分离自青海湖可培养耐盐真菌的多样性研究

青海湖位于青藏高原东北部,属于典型的高原盐湖沉积环境。作者以常规的分离培养方法对青海湖中的耐盐真菌多样性进行了研究。从黑马河、耳海和尕海共分离得到617株耐盐真菌,结合形态学和分子生物学的方法将菌株鉴定到18个属和45个种。子囊菌的菌株数比接合菌和担子菌多,枝孢菌和青霉菌为优势耐盐真菌。在这45种耐盐真菌中,轻度、中度和极端嗜盐真菌分别有19、25和1种。分离自黑马河地区的耐盐真菌,其物种丰富度指数Ma（3.12）、多样性指数H′（1.11）和均匀度指数J（0.267）均比耳海和尕海的高。从1.5%浓度下     

从天然蛹虫草不同部位分离菌株特性比较

以采集自泰山地区的6株野生蛹虫草为研究对象,分别通过菌核、子座组织分离和子囊孢子分离法共获得18个菌株,经ITS鉴定均为蛹虫草菌株后,进一步对18个菌株进行了菌丝生长速度、菌落形态、出草试验、主要活性物质（虫草素、腺苷、虫草多糖、β-类胡萝卜素、叶黄素）和交配型的测定分析。结果表明：在菌丝长势、出草产量和整齐度方面,从子囊孢子分离得到的菌株普遍优于组织分离菌株;而在活性物质含量方面,从子囊孢子和子座分离得到的菌株普遍优于菌核分离菌株。通过交配型测定发现,从菌核、子座和子囊孢子分离得到的18个菌株均不存在交配型缺失现象。     

蝉棒束孢内菌核、菌膜及其生境土壤真菌群落结构及生态功能分析

通过分析蝉棒束孢Isaria cicadae内菌核、菌膜及其生境土壤的真菌群落结构与核心真菌组的生态功能,初步探索蝉棒束孢自然生长过程中内环境真菌与其周围环境中真菌的关系。本研究利用高通量测序技术检测6株采自贵阳大将山和贵阳森林公园的蝉棒束孢及土壤样本,结果表明,蝉棒束孢内菌核共检测到43个真菌属,菌膜检测到58个真菌属,菌际土检测到260个真菌属,且内菌核和菌膜真菌群落结构相似。而两地生境土的真菌群落结构有差异,大将山以棒束孢属Isaria、被孢霉属Mortierella和一分类地位未知属占优势,而贵阳森林公园以被孢霉属、隐球酵母属Cryptococcus、红菇属Russula、棒束孢属占优势,且两地菌际土中棒束孢属的丰度均显著高于其他土样。核心真菌组检测分析显示,内菌核的核心真菌组有10个OTUs,菌膜的核心真菌组有5个OTUs,菌际土的核心真菌组有20个OTUs,3种样本的核心真菌组的生态功能群均检测到有与植物相关的功能群,内菌核中检测到外生菌根真菌群,菌膜中检测到植物病原真菌群,菌际土中检测到丛枝菌根真菌群和植物病原菌群。     

滨海湿地植物芦苇可培养内生真菌的多样性

内生真菌在植物生长以及抵御环境胁迫过程中起着非常重要的生态作用.本研究从黄河三角洲滨海湿地1350个芦苇组织切片中分离得到318株内生真菌,通过对rDNA ITS的分型、测序及系统进化分析,研究了该地区芦苇植株根、茎、叶中可培养内生真菌的种类组成及在不同盐度、不同组织中的分布情况.根据序列相似性(以98%为阈值),共获得12个真菌分类操作单元(OTUs).在门级分类水平上,子囊菌门为绝对优势菌群, 在各组织和站位中均有分布.芦苇根组织中分离得到的内生真菌OTU数相对较多,叶组织和茎组织中分离到的OTU数相同,且叶中的OTU在根中均存在.潮上区(低盐区)内生真菌OTU数最多,高潮区次之.根中可培养内生真菌的丰富度和多样性指数最高,叶中多样性最低;潮上区丰富度最高,中潮区多样性最高.尽管不同盐度梯度及不同芦苇组织中都有其特异的菌株,但总体看,土壤盐度及不同组织对可培养内生真菌的种类组成的影响并不显著(ANOSIM,P>0.05).链格孢属是所有样品共有的优势菌.     

