马尾松内生真菌产油脂菌株的分离、筛选和鉴定

采用组织块法从马尾松(Pinus massoniana Lamb.)根、茎和叶片中分离获得内生真菌,初步筛选出产油脂菌株且对其油脂含量进行了分析;并采用形态学及分子生物学方法对产油脂菌株进行了分类鉴定。结果显示:从马尾松植株中共获得21株内生真菌菌株,其中分离自根、茎和叶片的菌株分别为4株、6株和11株。有14株菌株的菌丝中有油滴;其中,分离自叶片的菌株ZP-1、分离自根的菌株ZP-2和分离自茎的菌株ZP-3的菌丝内油滴较多且油脂含量较高,平均油脂含量分别达到29.12%、25.03%和30.56%,差异极显著(P<0.01)。不同菌株的菌落颜色、菌丝和分生孢子形态特征明显不同,菌株ZP-1、ZP-2和ZP-3的形态分别与拟青霉属(Paecilomyces Bainier)、生赤壳菌属(Bionectria Speg.)和镰刀菌属(Fusarium Link)菌种的形态相似。与GenBank中相关真菌ITS序列的比对以及NJ系统树分析结果显示:菌株ZP-1与拟青霉属、菌株ZP-2与生赤壳菌属、菌株ZP-3与镰刀菌属间的ITS片段序列相似性均达到99%,在各自的NJ系统树上它们也分别聚在一起。初步确定菌株ZP-1、ZP-2和ZP-3分别属于拟青霉属、生赤壳菌属和镰刀菌属。     

青檀果内生真菌菌群的组成及多样性

为进一步了解我国古老特有的野生植物青檀内生真菌的组成特征,以安徽琅琊山自然保护区内的野生青檀果为研究对象,采用组织分离和形态学鉴定的方法对青檀果内生真菌进行了分离和鉴定.结果表明: 从500个组织块中共分离内生真菌428株,隶属于3目4科20属,未产孢的菌株暂归为无孢菌群(80株,18.7%).暗梗孢科和球壳孢科是青檀果内生真菌的优势科,分别占38.1%和32.9%,交链孢属和拟茎点霉属为其优势属,分别占27.1%和16.8%.青檀果不同部位内生真菌的组成存在差异,以种子中分离的内生真菌数量最多(19属181株,占42.3%),果翅中分离的内生真菌最少(7属88株,占20.6%).除了交链孢属为3个不同部位的共同优势属之外,拟茎点霉属和大茎点霉属分别是果柄和果翅的优势属.果柄和果翅内生真菌菌群的组成相似性最高(C_S=0.78),种子和果翅的相似性系数最低(C_S=0.50).分析显示,果柄与果翅在内生真菌菌群组成上无显著差异,而种子与果柄、种子与果翅在内生真菌菌群组成上均存在显著差异.     

广藿香内生真菌多样性及其对青枯菌的拮抗活性

【目的】对广藿香各部位内生真菌的类群结构及多样性进行系统分析,筛选出对青枯菌具拮抗活性的菌株。【方法】采用组织块分离法从广藿香健康植株的根、茎、叶中分离内生真菌,结合形态学和基于ITS-r DNA序列分析鉴定方法分析广藿香内生真菌的类群结构及多样性,并利用双层平板拮抗法筛选对青枯菌具有抑制活性的菌株。【结果】从广藿香中共分离获得313株内生真菌,隶属于30个属,其中链格孢属(占28.75%)、拟茎点霉属(占23.00%)和炭疽菌属(11.82%)为优势类群,此外还分离到炭皮菌属、弯孢聚壳属、Gibellulopsis、新萨托菌属、葡萄座腔菌属、篮状菌属等较为少见的类群。茎、叶内生真菌的定殖率和分离率明显高于根,而根部多样性指数为2.64,要高于茎(2.00)和叶(1.97);广藿香茎与叶、根与茎、根与叶之间的内生真菌相似性指数分别为0.35、0.20、0.19,均小于0.5,显示各部位之间的内生真菌组成不相似程度高。从313株广藿香内生真菌中通过拮抗实验筛选到16株对青枯菌有拮抗活性的菌株,其中GHXR07、GHXR27和GHXR29的拮抗活性尤为显著,分别被鉴定为Talaromyces sp.、Myrothecium roridum Tode和Talaromyces wortmannii(Kl?cker)Benjamin。【结论】广藿香内生真菌具有丰富的物种多样性且其类群分布具有一定的组织特异性,其中部分菌株对青枯菌具有明显的拮抗活性。     

