两种常见拟青霉的rRNA基因ITS区及5.8SrRNA基因的克隆测序

对粉拟青霉和细脚拟青霉 r RNA基因内转录间区 (ITS)和 5 .8Sr RNA基因进行了克隆和测序 ,并将之与在 Cen Bank中已登录的粉拟青霉和细脚拟青霉的同类序列比较 ,发现 Park etal.登录的粉拟青霉 (登录号 AF2 3 7664)是一个不正确的序列 ,实际是细脚拟青霉的序列。

从分子系统学角度分析拟盘多毛孢属及其近缘属的界限

拟盘多毛孢属和截盘多毛孢属的划分界限长期以来因分类学家的观点不一致而处于模糊状态,以核糖体DNA的内转录间区序列建立分子系统树的结果支持五细胞分生孢子类型应归入拟盘多毛孢属,四细胞分生孢子类型应归入截盘多毛孢属,分子系统树同时可以反映无性阶段和有性阶段之间的联系,显示出分子系统学对形态分类学的客观性的评判作用。

临床常见毛孢子菌的鉴定

为了探讨临床常见毛孢子菌(Trichosporonspp.)的鉴定方法,在形态学、营养生理学试验的基础上对临床常见的毛孢子菌———皮瘤毛孢子菌(Trichosporoninkin)、皮毛孢子菌(Trichosporoncutaneum)及阿萨希毛孢子菌(Trichosporonasahii)进行核糖体核糖核酸(rRNA)基因(rDNA)的内转录间区(ITS)扩增,用限制性内切酶Cfr13I、NcoI对聚合酶链反应(PCR)产物进行限制性片段长度多态性(RFLP)分析及序列测定,发现用Cfr13I做酶切,皮毛孢子菌的RFLP带型和阿萨希毛孢子菌、皮瘤毛孢子菌的不同;用NcoI做酶切,皮瘤毛孢子菌的带型与另两者不一致,序列分析的结果与形态学、营养生理学试验一致。结果提示,温度试验、API20C、RFLP及序列测定可望用于鉴定临床常见的毛孢子菌。

杂交稻亲本核糖体DNA的ITS序列比较

以典型粳稻(O.sativassp.japonica)秋光作为外类群,典型籼稻(O.sativassp.indica)南京11号作为对照,对两优培9、培两优288、汕优63三个籼型组合的6个亲本的核糖体基因(nrDNA)的内转录间隔区(ITS)的全序列进行了比较分析,结果表明:供试材料的ITS序列有较丰富的遗传差异,以ITS序列信息构建的系统树能反映出品种间的亲缘关系,3个组合亲本间遗传距离以超级稻两优培九的亲本9311与培矮64S遗传距离最远.

MALDI-TOF质谱仪进行临床酵母菌鉴定的方法学评价

目的探讨MALDI-TOF质谱仪用于临床酵母菌鉴定的优越性,为临床真菌感染提供早期诊断、治疗的依据。方法收集该院2015年8月至2016年3月临床分离酵母菌206株,分别用MALDI-TOF质谱仪、VITEK2 Compact微生物鉴定仪和显色培养法进行鉴定,以内转录间区序列测序结果为最终确认结果。比较MALDI-TOF质谱仪、VITEK2 Compact微生物鉴定仪和显色培养法鉴定的准确性。结果 3种方法鉴定酵母菌的准确率分别为VITEK2 Compact鉴定仪82.5%,显色培养法67.0%,MALDI-TOF质谱仪92.7%。MALDI-TOF质谱仪与VITEK2 Compact鉴定仪比较,差异有统计学意义(P=0.006),MALDI-TOF质谱仪与显色培养法比较,差异有统计学意义(P=0.001)。结论 MALDI-TOF质谱仪与VITEK2Compact鉴定仪、显色培养法相比,具有快速、操作简便、结果准确等优点。

Infrageneric and Sectional Relationships in the Genus Rhododendron (Ericaceae) Inferred from ITS Sequence Data

The internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (including 5.8S rRNA) of 15 Rhododendron, species, representing most sections of the genus, one Ledum species and Cassiope fastigiata were sequenced. Together with the ITS sequences of 13 selected Rhododendron species and Bejaria racemosa downloaded from GenBank, we explored the infrageneric and sectional relationships of this important North Temperate genus by employing maximum-parsimony analysis using PAUP software. C. fastigiata and B. racemosa were designated as outgroups. The ITS-based tree inferred that: (1) Rhododendron was a well-supported monophyletic group, while subg. Therorhodion was basal to the rest of the genus; (2) Ledum was a member of Rhododendron, and its close relationship with the lepidote rhododendron was confirmed; (3) the lepidote rhododendron plus Ledum formed a strongly-supported monophyletic clade which was sister to the rest of the elepidote rhododendron; (4) the elepidote rhododendron formed a weakly-supported clade within which the monophyly of subg. Hynwrianthes and subg. Tsutsusi were strongly supported, while subg. Pentanthera and subg. Azaleastrum were polyphyletic; and (5) the monophyly of sect. Choniastnini, (subg. Azaleastrum) was strongly-supported, while subg. Tsutsusi could be sister to a weakly-supported clade composed of two sampled species of sect. Azaleastrum (subg. Azaleastrum) together with R. sentibarbatum, of subg. Mumeazalea.

Screening of the Gene for Chlorella Identification and Identification of oil-producing Microalgae

[Objective] The aim was to select suitable gene for Chlorella identification and to identify the oil-producing microalgae.[Method] Four candidate gene sequences,the nuclear genomic rDNA of the 18S rRNA gene,internal transcribed spacer（ITS）,internal transcribed spacer Ⅱ（ITS Ⅱ）and the chloroplast rbcL gene,were selected for Chlorella molecular identification.Through these four candidate genes,the genetic variability and distinguish ability between intra-species and inter-species was analyzed to choose the right genes for identification of the high oil-content Chlorella.On this basis,application of these gene segments were classified and identified for five fresh-water isolated Chlorella,which oil-content is more than 30%.[Result] ITS gene was a suitable gene because of its high variation and short fragment length,meanwhile its genetic distance intra-species（0.439 6±0.135 9）was larger than inter-species（0.045 7±0.084 3）.Its sequence length varied between different species whereas highly conserved in the same species.By the application of ITS sequences,respectively,five high oil-content stains were identified as one C.vulgaris,two strains of C.sorokiniana and two strains of algae Chlorella sp.[Conclusion] This study had provided reference for the establishment of identification gene pool of Chlorella.

Phylogenetic analyses of four species of Ulva and Monostroma grevillei using ITS, rbcL and 18S rDNA sequence data

Chlorophyta species are common in the southern and northern coastal areas of China. In recent years, frequent green tide incidents in Chinese coastal waters have raised concerns and attracted the attention of scientists. In this paper, we sequenced the 18S rDNA genes, the internal transcribed spacer （ITS） regions and the rbcL genes in seven organisms and obtained 536-566 bp long ITS sequences, 1 377-I 407 bp long rbcL sequences and 1 718-1 761 bp long partial 18S rDNA sequences. The GC base pair content was highest in the ITS regions and lowest in the rbcL genes. The sequencing results showed that the three Ulvaprolifera （or U. pertusa） gene sequences from Qingdao and Nan＇ao Island were identical. The ITS, 18S rDNA and rbcL genes in U. prolifera and U. pertusa from different sea areas in China were unchanged by geographic distance. U.flexuosa had the least evolutionary distance from U. californica in both the ITS regions （0.009） and the 18S rDNA （0.002）. These data verified that Ulva and Enteromorpha are not separate genera.

