方法,发现肠道微生物与宿主表现出共进化的特点,肠道微生物群落及其基因组为宿主提供了互补的遗传和代谢功能,表现出互惠共生关系.但是,肠道微生物群落中影响宿主代谢表型的关键功能菌鉴定及其作用模式问题仍然悬而未决,综合运用多种高通量研究方法和多维数据分析方法可能成为解决这个问题的突破口.     

Phylogeny of Thalassinidea (Crustacea, Decapoda) inferred from three rDNA sequences: implications for morphological evolution and superfamily classification

The infraorder Thalassinidea is a group of cryptic marine burrowing decapods of which the higher taxonomy is often contentious. The present analysis attempts to reconstruct phylogenetic relationship among 12 of the 13 currently recognized families using partial nuclear 18S, 28S rDNA and mitochondrial 16S rDNA sequences. The infraorder is divided into two distinct clades, with the first clade consisting of Thalassinidae, Laomediidae, Axianassidae and Upogebiidae, and the second clade including Axiidae, Calocarididae, Eiconaxiidae, Callianassidae, Ctenochelidae, Micheleidae, Strahlaxiidae and Callianideidae. Within the first clade, the Upogebiidae is the basal family. The Axianassidae shows low affinity to other laomediid genera indicating that it is a valid family. The interfamilial relationships are less well resolved in the second clade. The Axiidae is paraphyletic with respect to Calocarididae and Eiconaxiidae. Thus, the status of these two latter families is not supported if the currently defined Axiidae is maintained. All three families appear to be basal in the thalassinidean clade. The Micheleidae is closely related to the Callianideidae and they form a sister group to the Strahlaxiidae. The monophyletic Callianassidae aligns with the Micheleidae + Callianideidae + Strahlaxiidae clade. The relationship among the Axiidae + Calocarididae + Eiconaxiidae clade, Callianassidae + Micheleidae + Callianideidae + Strahlaxiidae clade and the Ctenochelidae cannot be resolved which might be due to a rapid radiation of the three lineages. Our results do not support the generally used classification scheme of Thalassinidea and suggest that the infraorder might be divided into two superfamilies instead of three as suggested based on larval morphology, second pereiopod morphology in adults and gastric mill structure. The two superfamilies are Thalassinoidea (i.e. Thalassinidae, Laomediidae, Upogebiidae and Axianassidae) and Callianassoidea (i.e. Axioidea + Callianassoidea, as defined in Martin and Davis (2001) but excluding Laomediidae and Upogebiidae). It also appears that gill‐cleaning adaptations are important in thalassinidean evolution while the presence of linea thalassinica is a result of parallel evolution.     

抑黄曲霉乳酸菌的筛选及菌种鉴定

目的筛选具有抑制黄曲霉(Aspergillus flavus)生长的乳酸菌。方法以各地泡菜、实验室自制泡菜、豆浆渣以及新鲜猪肠、鸡肠道内容物为材料,采用牛津杯法筛选所需菌株。对筛选出的菌株进行生理生化及16S rRNA基因序列同源性分析。结果分离得到756株乳酸菌,其中有6株菌株对黄曲霉的生长有明显的抑制作用。结论实验获得的6株产酸菌,3株为植物乳杆菌(Lactobacillus plantarum),2株为消化乳杆菌(L.alimentari-us),1株为亚利桑那乳杆菌(L.arizonenensis)。     

双歧杆菌地高辛标记寡核苷酸基因探针制备和应用研究

目的探讨地高辛标记寡核苷酸基因探针应用于微生态研究的可行性和实用性。方法制备双歧杆菌属和部分种的地高辛标记16S rRNA寡核苷酸探针,初步应用于微生态制剂鉴定和临床肠道微生态检测,评价寡核苷酸探针杂交在肠道微生态研究和检测中的应用价值。结果地高辛标记寡核苷酸探针具有较好的特异性与灵敏度：地高辛标记的双歧杆菌属和种的共6种寡核苷酸基因探针与标准菌株杂交后灵敏度和特异度分别为属探针95％、75％,青春双歧87．5％、90％,两歧双歧87．5％、87．5％,短双歧87．5％、92．5％,婴儿双歧75％、95％,长双歧75％、100％。结论寡核苷酸基因探针用于肠道细菌的鉴定显示出一定前景,加大探针的种类与扩大调查范围有可能使该技术替代现有细菌培养技术。     

多环芳烃降解菌X20的鉴定及降解特性

从多环芳烃降解高效的混合菌群中分离筛选到1株多环芳烃降解菌X20,经形态观察和16SrRNA序列分析,属于假单胞菌(Pseudomonas sp.)。采用室内摇瓶培养的方法,研究了该菌在不同环境条件下对菲和芘的降解。结果表明:弱碱环境有利于菌株X20对菲和芘的降解,最适pH为8.0;葡萄糖对菲芘降解率的影响呈抛物线变化,当葡萄糖浓度为0.2%时,X20对菲和芘的降解达到最高;X20对菲和芘的降解率随其初始浓度的上升而降低,菲和芘在初始浓度为10、20和40mg.L-1时的7d降解率分别为56.3%、39.25%、29.75%和41.8%、29.55%、23.50%,芘对X20降解的抑制强度高于菲。本研究结果将为构建高效的多环芳烃降解菌群,提高多环芳烃原位污染土壤的生物修复效果奠定基础。     

