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菌物学报, 2021, 40(7): 1605-1616 doi: 10.13346/j.mycosystema.210001

研究论文

藏羚羊和藏野驴粪便真菌多样性比较研究

商振达1,2, 孔庆辉1,3, 谭占坤1,2, 商鹏1,4, 王宏辉1,2, 刘锁珠,1,2,*

1.西藏农牧学院动物科学学院 西藏 林芝 860000

2.西藏高原饲料加工工程研究中心 西藏 林芝 860000

3.华中农业大学动物医学院 湖北 武汉 430070

4.藏猪协作研究中心 西藏 林芝 860000

Comparative study on fecal fungal diversity between Tibetan antelope and Tibetan wild ass

SHANG Zhen-Da1,2, KONG Qing-Hui1,3, TAN Zhan-Kun1,2, SHANG Peng1,4, WANG Hong-Hui1,2, LIU Suo-Zhu,1,2,*

1. College of Animal Science, Tibet Agricultural & Animal Husbandry University, Nyingchi, Tibet 860000, China

2. Tibetan Plateau Feed Processing Research Center, Nyingchi, Tibet 860000, China

3. College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei 430070, China

4. Tibetan Pig Collaborative Research Center, Nyingchi, Tibet 860000, China

责任编辑: 王敏

收稿日期: 2021-01-4   接受日期: 2021-05-28  

基金资助: 西藏自治区厅校联合基金-重点项目(XZ2019ZRG-55(Z))
西藏高原饲料加工工程研究中心项目(XZJYT2018GCZX)

Corresponding authors: * E-mail: L-szh@163.com

Received: 2021-01-4   Accepted: 2021-05-28  

Fund supported: Tibet Autonomous Region Office and School Joint Fund-Key Project(XZ2019ZRG-55(Z))
Tibetan Plateau Feed Processing Research Center Project(XZJYT2018GCZX)

摘要

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。

关键词： 藏羚羊; 藏野驴; 粪便真菌; ITS1区

Abstract

Fungal diversity and community structure in the feces of ruminants (Tibetan antelope) and monogastric herbivores (Tibetan wild ass) in Tibet are investigated by using ITS1 high through-put sequencing method. Five phyla, 15 classes, 32 orders, 45 families and 56 genera were discovered in feces of Tibetan antelopes. Three phyla, 10 classes, 18 orders, 18 families and 28 genera were discovered in feces of Tibetan wild asses. Ascomycota was the dominant phylum, and the relative abundance accounted for 82.70% of the total fungal phyla. Thelebolus, Naganishia and Didymella were the dominant genera, and the relative abundance accounted for 43.91%, 7.38% and 7.03% of the total fungal genera, respectively. The comparison of facal fungal community structure between Tibetan antelope and Tibetan wild ass by using Metastats showed that the fungal community composition in the feces of the two animal species was different, in which the relative abundance of 34 genera showed significant differences.

Keywords： Tibetan antelope; Tibetan wild ass; fecal fungal; ITS1 region

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本文引用格式

商振达, 孔庆辉, 谭占坤, 商鹏, 王宏辉, 刘锁珠. 藏羚羊和藏野驴粪便真菌多样性比较研究[J]. 菌物学报, 2021, 40(7): 1605-1616 doi:10.13346/j.mycosystema.210001

SHANG Zhen-Da, KONG Qing-Hui, TAN Zhan-Kun, SHANG Peng, WANG Hong-Hui, LIU Suo-Zhu. Comparative study on fecal fungal diversity between Tibetan antelope and Tibetan wild ass[J]. Mycosystema, 2021, 40(7): 1605-1616 doi:10.13346/j.mycosystema.210001

