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。     

基于形态特征和ITS序列对7个鹅膏菌属菌株的分类鉴定

以采自浙江省丽水地区的7个鹅膏菌属菌株作为研究材料,在基于形态特征进行初步鉴定的基础上,对7种鹅膏菌的rDNAITS区段进行克隆测序和序列特征比较分析。进一步对ITS序列进行核酸序列数据库GenBank同源性检索比对,将从GenBank检索获得的9个最相似物种的ITS序列连同7种鹅膏菌的ITS序列一起作系统发育分析。结果表明:基于ITS序列对f6、f9和f493个菌株的分子鉴定支持了基于形态特征的鉴定结果,对f5的分子鉴定不支持形态鉴定的结果,f8为鹅膏菌属内某种,f66为鹅膏菌属内某种,并与Amanitafulva,A.atrofusca,A.orientifulva3种鹅膏菌的亲缘关系较近,f7与另外6种鹅膏菌的亲缘关系相差甚远。研究结果提示基于分子水平上的ITS序列分析不能单方面作为大型真菌分类鉴定的可靠依据,可以作为基于传统形态学分类鉴定的辅助参考依据。     

野生灵芝BSU01的分离鉴定、生物学特性及其木质纤维素酶活分析

灵芝作为传统中药,具有重要的医药和经济价值。本研究以一株野生灵芝属真菌BSU01为实验材料,通过形态特征和ITS序列聚类分析对其进行了鉴定,探讨了该菌株菌丝生长的最适碳源、氮源、C/N、pH及温度等生物学特征,以及该菌株产木质纤维降解素酶的能力。结果表明,菌株BSU01的形态特征与有柄灵芝相符;且与有柄灵芝ITS序列一致性达99%,并在系统发育树上聚在一起;菌株BSU01菌丝生长最适碳源为果糖或者葡萄糖,氮源为酵母提取物,C/N比为20/1到30/1,温度为30℃,pH值为5.5;菌株BSU01的漆酶、木质素过氧化物酶和Mn依赖过氧化物酶分别为12.13 U/L、0.52 U/L和0.33 U/L;CMCase和木聚糖酶酶活分别为7.14 U/mL和1.88 U/m L。本研究结果将为该菌的进一步开发利用提供数据参考。     

干皮孔菌属的一个新种和三个组合种

本文报道了产自斯里兰卡的干皮孔菌属一个新种,该种的主要特征是具有平伏反卷或具菌盖的子实体,很小的孔口（每毫米8-10个）,菌丝系统二系,生殖菌丝覆盖有刺状结晶,骨架菌丝橙棕色,细腊肠状担孢子（2.7-3.4×0.5-0.8μm）。基于ITS和nLSU序列的系统发育分析表明该新种属于干皮孔菌属的一个明确的分支。此外,将毛孔菌属组合到干皮孔菌属中,并报道了3个组合种,白边干皮孔菌、印度干皮孔菌和萨彦干皮孔菌。     

裂褶菌培养、鉴定及氨基酸组成分析

目的:获得裂褶菌的纯培养物,鉴定培养菌丝,并分析子实体及菌丝氨基酸含量.方法:利用孢子分离法、组织分离法培养菌丝,扫描电镜观测其菌丝特征,结合ITS区序列分析,并利用出菇试验分析其菌丝体,全自动氨基酸分析仪分别测定裂褶菌子实体、菌丝体氨基酸组成.结果: 菌丝体ITS区序列与子实体完全一致,出菇试验亦表明裂褶菌菌丝体培养成功,子实体及菌丝体均含有丰富的氨基酸成分,两者氨基酸种类及含量相当,菌丝体总氨基酸含量14.01%,子实体总氨基酸含量15.59%,其中菌丝体必需氨基酸的含量(38.60%)比子实体稍高(37.95%).结论:裂褶菌菌丝亦具有丰富的营养价值,可作为重要的氨基酸来源及功能性食品.     

南方灵芝ITS、EF1-α和RPB2序列多态性研究

内转录间隔区（ITS）、延伸因子1-α（EF1-α）与RNA聚合酶II第二大亚基（RPB2）是真菌系统学研究中常用的基因片段。已有研究发现,在同一个体内ITS也可能存在差异,但EF1-α和RPB2是否也存在同样的现象却鲜有报道。本研究通过克隆测序,分析了南方灵芝Ganoderma australe的ITS、EF1-α和RPB2在同一个体中的序列差异,并探讨这种序列差异是否会影响分子鉴定的准确性。结果表明,在南方灵芝供试样品中,ITS、EF1-α和RPB2序列都有可能存在个体内变异,但个体内变异程度不尽相同。基于供试样品和克隆测序结果发现,ITS序列在同一个体内的变异最高可达2.3%,而RPB2序列在同一个体内的差异仅0.5%,EF1-α序列在同一个体内序列差异也可高达1.8%。ITS和EF1-α序列在同一个体内的差异可能超过了灵芝属部分物种间的差异,对于部分灵芝属物种来说直接利用克隆序列进行分子鉴定或系统发育分析可能存在一些问题。     

