高寒草甸优势禾草-异针茅内生真菌的分离与鉴定

doi:10.11686/cyxb2018470

摘要/Abstract

摘要： 异针茅作为青藏高原地区重要的乡土草种之一,是高寒草甸草原主要的建群禾草种类。迄今为止,有关异针茅所感染内生真菌的种类和分类地位的研究尚未见报道。基于此,以青海高原地区两个地理种群的异针茅为研究对象,采用常规分离培养形态观测和管家基因扩增目的片段基因并与已公布的内生真菌序列构建系统发育树的方法,确定异针茅感染内生真菌的种类和分类地位。结果表明:异针茅分离出的内生真菌菌落形态、生长速度和分生孢子形态等均与Epichloё属内生真菌的形态特征相似。编码蛋白tub和肌动蛋白actin序列构建的系统发育树表明,异针茅感染的内生真菌种类分别为Epichloё gansuensis和Epichloё sylvatica,而转录延长因子tef序列构建的系统发育树发现异针茅感染的内生真菌种类分别为Epichloё inebrians和E. sylvatica。由此可见,异针茅感染的内生真菌分别为甘肃内生真菌E. gansuensis、醉马草内生真菌E. inebrians和欧洲短柄草内生真菌E. sylvatica;进一步揭示异针茅感染的内生真菌表现出多样性,异针茅与其感染的内生真菌间未形成严格的宿主特异性。

关键词: 异针茅, 青海高原, 内生真菌, 系统进化, 多样性, 宿主特异性

Abstract: Stipa aliena is one of the important native plant species and a dominant grass species in alpine meadows of the Qinghai-Tibet Plateau. There has been little study of the endophyte(s) of S. aliena; therefore, in order to determine colonisation morphology and taxonomic status of the endophytes of this grass, samples of S. aliena were isolated from two different geographical populations, and the morphological features of endophyte strains found were observed. In addition, three housekeeper genes (tub, actin, tef) were selected for amplification of specific gene fragments in order to construct phylogenetic trees for each housekeeper gene for the endophyte species recovered. The endophyte morphological traits, such as colonisation morphology, hyphal growth rate, spore morphology and size were consistent with the recovered endophytes being Epichloё sp. Phylogenetic trees for tub and actin indicated that endophytes isolated from S. aliena had a close relationship with Epichloё gansuensis and Epichloё sylvatica; however, phylogenetic trees for tef showed that endophytes isolated from S. aliena were closely related to E. gansuensis and E. inebrians. Therefore, the endophytes of S. aliena were preliminarily identified as E. gansuensis from China (Xinjiang), E. inebrians from China (Gansu) and E. sylvatica from Europe. Our results suggest that the host-specificity might not be strongly developed in endophytes infecting S. aliena, and that different Epichloё species have independently developed a symbiotic relationship with the host grass S. aliena.

Key words: Stipa aliena, Qinghai plateau, Epichloë, systematic evolution, diversity, host specificity

鲍根生, 俞永飞, 李春杰. 高寒草甸优势禾草-异针茅内生真菌的分离与鉴定[J]. 草业学报, 2019, 28(8): 150-160.

BAO Gen-sheng, YU Yong-fei, LI Chun-jie. Isolation and identification of Epichloё sp. infecting Stipa aliena in alpine meadows of the Qinghai-Tibet Plateau[J]. Acta Prataculturae Sinica, 2019, 28(8): 150-160.

