鄂西地区蜜环菌生物种的遗传多样性研究
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摘要
蜜环菌属Armillaria真菌是世界范围内广泛分布的担子菌,既是引起森林病害的重要病原菌,也是天麻和猪苓生长的必须共生真菌,具有重要的经济价值。本文对我国鄂西天麻主产区即神农架林区、大老岭自然保护区、宜昌市所属的夷陵区和五峰县、恩施自治州等地进行了系统的调查采样,共获得45份蜜环菌子实体样本及其二倍体菌株,其中通过孢子印分离获得约320个单孢菌株。在此基础上,采用IGS、ITS和elongation factor-1 alpha(EF1-α)分子标记对45份二倍体菌株进行测序和系统发育分析,对有单孢株的样本采用遗传互交不育性实验进行生物种鉴定。结果表明,鄂西地区的蜜环菌分为4个clade,共有6个不同的蜜环菌生物种,分别为CBS J,CBS L,CBS N,CBS O,CBS F和Armillaria mellea,具有丰富的遗传多样性。其中,24个样本鉴定为CBSL,是鄂西地区的优势种;9个样本鉴定为CBSJ;9个样本鉴定为CBS N;另有3个样本分别鉴定为CBS O,CBS F和Armillaria mellea。其中CBS L、CBS N、CBS O首次在湖北地区报道,扩大了这3个生物种的分布范围。本研究为了解鄂西地区蜜环菌的资源情况提供基础数据,对天麻生产蜜环菌菌种选育具有重要的参考价值。
Species of Armillaria belonging to basidiomycetes distributed worldwide, with special values in China,are important forest pathogens and necessary for culturing notable Chinese medicine Gastrodia elata and Polyporus umbellatus. A systematic investigation of the diversity of Armillaria species was performed in Shennongjia forest zone, Yiling County, Wufeng County, Enshi Prefecture and Lichuan City in western Hubei. In total, 45 fruiting bodies of Armillaria spp. were collected, and 45 diploid isolates and 320 single spore strains were isolated. The 45 isolates were sequenced and phylogenetically analysed based on molecular markers of IGS,ITS and elongation factor-1 alpha(EF1-α). The genetic intersterility experiments were performed to verify the molecular analysis. Our results suggested that there were four clades of Armillaria and six Armillaria species in the western Hubei Province. Further analysis indicated that 24 isolates belonged to CBS L, suggesting that it was dominant in western Hubei. Nine isolates were identified as CBS J, nine isolates CBS N, and the rest three isolates CBS O, CBS F and Armillaria mellea respectively. CBS L, CBS N and CBS O were first found in Hubei Province,which vastly expanded their distribution range in China. This study provides data for understanding the diversity of Armillaria in western Hubei Province, and breeding Armillaria strains for production of Gastrodia elata.
Species of Armillaria belonging to basidiomycetes distributed worldwide, with special values in China,are important forest pathogens and necessary for culturing notable Chinese medicine Gastrodia elata and Polyporus umbellatus. A systematic investigation of the diversity of Armillaria species was performed in Shennongjia forest zone, Yiling County, Wufeng County, Enshi Prefecture and Lichuan City in western Hubei. In total, 45 fruiting bodies of Armillaria spp. were collected, and 45 diploid isolates and 320 single spore strains were isolated. The 45 isolates were sequenced and phylogenetically analysed based on molecular markers of IGS,ITS and elongation factor-1 alpha(EF1-α). The genetic intersterility experiments were performed to verify the molecular analysis. Our results suggested that there were four clades of Armillaria and six Armillaria species in the western Hubei Province. Further analysis indicated that 24 isolates belonged to CBS L, suggesting that it was dominant in western Hubei. Nine isolates were identified as CBS J, nine isolates CBS N, and the rest three isolates CBS O, CBS F and Armillaria mellea respectively. CBS L, CBS N and CBS O were first found in Hubei Province,which vastly expanded their distribution range in China. This study provides data for understanding the diversity of Armillaria in western Hubei Province, and breeding Armillaria strains for production of Gastrodia elata.