天目山山胡椒不同部位内生真菌组成及多样性分析

采用表面消毒法,从浙江天目山野生山胡椒[ Lindera glauca( Sieb.et Zucc.) Blume]的茎、叶和树皮中分离出内生真菌,基于ITS序列分析进行分类鉴定;并以内生真菌的分离率、定殖率、分离频率、多样性指数(H')及相似性系数为指标,分析了山胡椒内生真菌的菌群组成及多样性.结果显示:在26株山胡椒样株的728块组织块中共分离得到328株内生真菌(茎、叶和树皮中分别有161、40和127株);共鉴定出44个分类单元(茎、叶和树皮中各有19、18和28个),其中25个分类单元鉴定到种、17个鉴定到属、2个鉴定到科,ITS序列的GenBank登录号从JF502420至JF502462.在44个分类单元中,有40个分类单元属于子囊菌(310株),存在于山胡椒的各个部位;仅有4个分类单元属于担子菌(18株),且仅存在于茎和树皮中.山胡椒茎、树皮和叶中内生真菌的定殖率分别为65％、60％和15％,分离率分别为0.77、0.61和0.19;叶和树皮中内生真菌的多样性指数均为2.63,远大于茎(H’=1.83).山胡椒内生真菌的优势属为Phomopsis、Paraconiothyrium、Phoma和Colletotrichum,大量存在于叶、茎和树皮中.山胡椒茎与树皮、茎与叶及叶与树皮间内生真菌的相似性系数分别为0.27、0.19和0.18,显示树皮和叶之间以及树皮和茎之间内生真菌的组成极不相似.研究结果表明:山胡椒体内存在大量的内生真菌,其茎、叶和树皮的内生真菌菌群组成具有一定程度的多样性和差异性,且内生真菌的分布具有组织特异性.     

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。     

广西柳树桑寄生内生真菌的分离鉴定与抗肿瘤活性菌株筛选

该研究从广西钦州市采集健康的柳树桑寄生的根、茎和叶中分离并纯化内生真菌,对真菌进行形态学鉴定,提取内生真菌的DNA,采用真菌ITS序列对内生真菌进行分子鉴定。利用A549和H460细胞作为抗肿瘤活性指示细胞,采用MTT法测定真菌乙酸乙酯提取物的抗肿瘤活性。经过初步分离分析,从柳树桑寄生中纯化出27株内生真菌,经鉴定它们分别属于7个目9个属15个种。拟盘多毛孢属和间座壳属为优势属,其中拟盘多毛孢属全部定植于寄生根;其次为新壳梭孢属、拟茎点霉属和球座菌属,各分离到3株;其他包括青霉属、镰刀菌属、炭疽菌属和派伦霉属,各分离到1株。抗肿瘤活性研究表明,有一株与Pestalotiopsis protearum的ITS序列相似性达100%的拟盘多毛孢属菌株Gen24表现有抑制肿瘤细胞A549和H460生长的特性,在真菌乙酸乙酯浸提物浓度为800μg·mL~(-1)时,对A549细胞的抑制率达到了56.92%,对H460细胞的抑制率达到了70.11%。该研究结果表明广西柳树桑寄生内生真菌较丰富,在寄主中的分布表现了一定的组织特异性,而且还存在一些具有抗肿瘤活性的菌株及其活性物质可供进一步深入研究。     

构树可培养内生真菌的多样性初探

构树Broussonetia papyrifera为桑科重要的资源植物。为调查逆境构树内生真菌的多样性,我们对铅锌矿区构树根、茎、叶和果实各部位进行内生真菌分离鉴定,从中分离得到187株内生真菌,根据形态学与分子生物学方法鉴定为20属,其中链格孢属Alternaria（50.27%）、镰孢属Fusarium（11.76%）为优势属。不同培养基的内生真菌分离率结果表明,马铃薯葡萄糖琼脂培养基（PDA）分离出的菌株数最多,占总数的73.96%;构树不同部位的菌株分离数目显示,叶中分离出的菌株较多,占38.02%。在属的分类水平上,根、茎、叶、果实不同部位的真菌数量也存在明显差异。Shannon-Wiener多样性指数和Simpson多样性指数分析结果提示不同样地的构树内生真菌的多样性也存在差异。Chao1指数和ACE指数也提示构树内生真菌丰富度与样地和组织有关。     