参考文献

[1] Han T H, Zhao Z, Wang A L, et al. Studies community composition and productivity in eastern edge of Qinghai-Tibetan Stipa alienagrassland. Acta Prataculturae Sinica, 2007, 16(6): 62-66.
韩天虎, 赵忠, 王安禄, 等. 青藏高原东缘异针茅草地群落组成及生产力研究. 草业学报, 2007, 16(6): 62-66.
[2] Yao T D, Chen F H, Cui P, et al. From Tibetan Plateau to third pole an pan-third pole. Bulletin of Chinese Academy of Sciences, 2017, 32(9): 924-931.
姚檀栋, 陈发虎, 崔鹏, 等. 从青藏高原到第三极和泛第三极. 中国科学院院刊, 2017, 32(9): 924-931.
[3] He T, Wu X M, Jia J F.Research advances in morphology and anatomy of alpine plants growing in the Qinghai-Tibet Plateau and their adaptations to environments. Acta Ecologica Sinica, 2007, 27(6): 2574-2583.
何涛, 吴学明, 贾敬芬. 青藏高原高山植物的形态和解剖结构及其对环境的适应性研究进展. 生态学报, 2007, 27(6): 2574-2583.
[4] Yang Y Q, Li X, Kong X X, et al. Transcriptome analysis reveals diversified adaptation of Stipa purpurea along a drought gradient on the Tibetan Plateau. Functional & Integrative Genomics, 2015, 15(3): 295-307.
[5] Schlaeppi K, Bulgarelli D.The plant microbiome at work. Molecular Plant Microbe Interact, 2015, 28(3): 212-217.
[6] De Z A, Alyousif M, Hirt H.Rhizosphere microbes as essential partners for plant stress tolerance. Molecular Plant, 2013, 6(2): 242-245.
[7] Vandenkoornhuyse P, Quaiser A, Duhamel M, et al. The importance of the microbiome of the plant holobiont. New Phytologist, 2015, 206(4): 1196-1206.
[8] Saikkonen K, Saari S, Helander M.Defensive mutualism between plants and endophytic fungi? Fungal Diversity, 2010, 41(1): 101-113.
[9] Sun H Q.Biodiversity analysis of Gramineae plants in Qinghai Province, China. Pratacultural Science, 2002, 19(2): 7-12.
孙海群. 青海禾本科植物的生物多样性分析. 草业科学, 2002, 19(2): 7-12.
[10] Bao G S, Zhang X X, Li X Z, et al. Incidence and isolation of endophyte in native grasses of Qinghai Plateau. Pratacultural Science, 2015, 32(12): 1997-2007.
鲍根生, 张兴旭, 李秀璋, 等. 青海高原禾草内生真菌资源的调查和分离. 草业科学, 2015, 32(12): 1997-2007.
[11] Clay K.Fungal endophytes of grasses. Current Opinion in Plant Biology, 1990, 21(4): 275-297.
[12] Leuchtmann A, Bacon C W, Schardl C L, et al. Nomenclatural realignment of Neotyphodium species with genus Epichloё. Mycologia, 2014, 106(2): 202-215.
[13] Saikkonen K, Young C A, Helander M, et al. Endophytic Epichloё species and their grass hosts: From evolution to applications. Plant Molecular Biology, 2016, 90(6): 665-675.
[14] Nan Z B, Li C J.Roles of the grass-Neotyphodium association in pastoral agriculture systems. Acta Ecologica Sinica, 2004, 24(3): 605-616.
南志标, 李春杰. 禾草-内生真菌共生体在草地农业系统中的作用. 生态学报, 2004, 24(3): 605-616.
[15] Song H, Nan Z B, Song Q Y, et al. Advances in research on Epichloё endophytes in Chinese native grasses. Frontiers in Microbiology, 2016, 7: 1399.
[16] Nan Z B.Incidence and distribution of endophytic fungi in seeds of some native and introduced grasses in China. Acta Prataculturae Sinica, 1996, 5(2): 1-8.
南志标. 内生真菌在我国部分国产和引进禾草品种种子中的分布. 草业学报, 1996, 5(2): 1-8.
[17] Wei Y K, Gao Y B, Xu H, et al. Occurrence of endophytes in grasses native to northern China. Grass & Forage Science, 2010, 61(4): 422-429.
[18] Li C J, Nan Z B, Paul V H, et al. A new Neotyphodium species symbiotic with drunken horse grass (Achnatherum inebrians) in China.Mycotaxon, 2004, 90(1): 141-147.