羌塘自然保护区是我国面积最大的国家级自然保护区,主要保护对象为保存完整的、独特的高寒生态系统及多种大型有蹄类动物 （Zhang et al. 2013）。生活在该区域的珍稀野生动物主要有藏羚羊Pantholops hodgsoni、藏野驴 Equus kiang和野牦牛Bos grunniens等青藏高原特有的国家级保护动物（Zhang & Jiang 2006;Chai et al. 2020）。近年来,由于野生动物栖息地遭到破坏,导致野生动物的种群数量逐渐减少（Harris et al. 1999）。因此,国内外越来越多的科研人开始关注和研究野生动物,但对西藏野生动物肠道微生物的研究相对较少。

动物的肠道微生物是一个庞大的、动态的系统。这些微生物依存于动物的肠道中,同时协助寄主完成多种生理生化功能（Wei et al. 2007;Yatsunenko et al. 2012）。肠道微生物不仅能够分泌多种物质,具有屏障病原体的功能,还可以影响肠道的发育,维持肠道的免疫系统,辅助肠道对各种营养物质进行消化和吸收（Macfarlane & Macfarlane 2007;Kim et al. 2012;Looft et al. 2012）。动物肠道中大部分微生物是细菌,但也存在一部分真菌。动物真菌的种类和数量受到宿主的饲粮类型、生活环境、品种以及生理阶段的影响（Liggenstoffer et al. 2010;Kittelmann et al. 2012;Sirohi et al. 2013）。真菌是动物胃肠道中主要的功能性厌氧微生物,具有强大的粗纤维降解能力,其能通过丰富的假根系统侵入植物的细胞壁中,可以产生纤维素酶、木质素酶和半纤维素酶等多种可以降解植物细胞壁的酶类,以分解纤维素等难于降解的物质,在提高草食类动物对粗饲料的利用率中具有重要意义（Joblin et al. 1989;Cheng et al. 2018）。

已有研究表明,动物的消化道组织结构不同会导致其肠道微生物不同。在对单胃草食动物和反刍动物肠道细菌菌群结构的研究中得到,厚壁菌门Firmicutes和拟杆菌门Bacteroidetes均为两类动物肠道菌群中最大的门,但反刍动物厚壁菌门的丰度显著高于单胃草食动物,且单胃草食动物拥有反刍动物没有的优势菌门（O’Donnell et al. 2017）,而对两类动物肠道真菌的对比研究较少。因此本研究采用ITS1区域测序方法对羌塘保护区中的藏羚羊和藏野驴的肠道真菌群落结构进行研究,为今后功能性优势菌株筛选提供理论依据,也为研究野生动物的消化机制做出贡献。

1 材料与方法

1.1 试验动物和样品采集

本研究以西藏羌塘自然保护区的藏羚羊和藏野驴为研究对象,该区域的平均海拔在5 000m以上;气候寒冷而干燥,空气稀薄,自然环境严酷。在保护区内四处地点（32°73ʹN,88°91ʹE,海拔4 969m;32°64ʹN,88°92ʹE,海拔4 878m;32°82ʹN,88°87ʹE,海拔4 952m;32°68ʹN,88°93ʹE,海拔4 903m）分别发现藏羚羊和藏野驴各2群（藏羚羊约10只、藏野驴约9只）。采用望远镜追踪观察野生动物的排粪,当动物离开后随机挑拣两种野生动物的新鲜粪样各5份,用灭菌牙签将粪便外层拨开,挑取里面没有污染的新鲜粪便装入20mL的冻存管中,并在试管表面进行标记:A组（A1、A2、A3、A4、A5）为5份藏羚羊粪样、B组（B1、B2、B3、B4、B5）为5份藏野驴粪样。所有粪样均放入车载冰箱中（-16℃）运回实验室进行粪便真菌检测。

