深圳大鹏湾六种珊瑚共附生真菌多样性研究

本研究使用6种不同培养基对深圳大鹏湾6种珊瑚上的共附生真菌进行平板涂布法分离培养,结合ITS-rDNA基因序列特征和形态学特征进行鉴定来研究深圳大鹏湾海域珊瑚共附生真菌的多样性。共分离培养出457株真菌菌株,分属于7个属,分别为红酵母属Rhodotorula（180株）、曲霉属Aspergillus（170株）、木霉属Trichoderma（50株）、青霉属Penicillium（34株）、枝孢属Cladosporium（21株）、透孢黑团壳属Massarina（1株）和弯颈霉属Tolypocladium（1株）,其中优势属为红酵母属Rhodotorula和曲霉属Aspergillus,占菌株分离总数的76.59%。单独鹿角珊瑚上分离到的共附生真菌数量和种类最多,为160株9种;在该种珊瑚上还分离出弯颈霉属Tolypocladium sp.,该属真菌此前从未在珊瑚共附生真菌的研究中分离出来。本研究还发现SDA培养基分离出的共附生真菌数量最多,CDA培养基的真菌数量最少,表明不同培养基分离的共附生真菌多样性存在差异。     

利用18S和ITS序列揭示8种鲇形目鱼类的系统发育

为了探讨鲇形目(Siluriformes)鱼类系统发育关系,本研究克隆了黄颡鱼(Pelteobagrus fulvidraco)、长吻(Leiocassis longirostris)、斑鳠(Mystus guttatus)、革胡子鲇(Clarias gariepinus)、鲇鱼(Silurus asotus)和斑点叉尾(Ictalurus punctatus)6种鱼类的18S和两个内转录间隔区(包括全长ITS1-5.8S-ITS2)基因,结合GenBank中双须缺鳍鲇(Kryptopterus bicirrhis)和脂鳍胡鲇(Dinotopterus cunningtoni)的同源序列进行比较分析,结果表明,(1)8种鱼18S的长度为1814～1842bp,同源性达97%以上,5.8S均为157bp,同源性也高达99.36%～100%;(2)8种鱼ITS1长度为335～620bp,其中,黄颡鱼的最长,为618～620bp,斑点叉尾的最短,为335～336bp;ITS2长度为265～459bp,其中,脂鳍胡鲇最长,为459bp,斑点叉尾的最短,约为270bp。ITS1序列的同源性为29.45%～88.21%,其中,革胡子鲇和脂鳍胡鲇同源性最高,鲇鱼和革胡子鲇同源性最低。ITS2序列的同源性为41.59%～94.07%,其中,革胡子鲇和脂鳍胡鲇同源性最高,鲇鱼和革胡子鲇同源性最低;(3)分别以鲤鱼(Cyprinus carpio)18S和ITS为外群,采用NJ法构建18S、ITS系统发育树,结果显示,鲇科与胡鲇科的关系最近,鲿科与这两科关系较远,科与另外3科关系最远。鲿科中属和黄颡鱼属的关系较鳠属更近;胡鲇科的胡鲇属和脂鳍胡鲇属是关系很近的两个属;鲇科的鲇属和缺鳍鲇属是关系较远的两属。     

广东湛江湾可培养丝状真菌分布状况的初步调查

通过一次航行对广东湛江湾19个站位的表层和底层海水进行采样。经过对样品的分离纯化,共获得253株丝状真菌菌株。通过测序获得了121个rDNA-ITS序列,并已提交给GenBank。经统计分析发现,湛江湾表层和底层海水真菌数量总体上表现为由湾内向湾口逐渐减少的水平分布格局;湛江湾表层丝状真菌的数量略多于底层,在海水表层和底层中真菌种类和数量的分布规律相似。根据形态特征及rDNA-ITS的Blast同源性分析,这些菌株分属于18个属和32个分类单元,其中包括7个海洋真菌新记录属。结果表明,湛江湾丝状真菌多样性Shannon指数达2.75,物种优势度变化范围为30.90%–0.02%。枝孢属Cladosporium的优势度最高,为湛江湾优势种群,其次是青霉属Penicillium、侧齿霉属Engyodontium、曲霉属Aspergillus、枝顶孢属Acremonium等。湛江湾表层和底层海水真菌群落的Jaccard相似系数为0.42。     