[19] Zhang X, Ren A Z, Wei Y K, et al. Taxonomy, diversity and origins of symbiotic endophytes of Achnatherum sibiricum in the Inner Mongolia Steppe of China. Fems Microbiology Letters, 2010, 301(1): 12-20.
[20] Zhu M J, Ren A Z, Wen W, et al. Diversity and taxonomy of endophytes from Leymus chinensis in the Inner Mongolia steppe of China. Fems Microbiology Letters, 2013, 340(2): 135-145.
[21] Kang Y, Ji Y L, Zhu K R, et al. A new Epichloё species with interspecific hybrid origins from Poa pratensis ssp. pratensis in Liyang, China. Mycologia, 2011, 103(6): 1341-1350.
[22] Kang Y, Ji Y L, Sun X H, et al. Taxonomy of Neotyphodium endophytes of Chinese native Roegneria plants. Mycologia, 2009, 101(2): 211-219.
[23] Ji Y L, Zhan L H, Kang Y, et al. A new stromata-producing Neotyphodium species symbiotic with clonal grass Calamagrostis epigeios (L.) Roth. grown in China. Mycologia, 2009, 101(2): 200-205.
[24] Kang Y, Ji Y L, Wang Y, et al. A Neotyphodium endophyte from Festuca myuros L. in Nanjing, China. Mycology, 2012, 3(3): 201-209.
[25] Chen Y G, Ji Y L, Yu H S, et al. A new Neotyphodium species from Festuca parvigluma Steud. grown in China. Mycologia, 2009, 101(5): 681-685.
[26] Zhang C W, Ji Y L, Yang S L, et al. Isolation and identification of a fungal endophyte obtained from Roegneria canina (L.) Nevski grown in Jilin, China. Journal of Nanjing Agricultural University, 2011, 34(2): 78-84.
张晨炜, 纪燕玲, 杨守玲, 等. 禾本科植物内生真菌研究12:吉林犬草内生真菌的分离和鉴定. 南京农业大学学报, 2011, 34(2): 78-84.
[27] Kuang Y, Wang J J, Xu W B, et al. Analysis of actin sequences from Epichloё endophyte in Festuca sinensis. Acta Prataculturae Sinica, 2016, 25(9): 125-131.
旷宇, 汪建军, 许文博, 等. 中华羊茅Epichloё内生真菌的actin序列分析. 草业学报, 2016, 25(9): 125-131.
[28] Bao G S, Li C J.Isolation and identification of endophytes infectiong Stipa purpurea, a dominant grass in meadows of the Qinghai-Tibet Plateau. Acta Prataculturae Sinica, 2016, 25(3): 32-42.
鲍根生, 李春杰. 青藏高原高寒草地优势禾草—紫花针茅内生真菌分离和鉴定. 草业学报, 2016, 25(3): 32-42.
[29] Song H, Nan Z B.Origin, divergence, and phylogeny of asexual Epichloё endophyte in Elymus species from western China. PLoS ONE, 2015, 10(5): e0127096.
[30] Ji Y L, Wang Z W, Yu H S, et al. Neotyphodium uncinatum, an endophytic fungus obtained from Festuca arundinacea Schreb. Journal of Nanjing Agricultural University, 2003, 26(2): 47-50.
纪燕玲, 王志伟, 于汉寿, 等. 分离自苇状羊茅(Festuca arundinacea Schreb.)的内生真菌Neotyphodium uncinatum. 南京农业大学学报, 2003, 26(2): 47-50.
[31] Yuan F R, Zhu Z H, Li Y N, et al. Research on compensatory growth of Stipa alina and impact factors under different habitats. Pratacultural Science, 2011, 28(11): 1972-1981.
袁芙蓉, 朱志红, 李英年, 等. 异针茅在不同生境下补偿生长特性及其影响因素的研究. 草业科学, 2011, 28(11): 1972-1981.
[32] Shi J J, Ma Y S, Wang L Y, et al. Study on cultivation and tame of Stipa aliena Keng. Chinese Journal of Grassland, 2006, 28(4): 84-86.
施建军, 马玉寿, 王柳英, 等. 异针茅栽培驯化初报. 中国草地学报, 2006, 28(4): 84-86.
[33] Swofford D L.Phylogenetic analysis using parsimony (Mac Version). Sunderland, Massachusetts: Sinauer, 1998.
[34] Kauppinen M, Saikkonen K, Helander M, et al. Epichloё grass endophytes in sustainable agriculture. Nature Plants, 2016, 2(2): 15224.