临床相关毛孢子菌的鉴定及体外药物敏感性研究

目的探讨临床相关毛孢子菌的鉴定方法及对常见抗真菌药物的体外敏感性。方法对48株临床分离的毛孢子菌分别通过形态学、API20C AUX、Vitek 2 Compact及核糖体rDNA ITS序列分析等方法鉴定到种;采用浓度梯度法（E-test）测定氟康唑、伏立康唑、伊曲康唑、两性霉素B及卡泊芬净对48株毛孢子菌的最低抑菌浓度。结果形态学和API20C AUX、Vitek 2 Compact不能准确区分不同种的毛孢子菌,以核糖体rDNA ITS序列分析将48株毛孢子菌鉴定为8个种：阿萨希毛孢子菌,星型毛孢子菌,皮瘤毛孢子菌,真皮毛孢子菌,皮肤毛孢子菌,赖巴克毛孢子菌,T.domesticum,T.jirovecii。体外药敏结果显示：卡泊芬净对毛孢子菌无体外活性,MIC〉32μg/mL;氟康唑和两性霉素B对毛孢子菌活性差,体外活性最好的药物是伏立康唑和伊曲康唑。结论常规方法不易将毛孢子菌准确鉴定到种的水平,ITS序列分析准确快速,可以辅助临床区分难鉴定毛孢子菌。毛孢子菌药敏谱不同于临床常见其他酵母菌,氟康唑和两性霉素B对其活性差,伏立康唑具有良好的体外抗菌活性。     

采自东南亚的变色卧孔菌一新种（英文）

报道了采自新加坡的多孔菌一新种,硫色变色卧孔菌,并对该种进行了描述和显微绘图。该种的主要特征是子实体一年生,平伏,新鲜时软,硫磺色,干后浅橄榄色至蜜黄色,单系菌丝系统,生殖菌丝具简单分隔,存在菌丝状囊状体和锥形拟囊状体,担孢子近球形至球形,具一大液泡。基于ITS和LSU序列的系统发育分析表明该新种属于变色卧孔菌属分支的一个明确的支系。     

297例浅部真菌病临床及病原菌菌种分析

目的 探讨皮肤浅部真菌病致病真菌菌种的构成.方法 对297例真菌涂片阳性和培养阳性的浅部真菌病患者,取标本进行分离培养及菌种鉴定,培养阳性标本在形态学上不能准确鉴定的,进行梅里埃API 20C AUX酵母菌鉴定试剂盒或核糖体DNA （rDNA） ITS区序列测定,确切鉴定菌种.使用SPSS 17.0统计软件对于结果进行统计分析.结果 共分离培养出致病菌13种,其中红色毛癣菌86株（29.0％）,须癣毛癣菌68株（22.9％）,念珠菌属59株（19.9％）,暗色真菌属13株（4.4％）,曲霉菌属13株（4.4％）,红酵母菌12株（4.0％）,青霉菌属9株（3.0％）,毛霉菌9株（3.0％）,犬小孢子菌5株（1.7％）,浅白隐球菌3株（1.0％）,毛孢子菌属2株（0.7％）,絮状表皮癣菌1株（0.3％）,混合感染17株（5.7％）.结论 本地区浅部真菌病以甲癣为主,主要致病真菌是红色毛癣菌,但其他种类真菌感染尤其是念珠菌属有明显上升趋势.     

胶陀螺(Bulgaria inquinans)的生药学研究

对胶陀螺药材性状、显微特点、理化特性和分子鉴定等方面研究结果表明,子实体初期呈黄褐色球形,并带有黄棕色麻点,顶端逐渐开裂,产生一至多个不等的裂口,裂口不断扩大,最后整个子实体表面呈黑色皱缩。子囊棒形。外囊盘被由圆形细胞组成,圆形细胞外侧生绒毛,细胞内含类似载色体的物质;中囊盘被由埋在胶质中的交错菌丝组成。采用子囊孢子弹射分离法所得菌丝有橘黄色菌丝和长有棒状等分生孢子的有隔菌丝无色;从子实体组织分离培养的菌丝状,无色,其中生有黑褐色或黄棕色的圆形细胞,呈念珠状。胶陀螺子实体DNA序列与菌丝DNA序列的同源性为99.65%,胶陀螺子实体的DNA序列与GenBank数据库中AY789345比对,其同源性为99.80%。     

