富硒矿区真菌群落特征初步研究

[目的]研究湖北恩施富硒区真菌群落特征。[方法]对环境样本提取总DNA,通过构建PCR扩增子真菌ITS文库,利用Illuminate Miseq双末端测序以及生物信息学软件分析样本的α-、β-生物多样性。[结果]5样本经Illuminate Miseq平台获得153 363条Filter reads,506个OTUs。样本中包含子囊菌门、罗兹菌门(Rozellomycota)、担子菌门、球囊菌门、结合菌门、壶菌门和未分类的真菌;子囊菌门为优势菌群,丰度在86%~94%之间;从属水平上相对丰度较高的前5个属为炭角菌属、拟青霉属、叉四枝孢属、杯梗孢属和明梭孢属,其中炭角菌属属为绝对优势菌群,丰度在37%~74%之间。[结论]恩施富硒区真菌群落结构与硒含量相关,炭角菌属、拟青霉属、叉四枝孢属、杯梗孢属和明梭孢属为该富硒区的优势真菌属。     

DNA分子标记技术在真菌系统学研究中的应用及影响

DNA分子标记技术为真菌系统进化研究提供了许多新的方法,真菌分子系统学已成为一门成熟的学科。简述了真菌分子系统学的发展简史和代表性的研究方法以及对真菌系统学的主要贡献,包括将广义的真菌划分为3个类群,粘菌和卵菌不再属于真菌界成员。真菌生命之树项目的研究结果对真菌界高阶分类系统作出重大调整,将先前的4个门(壶菌门、接合菌门、子囊菌门和担子菌门)变为7个门(微孢子虫门、壶菌门、新丽鞭毛菌门、芽枝霉门、球囊菌门、子囊菌门和担子菌门)和4个亚门,并对真菌各类群概念作出修订。此外,DNA分子标记技术对真菌种概念的认识、有性型-无性型关联及分子生态学等研究领域产生了重要影响。     

天山林区土壤真菌多样性及其群落结构

土壤真菌群落结构对森林生态系统功能的维持具有重要作用,目前对天山林区土壤微生物的种类和群落结构了解甚少。为了解天山不同植被类型下土壤真菌的群落动态及优势菌属,使用Illumina MiSeq高通量测序技术,研究天山林区根际土与非根际土中真菌群落组成及多样性。结果表明:(1)对有效序列进行OTU聚类(operational taxonomic unit)后共得到14121个OTUs,经注释划分为7个门、33个纲、109个目、255个科、444个属、677个种;(2)子囊菌门(Ascomycota)和担子囊菌门(Basidiomycota)是天山植物的优势菌门,灌丛中相对丰度较高的有帚枝霉属(Sarocladium)、鬼笔属(Lysurus)、Microidium;云杉林中相对丰度较高的属有镰刀属(Fusarium)、赤霉属(Gibberella)、丝盖伞属(Inocybe);(3)灌丛与云杉林土壤真菌的群落结构和丰富度有显著差异,灌丛土壤真菌的多样性和丰富度更高,云杉林中阴坡的土壤真菌丰富度较高而阳坡和林窗较低;(4)丝盖伞属(Inocybe)、被孢霉属(Mortierella)、赤霉属(Gibberella)、镰刀属(Fusarium)与云杉的生长可能具有密切的关系。本文推测,根际土壤真菌的多样性和丰富度较低可能是导致云杉幼苗成活率低等更新障碍的问题。     

基于rDNA ITS序列的油茶叶片内生真菌多样性研究(英文)

油茶是我国热带和亚热带地区广泛栽培的一类重要的经济树种,其种子可生产优质食用油(茶油)。为了比较全面地了解油茶的内生真菌多样性,采用基于rDNA ITS的免培养法从油茶叶片中提取了总DNA,再从总DNA样本中直接扩增了内生真菌核糖体RNA基因内转录间隔区序列(ITS),进而构建了ITS克隆文库。共计从50个克隆子中获得了22种不同的克隆序列,其中3个为嵌合体,7个归属为植物,其余克隆序列根据序列相似性和系统发育分析归为12个不同的分类操作单元(OTUs),全部为子囊菌,分属于4纲4目,其中Aspergillus属2种,Mycosphaerellaceae科1种,Sordariales目4种,Helotiales目5种,Aspergillus属真菌是优势菌。结果表明油茶叶片内生真菌的种类分布较广。     