不同化学型和季节变化对茅苍术内生真菌群落多样性的影响

对茅山型茅苍术(MA)和湖北型茅苍术(HA)的内生真菌多样性进行调查,分析二者之间优势菌群和特异菌群的差异。将两种化学型的茅苍术移栽至同一环境中,分别于春季、夏季、秋季3个季节采集叶片进行内生真菌的分离、鉴定和多样性分析。调查一共得到530株内生真菌,总分离率为58.89%,通过形态学特征和ITS序列鉴定归结为20个属。MA内生真菌分离率要高于HA,二者的真菌多样性构成有一定差异性。MA和HA内生真菌的优势菌群呈基本重合现象,主要集中于交链孢属、球座菌属、刺盘孢属等几个属;而二者之间的特异菌群显示出差异性,MA的特异菌群为Leptospora属、支顶孢属、壳针孢属等,HA特异菌群为白僵菌属、轮枝孢属、毛壳菌属等。随着季节的变化,内生真菌群落显示出一定的演替规律。夏季内生真菌的多样性高于春季和秋季。     

白及内生真菌多样性研究

白及( Bletilla striata)是兰科地生型多年生植物,也是我国传统中药材之一。利用菌根技术进行白及的保护和人工栽培,需要获得白及可培养的内生真菌。该研究以广西野生的白及根和叶为材料,采用分离培养法分离内生真菌,并结合真菌形态特征,及其核糖体的转录间隔区( ITS)序列分析,确定内生真菌的分类地位。结果表明：从2株白及植物90块组织中分离获得37株内生真菌,鉴定为15个分类单元,由9个属组成,分属于2门4纲7目8科,包括锤舌菌纲( Leotiomycetes)、座囊菌纲( Dothideomycetes)和粪壳菌纲( Sordariomy-cetes),伞菌纲( Agaricomycetes)。从根中分离获得内生真菌12种,蜡壳菌属为优势属;从叶中分离获得内生真菌3种,刺盘孢属为优势属;刺盘孢菌属( Colletotrichum)和蜡壳菌属( Sebacina)真菌的相对多度值均达到20％;4株担子菌均分布于根中,叶组织中未有分布。根组织中内生真菌的多样性指数(H＝1.863)高于叶组织(1.098)。该研究结果及其所分离培养的担子菌类真菌,为更好地利用菌根技术进行白及等兰科植物资源的保护与可持续利用奠定了基础。     

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.     

Diversity of fungal isolates from three Hawaiian marine sponges

Sponges harbor diverse prokaryotic and eukaryotic microbes. However, the nature of sponge-fungal association and diversity of sponge-derived fungi have barely been addressed. In this study, the cultivation-dependent approach was applied to study fungal diversity in the Hawaiian sponges Gelliodes fibrosa, Haliclona caerulea, and Mycale armata. The cultivated fungal isolates were representatives of 8 taxonomic orders, belonging to at least 25 genera of Ascomycota and 1 of Basidiomycota. A portion of these isolates (n=15, 17%) were closely affiliated with fungal isolates isolated from other marine habitats; the rest of the isolates had affiliation with terrestrial fungal strains. Cultivated fungal isolates were classified into 3 groups: 'sponge-generalists'-found in all sponge species, 'sponge-associates'-found in more than one sponge species, and 'sponge-specialists'-found only in one sponge species. Individuals of G. fibrosa collected at two different locations shared the same group of 'sponge-specialists'. Also, representatives of 15 genera were identified for the first time in marine sponges. Large-scale phylogenetic analysis of sponge-derived fungi may provide critical information to distinguish between 'resident fungi' and 'transient fungi' in sponges as it has been done in other marine microbial groups. This is the first report of the host specificity analysis of culturable fungal communities in marine sponges.     