利用CTAB法和子囊孢子破壁法提取冬虫夏草菌DNA

通过CTAB法从冬虫夏草菌株和天然冬虫夏草不同部位提取DNA,并用PCR扩增进行验证,证明了CTAB法适合从冬虫夏草子座、菌核和冬虫夏草菌培养物中提取DNA。首次报道1种将子囊孢子破壁直接进行PCR扩增的方法,并比较了该方法和CTAB法在提取冬虫夏草DNA方面的差异。2种方法获得的DNA用于PCR扩增均能得到较好的结果。     

南方红豆杉内生真菌的分离鉴定及其细胞毒活性研究

采用平板培养法对采自广东省的南方红豆杉进行内生真菌的分离,并通过形态学和分子生物学方法进行分类鉴定;共分离鉴定内生真菌22株,主要为刺盘孢属(Colletotrichum)、球座菌属(Guignardia)、座腔菌属(Botryosphaeria)、节菱孢属(Arthrinium)、炭角菌属(Xylaria)、毛壳菌属(Chaetomium)、交链孢属(Alternaria)、拟茎点霉属(Phomopsis)、长蠕孢属(Helminthosporium)等属。还采用MTT法测定了这些内生真菌发酵粗提物的细胞毒活性,活性研究发现有6个菌株的粗提物在作用浓度为100μg/mL时,对至少1种受试肿瘤细胞株的抑制率在50%以上,其中菌株A223和A240对人乳腺癌细胞MCF-7的抑制率在90%以上。表明南方红豆杉内生真菌多样性丰富,有些菌株具有良好的细胞毒活性,值得进一步深入研究。     

冬虫夏草不同发育时期蛋白质组iTRAQ质谱分析

本文选定冬虫夏草寄主昆虫幼虫（S1）、僵虫（S2）、子座初期（子座<1cm,S3）、子座早期（1cm<子座<3cm,S4）、成熟冬虫夏草（子座>7cm,S5）、商品冬虫夏草菌核（S6）、商品冬虫夏草子座（S7）及商品冬虫夏草（中期子座≈5cm,S8）8个样品,用定量蛋白质组学方法iTRAQ分析技术对冬虫夏草不同生长发育阶段的差异蛋白质组进行比较。共计获得9 924个不同肽段,鉴定到1 809个蛋白质,其中差异比值1.5倍以上,P<0.05蛋白个数506个,以商品冬虫夏草样本（S8）为参照,比较差异蛋白数量,寄主幼虫（S1）、僵虫（S2）、子座初期（S3）、子座早期（S4）、成熟冬虫夏草（S5）、商品冬虫夏草菌核（S6）及商品冬虫夏草子座（S7）阶段差异蛋白数分别为104、102、34、35、49、46和136个,说明昆虫幼虫、僵虫及子座与商品冬虫夏草样品差异显著。对鉴定蛋白质数据进行了主成分分析（principal component analysis,PCA）,在第三主成分（component 3）上的显著差异,表明成熟冬虫夏草（S5）的蛋白组成不同于商品虫草,说明成熟虫草品质下降。层次聚类（hierarchy clustering）分析结果表明所有样品分为两支,僵虫（S2）和商品冬虫夏草菌核（S6）与寄主幼虫（S1）聚在了一个分支上,而不同发育阶段样品（S3、S4和S5）与商品虫草子座（S7）聚在一个分支上,说明商品冬虫夏草菌核中还残留有寄主昆虫蛋白。冬虫夏草菌核部位的蛋白到子座部位的蛋白呈现由寄主幼虫蛋白向真菌蛋白发育过渡的聚类关系。子集嵌套关系同步子实体生长发育阶段蛋白组成变化随时间的程序性变化的规律。K-均值（K-means）聚类和Gene Ontology（GO）注释分析,提供了冬虫夏草成熟过程中能量代谢通路的变化趋势以及与真菌侵染昆虫和有性生殖相关蛋白质信息。研究结果为理解寄主昆虫对冬虫夏草功能的潜在贡献、子实体形成和发育的分子机制提供借鉴,并为蛋白质组作为冬虫夏草质量标准提供了科学参考。     

