内生真菌EPICOCCUM NIGRUM的形态与分子鉴定

在调查松属植物内生真菌过程中,从植物组织中分离到45株Epicoccum nigrum。根据它们在PDA培养基生长时的总体形态特征,划分为三组形态类型。从每组类型中随机选取三个代表菌株作进一步的分子鉴定。rDNA的ITS和5.8S基因序列分析结果表明,这9个菌株为E. nigrum。我们的研究结果也证明了前人提出的在Epicoccum菌种鉴定中,孢子大小和菌落颜色不能作为可靠的种级分类特征。     

一种新型生防细菌菌株13-1鉴定及其生物学特性

13-1 是从魔芋根际土壤中筛选得到的一株革兰氏阴性生防细菌, 抑菌试验结果表明它对7种植物病原细菌和8种病原真菌具有拮抗作用。形态和常规生理生化特征鉴定, 发现该菌株与溶杆菌属Lysobacter 特征很相近。Biolog 鉴定和16S rDNA基因序列分析表明, 菌株13-1与已报道的抗生素溶杆菌Lysobacter antibioticus DSM 2044(=ATCC29479)同源性高达99%。二者在所建系统发育树中处于同一分枝, 将13-1菌株鉴定为抗生素溶杆菌(L. antibioticus)。     

半夏内生真菌的初步研究

从健康无病害的半夏(Pinellia ternate)根、茎、叶和花组织中分离获得内生真菌共计61株,对其进行形态学鉴定后,针对不产孢的菌株,测定了ITSrDNA序列以进行分子鉴定。分离到的内生真菌分别来自11个属,以半知菌为主要群落,其中Hyphomycetes,Zygomycetes和Coelomycetes的比例分别为62.2%,18.1%和8.2%;镰刀菌属(Fusarium spp.)是半夏植物中分离率最高(6%)的属,其余主要优势属为Alternaria,Mucor,Epicoccum,Mortierella和Plectosphaerella。研究表明:从半夏球茎组织中分离到的内生真菌数量多于其他组织。     

千层塔内生真菌的分离与鉴定

目的：为从千层塔中分离具有药用价值的内生真菌奠定基础。方法：新鲜千层塔茎段,经酒精和升汞消毒后,接种于PDA平板培养基上进行内生真菌的分离、纯化;根据菌落形态和孢子等形态特征,结合核糖体基因居间序列（ITS序列）进行菌株鉴定。结果：从千层塔的茎中分离出4株内生真菌。内生真菌I菌落形态和孢子特征与枝状枝孢霉属的特征相符合,ITS序列与GenBank中多条属于枝状枝孢霉的ITS序列相似,鉴定该菌株属于枝状枝孢霉;内生真菌II菌落形态和孢子特征与黄青霉的特征相符合,鉴定该菌株属于黄青霉;内生真菌III菌落形态和孢子特征与尖孢镰刀菌的特征相符合,ITS序列与GenBank中多条属于尖孢镰刀菌的ITS序列相似程度高,鉴定该菌株属于尖孢镰刀菌;内生真菌Ⅳ菌落形态与盾壳霉相似,ITS序列与GenBank中6条属于盾壳霉的ITS序列具有较高的相似性,鉴定该菌株属于盾壳霉。结论：从千层塔中分离和鉴定出4株内生真菌,分别属于枝状枝孢霉、黄青霉、尖孢镰刀茵和盾壳霉。     

菌株S.tz的抗菌活性及其生物学研究

从沈阳天柱山土壤中分离到一种链霉菌S．tz,试验表明它所产生的水溶性抗生素不但对植物病毒有较好的防效．而且具有很强的抗真菌活性,对甜瓜枯萎病菌．番茄叶霉病菌,烟草赤星病菌等多种植物病原真菌表现强烈的抑菌效果,该菌株的形态特征．培养特征、生理生化特性等种的鉴定特征与不吸水链霉菌(Streptomyces ahygroscopicus)相似,初步鉴定该菌株是不吸水链霉菌的一个新的变种。     