1.2 粪便总DNA的提取和真菌ITS1区的扩增

使用QIAGEN公司的QIAamp Fast DNA Stool Mini试剂盒提取藏羚羊和藏野驴粪便的总DNA。通过紫外分光光度法和琼脂糖凝胶电泳对提取的DNA进行质量检测。将提取的DNA统一稀释到20ng/μL,不足的直接使用原液。以稀释后的DNA为模板,使用引物:ITS1f（5’-TCCGTAGGTGAACCTGCGG-3’）和ITS2（5’-TCCTCCGCTTATTGATATGC-3’）对真菌ITS1区进行PCR扩增。扩增体系（25μL）为:5×Reaction Buffer 5μL,5×GC缓冲液5μL,dNTP（2.5mmol/L）2μL,前引物（10μmol/L）1μL,后引物（10μmol/L）1μL,DNA模板2μL,ddH2O 8.75μL,Q5 DNA聚合酶0.25μL。扩增参数为:98℃预变性2min;98℃变性15s,55℃退火30s,72℃延伸30s,30个循环;72℃延伸5min;4℃保存。

1.3 扩增产物回收纯化和荧光定量

PCR扩增产物通过2%琼脂糖凝胶电泳进行检测,采用AXYGEN公司的凝胶回收试剂盒对目标片段进行切胶回收,参照电泳初步定量结果,利用Quant-iT PicoGreen dsDNA Assay Kit对扩增回收产物进行荧光定量。根据荧光定量结果,按照每个样本的测序量需求,对各样本按相应比例进行混合。

1.4 测序文库制备和高通量测序

采用Illumina公司的TruSeq Nano DNA LT Library Prep Kit制备测序文库,采用Agilent High Sensitivity DNA Kit对文库在Agilent Bioanalyzer上进行质检。采用Quant-iT PicoGreen dsDNA Assay Kit在Promega QuantiFluor荧光定量系统上对文库进行定量。将合格的各上机测序文库（Index序列不可重复）梯度稀释后,根据所需测序量按相应比例混合。通过琼脂糖凝胶电泳（2%）,对文库做最终的选择与纯化。利用Illumina MiSeq设备进行双端测序（上海派森诺生物科技股份有限公司）。

1.5 生物信息学和数据分析

本研究采用Illumina MiSeq平台对5份藏羚羊粪便和5份藏野驴粪便真菌的ITS1区域进行双端测序,对所获序列逐一作质量筛查,按照碱基平均测序准确率≥99%,序列长度≥150bp,且不允许存在模糊碱基的要求,剔除疑问序列。使用聚类程序VSEARCH（1.9.6）对获得的序列进行聚类,并以97%的序列相似性对OTU（operational taxonomic unit）进行归并划分（Bokulich et al. 2013）,通过与UNITE数据库的序列相比（Koljalg et al. 2013）,对OTU的代表序列进行分类地位鉴定,获取每个OTU所对应的分类学信息,将低于全体样本测序总量0.001%的OTU去除,并将去除了稀有OTU的多度矩阵用于后续分析;利用4种度量指数（Chao1、ACE、Shannon和Simpson）来计算真菌菌群的丰富度和均匀度;通过主成分分析（principal component analysis,PCA）考察不同样本之间群落结构的相似性（Ramette 2007）;使用IBM SPSS Statistics软件对试验数据进行单因素方差分析。

2 结果与分析

2.1 粪便真菌测序结果和各分类水平所获菌种数

本研究分别从藏羚羊和藏野驴中获得168 822条和194 063条有效序列,全部样本中序列的长度均分布于150-350bp之间。对每个样品可以鉴定为真菌的序列数进行抽平处理,按10个样品中最低样品的序列数34 214为依据,从每个样本中分别随机抽取一定数量的序列,使10个样品的序列数均达到34 214个,从而预测各样本在该测序深度下,所能观测到的OTUs及其相对丰度。藏羚羊和藏野驴各粪便样品在门、纲、目、科、属5种分类水平下所获得的OTUs数量见图1。藏羚羊中共鉴定出5个门、15个纲、32个目、45个科和56个属;藏野驴中共鉴定出3个门、10个纲、18个目、18个科和20个属。由各分类水平的真菌类群数统计结果可知,反刍动物（藏羚羊）粪便真菌在各分类水平的类群统计数均高于单胃草食动物（藏野驴）。由Venn图可知（图2）,藏羚羊和藏野驴粪便中共计获得731个OTUs,藏羚羊共有620个OTUs、藏野驴共有189个OTUs,藏羚羊和藏野驴的独有OTUs分别为542和111种,而共有OTUs为78种,独有OTUs数量均多于共有OTUs,表明藏羚羊和藏野驴粪便真菌菌群结构差异较大。