五味子根内生真菌的分离及其抑菌活性的研究

采用组织块分离方法对五味子根内生真菌进行分离鉴定,并以人参疫病病菌[Phytophthora cactorum(Leb.et Coh.)Schroet.]、人参黑斑病病菌(Alternaria panax Whetz.)为供试病原菌对分离的内生真菌进行了抑菌活性筛选。试验共分离得到74株内生真菌,根据形态特征鉴定出33株,分别隶属于半知菌亚门丛梗孢科的轮枝孢属(Verticillium)、柱隔孢属(Ramularia)和瘤座孢科的镰刀菌属(Fusarium)。通过抑菌试验筛选出5个内生真菌有抑制作用,其中3个内生真菌的发酵产物、乙醇提取液对2种病原菌均有一定的抑菌活性。     

金荞麦和苦荞麦抗菌活性内生真菌的筛选及鉴定

从药用植物金荞麦和苦荞麦的根、茎、叶、花中分离到62株内生真菌,并以金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)[CMCC(B)63501]、小麦赤霉病菌(Fusarium graminearum)、黄瓜枯萎病菌(Fusarium oxysporum f.sp.cu-cumerinum)和绵腐病菌(Pythium aphanidermatum)6种微生物为指示菌对分离到的内生真菌进行抗菌活性检测。结果发现,分离的内生真菌菌株KQH-01、KQH-02和JQY-1的发酵醇提取物具有较好的抑菌活性。形态学特征和分子鉴定确定菌株KQH-01为炭角菌属(Xylaria sp.)真菌,菌株KQH-02为球毛壳菌(Chaetomium globosum),菌株JQY-1为葡萄座腔菌(Botryosphaeria dothidea)。     

柱形虫草及其紫色野村菌无性型

报道安徽省滁州市琅琊山林场引起蜘蛛流行病的虫草真菌,经鉴定为柱形虫草Cordycepscylindrica,属中国首次报道。在子座柄部可见一小块灰紫色的分生孢子层,镜检表明:由此组织分离菌株的形态学特征与后来通过子囊孢子分离获得的菌株是一致的。柱形虫草无性型为紫色野村菌Nomuraeaatypicola,其培养特征也为国内首次描述。另外,在同一生境的地上叶背面,可见许多蜘蛛被紫色野村菌感染致死,虫尸被覆淡紫色菌膜,但未见孢梗束或子座。     

High diversity of the fungal community structure in naturally-occurring Ophiocordyceps sinensis

Background

Ophiocordyceps sinensis (syn. Cordyceps sinensis), which is a parasite of caterpillars and is endemic to alpine regions on the Tibetan Plateau, is one of the most valuable medicinal fungi in the world. “Natural O. sinensis specimens” harbor various other fungi. Several of these other fungi that have been isolated from natural O. sinensis specimens have similar chemical components and/or pharmaceutical effects as O. sinensis. Nevertheless, the mycobiota of natural O. sinensis specimens has not been investigated in detail.

