毛序棘豆内生真菌分离鉴定及系统进化分析
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摘要
为探明毛序棘豆中内生真菌的种属及种群分布情况,对采集于甘肃省通渭县毛序棘豆样品,运用表面消毒法消毒样品后进行内生真菌分离,采用形态学和ITS序列分析技术对所得菌株进行种属鉴定,并通过MEGA5.05软件进行系统进化分析。结果显示,从毛序棘豆(根、茎、叶)中共分离得到内生真菌29株,经鉴定,分属于4纲、5目、6科、10属;其中茎的分离率(39.13%)最高,根(23.91%)次之,叶(11.11%)最少;而链格孢属(Alternaria sp.)和镰刀菌属(Fusariumsp.)是毛序棘豆内生真菌的优势菌属,其相对分离频率分别为27.59%和17.24%;木霉菌属(Trichodermasp.)为毛序棘豆内分布最广泛的菌属,在根、茎、叶中均有分布;将所得菌株作系统进化分析,并根据其亲缘关系远近分为Ⅰ和Ⅱ两大种群。本试验结果表明,毛序棘豆内生真菌数量、种类与种属分布存在明显组织差异,茎是内生真菌侵染和定植的主要部位且真菌多样性最高。
To explore the species and population distribution of endophytic fungi fromOxytropis trichophora,Oxytropis trichophora samples in Tongwei county of Gansu province were collected,then were sterilized by using the method of surface disinfection.After that,endophytic fungi were insolated,and then the morphology techniques and ITS sequence analysis were used to identify their species,The phylogenetic relationship of endophytic fungi was analyzed by MEGA 5.05 software.The results showed that 29 species of endophytic fungi were isolated from Oxytropis trichophora belonging to 4 classes,5 orders,6 families and 10 genera,the isolation rate of stem was the highest(39.13%),followed with root(23.91%)and leaf was the least(11.11%).Alternariasp.and Fusariumsp.were the dominant species of endophytic fungi,relative frequency(RF)were 27.59%and 17.24%,respectively.Trichodermasp.was the most widely distributed in roots,stems and leaves of Oxytropis trichophora.The phylogenetic analysis of endophytic fungi isolated fromOxytropis trichophorashowed that they were divided into two groups according to their genetic relationship.The result indicated there was an obviously difference in quantity,species and population distribution of endophytic fungi between different parts of Oxytropistrichophora.Stem was the most vulnerable to infection and colonization by endophytic fungi,and the diversity was the maximum in stem of Oxytropis trichophora.
To explore the species and population distribution of endophytic fungi fromOxytropis trichophora,Oxytropis trichophora samples in Tongwei county of Gansu province were collected,then were sterilized by using the method of surface disinfection.After that,endophytic fungi were insolated,and then the morphology techniques and ITS sequence analysis were used to identify their species,The phylogenetic relationship of endophytic fungi was analyzed by MEGA 5.05 software.The results showed that 29 species of endophytic fungi were isolated from Oxytropis trichophora belonging to 4 classes,5 orders,6 families and 10 genera,the isolation rate of stem was the highest(39.13%),followed with root(23.91%)and leaf was the least(11.11%).Alternariasp.and Fusariumsp.were the dominant species of endophytic fungi,relative frequency(RF)were 27.59%and 17.24%,respectively.Trichodermasp.was the most widely distributed in roots,stems and leaves of Oxytropis trichophora.The phylogenetic analysis of endophytic fungi isolated fromOxytropis trichophorashowed that they were divided into two groups according to their genetic relationship.The result indicated there was an obviously difference in quantity,species and population distribution of endophytic fungi between different parts of Oxytropistrichophora.Stem was the most vulnerable to infection and colonization by endophytic fungi,and the diversity was the maximum in stem of Oxytropis trichophora.