图1

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图1   藏羚羊与藏野驴各分类等级的真菌类群数目

同一分类水平下,藏羚羊和藏野驴之间的差异显著性用星号表示,*P<0.05,**P<0.01

Fig. 1   Number of fungal taxa in feces of Tibetan antelope (A) and Tibetan wild ass (B) at different taxonomic level.

Asterisks represent significant differences between Tibetan antelope and Tibetan wild ass at the same taxonomic level, *P<0.05, **P<0.01.


图2

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图2   藏羚羊和藏野驴粪便中真菌OTUs的Venn图

Fig. 2   Venn analysis of fecal fungal OTUs of Tibetan antelopes (A) and Tibetan wild asses (B).


2.2 藏羚羊和藏野驴粪便真菌菌群多样性分析

藏羚羊和藏野驴粪便真菌的Alpha多样性指数见表1。Simpson指数在藏羚羊和藏野驴中分别为0.88和0.27,二者差异显著（P<0.05）;Shannon指数在2个试验组中分别为4.89和0.97,二者差异显著（P<0.05）。Chao1指数在藏羚羊和藏野驴中分别为188.30和77.00,藏羚羊中Chao1指数高于藏野驴,但差异不显著（P>0.05）;ACE指数在2个试验组中分别为188.99和78.06,藏羚羊中ACE指数高于藏野驴,但差异不显著（P>0.05）。主成分分析结果表明藏羚羊和藏野驴粪便中真菌群落组成存在明显差异（图3）。

表1   藏羚羊和藏野驴粪便中真菌菌群多样性指数

Table 1  Fungal diversity indexes in feces of Tibetan antelopes and Tibetan wild asses

分组
Group		Simpson指数
Simpson index		Chao1指数
Chao1 index		ACE指数
ACE index		Shannon指数
Shannon index		OTUs数
OTUs number
藏羚羊
Tibetan antelope		0.88±0.09a188.30±112.72a188.99±112.33a4.89±1.02a153.00±81.26a
藏野驴
Tibetan wild ass		0.27±0.12b77.00±15.63a78.06±16.71a0.97±0.40b73.60±13.72a

注:同列数字上的不同字母表示差异显著（P<0.05）

Note: The different letters indicate significant difference (P<0.05).

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图3

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图3   藏羚羊和藏野驴粪便样品真菌群落PCA图

Fig. 3   PCA analysis of fecal fungus community of Tibetan antelope and Tibetan wild ass.


2.3 藏羚羊和藏野驴粪便真菌菌群组成分析

在门分类水平上,藏羚羊和藏野驴粪便样品中共鉴定出5个真菌门,其中子囊菌门和担子菌门分别占总真菌序列数的82.70%和13.17%,4.13%为其他菌门和未鉴定的真菌（表2）。藏羚羊粪便中子囊菌门的相对多度低于藏野驴,而担子菌门的相对多度高于藏野驴,但差异均不显著（P>0.05）。在纲和目分类水平上,藏羚羊和藏野驴粪便样品中共鉴定出15个纲和32个目,其中有5个纲和6个目的相对多度大于1.0%（表2）。与藏野驴相比,藏羚羊粪便中锤舌菌纲Leotiomycetes、银耳菌纲Tremellomycetes、寡囊盘菌目Thelebolales和线黑粉菌目Filobasidiales的相对多度显著降低（P<0.05）;而座囊菌纲Dothideomycetes、粪壳菌纲Sordariomycetes、微球黑粉菌纲Microbotryomycetes、格孢菌目Pleosporales、肉座菌目Hypocreales、煤炱菌目Capnodiales和柔膜菌目Helotiales的相对多度显著升高（P<0.05）。在科分类水平上,两种野生动物粪便样品中共鉴定出45个科（表2）。藏羚羊粪便中寡囊盘菌科Thelebolaceae和线黑粉菌科Filobasidiaceae的相对多度显著低于藏野驴（P<0.05）;而小双腔菌科Didymellaceae、葡萄穗霉科Stachybotryaceae、荚孢腔菌科Sporormiaceae和虫草科Cordycipitaceae的相对多度显著高于藏野驴（P<0.05）。