Methodology/Principal Findings

Based on the technique of PCR-single-strand conformation polymorphism (PCR-SSCP), the mycobiota of three different sections (stromata, sclerotia, and mycelial cortices) from natural O. sinensis specimens were investigated using both culture-dependent and -independent methods. For the culture-dependent method, 572 fungal strains were isolated, and 92 putative operational taxonomic units (OTUs) were identified from 226 sequenced strains with the threshold of 97%. For the culture-independent method, 490 fungal clones were identified from about 3000 clones of ITS fragments from the whole-community DNA; based on PCR-SSCP analyses, 266 of these clones were selected to be sequenced, and 118 putative OTUs were detected. The overwhelming majority of isolates/clones and OTUs were detected from mycelial cortices; only a few were detected from stromata and sclerotia. The most common OTUs detected with both methods belonged to Ascomycota; however, only 13 OTUs were detected simultaneously by both methods. Potential novel lineages were detected by each of the two methods.

Conclusions/Significance

A great number of fungal species present in the mycobiota of naturally-occurring O. sinensis specimens were detected, and many of them may represent undescribed lineages. That only a few of the same OTUs were detected by both methods indicated that different methods should be used. This study increased our understanding about the fungal community structure of this valuable medicinal herb.     

Cloning and analysis of the MAT1-2-1 gene from the traditional Chinese medicinal fungus Ophiocordyceps sinensis

The entomopathogenic fungus Ophiocordyceps sinensis has been important in traditional Chinese medicine but has yet to be commercially cultivated. One bottleneck is the very low frequency of stromata formation from artificially infected moth larvae. The mating system of fungi is the determining factor for sexual reproduction, but mating-type genes of O. sinensis have not been previously investigated. In this study, the putative mating-type gene MAT1-2-1 within the MAT1-2 idiomorph was amplified by polymerase chain reaction (PCR) and was determined to consist of 859 nucleotides that encode 249 amino acids; genes within the MAT1-1 idiomorph, however, were not determined. The MAT1-2-1 gene contained the conserved high-mobility group (HMG) box, and MAT1-2-1 flanking sequences were subsequently obtained. Although no putative open reading frames of the MAT1-1 idiomorph were detected within the ca. 8-kb flanking sequences of MAT1-2-1, a putative DNA lyase gene (which is present next to both idiomorphs in some heterothallic fungi) was found ca. 3.0 kb downstream of MAT1-2-1. The intervening distance between MAT1-2-1 and the DNA lyase gene in O. sinensis is larger than that in Cordyceps militaris and Cordyceps takaomontana. In addition, O. sinensis showed low sequence similarities with C. militaris and C. takaomontana in both MAT1-2-1 and the DNA lyase gene. In the phylogenetic tree, different MAT1-2-1 haplotypes of O. sinensis clustered together with high bootstrap support. As a single-copy gene, MAT1-2-1 was detected in all examined O. sinensis isolates including tissue cultures and single-ascospore cultures. This report describes, for the first time, a mating-type gene of O. sinensis.     

冬虫夏草菌侵染及寄生阶段的生长发育研究

冬虫夏草菌侵染进入寄主幼虫血体腔后,断裂形成长梭形的菌体。菌体以顶端出芽的方式增殖,分隔分化,互相质配。质配菌体生长出菌丝充满寄主幼虫血体腔,使幼虫死亡并形成僵虫形的菌核,菌核萌生子座即形成冬虫夏草。     

Molecular and cultural characterization of Morchella spp. from disturbed environments of central-southern Chile