三硝酸丙三酯对厌氧真菌和甲烷菌代谢产物、纤维水解酶活性及菌群丰度的影响

【背景】近年来硝酸酯化合物抑制瘤胃甲烷生成的能力受到广泛关注,但鲜有研究关注其对瘤胃内主要纤维降解菌——厌氧真菌的影响。【目的】研究三硝酸丙三酯(Nitroglycerin,NG)对厌氧真菌和甲烷菌活性和代谢的影响。【方法】利用厌氧真菌(Piromyces sp.F1)纯培养及与甲烷菌共培养(Piromyces sp.F1+Methanobrevibacter sp.),比较不同剂量NG(0.0、6.6、13.2和19.8μmol/L)对厌氧真菌和甲烷菌的代谢终产物、主要纤维水解酶活性及菌群丰度的影响。【结果】厌氧真菌共培养组中,6.6μmol/L NG完全抑制了甲烷的生成,积累了氢气和甲酸(P0.05),降低了乙酸浓度以及木聚糖酶和羧甲基纤维素酶活性(P0.05),但未显著影响厌氧真菌和甲烷菌的丰度(P0.05)。厌氧真菌纯培养组中,6.6μmol/L NG显著降低了甲酸、乙酸、乙醇、木聚糖酶和羧甲基纤维素酶活性(P0.05),但总产气量和氢气产量没有显著差异(P0.05)。在2个培养体系中,随着剂量的升高,NG对厌氧真菌和甲烷菌的抑制增强。【结论】NG能够抑制甲烷菌的活性,但对厌氧真菌也有抑制作用。     

短花针茅荒漠草原土壤微生物群落组成及结构

为详细了解内蒙古短花针茅荒漠草原生态系统中土壤微生物群落组成与结构。对其土壤中微生物的总DNA提取后,采用高通量测序技术对土壤中细菌的16Sr DNA和真菌的ITS基因进行了序列测定,分析了短花针茅荒漠草原土壤中微生物群落结构特征。结果表明,共获得细菌OTUs13711个,真菌OTUs 5929个。物种分类显示,细菌种类隶属于29门57纲111目191科485属,其中优势类群为Gammaproteobacteria和Thermoleophilia,它们的相对丰度分别为32.68%和26.83%。真菌隶属于4门16纲45目78科105属,优势类群为Ascomycota和Basidiomycota,它们的相对丰度分别为35.76%和25.90%。土壤中细菌群落的多样性和丰富度均高于真菌。     

