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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。
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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)。
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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。
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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)。
表 1 三种园林植物叶际真菌群落多样性
Table 1. Diversity index of phyllosphere fungi community of three garden plants
样品编号
Sample IDChao1指数
Chao1 indexShannon指数
Shannon indexSimpson指数
Simpson index覆盖度
CoverageF 292.00±74.19 5.07±0.60 0.92±0.03 100% M 494.11±55.76 5.71±0.47 0.94±0.02 100% E 307.22±44.16 4.34±0.64 0.89±0.06 100% -
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)。
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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)。
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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Entomophthoromycota和Mucoromycota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。
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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,Keissleriella和Vuilleminia只在广玉兰(M. grandiflora)叶际检出;Phoma和Setophoma只在枇杷树(E. japonica)叶际出现;Simplicillium和Mycosphaerella仅在大青树(F. altissima)叶际检测到。
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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)。
表 2 不同大小叶片在门、属水平上的真菌群落组成
Table 2. Composition of phyllosphere fungal community from different leaf sizes at phylum level and genus level
Catalogue Ficus altissima Magnolia grandiflora Eriobotyra japonic L M S L M S L M S Phylum Ascomycota 0.963832 0.958881 0.969214 0.882193 0.890337 0.825276 0.910942 0.889770 0.938937 Basidiomycota 0.017317 0.016171 0.012430 0.034859 0.061321 0.054382 0.068762 0.072620 0.047372 Chytridiomycota 0.000080 0.000046 0.000035 0.058275 0.007122 0.091171 0.000333 0.006716 0.00034 Mortierellomycota 0.000017 0.000008 0 0.000055 0.000117 0.000013 0.000118 0.000033 Mucoromycota 0 0 0 0 0.000078 0 0 0 0 Zoopagomycota 0 0 0 0 0 0 0 0.000023 0 Entomophthoromycota 0 0 0 0.000008 0 0 0 0 0 Unspecified 0.018754 0.024894 0.018320 0.024610 0.041023 0.029158 0.019962 0.030753 0.013312 Genus Cladosporium 0.182855 0.303213 0.240443 0.184304 0.088225 0.092448 0.467896 0.292873 0.409869 Strelitziana 0.047890 0.022240 0.038646 0.043668 0.117051 0.070280 0.006179 0.012768 0.005901 Retiarius 0.029152 0.036326 0.039059 0.042633 0.070749 0.077404 0.002163 0.001783 0.010680 Zeloasperisporium 0.050754 0.129906 0.043830 0.027356 0.009263 0.021889 0.003357 0.003917 0.003005 Aspergillus 0.003013 0.001145 0.001718 0.171493 0.029346 0.049223 0.000702 0.000513 0.000293 Knufia 0.016333 0.011554 0.045202 0.004604 0.008368 0.010223 0.000574 0.000955 0.000438 Taphrina 0.000262 0.000069 0.000228 0.001495 0.002825 0.002418 0.018447 0.022059 0.025894 Exobasidium 0.000085 0.000575 0.000076 0.007484 0.014354 0.011351 0.001375 0.001611 0.004426 Kwoniella 0.000505 0.000020 0.000082 0.005623 0.007982 0.003839 0.006655 0.006203 0.002094 Orbilia 0.000009 0 0.000014 0.004003 0.009800 0.011977 0.001566 0.003288 0.002312 Alternaria 0.001010 0.000962 0.000544 0.006303 0.004594 0.002938 0.004541 0.003955 0.005473 Clinoconidium 0.000241 0.001587 0.000277 0.006954 0.009827 0.004342 0.001675 0.000319 0.001551 Blumeria 0.003260 0.001368 0.000791 0.006296 0.007723 0.003930 0.000550 0.000282 0.000730 Aureobasidium 0 0.000011 0.000102 0 0 0 0.011369 0.006291 0.004209 Tremella 0.002129 0.000462 0.000723 0.002263 0.001457 0.000649 0.002307 0.001654 0.005478 Ramularia 0.000009 0 0.000012 0.004089 0.003170 0.004256 0.000462 0.000309 0.000680 Nigrospora 0.000285 0.000347 0.000084 0.005497 0.001517 0.001513 0.000580 0.000356 0.000419 Zasmidium 0.003061 0.004342 0.000927 0.000392 0.000426 0.000196 0 0 0 Rachicladosporium 0.000690 0.000349 0.000377 0.001927 0.001349 0.000769 0.000409 0.001019 0.001166 Unspecified 0.007055 0.005072 0.001658 0.008774 0.013279 0.014680 0.003401 0.005349 0.008291 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.