广义虫草属二新记录种

【背景】广义虫草属是一类重要的虫生真菌资源。【目的】对我国西南地区虫生真菌及其相关真菌资源进行调查。【方法】在贵州省习水县和花溪区进行标本采集,采用含双抗的PDA培养基分离目的菌株;基于形态学特征和ITS rDNA序列系统发育分析相结合的方法进行菌株鉴定。【结果】从采集到的标本中分离获得5个菌株,菌株GY1113、GY90809和GY90810在形态特征上与马拉维白僵菌模式菌株非常相近;系统发育树中二者以高持率(ML/BI为99/1)聚成一个分支。菌株A1997在形态特征上与鳞翅目虫草模式菌株非常吻合;系统发育树中二者以较高支持率(ML/BI为78/0.95)聚成一个分支。菌株A1972在形态上与苏格兰白僵菌模式菌株的形态特征非常相近;系统发育树中二者以高支持率(ML/BI为99/1)聚成一个分支。因此分别鉴定为马拉维白僵菌、鳞翅目虫草及苏格兰白僵菌。【结论】马拉维白僵菌和鳞翅目虫草为中国新记录种。     

18种(1变型)鼠尾草属植物叶表皮及表皮毛微形态特征研究

鼠尾草属是唇形科中最大的属,全球有近1 000种。我国是鼠尾草属东亚分布中心,有84种,分布于全国各地,尤以西南地区最多。鼠尾草属植物具有很高的经济价值和观赏价值,但由于鼠尾草属植物分布广、种类多、形态变异大,给该属鉴定和分类学研究带来了很大的困难。此外,在采集和调查过程中发现弧隔鼠尾草亚属和荔枝草亚属的很多种类在营养生长期仅通过叶片很难鉴定。表皮毛是植物叶片上最常见的附属物,其分布特性和形态特征常作为植物鉴定和分类学研究的重要手段。该文利用光学显微镜和环境扫描电镜对18种和1变型共19份鼠尾草属植物的叶片类型、表皮细胞形状、垂周壁样式、气孔器类型和表皮毛等叶表皮微形态特征进行了观察和比较研究,为鼠尾草属的分类界定研究提供证据。结果表明:这19份鼠尾草属植物叶表皮和表皮毛形态特征在电子显微镜下表现出明显的多样性,叶表皮细胞形状为不规则形或多边形,垂周壁多数为浅波状或波状,少数为平直或弓形。气孔器为无规则型和不等细胞型,其中无规则型最为常见。样品叶表皮毛分为4个类型,盾状腺毛、头状腺毛、短非腺毛和长非腺毛。该属植物叶表皮的这些微形态特征,可为本属鉴定和分类学研究提供理论依据。     

一株内生真菌黑曲霉的活性检测及鉴定

从飞龙斩血内分离到一株产广谱、高活性抑菌物质的内生真菌F-001,经测定对细菌、植物病原真菌和皮肤致病真菌共26种病原微生物有不同程度的抑制作用。对其进行了系统鉴定和生物学研究,形态特征表明,该菌株与曲霉属Paecilomyces Bainier中的黑曲霉Paecilomyceslilacinus的特征基本一致,ITS序列分析显示本菌株与多个黑曲霉菌株同源性都在99%,因此将菌株F-001命名为黑曲霉Paecilomyces lilacinusF-001。该研究首次从核桃树皮内分离得到黑曲霉并进行了该菌株的抑菌活性检测。     