表2   藏羚羊和藏野驴粪便中不同真菌分类水平的相对多度

Table 2  The relative abundance of fungal community in feces of Tibetan antelopes and Tibetan wild asses at different taxonomic levels

分类水平
Taxonomic level		真菌
Fungus		分组Group
藏羚羊
Tibetan antelope		藏野驴
Tibetan wild ass
门
Phylum		子囊菌门 Ascomycota77.46±10.43a87.94±6.28a
担子菌门 Basidiomycota14.34±11.34a12.00±6.32a
被孢霉门 Mortierellomycota0.06±0.02b0.00±0.00a
丝足虫类 Cercozoa0.05±0.02b0.01±0.00a
毛霉门 Mucoromycota0.04±0.03b0.01±0.00a
纲
Class		锤舌菌纲 Leotiomycetes7.54±4.52a85.00±8.62b
座囊菌纲 Dothideomycetes40.80±10.26b1.60±1.44a
粪壳菌纲 Sordariomycetes17.52±12.77b0.38±0.21a
银耳菌纲 Tremellomycetes3.46±1.89a11.94±6.30b
微球黑粉菌纲 Microbotryomycetes8.80±6.95b0.00±0.00a
散囊菌纲 Eurotiomycetes0.94±0.64b0.06±0.04a
伞菌纲 Agaricomycetes0.54±0.36b0.01±0.00a
酵母菌纲 Saccharomycetes0.46±0.39b0.04±0.03a
马拉色菌纲 Malasseziomycetes0.33±0.06b0.01±0.00a
茶渍纲 Lecanoromycetes0.21±0.15b0.01±0.00a
沃勒曼菌纲 Wallemiomycetes0.19±0.12b0.01±0.00a
盘菌纲 Pezizomycetes0.06±0.03a0.11±0.07a
外担菌纲 Exobasidiomycetes0.17±0.16b0.01±0.00a
黑粉菌纲 Ustilaginomycetes0.10±0.07b0.02±0.01a
目
Order		寡囊盘菌目 Thelebolales3.28±2.25a84.54±8.52b
格孢菌目 Pleosporales35.06±7.57b1.58±1.40a
锁掷酵母目 Sporidiobolales14.76±9.09b0.00±0.00a
肉座菌目 Hypocreales14.50±11.95b0.10±0.07a
线黑粉菌目 Filobasidiales2.24±1.97a11.88±6.33b
煤炱菌目 Capnodiales3.78±3.10b0.00±0.00a
柔膜菌目 Helotiales3.00±1.35b0.16±0.11a
小丛壳目 Glomerellales1.68±0.62b0.01±0.00a
丝孢酵母目 Trichosporonales0.74±0.29b0.04±0.02a
白粉菌目 Erysiphales0.76±0.27b0.01±0.00a
粪壳菌目 Sordariales0.72±0.30b0.00±0.00a
禾本目 Poales0.62±0.31b0.00±0.00a
座囊菌纲未定目 Dothideomycetes ord. Incertae sedis0.60±0.53b0.00±0.00a
散囊菌目 Eurotiales0.47±0.45a0.06±0.03a
酵母菌目 Saccharomycetales0.46±0.44a0.04±0.02a
小囊菌目 Microascales0.44±0.32b0.01±0.00a
Coniochaetales0.20±0.09a0.19±0.16a
马拉色菌目 Malasseziales0.34±0.31a0.03±0.02a
刺盾炱目 Chaetothyriales0.15±0.02b0.00±0.00a
银耳菌目 Tremellales0.24±0.15b0.00±0.00a
Togniniales0.23±0.13b0.01±0.00a
座囊菌目 Dothideales0.22±0.13b0.00±0.00a
科
Family		寡囊盘菌科 Thelebolaceae3.28±2.25a84.54±8.52b
锁掷酵母科 Sporidiobolaceae14.76±9.09b0.00±0.00a
线黑粉菌科 Filobasidiaceae2.18±1.96a11.88±6.33b
小双腔菌科 Didymellaceae13.88±10.82b0.00±0.00a
葡萄穗霉科 Stachybotryaceae11.12±10.77b0.00±0.00a