引文
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[22]曹丹丹,路浩,李小蒙,等.茎直黄芪内生真菌分离鉴定及其多样性分析[J].草地学报,2015,23(6):1252-1258.CAO D D,LU H,LI X M,et al.Solation,identification and diversity analysis of fungal endophyte in Astragalus strictus[J].Acta Agrestia Sinica,2015,23(6):1252-1258.(in Chinese)
[23]俞大紱.中国镰刀菌属(Fusarium)菌种的初步名录[J].植物病理学报,1955,1(1):1-18.YU D F.A preliminary list of Fusariumin China[J].Acta Phytopathologica Sinica,1955,1(1):1-18.(in Chinese)
[24]唐岚,江厚利,王义勋,等.链格孢属真菌分类研究进展[J].湖北林业科技,2013,42(4):47-49,83.TANG L,JIANG H L,WANG Y X,et al.Advance on taxonomic studies of the genus Alternaria fungi[J].Hubei Forestry Science and Technology,2013,42(4):47-49,83.(in Chinese)
[2]傅立国,陈潭清,郎楷永,等.中国高等植物(第六卷)[M].青岛:青岛出版社,2003.FU L G,CHEN T Q,LANG K Y,et al.Higher plants of China(the sixth volume)[M].Qingdao:Qingdao Press,2003.(in Chinese)
[3]高淑静,孙启忠.棘豆属有毒植物及其对家畜的危害[J].牧草与饲料,1992(3):30-33.GAO S J,SUN Q Z.The Oxytropis poisonous plants and its harm to livestock[J].Journal of Forage&Feed,1992(3):30-33.(in Chinese)
[4]确生,才让卓玛.棘豆属植物研究综述[J].中国民族医药杂志,2006,12(5):102-104.QUE S,CAI R Z M.Research review of Oxytropis[J].Journal of Medicine&Pharmacy of Chinese Minorities,2006,12(5):102-104.(in Chinese)
[5]任清丹,杨金生,尹荣兰,等.疯草的研究现状与进展[J].中国畜禽种业,2013,9(5):40-41.REN Q D,YANG J S,YIN R L,et al.Research status and progress of locoweed[J].The Chinese Livestock and Poultry Breeding,2013,9(5):40-41.(in Chinese)
[6]李建科.中国疯草研究现状与展望[J].中国农业科学,2003,36(9):1091-1099.LI J K.The present situation and prospect of the studies on locoweed in China[J].Scientia Agricultura Sinica,2003,36(9):1091-1099.(in Chinese)
[7]任爱梅,张丽珂,孟宪刚.植物内生真菌研究进展与存在问题[J].广东农业科学,2010,37(2):103-106.REN A M,ZHANG L K,MENG X G.Research progress and existing problems of endophytic[J].Guangdong Agricultural Sciences,2010,37(2):103-106.(in Chinese)
[8]BRAUN K.Fungal endophyte infection and swainsonine toxicity in locoweed[D].Las Cruces,New Mexico:New Mexico State University,1999.
[9]COOK D,GARDNER D R,GRUM D,et al.Swainsonine and endophyte relationships in Astragalus mollissimus and Astragalus lentiginosus[J].J Agric Food Chem,2011,59(4):1281-1287.
[10]GAO X L,COOK D,RALPHS M H,et al.Detection of swainsonine and isolation of the endophyte Undifilum from the major locoweeds in Inner Mongolia[J].Biochem Syst Ecol,2012,45:79-85.
[11]GRUM D S,COOK D,BAUCOM D,et al.Production of the alkaloid swainsonine by a fungal endophyte in the host Swainsona canescens[J].J Nat Prod,2013,76(10):1984-1988.