具有细胞毒活性红树林真菌094811的鉴定

为鉴定合成细胞毒活性及多羟基甾醇脂肪酸酯新结构代谢产物的红树林真菌094811,采用形态观察、BIOLOG生理特征鉴定及核糖体18S rDNA测序对菌株094811进行了鉴定。菌株094811与模式菌株黑曲霉As3.40、泡盛曲霉As3.44形态特征极为相似,难以区分。扫描电镜下分别观察且比较菌株094811、模式菌株黑曲霉As3.40、泡盛曲霉As3.44分生孢子的形态,判断菌株094811为泡盛曲霉;根据BIOLOG微生物鉴定系统给出的相似菌种名称及综合形态和生理生化特征,推断菌株094811为泡盛曲霉;通过18S rDNA部分序列测序、GenBank比对,及系统演化分析进一步确认菌株094811为泡盛曲霉。菌株094811的形态、生理特征及其核糖体18S rDNA的序列特征给出了一致的结论。首次鉴定发现1株具有合成细胞毒活性及新的多羟基甾醇脂肪酸酯结构化合物的能力的泡盛曲霉。     

武汉梅花炭疽病病菌的多样性研究

炭疽病是梅花（Prunus mume）栽培中的重要病害,对梅花的栽培构成严重威胁。本研究从武汉发病的梅花叶片样品上分离、获得了170个炭疽病菌菌株,它们在形态特征、致病性、分子遗传水平等方面都表现出较大的差异。按菌落形态、色素分泌、拟菌核产生、分生孢子及孢子梗形态和大小等形态特征将梅树炭疽病菌分为7种类型,其中Ⅵ型和Ⅶ型菌株在PDA培养基上可以连续产生大量的有性后代。7种类型的菌株只能侵染梅花、樱树、梨树、苹果、桃树、杏树等蔷薇科园艺植物,并且存在着明显的致病力分化,但不侵染吉祥草、柑桔、大叶黄杨、豇豆、紫荆、高粱等供试的其它科植物。依据致病力可将梅树炭疽病菌分为强、中、弱3类。ITS序列表明它们均属于胶孢炭疽（Colletotrichum gloeosporioides）。对其中7种类型36个梅树炭疽病菌菌株的进行了RAPD聚类分析,在55%相似水平上,供试菌株可以分为3组,所聚类群与形态学类型和致病力分化所形成的强、中、弱3类没有明显的相关性。表明梅花炭疽病菌菌株间存在丰富的遗传多样性。     

金针菇rDNA序列结构特征分析

rDNA序列中的ITS作为DNA barcoding广泛应用于真菌的系统发育与物种辅助鉴定,IGS被认为可以用于种内水平不同菌株的鉴别。食用菌中还没有完整的rDNA序列的报道。本研究采用二代和三代测序技术分别对金针菇单核菌株“6-3”进行测序,用二代测序的数据对三代测序组装得到的基因组序列进行修正,得到一个在基因完整性、连续性和准确性均较好的基因组序列,对比Fibroporia vaillantii rDNA序列,获得金针菇完整的rDNA序列。金针菇rDNA序列结构分析表明,它有8个rDNA转录单元,长度均为5 903bp,有9个基因间隔区,其长度有较大差异,3 909-4 566bp。rDNA转录单元中,各元件的序列长度分别为：18S rDNA 1 796bp、ITS1 234bp、5.8S rDNA 173bp、ITS2 291bp、28S rDNA 3 410bp。基因间间隔区中,IGS1 1 351-1 399bp、5S rDNA 124bp、IGS2 2 435-3 092bp。金针菇的5S、5.8S、18S、28S rDNA序列准确性得到转录组数据的验证,也得到系统发育分析结果的支持。多序列比对发现,不同拷贝的基因间间隔区序列（IGS1和IGS2）存在丰富的多态性,多态性来源于SNP、InDel和TRS（串联重复序列）,而TRS来源于重复单元的类型和数量。9个基因间间隔区之间,IGS1只有少量的SNP和InDel,IGS2不仅有SNP和InDel,还有TRS。本研究结果提示,在应用IGS进行种内水平不同菌株之间的鉴别时,需要选取不同拷贝之间的保守IGS序列。     

Genotyping of a miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii, based on sequence analysis of the partial 26S ribosomal RNA gene and two internal transcribed spacers