引文
Anderson JB,Ullrich RC,1979.Biological species of Armillaria mellea in North America.Mycologia,71(2):402-414
Anderson JB,Stasovski E,1992.Molecular phylogeny of northern hemisphere species of Armillaria.Mycologia,84(4):505-516
Banik MT,Hhjr B,1998.Assessment of compatibility among Armillaria cepistipes,A.sinapina,and North American biological species X and XI,using culture morphology and molecular biology.Mycologia,90(5):798-805
Gunnell PS,Gubler WD,1992.Taxonomy and morphology of Colletotrichum species pathogenic to strawberry.Mycologia,84(2):157-165
Guo T,Han CW,Wan QX,Zhao J,Yang ZL,2016.Phylogenetic analyses of Armillaria reveal at least 15 phylogenetic lineages in China,seven of which are associated with cultivated Gastrodia elata.PLoS One,11(5):e0154794
Hintikka V,1973.A note on the polarity of Armillaria mellea.Karstenia,13:32-39
Harrington TC,Wingfield BD,1995.A PCR-based identification method for species of Armillaria.Mycologia,87(2):280-288
Hasegawa E,Ota Y,Hattori T,Kikuchi T,2010.Sequence-based identification of Japanese Armillaria species using the elongation factor-1 alpha gene.Mycologia,102(4):898-910
Korhonen K,Hintikka V,1974.Cytological evidence for somatic diploidization in dikaryotic cells of Armillaria mellea.Archives of Microbiology,95(1):187-192
Korhonen K,1978.Interfertility and clonal size in the Armillaria mellea complex.Karstenia,18:31-42
Li XQ,Huang YQ,Cheng DS,1998.The preleminary study of Armillaria biological species in northern China.Mycosystema,17(4):307-311(in Chinese)
Li HB,Wu XQ,Wang LW,Fu LZ,Wei HL,Wu QQ,2008.Pure culture isolation,cultivation and molecular identification of Armillaria luteo-virens from Tibet Plateau.Mycosystema,27(6):873-883(in Chinese)
Lochman J,Sery O,Mikes V,2004.The rapid identification of European Armillaria species from soil samples by nested PCR.FEMS Microbiology Letters,237(1):105
Men JX,Xing XK,Guo SX,2016.Biological species and identification methods of the genus Armillaria(Agaricales,Basidiomycota):a review.Mycosystema,35(11):1281-1302(in Chinese)
Men JX,Xing XK,Guo SX,2017.A comparison of Armillaria spp.associated with Polyporus umbellatus and Gastrodia elata in China.Mycosystema,36(8):1072-1082(in Chinese)
Maphosa L,Wingfield BD,Coetzee MPA,Mwenje E,Wingfied MJ,2006.Phylogenetic relationships among Armillaria,species inferred from partial elongation factor 1-alpha DNA sequence data.Australasian Plant Pathology,35(5):513-520
Peabody DC,Motta JJ,Therrien CD,1978.Cytophotometric evidence for heteroploidy in the life cycle of Armillaria mellea.Mycologia,70(3):487-498
Qin GF,Zhao J,Tian SM,Korhonen K,Dai YC,Liu CR,2000.Chinese Armillaria and new biological species.Mycosystema,19(4):509-516(in Chinese)