In Northwestern Patagonia (Chile), three species of Morchella from undisturbed environments have been identified to date: Morchella tridentina, Morchella andinensis and Morchella aysenina, all belonging to the Elata clade and associated mainly with Nothofagus forests. In this study, the search for Morchella specimens was extended to disturbed environments in Central-Southern Chile, to further explore Morchella species diversity in the country, which is still very limited. The Morchella specimens were identified through multilocus sequences analysis, and the mycelial cultures were characterized, establishing comparisons with specimens from undisturbed environments. To the best of our knowledge, these results reveal for the first time in Chile the presence of the species Morchella eximia and Morchella importuna, and in the case of the last one also the first record in South America. These species were found associated almost exclusively with harvested or burned coniferous plantations. The in vitro mycelial characterization revealed certain inter- and intra-specific patterns of the morphology, such as pigmentation, mycelium type, and development and formation of sclerotia, which varied according to growth media and incubation temperature. The growth rates (mm/day) and mycelial biomass (mg) were significantly influenced by the temperature (p < 0.05), with maximum rates (>10 mm/day) and biomass (approx. 20 mg) between 20 and 24 °C, while a significant growth reduction (70–90%) was observed at 28 °C, mainly in the species from undisturbed environments. Potato-dextrose (PDA) medium stimulated the greatest mycelial density and sclerotia formation in most of the isolates, mainly in M. eximia (UDEC-LAF 236 isolate) which recorded the best mycelial growth performance. Among isolates, UDEC-LAF 236 also showed the best performance in sclerotia production (>350 sclerotia/dish) in 10 days of growth. This study contributes to the knowledge of the diversity of Morchella species in Chile by broadening the species range to those from disturbed environments. It also provides molecular and morphological characterization of the in vitro cultures of different Morchella species. The report on M. eximia and M. importuna, species known as cultivable, adapted to local climatic and edaphic conditions could represent the first step to developing artificial Morchella cultivation methods in Chile.     

Diversity and host recurrence of fungi associated with the bleached leaf litter in a subtropical forest

Fungi that selectively remove lignin cause extensive bleaching of leaf litter, which results in the acceleration of litter decomposition. The taxonomic diversity and host recurrence of saprotrophic fungi associated with bleached leaf litter were investigated in a subtropical forest in southern Japan. A total of 211 fungal isolates and sporocarps were obtained from bleached portions of leaf litter of 33 plant species in 18 families and were classified into 83 operational taxonomic units (OTUs) at the 97% similarity level of the ITS rDNA sequence. These fungal OTUs belonged to Rhytismataceae and Xylariaceae in the Ascomycota, and Marasmiaceae, Mycenaceae, Lachnocladiaceae, and Physalacriaceae in the Basidiomycota. OTUs in Rhytismataceae and Marasmiaceae showed a significantly higher degree of recurrence for plant species than simulated networks where partners were associated randomly. In contrast, OTUs in Xylariaceae and Mycenaceae showed no significant recurrence for plant species. Such differing degrees of recurrence for plant species implied different roles of fungal families in leaf litter decomposition.     

三种杓兰根相关真菌多样性和生态功能

【背景】除了菌根真菌(Orchid mycorrhizal fungi,OrMF)外,兰科植物根中还有其它内生真菌,称为根相关真菌(Root-associated fungi,RAF)。【目的】采用分离培养的方法获得同一栖息地针叶林和灌木林两种不同生境西藏杓兰、黄花杓兰和无苞杓兰的RAF菌株,研究其真菌谱系、多样性和生态功能结构。【方法】从杓兰根碎屑中分离RAF,通过总DNA提取、PCR扩增及测序得到ITS(Internaltranscribedspacer)序列;进行系统发育和多样性分析,并通过NCBI数据库比对得到相似性最高序列的注释信息来分析RAF生态学特性。【结果】共分离得到278株RAF,25种OTU类型,包括23个子囊菌门OTU,2个毛霉菌门OTU。RAF物种丰富度分析发现西藏杓兰的较黄花杓兰高,不同生境没有显著差异;不同杓兰物种较不同生境的RAF群落分化程度高。生态功能分析显示25个OTU包括共生型、腐生型和致病型3种营养型,以及外生菌根菌群、植物病原菌群、内生真菌群、动物病原菌群、真菌寄生菌群、杜鹃花类菌根群、未定义的腐生菌群和不确定型8种共位群。【结论】阐明不同生境采集的不同杓兰中RAF的分布特点和生态功能,为未来研究RAF与杓兰属植物的共生关系奠定基础。     

Fungal communities on decaying leaves in streams: a comparison of two leaf species