We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), the internal transcribed spacer 1, the 5.8S ribosomal RNA gene, and the internal transcribed spacer 2 (the ITS sequence) from 46 strains of miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii and a closely related species, Z. mellis, for typing. Based on the 26S rDNA sequence analysis, the Z. rouxii strains were of two types, and the extent of sequence divergence between them was 2.6%. Based on the ITS sequence analysis, they were divided into seven types (I-VII). Between the type strain (type I) and type VI, in particular, a 12% difference was detected. The occurrence of these nine genotypes with a divergence of more than 1% in these two sequences suggests that Z. rouxii is a species complex including novel species and hybrids. Z. mellis strains were of two types (type alpha and type beta) based on the ITS sequence. Z. rouxii could clearly be distinguished from Z. mellis by 26S rDNA and ITS sequence analyses, but not by the 16% NaCl tolerance, when used as the sole key characteristic for differentiation between the two species.     

Epicoccum nigrum for biocontrol agents in vitro of plant fungal pathogens

Epicoccum nigrum strains were evaluated in vitro as potential biological agents for control of the growth of Fusarium avenaceum, F. graminearum, F. oxysporum and Botrytis cinerea. Individual biotic effects of five strains of E. nigrum on the various fungi were determined using the biotic series method elaborated for fungi, on potato dextrose agar (PDA - Difco) medium. Our results show that E. nigrum strains limited the growth of all isolates of Fusarium spp., but not of those of Botrytis cinerea.     

Description of a New Freshwater Ciliate Epistylis wuhanensis n. sp. (Ciliophora,Peritrichia) from China,with a Focus on Phylogenetic Relationships within Family Epistylididae

Two populations of Epistylis wuhanensis n. sp., a new freshwater peritrich ciliate, were isolated from different freshwater ponds located in Hubei, China. Their morphological characteristics were investigated using live observation, protargol impregnation, and scanning electron microscopy (SEM). Specimens from the two populations showed identical arrangement of the infraciliature and identical small subunit ribosomal RNA (SSU rRNA) gene and ITS1‐5.8S‐ITS2 sequences. The zooids present bell‐shaped and 90–175 × 27–54 μm in vivo. Macronucleus is variable in shape and located in the middle of cell. Pellicle is usually smooth with 139–154 and 97–105 striations above and below the trochal band, respectively. SSU rRNA gene and ITS1‐5.8S‐ITS2 sequences of E. wuhanensis n. sp. did not match any available sequences in GenBank. Phylogenetically, E. wuhanensis n. sp. clusters with the other Epistylis within the family Epistylididae, but is distinct from the major clades of Epistylis. Above all, the morphological characteristics and molecular analyses support that the present Epistylis is a new species. Expanded phylogenetic analyses of sessilids based on both SSU rRNA gene sequences and ITS1‐5.8S‐ITS2 sequences reveal that the genus Epistylis consists of Epistylis morphospecies and taxonomic revision of the genus is needed.     

The identification of vahlkampfiid amoebae by ITS sequencing

We have determined the internal transcribed spacer (ITS) sequences (including the 5.8S ribosomal DNA) of 30 strains of 14 species belonging to eight vahlkampfiid genera. Each previously described species has a specific ITS sequence, except for Tetramitus aberdonicus, Tetramitus thorntoni, and Tetramitus jugosus, which have identical ITS sequences. The latter three may therefore constitute a single species despite their apparent phenotypic differences. The ITS sequence appears to be conserved within a species. The species Willaertia magna appears to be ubiquitous. The 5.8S rDNA sequences of Singhamoeba horticola and Learamoeba waccamwensis indicate that they do not represent different genera, but both belong to the genus Tetramitus. The ITS sequences of 16 undescribed vahlkampfiid isolates were determined. Based on these sequences, seven isolates were identified as belonging to described species, while nine probably represent seven new species. Five of these presumed new species belong to the genus Tetramitus, and one each to the genera Vahlkampfia and Paravahlkampfia.     