南海长茎葡萄蕨藻共附生真菌的多样性

为了揭示南海长茎葡萄蕨藻Caulerpa lentillifera共附生真菌的群落结构,并分析自来水清洗对真菌群落多样性的影响,本研究以收集于深圳南海各月份长茎葡萄蕨藻为材料,利用Illumina MiSeq 2500测序平台进行真菌ITS1扩增子测序,分析自来水清洗前后真菌类群组成和多样性的差异。研究发现,从所有18个样品中总共获得914个OTUs,检测出真菌3门12纲32目50科74属106种,其中优势属为曲霉属Aspergillus、单胞瓶霉属Phialemonium、枝孢属Cladosporium、链座菌属Catenulostroma和茎点霉属Phoma;不同月份真菌Alpha多样性指数显示南海长茎葡萄蕨藻共附生真菌丰度和多样性均随着温度降低而降低;自来水清洗前后南海长茎葡萄蕨藻共附生真菌的多样性及群落结构存在一定差异,清洗后真菌OTUs和Alpha多样性指数均有所降低,其中Chao1和Ace指数为显著性下降,表明自来水清洗对真菌群落组成丰度及多样性影响显著,这对长茎葡萄蕨藻的食品安全具有积极意义。     

银杏内生真菌多样性研究(英文)

采用组织块分离法,从中国福建、江苏、贵州三省银杏Ginkgo biloba的根、茎、叶、树皮组织中分离内生真菌,利用形态学与ITS rDNA序列分析相结合的方法对所分离的菌株进行鉴定。结果表明从根、茎、叶和树皮分离出175株内生真菌,归为47类,每一类取代表菌株进行ITS测序及系统分析,分别属于子囊菌门的8个目,即Eurotiales、Hypocreales、Xylariales、Trichosphaeriales、Glomerellales、Diaporthales、Botryosphaeriales、Pleosporales,11科,16属。其中刺盘孢属Colletotrichum(19.75%)、链格孢属Alternaria(19.15%)、镰孢菌属Fusarium(10.64%)和拟茎点霉属Phomopsis(10.64%)为优势菌群;并且新丛赤壳属Neonectria和生赤壳属Bionectria为首次从银杏中分离出。Shannon-Wiener指数(H=2.4192)和Simpson指数(1-D=0.8856)的计算结果反应出所获得的银杏内生真菌菌群具有较高的多样性。     

Phylogenetic analysis of manganese-oxidizing fungi isolated from manganese-rich aquatic environments in Hokkaido, Japan

Three strains of Mn-oxidizing fungi were isolated from manganese-rich aquatic environments: sediment in a stream (Komanoyu) in Mori-machi and inflow to an artificial wetland in Kaminokuni-cho, Hokkaido, Japan. The characteristics of each strain were then established. Genetic analysis based on the ribosomal RNA (rRNA) gene was performed to clarify their classification. The sequences of the 18S rRNA and internal transcribed spacer (ITS1)-5.8S rRNA-ITS2 genes showed that all three strains are Ascomycetes. Based on its morphology, it seems probable that the KY-1 strain from Mori-machi belongs to the genus Phoma or Ampelomyces. The phylogenetic analysis indicates that this strain belongs to Phoma rather than Ampelomyces. Morphological identification of WL-1 and WL-2 strains from Kaminokuni-cho was impossible because of the lack of a sexual stage and specific organs. Phylogenetic analysis of the sequence in the ITS1-5.8S rRNA-ITS2 gene suggests that the WL-1 strain corresponds to Paraconyothyrium sporulosum and that WL-2 also belongs to the genus Paraconiothyrium. Because the ability to oxidize Mn has not been evaluated for most species of Phoma or Paraconiothyrium (Coniothyrium), further study is needed to confirm the status of these three strains.     