细菌鉴定方法

细菌的分类鉴定与细菌的研究和应用有着同等重要的地位。准确地鉴定细菌类别,根据其菌种种类不同,分别针对肠杆菌、阳性球菌、非发酵菌等菌种设计不同药敏板条的抗生素组合,可以指导临床应用用药。目前,细菌分类鉴定方法主要包括表型鉴定法和分子遗传学鉴定法两大类,针对这两大类鉴定方法,汇总其生理生化分类特征,简述各方法采用的关键技术,针对各技术讨论其优缺点。同时详细研究了表型鉴定法下数值分类法中自动化鉴定涉及的算法,结合目前细菌鉴定方法,对细菌分类鉴定的发展趋势进行了总结和展望。     

高山大黄的显微及分子鉴定研究

目的：对蓼科药用植物高山大黄进行显微及分子鉴定研究。方法：采用石蜡切片、粉末制片对高山大黄根及根茎的横切面组织结构和粉末特征进行显微研究,并采用matK基因序列分析技术对高山大黄进行了分子鉴定研究。结果：高山大黄根横切面中皮层宽,薄壁细胞中可见草酸钙簇晶,木质部宽广,导管径向排列;根茎髓部宽广,无异形维管束存在;粉末中草酸钙簇晶多,棱角大多短钝,导管木质化,多网纹导管,淀粉粒小而多。高山大黄matK序列全长1524 bp,在基因上游106、218、222、365、522、615、711、759、801、852处存在十处碱基替换,且在950－955处存在CAAGAA6个碱基插入。结论：本研究获得了高山大黄比较全面的鉴定信息,上述显微特征可作为高山大黄显微鉴定依据,其matK基因序列分析为高山大黄DNA条形码研究奠定基础。     

Individual and sexual identification for the wild black muntjac (Muntiacus crinifrons) based on fecal DNA

The black muntjac (Muntiacus crinifrons), endemic to China, was categorized as a Grade I National Key Protected Animal by the Ministry of Agriculture of China and classified as Vulnerable (VU) by IUCN. Recent years, studies had been conducted on this species mainly focusing on habitat selection, food habit, gene flow etc., only with a few reports on the population dynamics. Individual identification of wild animals is one of the most important subjects in the population dynamic research. Of various molecular markers, microsatellite DNA fingerprinting has been used most frequently and successfully on many kinds of animals. Here, we constructed identification system for the black muntjac using 8 microsatellite loci. 31 black muntjacs were identified from 141 fecal samples, whereas 43 samples could be used for PCR after repeated trials. Further, the sequencing for Cytb gene was also conducted for convincing us the identity of fecal samples. The results, highly consistent between sequencing consequence and sequence data from Genebank, implied that those experienced local people are of the convincing knowledge about wild animals, especially at the respects of identification to black muntjac’ feces pellets. Moreover, we detected the specificity of identification system to black muntjac. BM1225 was the only one locus that unsuccessful PCR for the muscle samples of Muntiacus reevesi was observed, which suggested that our identification system could be used for excluding the non-researched objects in some cases. Analyses using softwares CERVUS 3.0 and POPGENE 1.21 showed that the present identification system had strong discrimination power: 0.938 per loci (DP) or 0.999 in total (CDP). The mean observed number of alleles (Na), mean effective number of alleles (Ne), and mean excepted heterogosity (He) were 8.875, 6.375, and 0.829 respectively. Considering that the change of sex ratio in population could exert significant impact on population growth, density to some extent, we also analyzed the sex ratio of those individuals that had been identified based on fecal samples from filed using SRY (Sex-determining region Y) gene amplification, which identified 19 males and 12 females.     

滨海前胡生药组织学研究

采用性状鉴定及显微鉴定法对滨海前胡Peucedanum japonicum的生药组织学特征进行系统研究。结果表明,根木栓层中夹杂近方形或长圆形石细胞,油管呈环状散在;茎圆柱形有纵棱脊25～40条,棱脊内有厚角组织及油管,维管束于各棱脊处较发达;叶为等面叶,维管束上下方及韧皮部均有油管,薄壁细胞含有放射状或针簇状的橙皮苷结晶。以上生药组织学特征稳定、可靠,可作为滨海前胡的鉴别依据之一。     

Reassessment of species distribution and occurrence of mud crab (Scylla spp., Portunidae) in Malaysia through morphological and molecular identification