荚孢腔菌科 Sporormiaceae3.18±2.64b0.40±0.31a
虫草科 Cordycipitaceae1.92±1.05b0.06±0.05a
织球壳科 Plectosphaerellaceae1.44±0.87b0.01±0.00a
蛹孢假壳科 Leptosphaeriaceae1.62±0.96b0.06±0.05a
畸球腔菌科 Teratosphaeriaceae1.10±0.53b0.00±0.00a
球腔菌科 Mycosphaerellaceae0.99±0.37b0.00±0.00a
枝孢菌科 Cladosporiaceae0.96±0.42b0.00±0.00a
丛赤壳科 Nectriaceae0.81±0.27b0.00±0.00a
丝孢酵母科Trichosporonaceae0.74±0.29b0.04±0.02a
白粉菌科 Erysiphaceae0.76±0.36b0.00±0.00a
禾本科 Poaceae0.62±0.31b0.00±0.00a
座囊菌纲未定科 Dothideomycetes fam. Incertae sedis0.60±0.52b0.00±0.00a
暗球壳科Phaeosphaeriaceae0.54±0.27b0.00±0.00a
煤炱科 Capnodiaceae0.49±0.30b0.00±0.00a
晶杯菌科 Hyaloscyphaceae0.46±0.34b0.00±0.00a
生赤壳科 Bionectriaceae0.44±0.28b0.00±0.00a
曲霉科 Aspergillaceae0.38±0.36a0.05±0.03a
毛孢壳科 Coniochaetaceae0.19±0.03a0.22±0.20a
格孢腔菌科 Pleosporaceae0.32±0.20b0.00±0.00a
毛壳菌科 Chaetomiaceae0.31±0.15b0.00±0.00a
Togniniaceae0.23±0.19b0.01±0.00a
黏毛菌科 Myxotrichaceae0.23±0.09b0.00±0.00a
属
Genus		寡囊盘菌属 Thelebolus3.28±2.25a84.54±8.52b
掷孢酵母属 Sporobolomyces14.76±9.09b0.00±0.00a
Naganishia2.18±1.96a11.88±6.33b
亚隔孢壳属 Didymella13.80±10.85b0.00±0.00a
Plenodomus1.33±0.61b0.00±0.00a
蜡蚧轮枝菌属 Lecanicillium1.27±0.15b0.04±0.02a
织球菌属 Plectosphaerella1.19±0.43b0.00±0.00a
石菌属 Lapidomyces1.10±0.52b0.00±0.00a
枝孢菌属 Cladosporium0.96±0.42b0.01±0.00a
多胞小球壳属 Sphaerulina0.80±0.24b0.00±0.00a
小粉孢属 Microidium0.76±0.36b0.01±0.00a
皮生丝孢酵母属 Cutaneotrichosporon0.71±0.30b0.04±0.02a
简青霉属 Simplicillium0.71±0.25b0.02±0.01a
漆斑菌属 Myrothecium0.73±0.30b0.00±0.00a
门座菌属 Thyrostroma0.60±0.52b0.00±0.00a
小角炱属 Antennariella0.49±0.26b0.00±0.00a
粉红螺旋聚孢霉属 Clonostachys0.44±0.28b0.00±0.00a
轮枝菌属 Verticillium0.41±0.22b0.01±0.00a
Cistella0.42±0.36b0.00±0.00a
新壳多孢属 Neostagonospora0.37±0.24b0.01±0.00a
腐质霉属 Humicola0.25±0.16b0.00±0.00a
暗色枝顶孢属 Phaeoacremonium0.23±0.11b0.01±0.00a
马拉色氏霉菌属 Malassezia0.20±0.19b0.01±0.00a
镰刀菌属 Fusarium0.20±0.10b0.00±0.00a
树粉孢属 Oidiodendron0.18±0.13b0.00±0.00a
黑团孢属 Periconia0.18±0.08b0.00±0.00a
支顶孢属 Acremonium0.18±0.08b0.00±0.00a
顶囊菌属 Claussenomyces0.14±0.13b0.00±0.00a
金孢属 Chrysosporium0.13±0.08b0.00±0.00a
青霉菌属 Penicillium0.12±0.10b0.01±0.00a
盘菌属 Peziza0.00±0.00a0.11±0.09b
黑粉菌属 Ustilago0.03±0.02b0.00±0.00a