甘草内生真菌的分离及鉴定

植物体内普遍存在着内生菌,为了探寻传统药材甘草的质量与其内生菌之间可能存在的关系。对采自新疆的甘草根部进行了内生真菌的分离及鉴定。选用CYM真菌培养基,对其内生真菌进行分离及纯化,选择其中最占优势的两种菌落分别进行了菌落形态观察及菌丝形态(棉兰染色)观察,以及基因组DNA的18S rDNA和ITS rDNA序列鉴定和系统进化分析。本研究结果表明从甘草根部主要分离到两种内生真菌,分别属于Fusarium镰刀菌属和Gibberella赤霉菌属。     

双壳纲动物核糖体RNA 18S-ITS1序列及其在分子系统发育研究中的应用

测定了双壳纲不同科、属、种及种内共11个个体的核糖体RNA 18S-ITS1序列。结果表明,该序列在种间存在很高的多态性,长度从558bp到784bp不等,碱基差异百分比在10．7％～61．7％之间,ITS1序列同源性很低,有片段的插入与缺失。种间18S部分序列碱基差异百分比在0．9％～23．7％之间,变化主要是碱基的转换。用邻接法(NJ)构建了8个种的18S部分序列(约240bp)的系统发育树,与传统形态学分类结果相符。马氏珠母贝(Pinctada martrtensi)4个不同地域个体间的序列差异百分比在0．6％～1．9％之间。分析指出：18S基因可以作为双壳纲动物高阶元系统发育的分子标记;ITS1序列种间变化很大,可以应用于该纲物种的分类及鉴别．在亲缘关系相近种及种内变异相对较小,但核苷酸变异位点信息量丰富,可用于属内种间、亚种和群体间的遗传多样性研究。     

The diversity of fungi in aerobic sewage granules assessed by 18S rRNA gene and ITS sequence analyses

Aerobic sewage granules are dense, spherical biofilms, regarded as a useful and promising tool in wastewater treatment processes. Recent studies revealed that fungi can be implemented in biofilm formation. This study attempts to uncover the fungal diversity in aerobic granules by sequence analysis of the 18S and 5.8S rRNA genes and the internal transcribed spacer regions. For this purpose, appropriate PCR and sequencing primer sets were selected and an improved DNA isolation protocol was used. The sequences of 41 isolates were assigned to the taxonomic groups Pleosporaceae, Xylariales, Theleobolaceae, Claviceps, Aureobasidium, Candida boleticola , and Tremellomycetes within the fungi. It turned out that the fungal community composition in granules depended on the wastewater type and the phase of granule development.     

Diversity of the small subunit ribosomal RNA gene of the arbuscular mycorrhizal fungi colonizing Clintonia borealis from a mixed-wood boreal forest

Arbuscular mycorrhizal fungi (AMF) communities in Clintonia borealis roots from a boreal mixed forests in northwestern Québec were investigated. Roots were sampled from 100 m2 plots whose overstory was dominated by either trembling aspen (Populus tremuloides Michx.), white birch (Betula papyrifera Marsh.), or mixed white spruce (Picea glauca (Moench) Voss) and balsam fir (Abies balsamea (L.) Mill.).Part of the 18S ribosomal gene of the AMF was amplified and the resulting PCR products were cloned. Restriction analysis of the 576 resulting clones yielded 92 different restriction patterns which were then sequenced. Fifty-two sequences closely matched other Glomus sequences from Genbank. Phylogenetic analysis revealed 10 different AMF sequence types, most of which clustered with other uncultured AM sequences from plant roots from various field sites. Compared with other AMF communities from comparable studies, richness and diversity were higher than observed in an arable field, but lower than seen in a tropical forest and a temperate wetland. The AMF communities from Clintonia roots under the different canopy types did not differ significantly and the dominant sequence type, which clustered with AM sequences from a variety of environments and hosts at distant geographical locations, represented 66.9% of all the clones analyzed.     

Molecular taxonomy and biodiversity of rock fungal communities in an urban environment (Vienna, Austria)

The diversity of fungal communities on three different historical monuments in the city of Vienna (Austria) was analyzed and compared to the fungal diversity of microfungi on rock in the original quarry located in a rural area (Zogelsdorf, Austria). The fungal strains isolated were characterized by morphology and the complete rock fungal community was identified based on molecular data, that is, by sequencing parts of the small ribosomal subunit (18S) and internal transcribed spacer region 1 (ITS1). The genera Coniothyrium, Epicoccum and Phoma were found to be dominant {on} monument and rock surfaces. Additionally, black yeasts such as Exophiala species and microcolonial fungi like Sarcinomyces and Coniosporium which hitherto were regarded as typical rock inhabitants in semi-arid environments are frequently found on all rock surfaces in Vienna. The biodiversity of the fungi in the urban environment was much higher than on the same rock type in a rural environment, this difference can be attributed to the elevated organic pollution in the city.     

