大花杓兰根际土壤真菌及兰科菌根真菌多样性分析
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摘要
【目的】从分子水平上对兰科杓兰属大花杓兰根际土壤真菌群落组成及潜在兰科菌根真菌进行初步研究。【方法】采用克隆文库技术对大花杓兰根际土壤真菌的ITS片段进行PCR扩增、克隆测序及BLAST分析。【结果】共获得96个真菌克隆,其中44个克隆鉴定为未知真菌,52个真菌克隆基于97%的序列相似性被划分为24个操作分类单元(Operational Taxonomic Unit, OTU),属于5门6纲11目16科15属。被孢霉属Mortierella为大花杓兰根际土壤的主要真菌类群。综合兰科植物菌根真菌的研究报道,对24个OUTs进一步分析,发现4个OTUs为潜在的兰科菌根真菌,分别属于角担菌科Ceratobasidiaceae的丝核菌属Rhizoctonia(OTU1)和角担菌属Ceratobasidium(OTU2)及胶膜菌科Tulasnellaceae(OTU3和OTU4)。【结论】根际土壤真菌及潜在兰科菌根真菌类群的构成及多样性研究,对从土壤中分离筛选有效的大花杓兰菌根真菌及其野生种群的迁地保育均具有重要意义。
【Objective】The diversity of fungi and potential orchid mycorrhizas in Cypripedium macranthum rhizospheric soil collected from Baihuashan Natural Reserve in Beijing was preliminarily revealed at the molecular level.【Method】Clone library technique was used to analyze the composition of fungal community.【Result】96 rhizospheric fungi were cloned, of which 44 rhizospheric fungi cloned were unknown, and 52 rhizospheric fungi based on 97 % sequence similarity levels were divided into the total of 24 operational taxonomic units(OTUs), which belonged to 5 phyla, 6 calss, 11 order, 16 family and 15 genera. Among these fungi identified, Mortierella was dominant genera in rhizosphere soil of C. macranthum. According to reports on orchid mycorrhizal fungi, it was speculated that 4 OUTs might be potential mycorrhizal fungi, which were Rhizoctonia, Ceratobasidium, and Tulasnellaceae, respectively. 【Conclusion】The preliminary analysis on the composition of rhizosphere soil fungi and potential orchid mycorrhizas was of great significance for isolating and screening of certain mycorrhizal fungi that were associated with C. macranthum seed germination. Meanwhile, the research results were also beneficial for ex-situ conservation of wild C. macranthum.
【Objective】The diversity of fungi and potential orchid mycorrhizas in Cypripedium macranthum rhizospheric soil collected from Baihuashan Natural Reserve in Beijing was preliminarily revealed at the molecular level.【Method】Clone library technique was used to analyze the composition of fungal community.【Result】96 rhizospheric fungi were cloned, of which 44 rhizospheric fungi cloned were unknown, and 52 rhizospheric fungi based on 97 % sequence similarity levels were divided into the total of 24 operational taxonomic units(OTUs), which belonged to 5 phyla, 6 calss, 11 order, 16 family and 15 genera. Among these fungi identified, Mortierella was dominant genera in rhizosphere soil of C. macranthum. According to reports on orchid mycorrhizal fungi, it was speculated that 4 OUTs might be potential mycorrhizal fungi, which were Rhizoctonia, Ceratobasidium, and Tulasnellaceae, respectively. 【Conclusion】The preliminary analysis on the composition of rhizosphere soil fungi and potential orchid mycorrhizas was of great significance for isolating and screening of certain mycorrhizal fungi that were associated with C. macranthum seed germination. Meanwhile, the research results were also beneficial for ex-situ conservation of wild C. macranthum.
引文
[1]张毓,赵世伟,张启翔,等. 北京地区大花杓兰资源调查与生物学特性研究: 中国观赏园艺研究进展[C]. 北京: 中国林业出版社,2005:92-95.
[2]侯晓强,付亚娟,袁建平,等. 大花杓兰内生真菌多样性研究[J]. 湖北农业科学,2015,54(6): 1357-1360.
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[4]Shimura H, Sadamoto M, Matsuura M, et al. Characterization of mycorrhizal fungi isolated from the threatened Cypripedium macranthos, in a northern island of Japan: two phylogenetically distinct fungi associated with the orchid[J]. Mycorrhiza, 2009, 19(8): 525-534.