湛江红树林滩涂可培养真菌种群多样性分析

【目的】明确湛江地区红树林滩涂海洋真菌的种类及其分布,为海洋真菌的开发利用研究奠定基础。【方法】运用稀释平板法从湛江市高桥及特呈岛红树林滩涂不同的潮位带(低、中、高)和不同树种(白骨壤、桐花树、木榄、红海榄)采集淤泥样品550份,采用真菌形态学和ITS序列分析技术进行多样性研究【。结果】分离获得海洋真菌274株,共鉴定出19属39种真菌,以曲霉属Aspergillus、青霉属Penicillium和木霉属Trichoderma真菌分离频率高,为湛江红树林滩涂优势真菌种类,尤其在中潮位带真菌种类最多。此外,分离获得真菌Talaromyces helicus,为中国新记录种。【结论】湛江红树林滩涂海洋真菌的种类十分丰富,具有潜在的开发和利用前景。     

银杏叶部内生真菌多样性的研究

为了解我国特有植物银杏的叶部组织中内生真菌菌群的多样性和演替变化,分别于春、夏、秋季从江苏和山东两省的五个主要银杏栽培地采集健康叶片进行了内生真菌的研究。结果表明:所分离出的1971株内生真菌可归于32属,以交链孢属Alternaria(18.01%)、叶点霉属Phyllosticta(14.66%)和丝核菌属Rhizoctonia(14.56%)为优势菌群。在内生真菌的类群及组成上存在着部位、地区以及季节的差异性,也呈现一定的规律性。多样性指数的计算反映出所获得的银杏叶部内生真菌菌群具有不同的丰富度及均匀度,同时有一定的相似性。     

裸盖菇和斑褶菇属真菌菌种的有效分离方法

【目的】探讨获取裸盖菇属及斑褶菇属真菌纯培养的有效分离方法。【方法】采用菌褶接种法和孢子弹射法进行分离,以形态鉴定为基础,通过ITS区测序并与DNA序列库中已知序列进行比对的分子鉴定方法鉴别分离培养物的真伪,以确定分离方法的可靠性。【结果】对采自云南的28个裸盖菇属和斑褶菇属菌株进行了分离,菌褶接种法有24个菌株分离纯化成功,成功率达86%,而孢子弹射法仅有7个菌株分离成功,成功率为25%。【结论】菌褶接种法对于裸盖菇属和斑褶菇属真菌是一种有效而简便易行的分离方法,该法利用菌褶为产孢组织的优势,无需对菌褶进行表面消毒,易于纯化成功,值得在其他类似的腐生小型薄盖伞菌类群的分离中尝试应用。     

Isolation and characterization of endophytic huperzine A-producing fungi from Huperzia serrata

Huperzia serrata is a producer of huperzine A (HupA), a cholinesterase inhibitor (ChEI). Over 120 endophytic fungi were recovered from this plant and screened for Hup-A and nine were found. These nine represented seven different fungal genera with the most significant producer being Shiraia sp. A total of 127 endophytic fungi isolates obtained from the root, stem, and leaf segments of H. serrata were grouped into 19 genera based on their morphological traits and sequence analysis of the internal transcribed spacers (ITS1-5.8S-ITS2), indicating endophytic fungi in H. serrata are diverse and abundant. Aspergillus, Podospora, Penicillium, Colletotrichum, and Acremonium were the frequent genera, whereas the remaining genera were infrequent groups. Overall, 39 endophytic fungi isolates showed acetylcholinesterase (AChE) inhibition in vitro. Nine endophytic fungi isolates from seven distinct genera were capable of producing HupA verified by thin-layer chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Among the HupA-producing fungi, the yield of HupA produced by the Shiraia sp. Slf14 was 327.8 μg/l in potato dextrose broth, and the fungal HupA was further validated by mass spectrometry (ESI-MS). The present study demonstrated that H. serrata was a fascinating fungal reservoir for producing HupA and other ChEIs.     

凹叶厚朴内生真菌研究初报

从凹叶厚朴(Magnolia officinalissubsp.biloba)根、茎、叶中分离获得内生真菌共计28株,经形态观察,鉴定出13个属。研究表明凹叶厚朴不同部位内生真菌的数量、分布、种群及其组成存在差异。     

A proposed taxonomy of anaerobic fungi (class neocallimastigomycetes) suitable for large-scale sequence-based community structure analysis

