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YUAN C L, ZHANG C Z, DENG W, et al. Phyllosphere fungal diversity and community structure of three garden plants based on high-throughput sequencing[J]. Journal of Sichuan Forestry Science and Technology, 2023, 44(1): 24−31 doi: 10.12172/202203080001
Citation: YUAN C L, ZHANG C Z, DENG W, et al. Phyllosphere fungal diversity and community structure of three garden plants based on high-throughput sequencing[J]. Journal of Sichuan Forestry Science and Technology, 2023, 44(1): 24−31 doi: 10.12172/202203080001

Phyllosphere Fungal Diversity and Community Structure of Three Garden Plants Based on High-throughput Sequencing


doi: 10.12172/202203080001
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  • Corresponding author: yangxy@eastern-himalaya.cn
  • Received Date: 2022-03-08
    Available Online: 2023-01-11
  • Publish Date: 2023-02-25
  • The plant health is closely related to phyllosphere fungi, and their community structure and diversity are determined by the mutual activities of plant itself, the microorganisms and other environmental factors. In order to understand the differences of the community structure and diversity of the phyllosphere fungi which comes from different plants in the same habitat, and to determine the influences of different plants on the composition of phyllosphere fungi. The next generation sequencing method were applied to investigate the phyllosphere fungi community structures from three garden plants, including Ficus altissima, Magnolia grandiflora, Eriobotyra japonica, which were located within 50 meters in distance and around the campus of Dali University. The results showed that the alpha diversity of phyllosphere fungi was ranked as M. grandiflora > E. japonica > F. altissima. For the OTU abundance, the genus Cladosporium from phylum Ascomycota was the dominant taxon in all of the three plant phyllosphere fungi communities but the relative abundance was different. In addition, fungus communities from different plants had various endemic taxa, and the assemblies were distinct. The community structures and diversity of plant phyllosphere fungi were determined by the plant species, and the characteristics of different plants could shape their unique phyllosphere fungus communities.
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Phyllosphere Fungal Diversity and Community Structure of Three Garden Plants Based on High-throughput Sequencing

doi: 10.12172/202203080001
  • 1. Institute of Eastern-Himalaya Biodiversity Research, Dali University, Dali 671003, China
  • 2. Key Laboratory of Yunnan State Education Department on Er’hai Lake Basin Protection and the Sustainable Development Research, Dali 671003, China
  • 3. Provincial Innovation Team of Biodiversity Conservation and Utility of the Three Parallel Rivers Region from Dali University, Dali, 671003, China
  • 4. Collaborative Innovation Center for Biodiversity and Conservation in the Three Parallel Rivers Region of China, Dali, 671003, China
  • Corresponding author: yangxy@eastern-himalaya.cn

Abstract: The plant health is closely related to phyllosphere fungi, and their community structure and diversity are determined by the mutual activities of plant itself, the microorganisms and other environmental factors. In order to understand the differences of the community structure and diversity of the phyllosphere fungi which comes from different plants in the same habitat, and to determine the influences of different plants on the composition of phyllosphere fungi. The next generation sequencing method were applied to investigate the phyllosphere fungi community structures from three garden plants, including Ficus altissima, Magnolia grandiflora, Eriobotyra japonica, which were located within 50 meters in distance and around the campus of Dali University. The results showed that the alpha diversity of phyllosphere fungi was ranked as M. grandiflora > E. japonica > F. altissima. For the OTU abundance, the genus Cladosporium from phylum Ascomycota was the dominant taxon in all of the three plant phyllosphere fungi communities but the relative abundance was different. In addition, fungus communities from different plants had various endemic taxa, and the assemblies were distinct. The community structures and diversity of plant phyllosphere fungi were determined by the plant species, and the characteristics of different plants could shape their unique phyllosphere fungus communities.