[5]张亚平. 中国北方三种杓兰内生真菌多样性及其对原球茎生长的效应[D]. 雅安: 四川农业大学, 2013.
[6]Mccormick M K, Taylor D L, Whigham D F, et al. Germination patterns in three terrestrial orchids relate to abundance of mycorrhizal fungi[J]. Journal of Ecology, 2016, 104(3): 744-754.
[7]Waud M, Wiegand T, Brys R, et al. Nonrandom seedling establishment corresponds with distance-dependent decline in mycorrhizal abundance in two terrestrial orchids[J]. New Phytologist, 2016, 211(1): 255-264.
[8]Taylor D L, Mccormick M K. Internal transcribed spacer primers and sequences for improved characterization of basidiomycetous orchid mycorrhizas[J]. New Phytologist, 2008, 177(4): 1020-1033.
[9]Martos F, Munoz F, Pailler T, et al. The role of epiphytism in architecture and evolutionary constraint within mycorrhizal networks of tropical orchids[J]. Molecular Ecology, 2012, 21(20): 5098-5109.
[10]盖雪鸽,邢晓科,郭顺星. 长茎羊耳蒜菌根真菌类群的研究[J]. 菌物学报,2016,35(3):290-297.
[11]Dearnaley J W D, Martos F, Selosse M A. Orchid mycorrhizas: molecular ecology, physiology, evolution and conservation aspects[M]. Fungal Associations, Springer Berlin Heidelberg, 2012.
[12]McCormick M K, Jacquemyn H. What constrains the distribution of orchid populations?[J]. New Phytologist,2015, 202: 392-400.
[13]Shefferson R P, Cowden C C, Mccormick M K, et al. Evolution of host breadth in broad interactions: mycorrhizal specificity in East Asian and North American rattlesnake plantains (Goodyera spp.) and their fungal hosts[J]. Molecular Ecology, 2010, 19(14): 3008-3017.
[14]缪福俊,蒋宏,王宏虬,等. 黄花杓兰菌根真菌rDNA ITS的多样性[J]. 浙江农林大学学报,2015,32(5):815-820.
[15]Jacquemyn H, Honnay O, Cammue B P A, et al. Low specificity and nested subset structure characterize mycorrhizal associations in five closely-related species of the genus Orchids[J]. Molecular Ecology, 2010, 19(18): 4086-4095.
[16]Esposito F, Jacquemyn H, Waud M, et al. Mycorrhizal fungal diversity and community composition in two closely related Platanthera (Orchidaceae) species[J]. PLoS One, 2016, 11: e0164108.
[17]Oja J, Vahtra J, Bahram M, et al. Local-scale spatial structure and community composition of orchid mycorrhizal fungi in semi-natural grasslands[J]. Mycorrhiza, 2017, 27(4): 355-367.
[18]Huang C L, Jian F Y, Huang H J, et al. Deciphering mycorrhizal fungi in cultivated Phalaenopsis, microbiome with next-generation sequencing of multiple barcodes[J]. Fungal Diversity, 2014, 66(1): 77-88.
[19]Jacquemyn H, Brys R, Waud M, et al. Mycorrhizal networks and coexistence in species-rich orchid communities[J]. New Phytologist, 2015, 206(3): 1127-1134.
[20]Ochora J, Stock W D, Linder H P, et al. Symbiotic seed germination in twelve Kenyan orchid species[J]. Systematics & Geography of Plants, 2001, 71(2): 585-596.
[21]Mulyani R B, Sastrahidayat I R, Abadi A L, et al. Exploring ectomycorrhiza in peat swamp forest of Nyaru Menteng Palangka Raya Central Borneo[J]. Journal of Biodiversity and Environmental Sciences, 2014, 5(6): 133-145.
[22]Graf F, Brunner I. Natural and synthesized ectomycorrhizas of the alpine dwarf willow Salix herbacea[J]. Mycorrhiza, 1996(6): 227-235.
[23]Voyron S, Ercole E, Ghignone S, et al. Fine-scale spatial distribution of orchid mycorrhizal fungi in the soil of host-rich grasslands[J]. New Phytologist, 2017, 213: 1428-1439.
[2]侯晓强,付亚娟,袁建平,等. 大花杓兰内生真菌多样性研究[J]. 湖北农业科学,2015,54(6): 1357-1360.
[3]张毓, 赵世伟. 一种促进大花杓兰种子共生萌发的真菌及其应用[P]. 中国:CN201110402377.6, 2011-11-06.