Qin GF,Zhao J,Tian SM,Hantula J,2001.Genetic diversity and molecular identification of northern hemisphere species of Armillaria gallica.Scientia Silvae Sinicae,37(2):61-68(in Chinese)
Qin GF,2002.The genetics and systematics research of Chinese Armillaria.PhD Dissertation,Chinese Academy of Forestry Sciences,Beijing.1-209(in Chinese)
Ross-Davis AL,Hanna JW,Kim MS,Klopfenstein NB,2012.Advances toward DNA-based identification and phylogeny of North American Armillaria species using elongation factor-1 alpha gene.Mycoscience,53(2):161-165
Schulze S,Bahnweg G,Tesche M,Sandermann Jr H,2010.Identification of European Armillaria species by restriction-fragment-length polymorphisms of ribosomal DNA.Forest Pathology,25(4):214-223
Sun LF,Zhang YH,Pei KQ,2009.A rapid extraction of genomic DNA from fungi.Mycosystema,28(2):299-302(in Chinese)
Tsykun T,Rigling D,Prospero S,2013.A new multilocus approach for a reliable DNA-based identification of Armillaria species.Mycologia,105(4):1059-1076
Wang HC,2010.The research of the worldwide Armillaria systematics.Journal of Chongqing Normal University(Natural Science),27(1):61-68(in Chinese)
Wang L,Yang ZL,2003.The genus Armillaria fungi of southwest China.Edible Fungi of China,22(5):4-6(in Chinese)
Wang R,Zhang SY,Mu Q,2016.Research progress in chemical constituents and biological activities of Armillaria mellea.Chinese Traditional and Herbal Drugs,47(11):1992-1999(in Chinese)
Xu JT,Guo SX,1992.The relationship between Polyporus umbellatus and Armillariella mellea.Acta Mycologica Sinica,11(2):142-145(in Chinese)
Zhao J,Tian SM,Wang YL,Qin GF,1999.A method of single spore isolation and culture for Armillaria genetic test.Microbiology,26(3):207-209(in Chinese)
Zhao J,Qin GF,Dai YC,2005.The new biological species of Chinese Armillaria.Mycosystema,24(2):164-173(in Chinese)
Zhao J,Dai YC,Qin GF,Wei YL,Wang HC,Yuan HS,2008.New biological species of Armillaria from China.Mycosystema,27(2):156-170(in Chinese)
李向前,黄永青,程东升,1998.中国北方地区蜜环菌生物种的初步研究.菌物系统,4:307-311
李海波,吴学谦,王立武,付立忠,魏海龙,吴庆其,2008.青藏高原黄绿蜜环菌纯培养菌种的分离培养及分子鉴定.菌物学报,27(6):873-883
门金鑫,邢晓科,郭顺星,2016.蜜环菌生物种及鉴定方法研究进展.菌物学报,35(11):1281-1302
门金鑫,邢晓科,郭顺星,2017.猪苓与天麻共生蜜环菌类群的比较.菌物学报,36(8):1072-1082
秦国夫,赵俊,田淑敏,Korhonen K,戴玉成,刘春如,2000.中国蜜环菌的新生物种.菌物学报,19(4):509-516
秦国夫,赵俊,田淑敏,Jarkko Hantula,2001.北半球高卢蜜环菌的遗传多样性与分子鉴定.林业科学,37(2):61-68
秦国夫,2002.中国蜜环菌的遗传学与系统学研究.中国林业科学研究院博士论文,北京.1-209
孙立夫,张艳华,裴克全,2009.一种高效提取真菌总DNA的方法.菌物学报,28(2):299-302
王汉臣,2010.世界蜜环菌属系统学研究进展.重庆师范大学学报(自然科学版),27(1):61-68
王岚,杨祝良,2003.中国西南的蜜环菌属真菌.中国食用菌,22(5):4-6
王锐,张诗悦,穆青,2016.蜜环菌的化学成分及生物活性研究进展.中草药,47(11):1992-1999
徐锦堂,郭顺星,1992.猪苓与蜜环菌的关系.菌物学报,2:142-145
赵俊,田淑敏,王玉玲,秦国夫,1999.蜜环菌遗传测定的单孢分离和培养方法.微生物学通报,26(3):207-209
赵俊,秦国夫,戴玉成,2005.中国蜜环菌生物种新记录.菌物学报,24(2):164-173