This study utilized genetic and morphometric approaches to assess the molecular and morphometric differentiation among commercially important species of mud crab. Molecular investigations were derived from 542 bp mitochondrial DNA COI on 249 individuals within genus Scylla from nine states in Malaysia represents four marine regions; South China Sea, Sulu Sea, Straits of Singapore and Straits of Malacca. Four specimens were obtained from Indonesia to give a robust analysis in this study. For species delimitation, Automatic Barcode Gap Discovery (ABGD) method on a web interface was employed. Analysis on phylogenetics was implemented utilizing Neighbour joining (NJ) and Maximum Parsimony (MP) methods. The inter- and intraspecies genetic distances (D_s) was computed using Kimura 2-parameter distance and executed in MEGA version 5.05. All samples were genetically and morphologically identified and clustered into four distinct species. Among the species, S. olivacea was the most abundant (n = 111), on the other hand the occurrence of S. paramamosain in Malaysia was very low (n = 29). No single individual of S. serrata from Malaysia was recorded in this study. Both genetic distance and phylogenetic approaches exhibited a correlative monophyletic association among all specimens analysed. This present study is crucial as it reports the reassessment of all species within genus Scylla in Malaysia, eventually could be employed as a reference source for subsequent research mainly on mariculture and other conservation efforts for the species.     

Immunoelectrophorecal differences between phytopathogenic Fusarium species

Immunoelectrograms of certain tested Fusarium spp revealed that each has a characteristic pattern and showed the relatedness between F. oxysporum on the one hand and F. culmorum and F. semitectum on the other. The B₄ protein fraction is common to the former pair, while α_1a protein fraction links the second.     

Mnemonics are an Effective Tool for Adult Beginners Learning Plant Identification

Most beginners are introduced to plant diversity through identification keys, which develop differentiation skills but not species memorisation. We propose that mnemonics, memorable ‘name clues’ linking a species name with morphological characters, are a complementary learning tool for promoting species memorisation. In the first of two experiments, 64 adults in a group-learning environment were taught species identification using mnemonics, an educational card game and a text-based dichotomous key. In the second experiment, 43 adults in a self-directed learning environment were taught species identification using mnemonics and a pictorial dichotomous key. In both experiments, mnemonics produced the highest retention rates of species identification based on vegetative characters. The educational value of these findings is discussed for vegetative plant identification and broader applications.     

Species identification of polygonati rhizoma in China by both morphological and molecular marker methods

Morphological markers as well as two types of molecular markers, inter-sample sequence repeat (ISSR) and start codon targeted (SCoT) are suitable for species identification of the polygonati rhizoma germplasms. In this paper, we adopted these methods for the identification of rhizomes collected from 47 areas in China. Based on their morphological characters, the collected germplasms were classified into two populations, one with alternate leaf arrangement and the other with verticillate leaf arrangement, and they were comprised of five species and fourteen subgroups. Of the five species identified: Polygonatum kingianum, P. cirrhifolium, P. alternicirrhosum, and P. sibiricum belonged to one cluster, and P. cyrtonema belonged to a different cluster. According to the analysis of both ISSR and SCoT markers, all germplasms with greater genetic similarity were classified into one group. Especially, P. sibiricum and P. cirrhifolium, which shared ～80% similarity, were clustered together, whereas the germplasms identified as P. kingianum with ～86% similarity formed a separate clade. P. kingianum showed a much greater genetic similarity with P. cyrtonema than with P. sibiricum. The multidimensional scaling analysis further verified the accuracy and reliability of the molecular marker-based results. Thus, both morphological and molecular methods should be combined for the differentiation of germplasms such as those of polygonati rhizoma.     

A single multiplex of twelve microsatellite markers for the simultaneous study of the brown hare (Lepus europaeus) and the mountain hare (Lepus timidus)

The management of hunted species is challenging, as it must conciliate the conservation of species and their sustainable exploitation. Nongenetic tools are widely used in this context but they may present limitations notably when species can hybridize or when large‐scale spatial monitoring is required to establish optimal management actions. This is why genetic tools have been more and more integrated in wildlife management practices. However, the markers proposed are often amplified in small multiplexes when larger ones could allow to better cope with the small quantities of DNA obtained with noninvasive sampling methods. Here, we propose a unique multiplex of 12 autosomal microsatellite markers for the study of two hare species that exist in sympatry in some areas in Europe and are hunted notably in France: the brown hare Lepus europaeus and the mountain hare L. timidus. We tested 17 markers previously used in these two species or other lagomorph species, from which 12 were included in this single multiplex. Diversity was between 4 and 30 alleles per locus totaling 126 alleles, and we showed that these markers possess appropriate genetic resolution for individual and species identification for the populations under study. This multiplex panel represents the largest number of microsatellites amplified in one reaction proposed for these two hare species and provides a cost‐effective and valuable tool for further hybridization studies and the management of hares.