拟双角斯氏线虫共生细菌的分离与鉴定

从我国东北地区采集的拟双角斯氏线虫(Steinernema ceratophorum)肠道内分离到1株具有较强生物活性功能的致病杆菌菌株CB43。形态特征及生理生化特征测定结果表明,CB43菌株与致病杆菌属(Xe-norhabdus)的基本特性相似;对寄主线虫的产量有明显的促进作用,其代谢物对细菌和真菌均有较强的抑制活性,符合线虫共生菌的基本特性和功能。16S rRNA序列及根据16S rRNA序列构建的系统发育树中,CB43菌株与Xenorhabdus budapestensis序列同源性最高,形成一个类群。但CB43菌株不能产生吲哚,在麦芽糖、海藻糖、D-葡萄糖酸和乙酸利用等生化特征与X.budapestensis存在一定的差异,可能是由于菌株的生态差异造成的。根据形态及生理生化特征,结合16S rRNA序列分析,CB43菌株属于Xenorhabdus budapestensis。     

Pierce's Disease of Grapevines in Taiwan: Isolation,Cultivation and Pathogenicity of Xylella fastidiosa

Characteristic symptoms of Pierce's disease (PD) in grapevines (Vitis vinifera L.) were observed in 2002 in the major grape production fields of central Taiwan. Disease severity in vineyards varied, and all investigated grape cultivars were affected. Diseased tissues were collected from fields for subsequent isolation and characterization of the causal agent of the disease (Xylella fastidiosa). Koch's postulates were fulfilled by artificially inoculating two purified PD bacteria to grape cultivars Kyoho, Honey Red and Golden Muscat. The inoculated plants developed typical leaf‐scorching symptoms, and similar disease severity developed in the three cultivars from which the bacterium was readily re‐isolated, proving that the leaf scorch of grapevines in Taiwan is caused by the fastidious X. fastidiosa. This confirmed PD of grapevines is also the first report from the Asian Continent. Phylogenetic analyses were performed by comparing the 16S rRNA gene and 16S‐23S rRNA internal transcribed spacer region (16S‐23S ITS) of 12 PD strains from Taiwan with the sequences of 13 X. fastidiosa strains from different hosts and different geographical areas. Results showed that the PD strains of Taiwan were closely related to the American X. fastidiosa grape strains but not to the pear strains of Taiwan, suggesting that the X. fastidiosa grape and pear strains of Taiwan may have evolved independently from each other.     

基于形态特征和部分基因序列的中国蚱科四个种的分类订正

为了澄清形态相似种锯齿股蚱Tetrix dentifemura和粗体蚱T. grossus及红背悠背蚱Euparatettix erythronotus和九万山悠背蚱E. jiuwanshanensis的分类问题,我们分别从形态学分类和DNA数据对这4种蚱进行了分析。形态学特征分析结果显示：锯齿股蚱和粗体蚱形态相同之处非常多,而形态的不同之处主要表现在两者后翅的长度差异相对较大;红背悠背蚱和九万山悠背蚱在形态上共同之处很多,仅在头顶与颜面隆起的形状、触角与侧单眼着生位置以及后足股节长度等方面有微小差异。对蚱科2属4种昆虫的Cyt b,16S rRNA和18S rRNA基因部分序列进行了测定,3个片段共2 902 bp。发现蚱属的锯齿股蚱和粗体蚱的这三个基因同源序列完全一致,悠背蚱属的红背悠背蚱和九万山悠背蚱的同源序列也完全一致。结合形态学比较和基因序列分析结果,提出粗体蚱和九万山悠背蚱分别是齿股蚱和红背悠背蚱的同物异名。     

产中性纤维素酶耐碱芽孢杆菌的筛选及酶学性质研究

采用CMC碱性平板筛选方法,从造纸厂碱性淤泥中获得产中性纤维素酶的耐碱枯草芽孢杆菌C3004。根据其形态特征、生理生化特性和16S rRNA序列分析鉴定该菌株为枯草芽孢杆菌,并命名为Bacillus subtilis C3004。液体摇瓶培养24 h产生CMC酶活力达46.6 U/mL。酶学性质初步研究显示,CMC酶反应的pH值以7.0左右为适;在弱酸和碱性条件下也具有较高的酶活和一定的稳定性;反应温度以50℃左右为宜;且具有较好的热稳定性。Mn~(2+)与Fe~(3+)对酶反应有促进作用,Cu~(2+)、Mg~(2+)和Zn~(2+)对酶反应有抑制作用。该菌可在碱性(pH 8.5～10)条件下培养,具有不易被杂菌污染的特点。     