赵俊,戴玉成,秦国夫,魏玉莲,王汉臣,袁海生,2008.新的蜜环菌生物种.菌物学报,27(2):156-170
Anderson JB,Stasovski E,1992.Molecular phylogeny of northern hemisphere species of Armillaria.Mycologia,84(4):505-516
Banik MT,Hhjr B,1998.Assessment of compatibility among Armillaria cepistipes,A.sinapina,and North American biological species X and XI,using culture morphology and molecular biology.Mycologia,90(5):798-805
Gunnell PS,Gubler WD,1992.Taxonomy and morphology of Colletotrichum species pathogenic to strawberry.Mycologia,84(2):157-165
Guo T,Han CW,Wan QX,Zhao J,Yang ZL,2016.Phylogenetic analyses of Armillaria reveal at least 15 phylogenetic lineages in China,seven of which are associated with cultivated Gastrodia elata.PLoS One,11(5):e0154794
Hintikka V,1973.A note on the polarity of Armillaria mellea.Karstenia,13:32-39
Harrington TC,Wingfield BD,1995.A PCR-based identification method for species of Armillaria.Mycologia,87(2):280-288
Hasegawa E,Ota Y,Hattori T,Kikuchi T,2010.Sequence-based identification of Japanese Armillaria species using the elongation factor-1 alpha gene.Mycologia,102(4):898-910
Korhonen K,Hintikka V,1974.Cytological evidence for somatic diploidization in dikaryotic cells of Armillaria mellea.Archives of Microbiology,95(1):187-192
Korhonen K,1978.Interfertility and clonal size in the Armillaria mellea complex.Karstenia,18:31-42
Li XQ,Huang YQ,Cheng DS,1998.The preleminary study of Armillaria biological species in northern China.Mycosystema,17(4):307-311(in Chinese)
Li HB,Wu XQ,Wang LW,Fu LZ,Wei HL,Wu QQ,2008.Pure culture isolation,cultivation and molecular identification of Armillaria luteo-virens from Tibet Plateau.Mycosystema,27(6):873-883(in Chinese)
Lochman J,Sery O,Mikes V,2004.The rapid identification of European Armillaria species from soil samples by nested PCR.FEMS Microbiology Letters,237(1):105
Men JX,Xing XK,Guo SX,2016.Biological species and identification methods of the genus Armillaria(Agaricales,Basidiomycota):a review.Mycosystema,35(11):1281-1302(in Chinese)
Men JX,Xing XK,Guo SX,2017.A comparison of Armillaria spp.associated with Polyporus umbellatus and Gastrodia elata in China.Mycosystema,36(8):1072-1082(in Chinese)
Maphosa L,Wingfield BD,Coetzee MPA,Mwenje E,Wingfied MJ,2006.Phylogenetic relationships among Armillaria,species inferred from partial elongation factor 1-alpha DNA sequence data.Australasian Plant Pathology,35(5):513-520
Peabody DC,Motta JJ,Therrien CD,1978.Cytophotometric evidence for heteroploidy in the life cycle of Armillaria mellea.Mycologia,70(3):487-498
Qin GF,Zhao J,Tian SM,Korhonen K,Dai YC,Liu CR,2000.Chinese Armillaria and new biological species.Mycosystema,19(4):509-516(in Chinese)