Decomposition of leaf litter is a microbial mediated process that helps to transfer energy and nutrients from leaves to higher trophic levels in woodland streams. Generally, aquatic hyphomycetes are viewed as the major fungal group responsible for leaf litter decomposition. In this study, traditional microscopic examination (based on identification of released conidia) and phylogenetic analysis of 18S rRNA genes from cultivated fungi were used to compare fungal community composition on decomposing leaves of two species (sugar maple and white oak) from a NE Ohio stream. No significant differences were found in sporulation rates between maple and oak leaves and both had similar species diversity. From the 18S rRNA gene sequence data, identification was achieved for 12 isolates and taxonomic affiliation of 12 of the remaining 14 isolates could be obtained. A neighbor-joining tree (with bootstrap values) was constructed to examine the taxonomic distribution of the isolates relative to sequences of known operational taxonomic units (OTUs). Surprisingly, only 2 of the isolates obtained were aquatic hyphomycetes based on phylogenetic analysis. Overall, there were no differences between the two leaf types and a higher diversity was observed via culturing and subsequent 18S rRNA gene sequencing than by conidia staining. These differences resulted from the fact that traditional microscopy provides estimates of aquatic hyphomycete diversity while the other approach revealed the presence of both aquatic hyphomycete and non-aquatic hyphomycete taxa. The presence of this broad array of taxa suggests that the role of aquatic hyphomycetes relative to other fungi be re-evaluated. Even though the functional role of these non-aquatic hyphomycetes taxa is unknown, their presence and diversity demonstrates the need to delve further into fungal community structure on decomposing leaves.     

Diversity and dynamics of fungal endophytes in leaves, stems and roots of Stellera chamaejasme L. in northwestern China

This study was conducted to explore fungal endophyte communities inhabiting a toxic weed (Stellera chamaejasme L.) from meadows of northwestern China. The effects of plant tissue and growth stage on endophyte assemblages were characterized. Endophytes were recovered from 50 % of the samples, with a total of 714 isolates. 41 operational taxonomical units (OTUs) were identified, consisting of 40 OTUs belonging primarily to Ascomycota and 1 OTU belonging to Basidiomycota. Pleosporales and Hypocreales were the orders contributing the most species to the endophytic assemblages. The total colonization frequency and species richness of endophytic fungi were higher in roots than in leaves and stems. In addition, for the plant tissues, the structure of fungal communities differed significantly by growth stages of leaf emergence and dormancy; for the plant growth stages, the structure of fungal communities differed significantly by plant tissues. This study demonstrates that S. chamaejasme serves as a reservoir for a wide variety of fungal endophytes that can be isolated from various plant tissues.     

Endophytic fungi associated with leaves of Betulaceae in Japan

Diversity and species composition of endophytic fungi on leaves of 11 tree species in Betulaceae were studied, with reference to climatic, tree species, and seasonal variations. A total of 186 fungal isolates were obtained from 190 leaves collected in a subalpine forest, a cool temperate forest, and a subtropical forest in Japan, and were divided into 46 operational taxonomic units (OTUs) according to the base sequences of D1-D2 region of large subunit rDNA. The 2 most frequent OTUs were Muscodor sp. and Nemania sp. in Xylariaceae, followed by Gnomonia sp., Glomerella acutata , Apiosporopsis sp., Asteroma sp., and Cladosporium cladosporioides . The similarities of OTU composition in endophytic fungal assemblages on leaves of Betulaceae were generally low among the forests of different climatic regions. Fungal OTU compositions were relatively similar between 2 Betula species in the subalpine forest, whereas 7 tree species in the cool temperate forest were divided into 3 groups according to the similarity of endophytic fungal assemblages on the leaves, with 4 Carpinus species assigned into 2 of the 3 groups. The similarity of endophytic fungal assemblages between August and October was relatively high in the subalpine forest, whereas the seasonal changes were generally greater (i.e., the similarities among sampling dates were lower) in the cool temperate forest.