注:同列数字上不同字母表示差异显著（P<0.05）

Note: The different letters in the same column indicate significant difference (P<0.05).

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比较分析藏羚羊和藏野驴在真菌属分类水平下的差异,两种野生动物共有34属真菌的相对多度存在显著性差异（P<0.05,图4）。其中,Antennariella、枝顶孢属Acremonium、黑团孢霉属Periconia、Plectosphaerella、丰屋菌属Plenodomus、漆斑菌属Myrothecium、Oidiodendron、金孢菌属Chrysosporium、Hypomyces、Clonostachys、多胞小球壳属Sphaerulina、Thyrostroma、镰刀菌属Fusarium、Lapidomyces、Termitomyces和Cistella只在藏羚羊中存在;盘菌属Peziza和Stagonospora只在藏野驴中存在。除此之外,藏野驴粪便中寡囊盘菌属和Naganishia的相对多度显著高于藏羚羊（P<0.05）;而蜡蚧轮枝菌属Lecanicillium、轮枝菌属Verticillium、Cutaneotrichosporon、亚隔孢壳属、简青霉菌属Simplicillium、Neostagonospora、枝孢菌属Cladosporium、黑粉菌属Ustilago、马拉色氏霉菌属Malassezia、微杆菌属Microidium、腐质霉菌属Humicola、Phaeoacremonium、Claussenomyces和青霉菌属Penicillium的相对多度显著低于藏羚羊（P<0.05）。

图4

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图4   藏羚羊和藏野驴的真菌属的相对多度比较

Fig. 4   Relative abundance of fungal genera in feces of Tibetan antelopes and Tibetan wild asses.


3 讨论

动物的粪便真菌作为一类新型功能性微生物,可以生产酶制剂、抗真菌剂、抗细菌剂等多种产品,在工业上具有重要的潜在应用价值（Weber & Gloer 1991;Soman et al. 1999;Farouq et al. 2013）。已有研究表明,动物真菌具有强大的降解粗纤维的能力,可以有效地提高动物对粗饲料的利用率,因此研究动物粪便真菌对开发新的微生物来源具有重要意义。因此本研究对同一生活环境中的反刍动物（藏羚羊）和单胃草食动物（藏野驴）的粪便真菌进行研究,可以为阐明动物消化方式对真菌菌群结构的影响奠定基础。