猪蛔虫个体内ITS1序列差异比较

内转录间陋区1(ITS1)序列常被用于基因分型、物种鉴定和系统发育研究。论文比较猪蛔虫(Ascaris suum)个体ITS1序列差异。对采自江西的猪蛔虫样本进行DNA提取、PCR扩增rDNA的ITS1片段,并对该片段进行分子克隆、测序,使用MEGA5.0对测序结果进行比较。结果表明,同一个猪蛔虫个体的ITS1序列拷贝并不完全一致,总是会有个别的碱基差异,在polyA和polyT之间尤为明显,同一个猪蛔虫样本中含有多种基因型或单倍型,而且在试验中还发现了新的单倍型(HC1-HC5)。因此,将ITS1序列作为区分猪蛔虫基因型的依据值得商榷。

First record of Biecheleria cincta (Dinophyceae) from Chinese coasts, with morphological and molecular characterization of the strains

The presence ofBiecheleria cincta （=Woloszynskia cincta） in the Chinese coasts is reported for the first time. In scanning electron microscope, three to five series of vesicles and an elongated apical vesicle （EAV） were visible in the epicone, and both the hypocone and the cingulum had three series of vesicles each. Thin sections revealed that B. cincta possesses stalked pyrenoids and an unusual eyespot consisting of a stack of cistemae with brick-like materials （type E）, thus supporting its transfer from Woloszynskia to Biecheleria. Spiny cysts formed spontaneously in culture, with an encystment rate of around 20%. Both large subunit ribosomal DNA （LSU rDNA） and internal transcribed spacer region （ITS） sequences in 12 strains from the Chinese coasts were determined. Phylogenetic analyses based on LSU rDNA and ITS sequences using Bayesian inference and maximum likelihood revealed two distinct ribotypes （referred to as ribotype A and B） in B. cincta. ITS region pairwise distances within B. eincta ranged from 0.024 to 0.072, suggesting the existence of a complex of cryptic species.

Molecular identification based on ITS sequences for Kappaphycus and Eucheuma cultivated in China

The systematic classification of the Eucheumatoideae is difficult because of their variable morphology and interpretation of reproductive structures. Kappaphycus and Eucheuma specimens cultivated on the Hainan and Fujian coast of China were introduced from Vietnam, the Philippines and Indonesia. Combined with morphological characteristics, all Kappaphycus and Eucheuma cultivated strains were identified by internal transcribed spacer (ITS) sequences. The phylogenetic tree was constructed using neighbor-joining and maximum likelihood methods. The results indicate that different ITS sequence lengths occurred in the different genera and species. An obvious difference in morphology could be found in the protuberance shape between Kappaphycus and Eucheuma. The protuberance in Eucheuma was thorn-like and in Kappaphycus was wartlike or papillate. Their ITS sequence lengths differed significantly in nucleotide variation rates up to 58.55%-63.90%. All nucleotide variations occurred in the ITS1 and ITS2 regions except for five nucleotide transversions in the 5.8S rDNA region. In addition, the difference was at the branches among congeneric species. Kappaphycus sp. had branches with small buds, while K. alvarezii did not have such a feature. The nucleotide variation rates varied from 7.02% to 7.48% among species; within the same species of the clades it was <1.20%. Eucheumatoideae algae cultivated in China consisted of three clades, K. alvarezii, Kappaphycus sp., and E. denticulatum. The results indicate that ITS sequence analysis was an effective way for identification of interspecies and intraspecies phylogenetic relationships and might provide a clue for molecular identification of algal Eucheumatoideae.

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.

Phylogeography of Batrachospermum arcuatum in North China based on ITS sequence data

Batrachospermum arcuatum Kylin is typically dioecious,although some monoecious specimens have been collected from four locations in North China.In this study,B.arcuatum populations,including monoecious and dioecious thalli,were collected from seven stream segments.The nuclear DNA internal transcribed spacer(ITS)region was sequenced for the seven populations,and a phylogenetic tree was constructed using Bayesian inference to assess intraspecifi c relationships.A haplotype network was also created.The ITS region in B.arcuatum from North China comprised 822–853 bp,with 10 haplotypes detected from the seven locations.The results of this study support the inclusion of monoecious individuals in the species B.arcuatum.