基于高通量测序分析桃蚜体内微生物群落结构及多样性

为探明桃蚜Myzus persicae体内微生物群落结构及其种类多样性,采用Illumina HiSeq二代测序技术检测桃蚜体内细菌16S rRNA基因和真菌ITS基因序列的方法,分析取食白菜Brassica pekinensis和甘蓝Brassica oleracea的无翅孤雌桃蚜成虫体内微生物群落结构及多样性。研究结果获得桃蚜体内细菌16S rDNA和真菌ITS1优质序列分别为473 750条和472 980条,并根据序列相似性对其进行聚类分析,分别获得959个和1 424个OTUs。基于OTUs分类结果,共注释鉴定细菌类群26个门、55个纲、128个目、227个科、419属、451种,真菌类群10个门、31个纲、77个目、172个科、343属、441种。其中,在门级水平上,取食白菜和甘蓝的桃蚜体内细菌类群均以变形菌门Proteobacteria内的细菌（占73.11%,80.10%）为优势菌;真菌类群均以子囊菌门Ascomycota真菌（占51.91%,50.98%）为优势菌。在属级水平上,取食白菜和甘蓝的桃蚜体内细菌均以布赫纳氏菌属Buchnera（占60.82%,56.11%）为优势属,而其次优势细菌属则分别为未培养的叶绿体菌Chloroplas（占2.84%）和立克次氏小体属Rickettsiella（占10.04%）;取食白菜的桃蚜体内真菌优势属为青霉属Penicillium（占5.15%）,次优势菌属为被孢霉属Mortierella（占3.83%）,而取食甘蓝的桃蚜体内真菌优势属为曲霉属Aspergillus（占3.93%）,次优势菌属为被孢霉属Mortierella（占3.86%）。以上研究结果表明取食不同植物的桃蚜体内微生物群落结构和种类多样性存在一定差异。α多样性指数表明桃蚜体内微生物群落结构具有较高的丰富度和多样性。研究结果为进一步挖掘桃蚜体内关键微生物,并解析其生物学功能及其利用等方面奠定了重要基础。     

灵芝连作土壤真菌群落分析

本研究通过Illumina MiSeq平台对邻近野生土壤（GL0）、种植1年（GL1）、2年（GL2）和4年（GL4）的灵芝覆土进行ITS1扩增子测序,分析灵芝连作覆土中真菌群落的变化,为揭示灵芝连作障碍的机理提供理论依据。研究结果表明,12个灵芝土壤样本一共获得349 642条有效序列,经聚类得到2 426个OTU,分别隶属于8门、28纲、64目、127科、233属和449种。在门水平上,随着连作年限的增加,灵芝覆土中真菌的多样性水平逐渐减低,在GL4组中只含有担子菌门（Basidiomycota,占85.03%）、子囊菌门（Ascomycota,占14.77%）和少量被孢霉门（Mortierellomycota,占0.20%）。其中,担子菌门的相对丰度随着连作年限的增加显著增加,子囊菌门的相对丰度显著减少,而被孢霉门的相对丰度无显著性差异。在属水平上,担子菌门灵芝属Ganoderma的相对丰度随着连作年限的增加而极显著增加,子囊菌门仅青霉属Penicillium菌群的相对丰度呈现先减少后增加的趋势,在GL4组中相对于GL0组其相对丰度增加了57.92%,表明青霉属可能是引发该地区灵芝连作障碍的重要菌群,该研究为探索灵芝连作障碍的分子机制和生态防控提供了理论依据。     

枯草芽孢杆菌J-15抗大丽轮枝菌次生代谢产物对棉田土壤真菌多样性的影响

【背景】目前利用拮抗菌进行作物病害防治的研究较多,但拮抗菌次生代谢产物如何影响棉花根际土壤微生物群落相关的研究较少。【目的】探讨枯草芽孢杆菌J-15抗大丽轮枝菌次生代谢产物对棉田土壤真菌多样性的影响,为利用枯草芽孢杆菌J-15及其次生代谢产物防治棉花黄萎病的土壤微生物生态安全进行评估。【方法】以新疆北部玛纳斯地区棉田为土壤采样点,随机选取10个点进行采样后混合,经枯草芽孢杆菌J-15抗大丽轮枝菌次生代谢产物处理一定时间后,提取土样总DNA,利用Illumina HiSeq高通量技术,对样品中真菌ITS1-ITS2区进行高通量测序,分析J-15抗大丽轮枝菌次生代谢产物处理对土样真菌多样性的影响。【结果】在97%相似度水平下,处理10、30d后,样品中真菌的OTU数量、Chao1和ACE丰度指数均分别高于相同时间放置的未处理的对照组,而Simpson指数低于其对照组。从群落组成分析来看,与对照组相比,受J-15次生代谢产物处理的土壤样品,子囊菌门(Ascomycota)的盘菌属(Tricharina)和被孢霉门(Mortierellomycota)的被孢霉属(Mortierella)等优势真菌相对丰度提高,而丰度高于1%的2类病原真菌轮枝孢属(Verticillium)、镰孢霉属(Fusarmm)的丰度显著降低。【结论】J-15抗大丽轮枝菌次生代谢产物对棉田土壤真菌群落及丰度有显著影响,但不改变影响农业生产的土壤真菌群落的结构。     