研究结果表明,藏羚羊和藏野驴粪便中有多种真菌分布,在藏羚羊粪便中共鉴定到56属,而在藏野驴粪便中共鉴定到20属,且藏羚羊粪便真菌的平均物种丰富度和真菌多样性均高于藏野驴,这与之前对反刍动物和兔形目动物粪便真菌多样性的研究结果相一致（Richardson 2001）。这可能与反刍动物主要依靠瘤胃对粗饲料进行微生物消化（Jiao et al. 2013）,而单胃草食动物肠道中缺少可以消化粗纤维的酶类,其主要依靠肠道内的微生物降解粗纤维（Jones 2013）,且动物粪便真菌会受消化道结构的影响有关。研究得到,锤舌菌纲、座囊菌纲、粪壳菌纲、银耳菌纲和微球黑粉菌纲为两种野生动物的主要优势菌群,这与对中国云南（Hu et al. 2008）、意大利（Caretta et al. 1994）、波多黎各（Richardson 2008）、福克兰群岛（Watling & Richardson 2010）、非洲（Ebersohn & Eicker 1992）等地的动物粪便真菌的研究结果不一致,这可能是由于西藏羌塘国家自然保护区高原低氧的特殊环境,以及粪便真菌受饲养环境的影响造成。

本研究还发现,藏羚羊与藏野驴粪便真菌菌群结构具有显著差异,藏羚羊粪便中有16个属在藏野驴中没有鉴定到,而藏野驴粪便中有2个属在藏羚羊粪便中没有鉴定到。除此之外藏野驴粪便中2种真菌的丰度高于藏羚羊,而12种真菌的丰度低于藏羚羊,由此表明反刍动物与单胃草食动物所利用的功能性真菌的种类不同。在对西藏羌塘地区牧草种类的调查中发现,该地区大部分面积生长着紫花针茅等植物,紫花针茅的草质较硬,粗纤维含量高,可能是藏羚羊和藏野驴等野生动物的主要牧草。已有研究表明,Necallimastix、Anaeromyces、Orpinomyces以及Piromyces（Freelove et al. 2001;James et al. 2006）等属真菌可以通过假根对植物组织的细胞壁进行分解,进而降解粗饲料中的粗纤维等物质;Caeocomyces和Cyllamyces（Chen et al. 2007）等属真菌可以通过黏附和破坏植物组织分解粗纤维。本研究在两种野生动物中均未鉴定到以上菌属,因此推断,藏羚羊和藏野驴胃肠道中可能有其他具有降解粗纤维功能的菌种,但具体菌种,有待进一步研究。Lecanicillium是害虫防治中的一种重要的生防真菌,寄主范围十分广泛,作为生防制剂已被商品化生产（Gurulingappa et al. 2011）;Simplicillium代谢产生的抗生素对金黄色葡萄球菌、枯草芽孢杆菌、藤黄微球菌等革兰氏阳性菌具有较强的抑制作用,而对大肠杆菌和鼠伤寒沙门氏菌等革兰氏阴性菌无抑制作用（Chen et al. 2008;Dong et al. 2014）;研究得到,藏羚羊和藏野驴粪便中均有Lecanicillium和Simplicillium等多种有益真菌,但有益真菌在两种野生动物中的丰度明显不同,这可能说明动物的免疫机制也与动物种类有关。除此之外,本研究还在两种野生动物粪便中鉴定到黑团孢霉属、轮枝菌属、Cutaneotrichosporon、黑粉菌属、Malassezia、腐质霉菌属和青霉菌属等多种致病菌,由此推断,即使是生活于高原低氧的环境下,藏羚羊和藏野驴等野生动物在生长过程中也会滋生各种条件性致病真菌。

综上所述,本研究对藏羚羊和藏野驴粪便真菌菌群多样性进行测定和分析,既可以为探究动物消化结构对粪便真菌菌群结构的影响和挖掘功能性真菌奠定基础,又可以为进一步揭示青藏高原野生动物源真菌的分布和鉴定提供一定的理论依据和技术方法。

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