  • 叶际(Phyllosphere)指包括叶片在内的植物的空中表面部分,它是地球上最大的微生物栖息地之一,在其上生存和定殖的微生物称为叶际微生物[1-4]。叶际微生物中不仅有阻碍植物生长发育、危害植物健康的病原微生物[5],还有相当一部分非病原微生物,它们在促进植物生长[6]、降低植物病虫害[7]、分解植物体内残留农药[8]等方面发挥着重要作用,具有非常重要的生态学功能[7]。叶际微生物多样性高、群落结构复杂、生物量大[9, 10],与植物的相互作用及其生态学功能是植物学和微生物学交叉学科的一个新的研究领域[11],但相关研究尚未得到足够重视,远远落后于根际微生物的研究[5, 12]。仅有少量的研究关注到了叶际微生物对植物抗性的刺激[11]、对植物病原的防治[12]以及对植物呼吸的调节[13]等作用,尚不足以支撑叶际微生物与植物互作关系和叶际微生物功能的认识。因此,全面认识不同植物叶际微生物群落结构差异及其驱动机制是了解植物与叶际微生物互作关系的基础。

    真菌作为植物叶际微生物中一个十分重要的类群,在植物生长过程中发挥着不可或缺的作用[14]。一方面,许多叶际真菌是主要的植物病原菌[10],它们所形成的病害已经成为农作物、经济作物、高产作物稳产和优质的主要障碍;另一方面,叶际真菌对植物生长发育也有着积极影响,在降低植物病害方面发挥着重要作用[15]。此外,叶际真菌还可以影响植物的固氮作用,植物病原体的自然控制以及对有机污染物的生物修复[16]。目前大部分研究都以染病植株叶片的真菌群落结构为主,对不同植物健康叶片的叶际真菌的群落结构组成研究很少。因此,了解不同植物叶际真菌群落结构不仅有助于理解叶际微生物与植物的相互关系,对植物真菌病害预防以及植物病原菌拮抗菌的开发应用也至关重要。

    目前实验中使用的纯培养方法存在可培养微生物少、分析精度低等缺点[17],而利用高通量测序技术可以有效地避免培养方法造成的微生物多样性丢失,能够更有效更直接地反映环境中微生物的原始组成[18, 19]。同时,植物叶际微生物受地理位置、季节、降水、光照及其他环境因素的影响[20],要了解不同植物叶际微生物群落结构差异,就需要在较小空间尺度下采集样本。因此本研究选取了三种在50 m范围内分布的园林植物不同大小的叶片,采用高通量测序技术探究其真菌群落组成,比较三种园林植物叶片真菌多样性,了解不同植物间叶际真菌的多样性及结构差异。

    • 以大理大学古城校区(25°40′19′′ N,100°9′17′′ E)最大的一棵大青树(Ficus altissima)为中心,在其周围50 m半径内选择大青树(F. altissima)、广玉兰(Magnolia grandiflora)、枇杷树(Eriobotyra japonica)各一棵,使用无菌剪刀和镊子分别采集朝向相同(均为背阴面)的叶片。叶片分为大中小三个等级(长度≥15 cm:大;长度10—15 cm:中;长度5—10 cm:小),不同等级叶片各采3片,共采集27份叶片样本。叶片剪下后置于无菌自封袋中,贴上标签后立即送至实验室对样品进行处理。

    • 分别向装有各样品的自封袋中加入25 mL PBS缓冲液(pH值=8),置于超声波清洗机中洗涤1 min,涡旋10 s,将清洗液倒入50 mL离心管中。重复洗涤1次,2次洗涤液合并至50 ml离心管中,13000 r·min−1离心10 min,收集各叶片的离心沉淀,干冰保存,立即送样至深圳微科盟科技集团有限公司进行测序分析。

    • 根据DNA试剂盒Power Soil® DNA Isolation Kit (Omega Bio-tek, Norcross, GA, U.S.)说明书进行样品总DNA提取,按生产商提供的步骤快速提取内生真菌基因组DNA,利用NanoDrop2000检测DNA提取浓度和纯度,利用2 %琼脂糖凝胶电泳检测DNA提取质量。

    • 序列扩增及测序将提取的总DNA作为模板,以ITS1-1F-F (5′-CTTGGTCATTTAGAGGAAGTAA-3′), ITS-1F-R (5′-GCTGCGTTCTTCATCGATGC-3′) 为引物对真菌ITS1区进行PCR扩增。PCR反应条件为:98° C初始变性1 min,98°C变性30次,10 s,引物在50 °C退火30 s,72° C延伸30 s。在72 °C处添加5 min的延伸步骤,以确保目标区域的完全扩增。PCR产物从2 %琼脂糖凝胶中提取,并使用AxyPrepDNA凝胶提取试剂盒。PCR扩增及测序工作由深圳微科盟科技集团有限公司完成,测序平台为NovaSeq 6000 PE250平台。