黑老虎内生真菌及根际土壤真菌的群落结构与生态功能分析

为探讨黑老虎(Kadsura coccinea)根际土壤和组织内生真菌菌群的组成及其生态功能，该研究采用ITS高通量测序技术对成熟黑老虎(根、茎、叶)内生真菌及根际土壤真菌群落结构、多样性和生态功能进行了分析。结果表明：(1)从12个样品中共获得2 241个可操作分类单元(OTU),涉及10门、41纲、95目、212科、367属，内生真菌(根、茎、叶)和根际土壤真菌OTU数分别为386、536、258、1 435个，其中共有的OTU为18个。在门水平上，黑老虎内生真菌及根际土壤真菌优势群落均为子囊菌门和担子菌门，其中子囊菌门在叶和茎中占比分别高达96.99%和95.37%;在属水平上，黑老虎根际土壤真菌中腐生真菌被孢霉属占比较高(为13.5%),叶和茎等生长旺盛的组织中子囊菌门未分类属和痂囊腔菌属占比较高。(2)α多样性分析结果显示，黑老虎根际土壤真菌群落的丰度和多样性明显高于内生真菌，茎中内生真菌丰度显著高于根和叶，而根、茎和叶组织间内生真菌多样性差异不显著；PCoA分析结果显示，叶和茎的真菌群落结构相似性更高。(3)利用FUNGuild数据库进行的功能预测分析结果显示，黑老虎根际土...     

藏仔猪粪便真菌菌群多样性分析

为探究西藏地区藏仔猪粪便中真菌菌群结构,本研究采用ITS1序列分析新生藏仔猪和断奶藏仔猪新鲜粪便中真菌的多样性。结果表明：从5头新生藏仔猪新鲜粪便中,共鉴定出5个门、13个纲、 25个目、33个科和38个属的真菌;从5头断奶藏仔猪新鲜粪便中,共鉴定出5个门、12个纲、20个目、24个科和28个属的真菌。子囊菌门和担子菌门为优势门,占门级分类的86.35%;布勒担菌科、虫草科、丛赤壳科、曲霉科、丝孢酵母菌科、肉杯菌科、酵母菌科和毕赤酵母菌科为优势科,占科级分类的54.38%。随着藏仔猪的生长,其粪便中真菌菌群的数量和优势真菌的相对丰度均发生变化,这说明不同生长发育阶段会影响藏仔猪粪便中真菌的多样性。     

不同扩增引物对高通量测序分析盐角草内生真菌多样性的影响

【背景】高通量测序分析作为深入了解环境微生物群落组成的重要方法,已成为植物内生真菌多样性研究的有效手段,然而由于引物的扩增差异,采用不同引物可对实验结果分析造成影响。同时,盐角草作为世界上最耐盐的植物之一,存在着多种功能性的内生真菌,而较为全面介绍其内生真菌组成和多样性的报道鲜见。【目的】为了揭示盐角草内生真菌的多样性,解析不同扩增引物对内生菌多样性分析的影响。【方法】分别采用真菌高通量测序常用引物对ITS1-5F、ITS1-1F、ITS2对采自乌鲁木齐达坂城盐湖的盐角草内生真菌进行扩增,开展其内生真菌OTU的分析。【结果】通过不同引物对扩增并测序共获得102个盐角草内生真菌OTU,涉及真菌界8个门和未分类菌群,其中子囊菌门(Ascomycota)占绝对优势,其次为担子菌门(Basidiomycota);在属层次上,盐角草内生真菌共涉及64个属及20个未分类属,其中Alternaria、Cladosporium、Podospora等3个属为盐角草内生真菌优势菌群。对不同引物对扩增测序结果分析表明,不同引物对扩增对分析内生真菌OTU数量和种类具有明显的影响,在全部所得的102个OTU中,ITS1-5F引物对获得44个OTU、ITS1-1F引物对获得55个OTU、ITS2引物对获得25个OTU,但以上3对引物扩增均检测到的OTU数仅为5个。物种组成和多样性分析表明,内生真菌多样性分析中采用以ITS1-1F为主,ITS1-5F为辅的分析策略,可较为全面地展现内生真菌的多样性。【结论】盐角草存在较为丰富的内生真菌资源,不同扩增引物对高通量分析盐角草内生真菌组成和分布具有明显的影响。     