[12]路浩,马尧,赵宝玉,等.急弯棘豆内生真菌的分离与鉴定[J].畜牧兽医学报,2010,41(7):902-908.LU H,MA Y,ZHAO B Y,et al.Isolation and identification of endophytic fungus from Oxytropis deflexa[J].Acta Veterinaria et Zootechnica Sinica,2010,41(7):902-908.(in Chinese)
[13]陈基萍,赵宝玉,路浩,等.中国主要疯草内生真菌分离鉴定及其抑菌活性研究[J].畜牧兽医学报,2012,43(9):1471-1478.CHEN J P,ZHAO B Y,LU H,et al.Study on isolation,identification and the antibacterial activity of endophytic fungi from the major locoweed in China[J].Acta Veterinaria et Zootechnica Sinica,2012,43(9):1471-1478.(in Chinese)
[14]周启武,路浩,何平,等.小花棘豆(Oxytropis glabra DC)内生真菌的分布特点[J].微生物学报,2013,53(5):478-485.ZHOU Q W,LU H,HE P,et al.Distribution of endophytic fungi in Oxytropis glabra DC[J].Acta Microbiologica Sinica,2013,53(5):478-485.(in Chinese)
[15]路浩,李国中,杨晓雯,等.甘肃棘豆内生真菌种群多样性[J].中国兽医学报,2014,34(7):1094-1099.LU H,LI G Z,YANG X W,et al.Diversity of endophytic fungi from Oxytropis kansuensis[J].Chinese Journal of Veterinary Science,2014,34(7):1094-1099.(in Chinese)
[16]新疆八一农学院.新疆植物检索表(第三册)[M].乌鲁木齐:新疆人民出版社,1983.Xinjiang Bayi Agricultural College.The retrieval list of Xinjiang plant(the third book)[M].Urumchi:Xinjiang People's Press,1983.(in Chinese)
[17]崔鸿宾,张振万,徐朗然,等.中国植物志(第四十二卷)[M].北京:科学出版社,1998:1.CUI H B,ZHANG Z W,XU L R,et al.Flora of China(the forty second volume)[M].Beijing:Science Press,1998:1.(in Chinese)
[18]魏景超.真菌鉴定手册[M].上海:上海科学技术出版社,1979.WEI J C.Fungal identification manual[M].Shanghai:Shanghai Science and Technology Press,1979.(in Chinese)
[19]孙桂丽,陈有为,张琦,等.一株有抗菌活性的云南重楼植物内生真菌的鉴定[J].云南大学学报:自然科学版,2006,28(S1):347-351.SUN G L,CHEN Y W,ZHANG Q,et al.The identify of one endophytic fungi isolated from Paris polyphylla Smith var.yunnnanensis[J].Journal of Yunnan University:Natural Sciences Edition,2006,28(S1):347-351.(in Chinese)
[20]TAMURA K,PETERSON D,PETERSON N,et al.MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Mol Biol Evol,2011,28(10):2731-2739.
[21]曹丹丹,路浩,赵宝玉,等.变异黄芪内生真菌种群分布特征[J].畜牧兽医学报,2014,45(12):2057-2066.CAO D D,LU H,ZHAO B Y,et al.The species distribution characteristics of endophyte in Astragalus variabilis[J].Acta Veterinaria et Zootechnica Sinica,2014,45(12):2057-2066.(in Chinese)
[22]曹丹丹,路浩,李小蒙,等.茎直黄芪内生真菌分离鉴定及其多样性分析[J].草地学报,2015,23(6):1252-1258.CAO D D,LU H,LI X M,et al.Solation,identification and diversity analysis of fungal endophyte in Astragalus strictus[J].Acta Agrestia Sinica,2015,23(6):1252-1258.(in Chinese)
[23]俞大紱.中国镰刀菌属(Fusarium)菌种的初步名录[J].植物病理学报,1955,1(1):1-18.YU D F.A preliminary list of Fusariumin China[J].Acta Phytopathologica Sinica,1955,1(1):1-18.(in Chinese)
[24]唐岚,江厚利,王义勋,等.链格孢属真菌分类研究进展[J].湖北林业科技,2013,42(4):47-49,83.TANG L,JIANG H L,WANG Y X,et al.Advance on taxonomic studies of the genus Alternaria fungi[J].Hubei Forestry Science and Technology,2013,42(4):47-49,83.(in Chinese)