    • 使用2 %琼脂糖凝胶回收PCR产物,利用AxyPrep DNA Gel Extraction Kit(Axygen Biosciences, Union City, CA, USA)对PCR产物进行纯化,Tris-HCl洗脱,2 %琼脂糖电泳检测。使用Illumina公司TruSeqDNA PCR-Free Library Preparation Kit(Axygen Biosciences, Union City, CA, USA)建库试剂盒进行文库的构建,构建好的文库经过Qubit定量和文库检测,合格后,使用NovaSeq 6000 PE250平台上机测序。

    • 运用Qiime2 dada2插件对测序结果序列进行质控,修剪,去噪,拼接,以及去除嵌合体后,得到了最终的ASV表格[21]。接着将ASV的代表序列比对到Greengenes数据库得到物种的分类信息表[22]。之后用Qiime2 feature-table插件剔除了所有非真菌序列,再使用Qiime2 Core-Diversity[23]进行Venn图绘制、Alpha多样性、Beta多样性等分析。

    2.   结果与分析
    • 所有叶片样品的真菌共获得2184791条高质量序列,平均每个叶片样品检测到80918条序列。大青树(F. altissima)叶片、广玉兰(M. grandiflora)叶片和枇杷树(E. japonica)叶片所获得的序列总数分别为691459条、764235条和729097条,各处理样品文库覆盖率均达到100 %。随机抽取检测样本序列,将抽取的序列数与其代表的ASV数绘制稀释曲线(Rarefaction curve)。随着序列数的增加,曲线逐渐趋于平坦,并在40000条序列时达到平台期,继续增加检测样本没有产生更多新的ASV,说明本研究测序深度是合适的(见图1)。

      Figure 1.  Rarefaction curve

    • 通过将ASV的代表序列与微生物参考数据库进行比对注释,得到每个ASV对应的物种分类信息,共注释到8门,33纲,97目,224科,362属,395种。三种园林植物叶际真菌群落中注释到各阶元的比例如图2所示。从图中可以看出,广玉兰(M. grandiflora)注释的比例最高(门到种的注释比例顺序依次为75 %,84.38 %,81.52 %,72.73 %,55.85 %,13.40 %),其次为枇杷树(E. japonica),大青树(F. altissima)注释比例最低。

      Figure 2.  Species-annotable proportion of phyllosphere fungal communities of three garden plants in each order.

    • 三种园林植物叶际真菌群落Alpha多样性分析结果如表1所示。Chao1指数数值由高到低依次是广玉兰(M. grandiflora),枇杷树(E. japonica),大青树(F. altissima),Shannon指数和Simpson指数数值依次是广玉兰(M. grandiflora),大青树(F. altissima),枇杷树(E. japonica)。综合来看,真菌群落多样性最高的是广玉兰(M. grandiflora),其次是枇杷树(E. japonica),最低的为大青树(F. altissima)。

      样品编号
      Sample ID
      Chao1指数
      Chao1 index
      Shannon指数
      Shannon index
      Simpson指数
      Simpson index
      覆盖度
      Coverage
      F292.00±74.195.07±0.600.92±0.03100%
      M494.11±55.765.71±0.470.94±0.02100%
      E307.22±44.164.34±0.640.89±0.06100%

      Table 1.  Diversity index of phyllosphere fungi community of three garden plants

    • 三种植物的共有类群为256种,广玉兰(M. grandiflora)、枇杷树(E. japonica)和大青树(F. altissima)的特有类群依次为1739种、989种,893种。三种植物叶际真菌类群组成存在明显差异,群落结构也较复杂(见图3)。

      Figure 3.  Species composition difference of phyllosphere fungal community of three garden plants

    • 基于Beta多样性分析的PCoA真菌群落分析结果显示,同一植物叶片聚集在一起,不同植物分布于不同象限。大青树(F. altissima)与枇杷树(E. japonica)样品的叶际真菌群落较为相似;而广玉兰(M. grandiflora)则与大青树(F. altissima)和枇杷树(E. japonica)的叶际真菌群落差异较大(见图4)。