Changes in the Content of Modified Nucleotides in Wheat rRNA during Greening

The modified nucleotide content of the ribosomal RNAs in wheat is greatly influenced by light. The rRNAs of etiolated seedlings contain far fewer modified derivatives. The modified nucleotide composition characteristic of green plants develops gradually as a result of irradiation. In the course of the experiments changes in the state of modification of 5.8S and 18S rRNAs were examined during the greening of etiolated wheat seedlings. Three types of minor nucleotides, O^2′-methyladenosine, O^2′-methylguanosine and pseudouridine were found in the 5.8S rRNA of green wheat leaves, none of which was detected in etiolated wheat. The minor nucleotides appeared in the 5.8S rRNA only after 48 h irradiation. The sequences of 5.8S rDNA, TTS1, ITS2 and 18S rDNA were also determined and the presence of the hyper-modified nucleotide 1-methyl-3-(α-amino-α-carboxypropyl)-pseudouridine was detected in green wheat 18S rRNA. This minor component was not demonstrable in etiolated wheat 18S rRNA, but appeared after irradiation for 48 h. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ribosomal variation in six species of shape Fusarium

Eighteen isolates representing six Fusarium species from diverse hosts and geographical origins were evaluated to determine ribosomal DNA variation using polymerase chain reaction and restriction fragment length polymorphisms. No length variation was observed for amplified 18S and 28S regions. However, amplification of the ITS region showed one isolate, a F. oxysporum, to be about 120 bp larger than the remaining 17. Restriction digestions in the 18S region revealed polymorphisms within species of F. oxysporum and F. solani. An amplified variable stretch of the 28S gene showed restriction site differences between F. avenecum, F. sambucinum and F. sporotrichioides. A large degree of polymorphism was observed both between and within species in the ITS region. Therefore, entire sequences of the ITS and the 5.8S subunit were obtained for 17 of the 18 isolates. These sequences, along with those from eight additional isolates, were analysed using PAUP to assess the occurrence of DNA sequence divergence within the ITS region. The lack of correlation between molecular-based relationships and species affinities inferred from morphology for some isolates indicates that species designation can be unreliable using morphological data alone. Possible reasons for the discordance of the sequence and morphological data are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

产紫杉醇内生真菌TMS-26的分离和鉴定

为丰富产紫杉醇植物内生真菌资源库,从曼地亚红豆杉Taxus media茎中分离得到一株产紫杉醇的内生真菌TMS-26。通过对TMS-26的发酵提取物进行高效液相色谱分析,发现其具有与紫杉醇标准品(4.545 min)相近的色谱特征峰。进一步通过液质联用仪检测发现,内生真菌TMS-26的发酵提取物中具有与紫杉醇标准品((M+Na)+=876)相近的质谱特征峰,表明内生真菌TMS-26能够产生紫杉醇。同时通过传统形态学分类鉴定方法和18S r DNA序列分析、Internal-transcribed spacer(ITS)序列分析等现代分子生物学分类鉴定方法,最终将内生真菌TMS-26鉴定为曲霉属烟曲霉Aspergillus fumigatus,并命名为\"烟曲霉TMS-26\"。     

苹果炭疽菌的分子鉴定与检测

测定苹果炭疽菌rDNA全序列,比对苹果炭疽菌和其它炭疽菌ITS序列以及构建系统关系树,发现苹果炭疽菌与胶孢炭疽菌的ITS序列相似性高达99．8％,并与胶孢炭疽菌聚在一起,可以明确苹果炭疽菌应属于胶孢炭疽菌。进一步的序列比对发现,苹果炭疽菌的18S rDNA3’端比其它胶孢炭疽菌多出一段379bp的序列,根据这一特有片段设计引物CgF1与通用引物ITS4配对,结果仅能从苹果炭疽菌中扩增出1232bp的特异性条带。用苹果炭疽菌接种离体苹果,以接种发病的病组织总DNA为模板,利用引物CgF1／ITS4进行PCR扩增,同样可以扩增出1232bp的特异性条带,而健康苹果组织DNA中未能扩增出任何条带,表明该方法可用于苹果炭疽菌的鉴定和快速检测。