[35] Zhou H K, Tang Y H, Zhao X Q, et al. Long-term grazing alters species composition and biomass of a shrub meadow on the Qinghai-Tibet Plateau. Pakistan Journal of Botany, 2006, 38(4): 1055-1069.
[36] Saikkonen K, Wäli P, Helander M, et al. Evolution of endophyte-plant symbioses. Trends in Plant Science, 2004, 9(6): 275-280.
[37] Górzyńska K, Ryszka P, Anielska T, et al. Effect of Epichloё typhina fungal endophyte on the diversity and incidence of other fungi in Puccinellia distans wild grass seeds. Flora, 2017, 228: 60-64.
[38] Moon C D, Guillaumin J J, Ravel C, et al. New Neotyphodium endophyte species from the grass tribes Stipeae and Meliceae. Mycologia, 2007, 99(6): 895-905.
[39] Zhou L Y, Li C J, Zhang X X, et al. Effects of cold shocked Epichloё infected Festuca sinensis on ergot alkaloid accumulation. Fungal Ecology, 2015, 14: 99-104.
[40] Hu X W, Fan Y, Baskin C C, et al. Comparison of the effects of temperature and water potential on seed germination of Fabaceae species from desert and subalpine grassland. American Journal of Botany, 2015, 102(5): 649-660.
[41] Chen Z, Guan Y Z, Liang X P, et al. Effects of altitude on the morphological traits of Elymus species. Acta Agrestia Sinica, 2015, 23(5): 897-904.
陈钊, 管永卓, 梁新平, 等. 海拔高度对披碱草属植物形态特征的可塑性. 草地学报, 2015, 23(5): 897-904.
[42] Tadych M, Bergen M S, White J F.Epichloё spp. associated with grasses: New insights on life cycles, dissemination and evolution. Mycologia, 2014, 106(2): 181-201.
[43] Moore J R, Pratley J E, Mace W J, et al. Variation in alkaloid production from genetically diverse Lolium accessions infected with Epichloё species. Journal of Agricultural & Food Chemistry, 2015, 63(48): 10355-10365.
[44] Li W, Ji Y L, Yu H S, et al. Genetic diversity and rDNS-ITS sequence analysis of endophytic fungi isolated from gramineous plants in China. Mycosystema, 2006, 25(2): 217-226.
李伟, 纪燕玲, 于汉寿, 等. 中国产禾本科植物内生真菌的遗传多样性及rDNA-ITS系统发育分析. 菌物学报, 2006, 25(2): 217-226.
[45] Chen Y G, Ji Y L, Kang Y, et al. Grass endophyte researches 5. Genetic diversity in some Chinese Epichloё endophytes. Mycosystema, 2008, 27(6): 863-872.
陈永敢, 纪燕玲, 亢燕, 等. 禾本科植物内生真菌研究 5.中国产部分epichloё内生真菌的遗传多样性. 菌物学报, 2008, 27(6): 863-872.
[46] Tadych M, Ambrose K V, Bergen M S, et al. Taxonomic placement of Epichloё poae sp. nov. and horizontal dissemination to seedlings via conidia. Fungal Diversity, 2012, 54(1): 117-131.
[47] Yao X, Christensen M J, Bao G S, et al. A toxic endophyte-infected grass helps reverse degradation and loss of biodiversity of over-grazed grasslands in northwest China. Scientific Reports, 2015, 5: 18527.
[48] Oberhofer M, Leuchtmann A.Genetic diversity in epichloid endophytes of Hordelymus europaeus suggests repeated host jumps and interspecific hybridizations. Molecular Ecology, 2012, 21(11): 2713-2726.
[49] Li H F, Sun Q X.Advances in the research of model plant Brachypodium distachyon. Acta Botanica Boreali-Occidentalia Sinica, 2012, 32(3): 633-638.
李海峰, 孙其信. 模式植物短柄草研究新进展. 西北植物学报, 2012, 32(3): 633-638.
[50] Clay K, Schardl C L.Evolutionary origins and ecological consequences of endophyte symbiosis with grasses. American Naturalist, 2002, 160(S4): 99-127.