      Figure 4.  PCoA analysis chart of thphyllosphere fungi of three garden plants

    • 不同植物叶际真菌群落在门水平存在明显不同(见图5):3种植物叶片上共检测出8个真菌菌门,其共有优势门均为Ascomycota,其相对丰度均大于85 %,Chytridiomycota在广玉兰(M. grandiflora)、枇杷树(E. japonica)和大青树(F. altissima)的占比依次为46.97 %、2.22 %和0.049 %;而EntomophthoromycotaMucoromycota仅在广玉兰(M. grandiflora)叶际真菌群落中被检测到,Zoopagomycota只在枇杷树(E. japonica)叶际真菌群落被检测到。

      Figure 5.  Composition of phyllosphere fungal communities of three garden plants at phylum level

    • 不同植物叶际真菌群落在属水平同样存在明显不同(见图6):3种植物叶片上共检测出362属真菌,相对丰度排名前五的属为Cladosporium, Strelitziana, Retiarius, Zeloasperisporium, Aspergillus; Cladosporium在枇杷树(E. japonica)、大青树(F. altissima)和广玉兰(M. grandiflora)叶际真菌群落中的含量依次为39.02 %、24.22 %和12.17 %;此外,KeissleriellaVuilleminia只在广玉兰(M. grandiflora)叶际检出;PhomaSetophoma只在枇杷树(E. japonica)叶际出现;SimplicilliumMycosphaerella仅在大青树(F. altissima)叶际检测到。

      Figure 6.  Composition of phyllosphere fungal communities of three garden plants at genus level

    • 总的来说,同一植物不同大小叶片面积之间的叶际真菌组成相同,含量的相对丰度差异不大(见表2)。

      CatalogueFicus altissimaMagnolia grandifloraEriobotyra japonic
      LMSLMSLMS
      PhylumAscomycota0.9638320.9588810.9692140.8821930.8903370.8252760.9109420.8897700.938937
      Basidiomycota0.0173170.0161710.0124300.0348590.0613210.0543820.0687620.0726200.047372
      Chytridiomycota0.0000800.0000460.0000350.0582750.0071220.0911710.0003330.0067160.00034
      Mortierellomycota0.0000170.00000800.0000550.0001170.0000130.0001180.000033
      Mucoromycota00000.0000780000
      Zoopagomycota00000000.0000230
      Entomophthoromycota0000.00000800000
      Unspecified0.0187540.0248940.0183200.0246100.0410230.0291580.0199620.0307530.013312
      GenusCladosporium0.1828550.3032130.2404430.1843040.0882250.0924480.4678960.2928730.409869
      Strelitziana0.0478900.0222400.0386460.0436680.1170510.0702800.0061790.0127680.005901
      Retiarius0.0291520.0363260.0390590.0426330.0707490.0774040.0021630.0017830.010680
      Zeloasperisporium0.0507540.1299060.0438300.0273560.0092630.0218890.0033570.0039170.003005
      Aspergillus0.0030130.0011450.0017180.1714930.0293460.0492230.0007020.0005130.000293
      Knufia0.0163330.0115540.0452020.0046040.0083680.0102230.0005740.0009550.000438
      Taphrina0.0002620.0000690.0002280.0014950.0028250.0024180.0184470.0220590.025894
      Exobasidium0.0000850.0005750.0000760.0074840.0143540.0113510.0013750.0016110.004426
      Kwoniella0.0005050.0000200.0000820.0056230.0079820.0038390.0066550.0062030.002094
      Orbilia0.00000900.0000140.0040030.0098000.0119770.0015660.0032880.002312
      Alternaria0.0010100.0009620.0005440.0063030.0045940.0029380.0045410.0039550.005473
      Clinoconidium0.0002410.0015870.0002770.0069540.0098270.0043420.0016750.0003190.001551
      Blumeria0.0032600.0013680.0007910.0062960.0077230.0039300.0005500.0002820.000730
      Aureobasidium00.0000110.0001020000.0113690.0062910.004209
      Tremella0.0021290.0004620.0007230.0022630.0014570.0006490.0023070.0016540.005478
      Ramularia0.00000900.0000120.0040890.0031700.0042560.0004620.0003090.000680
      Nigrospora0.0002850.0003470.0000840.0054970.0015170.0015130.0005800.0003560.000419
      Zasmidium0.0030610.0043420.0009270.0003920.0004260.000196000
      Rachicladosporium0.0006900.0003490.0003770.0019270.0013490.0007690.0004090.0010190.001166
      Unspecified0.0070550.0050720.0016580.0087740.0132790.0146800.0034010.0053490.008291
        注: L,M,S分别表示叶片面积大小为大,中,小等级。
        Note: L, M and S indicate that the leaf area size is large, medium and small respectively.