[4]Shimura H, Sadamoto M, Matsuura M, et al. Characterization of mycorrhizal fungi isolated from the threatened Cypripedium macranthos, in a northern island of Japan: two phylogenetically distinct fungi associated with the orchid[J]. Mycorrhiza, 2009, 19(8): 525-534.
[5]张亚平. 中国北方三种杓兰内生真菌多样性及其对原球茎生长的效应[D]. 雅安: 四川农业大学, 2013.
[6]Mccormick M K, Taylor D L, Whigham D F, et al. Germination patterns in three terrestrial orchids relate to abundance of mycorrhizal fungi[J]. Journal of Ecology, 2016, 104(3): 744-754.
[7]Waud M, Wiegand T, Brys R, et al. Nonrandom seedling establishment corresponds with distance-dependent decline in mycorrhizal abundance in two terrestrial orchids[J]. New Phytologist, 2016, 211(1): 255-264.
[8]Taylor D L, Mccormick M K. Internal transcribed spacer primers and sequences for improved characterization of basidiomycetous orchid mycorrhizas[J]. New Phytologist, 2008, 177(4): 1020-1033.
[9]Martos F, Munoz F, Pailler T, et al. The role of epiphytism in architecture and evolutionary constraint within mycorrhizal networks of tropical orchids[J]. Molecular Ecology, 2012, 21(20): 5098-5109.
[10]盖雪鸽,邢晓科,郭顺星. 长茎羊耳蒜菌根真菌类群的研究[J]. 菌物学报,2016,35(3):290-297.
[11]Dearnaley J W D, Martos F, Selosse M A. Orchid mycorrhizas: molecular ecology, physiology, evolution and conservation aspects[M]. Fungal Associations, Springer Berlin Heidelberg, 2012.
[12]McCormick M K, Jacquemyn H. What constrains the distribution of orchid populations?[J]. New Phytologist,2015, 202: 392-400.
[13]Shefferson R P, Cowden C C, Mccormick M K, et al. Evolution of host breadth in broad interactions: mycorrhizal specificity in East Asian and North American rattlesnake plantains (Goodyera spp.) and their fungal hosts[J]. Molecular Ecology, 2010, 19(14): 3008-3017.
[14]缪福俊,蒋宏,王宏虬,等. 黄花杓兰菌根真菌rDNA ITS的多样性[J]. 浙江农林大学学报,2015,32(5):815-820.
[15]Jacquemyn H, Honnay O, Cammue B P A, et al. Low specificity and nested subset structure characterize mycorrhizal associations in five closely-related species of the genus Orchids[J]. Molecular Ecology, 2010, 19(18): 4086-4095.
[16]Esposito F, Jacquemyn H, Waud M, et al. Mycorrhizal fungal diversity and community composition in two closely related Platanthera (Orchidaceae) species[J]. PLoS One, 2016, 11: e0164108.
[17]Oja J, Vahtra J, Bahram M, et al. Local-scale spatial structure and community composition of orchid mycorrhizal fungi in semi-natural grasslands[J]. Mycorrhiza, 2017, 27(4): 355-367.
[18]Huang C L, Jian F Y, Huang H J, et al. Deciphering mycorrhizal fungi in cultivated Phalaenopsis, microbiome with next-generation sequencing of multiple barcodes[J]. Fungal Diversity, 2014, 66(1): 77-88.
[19]Jacquemyn H, Brys R, Waud M, et al. Mycorrhizal networks and coexistence in species-rich orchid communities[J]. New Phytologist, 2015, 206(3): 1127-1134.
[20]Ochora J, Stock W D, Linder H P, et al. Symbiotic seed germination in twelve Kenyan orchid species[J]. Systematics & Geography of Plants, 2001, 71(2): 585-596.
[21]Mulyani R B, Sastrahidayat I R, Abadi A L, et al. Exploring ectomycorrhiza in peat swamp forest of Nyaru Menteng Palangka Raya Central Borneo[J]. Journal of Biodiversity and Environmental Sciences, 2014, 5(6): 133-145.
[22]Graf F, Brunner I. Natural and synthesized ectomycorrhizas of the alpine dwarf willow Salix herbacea[J]. Mycorrhiza, 1996(6): 227-235.
[23]Voyron S, Ercole E, Ghignone S, et al. Fine-scale spatial distribution of orchid mycorrhizal fungi in the soil of host-rich grasslands[J]. New Phytologist, 2017, 213: 1428-1439.