Assessment of fungal diversity in deep-sea sediments by multiple primer approach

Increasing evidence of the fungal diversity in deep-sea sediments has come from amplification of environmental DNA with fungal specific or eukaryote primer sets. In order to assess the fungal diversity in deep-sea sediments of the Central Indian Basin (CIB) at ~5,000 m depth, we amplified sediment DNA with four different primer sets. These were fungal-specific primer pair ITS1F/ITS4 (internal transcribed spacers), universal 18S rDNA primers NS1/NS2, Euk18S-42F/Euk18S-1492R and Euk18S-555F/Euk18S-1269R. One environmental library was constructed with each of the primer pairs, and 48 clones were sequenced per library. These sequences resulted in 8 fungal Operational Taxonomic Units (OTUs) with ITS and 19 OTUs with 18S rDNA primer sets respectively by taking into account the 2% sequence divergence cut-off for species delineation. These OTUs belonged to 20 distinct fungal genera of the phyla Ascomycota and Basidiomycota. Seven sequences were found to be divergent by 79–97% from the known sequences of the existing database and may be novel. A majority of the sequences clustered with known sequences of the existing taxa. The phylogenetic affiliation of a few fungal sequences with known environmental sequences from marine and hypersaline habitat suggests their autochthonous nature or adaptation to marine habitat. The amplification of sequences belonging to Exobasidiomycetes and Cystobasidiomycetes from deep-sea is being reported for the first time in this study. Amplification of fungal sequences with eukaryotic as well as fungal specific primers indicates that among eukaryotes, fungi appear to be a dominant group in the sampling site of the CIB.     

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.     

藏羚羊和藏野驴粪便真菌多样性比较研究

为探究西藏地区反刍动物（藏羚羊）和单胃草食动物（藏野驴）粪便中真菌群落结构,本研究采用ITS1区高通量测序方法分析西藏羌塘国家自然保护区藏羚羊和藏野驴新鲜粪便中真菌的多样性。结果表明:从5头藏羚羊新鲜粪便中共鉴定出5个门、15个纲、32个目、45个科和56个属的真菌;从5头藏野驴新鲜粪便中共鉴定出3个门、10个纲、18个目、18个科和20个属的真菌。子囊菌门Ascomycota为优势门,相对多度占所有真菌门数的82.70%;寡囊盘菌属Thelebolus、Naganishia和亚隔孢壳属Didymella为优势属,3个属的相对多度依次占所有真菌属数的43.91%、7.38%和7.03%。藏羚羊和藏野驴粪便真菌菌群存在明显差异,其中34个属的多度在两种野生动物新鲜粪便中有显著差异,这说明动物的种类会影响其粪便中真菌多样性。     

Massive parallel 16S rRNA gene pyrosequencing reveals highly diverse fecal bacterial and fungal communities in healthy dogs and cats