茜草内生菌的分离鉴定及其抗菌活性物质研究

对中药植物茜草(Rubia cordifoliaL)的内生菌进行了分离和抗菌活性筛选,获得一株具有广谱抗菌活性的内生细菌。该细菌对常见的3种人类病原菌和4种植物病原菌具有拮抗作用。传统分类学和基于16S rRNA基因的分子分类学证据表明,该内生细菌为一株新的枯草芽孢杆菌,命名为Bacillus subtilisRC4。B.subtilisRC4在综合马铃薯培养基(pH值5.0)中于28℃振荡培养60h,产生的代谢物对白色念珠菌的抗菌活性最强。抗菌活性物质在100℃受热20min,活性维持80%以上,且在pH值2.0~11.0范围内稳定。经硅胶柱层析和高效液相色谱分离,得到主要抗菌活性化合物,质谱分析表明其分子量约为288Da。     

鸡球虫18S rRNA基因序列的测定与分析

为了利用18S rRNA基因进行鸡球虫系统进化分析,对巨型艾美耳球虫(Eimeria maxima)、柔嫩艾美耳球虫(E.tenella)、堆形艾美耳球虫(E.acervulina)3种共8个不同来源的虫株,分别提取总DNA进行18S rRNA基因的扩增和测序;将得到的序列登录GenBank进行同源性和趋异性分析,并结合GenBank中其它原虫的18S rRNA基因序列构建进化树.结果显示扩增获得8株鸡球虫18S rRNA基因长度为1746～1756 bp,序列比对显示同种不同株间的同源性大于不同种间的同源性,其中3株E.maxima株间同源性在98.7%～99.3%之间,4株E.tenella株间同源性在99.7%～99.9%之间;不同种间同源性为96.5%～98.1%,其中E.maxima与E.tenclla的遗传距离最大,为0.038;E.maxima与E.acervulina的遗传距离最小,为0.021.顶复器门9个不同属所构建的进化树结果显示,E.imeria和等孢属(Isospora)聚为一支,说明亲缘关系比较近.与GertBank中其它5株不同鸡球虫的18S rRNA基因共同构建的进化树显示,3株E.maxima聚为一支,与E.brunetti、E.mitis、E.mivati、E.praecox和E.acervulina聚为一大分支;4株E.tenella与1株E.necatrix共同形成一个分支,说明E.tenella与E.necattix的亲缘关系最近.本研究证实了在鸡球虫系统进化研究中,18S rRNA基因不仅可以区分不同种,而且有可能成为区分同种不同株的理想靶基因.     

嗜盐菌HBCC-2的16S rRNA基因测序分析及其培养特性

从连云港台南盐场海盐生产区中分离纯化到一株嗜盐古菌HBCC-2,该菌株经PCR扩增后,测定其16S rRNA基因序列,采用BLAST软件对基因库中基因序列进行同源性比较,选取其相似性序列,采用Clustalx1.8和MEGA3.1软件对其16S rDNA序列进行了系统发育分析研究,结果表明HBCC-2菌株与菌株Halorubrum sp.GSL5.48的相似性达99%,结合其形态观察及生理生化反应特性,初步确定该菌株属于嗜盐红菌属(Halorubrum),菌株HBCC-2的16S rDNA序列已登陆到GenBank,其序列号为EF687739.通过比较不同NaCl浓度、pH和培养温度对该菌株生长的影响情况,研究了该菌株的生长特性,结果表明NaCl浓度为4mol/L、温度为35℃和pH为7.0的培养条件下其生长最佳.     

基于18S rRNA的中国大陆沿海石磺科贝类分类的初步分析

对采自上海崇明、福建宁德、海南海口等沿海地区9个群体的石磺科贝类进行外部形态特征差异分析和内部结构比较,在初步分类基础上利用核糖体小亚基18S rRNA基因部分序列对9个群体进行系统发育分析,以菊花螺为外群,结合GenBank上石磺科4个18S rRNA基因序列构建系统发生树来探讨我国大陆沿海石磺科属种间的亲缘关系.结果显示:我国石磺科贝类南方沿海种类多于北方沿海;除报道的瘤背石磺(Onchidium struma)和石磺(O.verruculatum)外,可能还有新记录5种:Onchidium属1种、Platevindex属2种、Peronia属1种和Paraoncidium属1种.分子系统发生树显示,我囝大陆沿海石磺科9个群体可分为4个亚群,分别为Onchidium、Platevindex、Paraoncidium、Peronia,其中Peronia亚群的置信度较高;Onchidium verruculatum应更名为Peronia verruculata.     

枯草菌素产生菌芽孢杆菌FB123的发酵条件优化及其16S rRNA序列分析

对芽孢杆茵FB123产枯草菌素发酵条件进行了优化并对菌株进行了16S rRNA分子鉴定.采用不同培养基配方、单因素及正交设计等试验对FBl23培养基、发酵条件进行了优化,FBl23的枯草菌素产量(透明圈直径:cm)从1.1 cm增加到1.67 cm;生物量提高了2.5倍.最佳培养基为(%):葡萄糖0.5,蛋白胨1.0,牛肉膏0.5,酵母膏0.1,pH 7.5;培养条件:培养温度28℃,培养时间32 h.进一步克隆测定了该茵16S rRNA基因序列,系统进化树分析表明该茵与枯草芽孢杆菌(Bacillus subtilis)具有最紧密亲缘关系.