Phyllosphere Fungal Diversity and Community Structure of Three Garden Plants Based on High-throughput Sequencing
More Information-
摘要: 植物叶际真菌与植物健康息息相关,其群落结构和多样性是植物、微生物与其他环境因子相互作用的结果。为了解同一生境下,不同植物叶际真菌群落和多样性差异,确定不同植物对叶际真菌组成的影响,采用高通量测序技术,调查大理大学古城校区校园内50m范围内的大青树(Ficus altissima)、广玉兰(Magnolia grandiflora)和枇杷树(Eriobotyra japonica)三种园林植物叶际真菌群落结构组成。叶际真菌群落Alpha多样性由高到低依次为广玉兰(M. grandiflora)、枇杷树(E. japonica)和大青树(F. altissima)。三种园林植物叶际真菌群落注释阶元中,Ascomycota门,Cladosporium属皆为三种植物叶际真菌群落的优势类群,其相对丰富度略有不同。此外,不同植物也有其特有物种,三种植物叶际真菌群落组成存在明显差异。植物种类决定叶际真菌群落组成及其多样性,不同植物自身的特性会塑造其独特的叶际真菌群落。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.
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表 1 三种园林植物叶际真菌群落多样性
Tab. 1 Diversity index of phyllosphere fungi community of three garden plants
样品编号
Sample IDChao1指数
Chao1 indexShannon指数
Shannon indexSimpson指数
Simpson index覆盖度
CoverageF 292.00±74.19 5.07±0.60 0.92±0.03 100% M 494.11±55.76 5.71±0.47 0.94±0.02 100% E 307.22±44.16 4.34±0.64 0.89±0.06 100% 表 2 不同大小叶片在门、属水平上的真菌群落组成
Tab. 2 Composition of phyllosphere fungal community from different leaf sizes at phylum level and genus level
Catalogue Ficus altissima Magnolia grandiflora Eriobotyra japonic L M S L M S L M S Phylum Ascomycota 0.963832 0.958881 0.969214 0.882193 0.890337 0.825276 0.910942 0.889770 0.938937 Basidiomycota 0.017317 0.016171 0.012430 0.034859 0.061321 0.054382 0.068762 0.072620 0.047372 Chytridiomycota 0.000080 0.000046 0.000035 0.058275 0.007122 0.091171 0.000333 0.006716 0.00034 Mortierellomycota 0.000017 0.000008 0 0.000055 0.000117 0.000013 0.000118 0.000033 Mucoromycota 0 0 0 0 0.000078 0 0 0 0 Zoopagomycota 0 0 0 0 0 0 0 0.000023 0 Entomophthoromycota 0 0 0 0.000008 0 0 0 0 0 Unspecified 0.018754 0.024894 0.018320 0.024610 0.041023 0.029158 0.019962 0.030753 0.013312 Genus Cladosporium 0.182855 0.303213 0.240443 0.184304 0.088225 0.092448 0.467896 0.292873 0.409869 Strelitziana 0.047890 0.022240 0.038646 0.043668 0.117051 0.070280 0.006179 0.012768 0.005901 Retiarius 0.029152 0.036326 0.039059 0.042633 0.070749 0.077404 0.002163 0.001783 0.010680 Zeloasperisporium 0.050754 0.129906 0.043830 0.027356 0.009263 0.021889 0.003357 0.003917 0.003005 Aspergillus 0.003013 0.001145 0.001718 0.171493 0.029346 0.049223 0.000702 0.000513 0.000293 Knufia 0.016333 0.011554 0.045202 0.004604 0.008368 0.010223 0.000574 0.000955 0.000438 Taphrina 0.000262 0.000069 0.000228 0.001495 0.002825 0.002418 0.018447 0.022059 0.025894 Exobasidium 0.000085 0.000575 0.000076 0.007484 0.014354 0.011351 0.001375 0.001611 0.004426 Kwoniella 0.000505 0.000020 0.000082 0.005623 0.007982 0.003839 0.006655 0.006203 0.002094 Orbilia 0.000009 0 0.000014 0.004003 0.009800 0.011977 0.001566 0.003288 0.002312 Alternaria 0.001010 0.000962 0.000544 0.006303 0.004594 0.002938 0.004541 0.003955 0.005473 Clinoconidium 0.000241 0.001587 0.000277 0.006954 0.009827 0.004342 0.001675 0.000319 0.001551 Blumeria 0.003260 0.001368 0.000791 0.006296 0.007723 0.003930 0.000550 0.000282 0.000730 Aureobasidium 0 0.000011 0.000102 0 0 0 0.011369 0.006291 0.004209 Tremella 0.002129 0.000462 0.000723 0.002263 0.001457 0.000649 0.002307 0.001654 0.005478 Ramularia 0.000009 0 0.000012 0.004089 0.003170 0.004256 0.000462 0.000309 0.000680 Nigrospora 0.000285 0.000347 0.000084 0.005497 0.001517 0.001513 0.000580 0.000356 0.000419 Zasmidium 0.003061 0.004342 0.000927 0.000392 0.000426 0.000196 0 0 0 Rachicladosporium 0.000690 0.000349 0.000377 0.001927 0.001349 0.000769 0.000409 0.001019 0.001166 Unspecified 0.007055 0.005072 0.001658 0.008774 0.013279 0.014680 0.003401 0.005349 0.008291 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. -
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