      Table 2.  Composition of phyllosphere fungal community from different leaf sizes at phylum level and genus level

    3.   讨论与结论
    • 为了排除地理位置、季节、空间距离等环境因素对研究结果的影响,以大理大学校园50 m范围内同一朝向的三种园林植物叶片为研究对象,比较不同植物种类叶际真菌群落结构和多样性的差异,聚焦植物种类对叶际真菌群落组成的影响。结果显示,同一植物不同大小叶片面积之间的叶际真菌组成和含量的相对丰度差异不大,三种园林植物叶际真菌的群落结构和多样性确实是存在差异的,这说明植物自身的特性会塑造不同的真菌群落。

      已有研究表明,植物叶片蜡质层厚度的不同会影响微生物的吸附能力,尤其是对真菌孢子的吸附,蜡质层越厚的叶片其表面吸附的真菌孢子就越少,蜡质层越薄的叶片其表面吸附的真菌孢子就越多[24]。本研究所选取的植物中,广玉兰叶片蜡质层最厚。此外,广玉兰(M. grandiflora)和枇杷树(E. japonica)叶片背面都覆有绒毛,广玉兰(M. grandiflora)叶片的绒毛更加小而密;大青树(F. altissima)叶片没有绒毛,说明三种植物叶片的形态结构的差异可能是造成其叶际真菌群落差异的主要原因。其次,大青树(F. altissima)属于落叶植物,而广玉兰(M. grandiflora)和枇杷树(E. japonica)属于常绿植物;随着植物叶片的生长,叶际微生物会不断累积,不同生长时间的叶片微生物累积量也会有所不同,因此叶片的生长周期也可能造成这种差异。基于此可以确定植物种类决定叶际真菌群落组成及其多样性。

    • 选取的三种园林植物的优势菌门为Ascomycota,优势菌属是Cladosporium,与以往相关研究结果一致[25],这说明Cladosporium属真菌对植物叶际环境的适应性和传播能力最强[26],是很多高等植物叶际的优势菌群。Cladosporium属这种较强的适应及传播特性可能为我们相关叶际真菌资源的开发和利用提供导向。除优势属Cladosporium外,AspergillusRamularia也是三种园林植物的共有真菌。此外,广玉兰(M. grandiflora)特有Vuilleminia,枇杷树(E. japonica)特有Phoma,大青树(F. altissima)特有Mycosphaerella等真菌,这些特有真菌中一部分是对植物有害的,它们可以使植物患病,同时一部分的特有真菌又能分泌或产生拮抗物质来抑制疾病。植物叶际大量的特有真菌说明了未来要针对植物种类开展群落结构研究,进而对其进行资源开发和利用。

    • 已有研究发现一些叶际真菌群落通过刺激植物抗性,加强抗植物病原体的生物抵抗作用来促进植物健康[11],但是有益微生物在叶际中的作用仅被部分了解,进一步探究植物和叶际微生物相互作用的关系可能鼓励开发新的植物保护方法。

      三种植物叶际的真菌资源极其丰富,但其注释比例极小,在种水平上的注释度仅为10 %左右。一方面,这可能是高通量测序技术在物种鉴定中所存在的局限性所致。高通量测序技术中的物种鉴定是基于已有微生物基因数据库,而已有微生物基因数据库中的基因与物种的关联信息是基于对微生物纯培养物所得,但到目前为止,自然界中能够被培养的微生物只有1 %[27, 28]。另一方面,这也表明叶际中确实有极大的真菌资源有待研究和开发。因此,在未来的研究中,要加大纯培养技术的研究进程,为微生物基因库提供更多的数据,丰富高通量技术的可注释比例。此外,也要加大对叶际真菌群落的研究力度,叶际真菌中有极为丰富的资源和极大的研究价值和潜力,可能会成为继根际微生物后又一研究热点问题。

      致谢:感谢云岭滇金丝猴云南省野外观测研究站为本研究顺利实施提供的支持;感谢大理大学东喜玛拉雅研究院肖文研究员和刘硕然副教授为本文撰写提供的建议。

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