Anaerobic fungi are key players in the breakdown of fibrous plant material in the rumen, but not much is known about the composition and stability of fungal communities in ruminants. We analyzed anaerobic fungi in 53 rumen samples from farmed sheep (4 different flocks), cattle, and deer feeding on a variety of diets. Denaturing gradient gel electrophoresis fingerprinting of the internal transcribed spacer 1 (ITS1) region of the rrn operon revealed a high diversity of anaerobic fungal phylotypes across all samples. Clone libraries of the ITS1 region were constructed from DNA from 11 rumen samples that had distinctly different fungal communities. A total of 417 new sequences were generated to expand the number and diversity of ITS1 sequences available. Major phylogenetic groups of anaerobic fungi in New Zealand ruminants belonged to the genera Piromyces, Neocallimastix, Caecomyces and Orpinomyces. In addition, sequences forming four novel clades were obtained, which may represent so far undetected genera or species of anaerobic fungi. We propose a revised phylogeny and pragmatic taxonomy for anaerobic fungi, which was tested and proved suitable for analysis of datasets stemming from high-throughput next-generation sequencing methods. Comparing our revised taxonomy to the taxonomic assignment of sequences deposited in the GenBank database, we believe that >29% of ITS1 sequences derived from anaerobic fungal isolates or clones are misnamed at the genus level.     

银杏内生真菌的生态分布

2004—2005年春夏秋3季,在江西省九江地区4个地点分别采集健康的银杏(Ginkgo biloba)植株,分离得到内生真菌198株,经形态学分类鉴定为6目、7科、22属。研究结果表明,银杏内生真菌的分布存在季节差异、组织部位差异、年龄差异;优势菌群的组成与数量随采样地点、分离组织部位、不同生长时期内生真菌的不同而变化。与中国其他地区的银杏植株内生真菌进行比较,结果表明,植物内生真菌的分布与地域具有一定的相关性,体现了内生真菌生态分布的多样性。     

Phylogenetic diversity of culturable fungi associated with the Hawaiian Sponges Suberites zeteki and Gelliodes fibrosa

Sponges are well documented to harbor large amounts of microbes. Both culture-dependent and molecular approaches have revealed remarkable bacterial diversity in marine sponges. Fungi are commonly isolated from marine sponges, yet no reports on phylogenetic diversity of sponge-inhabiting fungi exist. In this report, we investigated the phylogenetic diversity of culturable fungi from the Hawaiian alien marine sponges Suberites zeteki and Gelliodes fibrosa. A total of 44 independent isolates were recovered from these two sponge species, representing 7 orders and 22 genera of Ascomycota. The majority (58%) of fungal isolates from S. zeteki resided in the Pleosporales group, while the predominant isolates (52%) from G. fibrosa were members of the Hypocreales group. Though differing in fungal species composition and structure, culturable communities of these two sponges displayed similar phylogenetic diversity. At the genus level, only two genera Penicillium and Trichoderma in the Eurotiales and Hypocreales orders, respectively, were present in both sponge species. The other genera of the fungal isolates were associated with either S. zeteki or G. fibrosa. Some of these fungal genera had been isolated from sponges collected in other marine habitats, but more than half of these genera were identified for the first time in these two marine sponges. Overall, the diversity of culturable fungal communities from these two sponge species is much higher than that observed in studies of marine sponges from other areas. This is the first report of phylogenetic diversity of marine sponge-associated fungi and adds one more dimension to our current understanding of the phylogenetic diversity of sponge-symbiotic microbes.     

Diverse Deep-Sea Fungi from the South China Sea and Their Antimicrobial Activity

We investigated the diversity of fungal communities in nine different deep-sea sediment samples of the South China Sea by culture-dependent methods followed by analysis of fungal internal transcribed spacer (ITS) sequences. Although 14 out of 27 identified species were reported in a previous study, 13 species were isolated from sediments of deep-sea environments for the first report. Moreover, these ITS sequences of six isolates shared 84–92 % similarity with their closest matches in GenBank, which suggested that they might be novel phylotypes of genera Ajellomyces, Podosordaria, Torula, and Xylaria. The antimicrobial activities of these fungal isolates were explored using a double-layer technique. A relatively high proportion (56 %) of fungal isolates exhibited antimicrobial activity against at least one pathogenic bacterium or fungus among four marine pathogenic microbes (Micrococcus luteus, Pseudoaltermonas piscida, Aspergerillus versicolor, and A. sydowii). Out of these antimicrobial fungi, the genera Arthrinium, Aspergillus, and Penicillium exhibited antibacterial and antifungal activities, while genus Aureobasidium displayed only antibacterial activity, and genera Acremonium, Cladosporium, Geomyces, and Phaeosphaeriopsis displayed only antifungal activity. To our knowledge, this is the first report to investigate the diversity and antimicrobial activity of culturable deep-sea-derived fungi in the South China Sea. These results suggest that diverse deep-sea fungi from the South China Sea are a potential source for antibiotics’ discovery and further increase the pool of fungi available for natural bioactive product screening.