Torulaspora maleeae sp. nov., a novel ascomycetous yeast species from Japan and Thailand

Nine strains of a new Torulaspora species were isolated from natural samples collected in Japan and Thailand including one strain obtained from a leaf of Rhizophora stylosa (NBRC 11061T), one strain from soil (NBRC 11062), six strains from mosses (ST-14, ST-266, ST-510, ST-511, ST-513 and ST-581) and one strain from sediment in mangrove forest (RV-51). On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, and the sequence analyses of the D1/D2 domain of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) (ITS1-5.8S rRNA gene-ITS2) region, the nine strains were found to represent a single novel species of the genus Torulaspora, which were named Torulaspora maleeae sp. nov. The type strain is NBRC 11061T (BCC 25515T=CBS 10694T). In the phylogenetic trees based on the sequences of the D1/D2 domain of the LSU rRNA gene, T. maleeae showed a close relationship with the five recognized species of the genus Torulaspora, Torulaspora delbrueckii, Torulaspora franciscae, Torulaspora globosa, Torulaspora microellipsoides and Torulaspora pretoriensis. Torulaspora maleeae differed from the five recognized species of the genus Torulaspora by six to 12 nucleotide substitutions (1.1-2.1%) in the D1/D2 domain of the LSU rRNA gene and by 6.4-11.7% nucleotide substitutions in the ITS (ITS1-5.8S rRNA gene-ITS2) region.     

亚稀褶黑菇和稀褶黑菇的ITS序列分析

对亚稀褶黑菇和稀褶黑菇的ITS全序列进行了测定和比较,首次报道了亚稀褶黑菇的ITS1和5.8S区域.在ITS区域中,不同采集地的亚稀褶黑菇与稀褶黑菇的5.8S rDNA具有100%的同源性,而两侧ITS之间表现出种内和种间的多态性,种内的差异均不超过5%,种间的差异达10%左右.ITS序列分析方法可以作为两者的鉴定方法.     

亚稀褶黑菇和稀褶黑菇的ITS序列分析

对亚稀褶黑菇和稀褶黑菇的ITS全序列进行了测定和比较,首次报道了亚稀褶黑菇的ITS1和5.8S区域。在ITS区域中,不同采集地的亚稀褶黑菇与稀褶黑菇的5.8S rDNA具有100%的同源性,而两侧ITS之间表现出种内和种间的多态性,种内的差异均不超过5%,种间的差异达10%左右。ITS序列分析方法可以作为两者的鉴定方法。     

Sequence Variation of Ribosomal Internal Transcribed Spacers (ITS) in Commercially Important Phytoseiidae Mites

Preliminary work is needed to assess the usefulness of different markers at different taxonomic scales when a new group is analyzed, such as the commercially important Phytoseiidae mites. We investigate here the level of sequence variation of the nuclear ribosomal spacers ITS 1 and 2 and the 5.8S gene in six species of Phytoseiidae: Neoseiulus californicus, N. fallacis, Euseius concordis, Metaseiulus occidentalis, Typhlodromus pyri and Phytoseiulus persimilis. As expected, the 5.8S gene (148 base pairs) is markedly conserved and displays little variation in between genera comparisons. ITS1 and ITS2 show contrasting patterns: while the ITS2 is short (80–89 bp) and shows little variation, the ITS1 is longer (303–404 bp) and is very variable in sequence. This fact compromises reliable nucleotide homologies when comparing the genera. The comparison of ITS1 sequence similarity at the species level might be useful for species identification, however, the value of ITS in taxonomic studies does not extend to the level of the family. The intraspecific variations of ITS were investigated in three species: N. californicus, N. fallacis and E. concordis. The first species has identical ITS1 sequences and the last two display low polymorphism (2 nucleotide substitutions). The ITS2 and 5.8S sequences were identical in all three subspecies comparisons.