Morphology and Molecular Identification of Ulva Forming Green Tides in Qingdao,China

Green tides are caused by the proliferation of chlorophytes under suitable hydrographic conditions.These blooms lead to environmental degradation and negatively impact the waters and seagrass beds,as well as fishing and other recreational activities in the bay.A comprehensive ecological understanding of the bloom dynamics,including the origin and persistence,is needed to foster management decisions.The algae in the great majority of green tide blooms usually belong to two genera of Ulvophyceae,Ulva and Enteromorpha.Ulva has been observed more often in recent years.In China,green tides occurred for the first time in the middle area of the Yellow Sea in 2007,and a large-scale algae blooming broke out in the middle and southern areas of the Yellow Sea in late May 2008.We identified them as Ulva prolifera by comparative analysis of the rDNA internal transcribed spacer 1 (ITS1),5.8S and ITS2 sequences in combination with microscopic observation.Morphological differences were found between the free-floating algae and the attached thalli.Various reproduction patterns of the free-floating algae include sexual,asexual and vegetative propagations,which played important roles in the long-term green tide persistence in China.The ITS sequences of the blooming algae were identical to those of the samples from the Lianyungang sea area but were different from the attached samples from the Qingdao sea area.The results infer that the blooms are originated from other sea areas rather than from the local attached populations.

Molecular Characterization of Indian Species of Steinernema （Nematoda： Steinernematidae） Based on Restriction Fragment Length Polymorphism Profile of Internal Transcribed Spacer Region of Ribosomal DNA

Restriction fragment length polymorphism （RFLP） profiles of the amplified products of Internal Transcribed Spacer （ITS） region of rDNA using four restriction enzymes （Alul, Rsal, HinfI and HhaI） revealed distinctness of six Indian isolates of Steinernema one each from Maharashtra （IARI-EPN-mh）, Himachal Pradesh （IARI-EPN-hp）, Dehradun （IARI-EPN-dhdl）, Jharkhand （IARI-EPN-jhl） and two from Madhya Pradesh （IARI-EPN-bpll ＆ IARI-EPN-gwll）, when compared with the only native species Steinernema thermophilum. One of the restriction enzyme, Rsal could differentiate all the six species/strains from one another. The three restriction enzymes yielded patterns which were of diagnostic value but Rsal appeared to be the best diagnostic marker for differentiating these isolates. A tree constructed based upon the band sharing amongst the isolates, produced trichotomy which placed strains from Madhya Pradesh and Jharkhand in one group showing 94% homology, one strain from Bhopal （M.P） formed separate clade along with S. thermophilum with 72% similarity. These isolates, from Maharashtra, Himachal Pradesh and Dehradun, showed only 51% similarity with the S. thermophilum by forming separate clade.

Molecular Characterization of Pythium Spp. Isolated from Tomato Seedlings in the Syrian Coast

Tomato seedlings damping-off is a limiting factor in commercial greenhouse production. To determine the causal agents of disease, sampling and fungal isolation were performed during 2012. Samples were collected from infected seedlings growing in greenhouses in the Syrian coastal region. Isolation of fungi was done in the laboratories of the Agronomical Reaserch Center, in Lattakia and the molecular analyses were done in the Biotechnology Center at Tishreen University, Lattakia, Syria, during the years 2012, 2013. Eight isolates ofPythium sp. obtained were purified using hyphal tip method （named P1, P2, P3, P4, P5, P6, P7 and P8）. Isolates were morphologically identified by optical microscope, then molecularly Characterized using genus specific ITS primers. The results of morphological characterization of pathogenic species suggested the detection of Pythium aphanidermatum, P. ultimum. The analysis of DNAs from the different isolates with ITS primers, recognizing the inter transcript spacer of nuclear ribosomal DNA proved that the eight, isolates were belonging to the species P. ultimum. The complete sequences of ribosomal DNA internal transcribed spacers regions of selected isolates were determined and submitted to GenBank. The GenBank-BLAST homology search revealed P. ultimum as the most similar sequence （〉 96% identity） with GenBank entry AB355596.