Qin GF,Zhao J,Tian SM,Hantula J,2001.Genetic diversity and molecular identification of northern hemisphere species of Armillaria gallica.Scientia Silvae Sinicae,37(2):61-68(in Chinese)
Qin GF,2002.The genetics and systematics research of Chinese Armillaria.PhD Dissertation,Chinese Academy of Forestry Sciences,Beijing.1-209(in Chinese)
Ross-Davis AL,Hanna JW,Kim MS,Klopfenstein NB,2012.Advances toward DNA-based identification and phylogeny of North American Armillaria species using elongation factor-1 alpha gene.Mycoscience,53(2):161-165
Schulze S,Bahnweg G,Tesche M,Sandermann Jr H,2010.Identification of European Armillaria species by restriction-fragment-length polymorphisms of ribosomal DNA.Forest Pathology,25(4):214-223
Sun LF,Zhang YH,Pei KQ,2009.A rapid extraction of genomic DNA from fungi.Mycosystema,28(2):299-302(in Chinese)
Tsykun T,Rigling D,Prospero S,2013.A new multilocus approach for a reliable DNA-based identification of Armillaria species.Mycologia,105(4):1059-1076
Wang HC,2010.The research of the worldwide Armillaria systematics.Journal of Chongqing Normal University(Natural Science),27(1):61-68(in Chinese)
Wang L,Yang ZL,2003.The genus Armillaria fungi of southwest China.Edible Fungi of China,22(5):4-6(in Chinese)
Wang R,Zhang SY,Mu Q,2016.Research progress in chemical constituents and biological activities of Armillaria mellea.Chinese Traditional and Herbal Drugs,47(11):1992-1999(in Chinese)
Xu JT,Guo SX,1992.The relationship between Polyporus umbellatus and Armillariella mellea.Acta Mycologica Sinica,11(2):142-145(in Chinese)
Zhao J,Tian SM,Wang YL,Qin GF,1999.A method of single spore isolation and culture for Armillaria genetic test.Microbiology,26(3):207-209(in Chinese)
Zhao J,Qin GF,Dai YC,2005.The new biological species of Chinese Armillaria.Mycosystema,24(2):164-173(in Chinese)
Zhao J,Dai YC,Qin GF,Wei YL,Wang HC,Yuan HS,2008.New biological species of Armillaria from China.Mycosystema,27(2):156-170(in Chinese)
李向前,黄永青,程东升,1998.中国北方地区蜜环菌生物种的初步研究.菌物系统,4:307-311
李海波,吴学谦,王立武,付立忠,魏海龙,吴庆其,2008.青藏高原黄绿蜜环菌纯培养菌种的分离培养及分子鉴定.菌物学报,27(6):873-883
门金鑫,邢晓科,郭顺星,2016.蜜环菌生物种及鉴定方法研究进展.菌物学报,35(11):1281-1302
门金鑫,邢晓科,郭顺星,2017.猪苓与天麻共生蜜环菌类群的比较.菌物学报,36(8):1072-1082
秦国夫,赵俊,田淑敏,Korhonen K,戴玉成,刘春如,2000.中国蜜环菌的新生物种.菌物学报,19(4):509-516
秦国夫,赵俊,田淑敏,Jarkko Hantula,2001.北半球高卢蜜环菌的遗传多样性与分子鉴定.林业科学,37(2):61-68
秦国夫,2002.中国蜜环菌的遗传学与系统学研究.中国林业科学研究院博士论文,北京.1-209
孙立夫,张艳华,裴克全,2009.一种高效提取真菌总DNA的方法.菌物学报,28(2):299-302
王汉臣,2010.世界蜜环菌属系统学研究进展.重庆师范大学学报(自然科学版),27(1):61-68
王岚,杨祝良,2003.中国西南的蜜环菌属真菌.中国食用菌,22(5):4-6
王锐,张诗悦,穆青,2016.蜜环菌的化学成分及生物活性研究进展.中草药,47(11):1992-1999
徐锦堂,郭顺星,1992.猪苓与蜜环菌的关系.菌物学报,2:142-145
赵俊,田淑敏,王玉玲,秦国夫,1999.蜜环菌遗传测定的单孢分离和培养方法.微生物学通报,26(3):207-209
赵俊,秦国夫,戴玉成,2005.中国蜜环菌生物种新记录.菌物学报,24(2):164-173
赵俊,戴玉成,秦国夫,魏玉莲,王汉臣,袁海生,2008.新的蜜环菌生物种.菌物学报,27(2):156-170