This study evaluated the fecal microbiota of 12 healthy pet dogs and 12 pet cats using bacterial and fungal tag-encoded FLX-Titanium amplicon pyrosequencing. A total of 120,406 pyrosequencing reads for bacteria (mean 5017) and 5359 sequences (one pool each for dogs and cats) for fungi were analyzed. Additionally, group-specific 16S rRNA gene clone libraries for Bifidobacterium spp. and lactic acid-producing bacteria (LAB) were constructed. The most abundant bacterial phylum was Firmicutes, followed by Bacteroidetes in dogs and Actinobacteria in cats. The most prevalent bacterial class in dogs and cats was Clostridia, dominated by the genera Clostridium (clusters XIVa and XI) and Ruminococcus. At the genus level, 85 operational taxonomic units (OTUs) were identified in dogs and 113 OTUs in cats. Seventeen LAB and eight Bifidobacterium spp. were detected in canine feces. Ascomycota was the only fungal phylum detected in cats, while Ascomycota, Basidiomycota, Glomeromycota, and Zygomycota were identified in dogs. Nacaseomyces was the most abundant fungal genus in dogs; Saccharomyces and Aspergillus were predominant in cats. At the genus level, 33 different fungal OTUs were observed in dogs and 17 OTUs in cats. In conclusion, this study revealed a highly diverse bacterial and fungal microbiota in canine and feline feces.     

Community dynamics of Neocallimastigomycetes in the rumen of yak feeding on wheat straw revealed by different primer sets

Anaerobic fungi (Neocallimastigomycetes) play an important role in fermenting lignin-rich plant biomass into sugars in the rumen of animals, representing a very promising enzyme resource to contribute to the conversion of plant biomass into biofuels. However, current studies about their functions mainly focus on limited species, and little is known about the coordination of different members of the anaerobic fungi in the digestion process of plant fibres. In this study, the community composition of anaerobic fungi in the rumens of yaks at five different time points (1, 3, 5, 7.5 and 24 h after feeding wheat straw) was investigated employing a cultivation-independent method using ITS clone libraries. Comparison of five pairs of primers showed that PCR primer sets could have clear amplification bias and therefore potentially affect the interpretation of the resulting fungal community structure; then two primer sets GM1/MNGM2 and ITS1/ITS4 were selected. Among the 398 sequences from 10 clone libraries, 18 operational taxonomic units (OTUs) of Neocallimastigomycetes were obtained, covering five known genera and one yet uncultured lineage. OTUs belonging to the bulbous-type morphotype (Caecomyces- or Cyllamyces-related) and the rhizoidal genus Neocallimastix were abundant and predominantly present, representing 62.7% and 19.3% OTUs respectively. In all the later samples taken from 3 h to 24 Neocallimastigomycetes h after feeding, a relatively stable community composition was revealed: members of Neocallimastix increased to represent 43.4–49.4% and the bulbous-type morphotype declined to represent 39.5–42.7%. This implies a substantial turnover and synergy between bulbous and rhizoidal morphotypes of anaerobic fungi during the process of fibre digestion. Our study provided the first insight into the in vivo temporal change in the anaerobic fungal community, and the role of Neocallimastigomycetes with a bulbous morphotype in the degradation of plant cell wall in the yak rumen.     

Endophytic Fungal Communities Associated with Vascular Plants in the High Arctic Zone Are Highly Diverse and Host-Plant Specific

This study assessed the diversity and distribution of endophytic fungal communities associated with the leaves and stems of four vascular plant species in the High Arctic using 454 pyrosequencing with fungal-specific primers targeting the ITS region. Endophytic fungal communities showed high diversity. The 76,691 sequences obtained belonged to 250 operational taxonomic units (OTUs). Of these OTUs, 190 belonged to Ascomycota, 50 to Basidiomycota, 1 to Chytridiomycota, and 9 to unknown fungi. The dominant orders were Helotiales, Pleosporales, Capnodiales, and Tremellales, whereas the common known fungal genera were Cryptococcus, Rhizosphaera, Mycopappus, Melampsora, Tetracladium, Phaeosphaeria, Mrakia, Venturia, and Leptosphaeria. Both the climate and host-related factors might shape the fungal communities associated with the four Arctic plant species in this region. These results suggested the presence of an interesting endophytic fungal community and could improve our understanding of fungal evolution and ecology in the Arctic terrestrial ecosystems.