广东省柑橘炭疽病病原菌的形态与分子鉴定

炭疽病是柑橘的主要真菌性病害之一。2007年春,广东省德庆县名优柑橘品种贡柑炭疽病暴发流行。为了明确该县及广东省其他地区柑橘炭疽病菌的种类,为防治提供依据,对采集自广东省6个地区柑橘属10个栽培品种上的炭疽病样本进行病原菌分离,共获得柑橘炭疽病菌单孢菌株75株,对其中10株代表性的菌株进行了种类鉴定。通过培养性状和形态学特征观测、核糖体DNA(rDNA)内转录间区(ITS)序列分析、ITS区特异性引物PCR检测和系统发育关系比较等方面的研究,结果表明:10个柑橘炭疽病菌菌株均为盘长孢状刺盘孢Colletotrichum gloeosporioides,未发现国际上其他国家报道的严重危害柑橘花器和幼果部位的柑橘花后落果病病原菌——尖刺盘孢C.acutatum。

器官移植术后少见真菌感染的临床分析

目的掌握移植术后少见真菌(马内菲青霉菌、曲霉菌等非念珠菌类)感染的特点,以提高其早期诊断率及降低受体的死亡率。方法回顾性总结我器官移植中心2008年4月至2010年4月移植后7例少见真菌感染的临床表现、影像学、实验室检查特点及治疗情况等。结果曲霉菌或马内菲青霉菌感染的临床表现一般不具典型性,但曲霉菌感染肺部时,CT上可发现毛玻璃样改变(2/4)。G、GM试验检测少见真菌感染速度较快,但假阴性率较高(6/7、2/4)。真菌培养诊断少见真菌感染时间较长,均需2周左右,ITS测序确诊少见真菌感染只需3～5d。治疗上运用伊曲康唑、卡泊芬净等为代表的抗真菌药物,5例治愈(5/5)。结论内转录间区(ITS)测序既快又准确,结合临床表现、影像学特征,可提高器官移植术后少见真菌感染的早期诊治率。

Four new species of the genus Hymenoscyphus (fungi) based on morphology and molecular data

Four new species of Hymenoscyphus (H. brevicellulus, H. hyaloexcipulus, H. microcaudatus, and H. subsymmetricus) and a new Chinese record (H. subpallescens) are described. These five species share common characteristics: small apothecia (<3mm in diameter); hymenium whitish, pale yellow, to yellow in color; ectal excipulum of textura prismatica; asci arising from simple septa; ascospores scutuloid and guttulate; saprophytic nutrition; and leaf habitats, except for H. subsymmetricus, which grows on herbaceous stems. Phylogenetic analyses of internal transcribed spacer nuclear ribosomal DNA sequences, the universal DNA barcode for fungi, for 16 species in the genus indicated that these taxa were closely related to H. microserotinus, in accordance with their morphological features, but represented independent species. The distinguishing features of each new species from its relatives are discussed, and their phylogenetic relationships explored.

Molecular identification of Sporothrix clinical isolates in China

In this study, we investigated the molecular phylogeny of 64 clinical isolates which were identified as Sporothrix schenckii sensu lato by morphological identification. All of the strains were isolates from patients from several provinces in China. The phylogeny was inferred by DNA sequence analyses based on datasets of the ribo- somal internal transcribed spacer （ITS） and a combined ITS and partial 13-tubulin region. Reference sequences were retrieved from GenBank. Results showed that all of the isolates were clustered in a distinct clade with a type of Spo- rothrix globosa. Our analysis showed that S. globosa is the causal agent of the tested sporotrichosis in China, rather than S. schenckii that was generally believed to be the case. The existence of S. schenckii in China remains to be confirmed. This study improved our understanding of the distribution of the species in S. schenckii complex.