西藏自治区色季拉山高寒草甸牧草根腐线虫的鉴定
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摘要
在西藏自治区色季拉山高寒草甸土壤线虫群落调查期间,从高山嵩草(Kobresia pygmaea)根际土壤中分离得到一种根腐线虫。通过形态特征观察和分子序列比对,将其鉴定为刻痕短体线虫(Pratylenchus crenatus)。其主要特征:唇环3个,口针长15.6～16.2μm,口针基部球前缘略凹,侧线6条,V值为82.5～83.2,尾圆柱形,末端环纹清晰。对该线虫rDNA的3个区段(18S部分序列、28SD2D3区和ITS区)进行特异性扩增和测序,分别得到902、774和1 013bp的序列。将所获得的序列在GenBank数据库上进行BLAST比对,结果显示:此线虫群体与刻痕短体线虫群体的相似度分别为97.4%～99.8%、97.4%～97.8%和90.2%～99.8%。贝叶斯推断法构建的系统进化树显示:本研究的线虫群体与刻痕短体线虫聚为高度支持的分支,进一步确定此线虫群体为刻痕短体线虫。本研究获得刻痕短体线虫中国群体的分子数据。该刻痕短体线虫是西藏自治区的新纪录种。高山嵩草是刻痕短体线虫的新寄主。
During the survey of soil nematodes in an alpine cold meadow of Color Gerahs Mountain,Tibet,a species of root lesion nematode was obtained from Kobresia pygmaearhizosphere soil.Based on morphological characterization and molecular analysis,the species was identified as Pratylenchus crenatus.The nematode was characterized by a flatten lip with 3 annuli,stylet length of 15.6-16.2μm,stylet knobs with concave anterior face,lateral fields with 6 lines,vulva position at 82.5%-83.2% of body length,and a cylindrical tail with distinct striated terminus.Amplification of 3 rDNA segments(partial18 S,28 SD2 D3 and ITS sequences)generated PCR fragments with sizes of 902 bp,774 bp and 1 013 bp,respectively.The similarities between the 3 rDNA segments in this study and those deposited in the GenBank database were 97.4%-99.8%,97.4%-97.8%and 90.2%-99.8%,respectively.Bayesian inference method was used to construct the phylogenetic relationship of P.crenatus in China and other regions,as well as other associated species.All sequences of P.crenatus including that of this study formed a highly supported clade,which further confirmed that this population was P.crenatus.Molecular information of Chinese P.crenatus population was obtained in this study.P.crenatus was a new record in Tibet,southwest China,and K.pygmaea was founded to be a new host plant of P.crenatus.
During the survey of soil nematodes in an alpine cold meadow of Color Gerahs Mountain,Tibet,a species of root lesion nematode was obtained from Kobresia pygmaearhizosphere soil.Based on morphological characterization and molecular analysis,the species was identified as Pratylenchus crenatus.The nematode was characterized by a flatten lip with 3 annuli,stylet length of 15.6-16.2μm,stylet knobs with concave anterior face,lateral fields with 6 lines,vulva position at 82.5%-83.2% of body length,and a cylindrical tail with distinct striated terminus.Amplification of 3 rDNA segments(partial18 S,28 SD2 D3 and ITS sequences)generated PCR fragments with sizes of 902 bp,774 bp and 1 013 bp,respectively.The similarities between the 3 rDNA segments in this study and those deposited in the GenBank database were 97.4%-99.8%,97.4%-97.8%and 90.2%-99.8%,respectively.Bayesian inference method was used to construct the phylogenetic relationship of P.crenatus in China and other regions,as well as other associated species.All sequences of P.crenatus including that of this study formed a highly supported clade,which further confirmed that this population was P.crenatus.Molecular information of Chinese P.crenatus population was obtained in this study.P.crenatus was a new record in Tibet,southwest China,and K.pygmaea was founded to be a new host plant of P.crenatus.
引文
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[2]CASTILLO P,VOVLAS N.Pratylenchus(Nematoda:Pratylenchidae):diagnosis,biology,pathogenicity and management[M].Leiden:Brill,2007.
[3]杨意伯,张绍升.番石榴根部短尾短体线虫的鉴定[J].热带作物学报,2013,34(8):1557-1563.
[4]章淑玲,王宏毅,金亮,等.寄生台湾春兰的短体线虫种类鉴定[J].热带作物学报,2013,34(12):2463-2466.
[5]刘星彤,林柏荣,廖金铃,等.环介导等温扩增法(LAMP)检测最短尾短体线虫[J].华中农业大学学报,2018,37(6):17-24.
[6]王宏洪,黄少彬,杨晓朱,等.地稔根际根腐线虫的形态和分子鉴定[J].热带作物学报,2018,39(9):1820-1826.
[7]冯志新.植物线虫学[M].北京:中国农业出版社,2001.
[8]LOOF P A A.Taxonomic studies on the genus Pratylenchus(Nematoda)[J].Tijdschrift over plantenziekten,1960,66(2):29-90.
[9]WANG H H,ZHUO K,YE W M,et al.Morphological and molecular charaterisation of Pratylenchus parazeae n.sp.(Nematoda:Pratylenchidae)parasitizing sugarcane in China[J].European journal of plant pathology,2015,143(1):173-191.
[10]MUNDO-OCAMPO M,TROCCOLI A,SUBBOTIN S A,et al.Synonymy of Afenestrata with Heterodera supported by phylogenetics with molecular and morphological characterisation of H.koreanacomb.n.and H.orientalis comb.n.(Tylenchida:Heteroderidae)[J].Nematology,2008,10(5):611-632.
[11]CHIZHOV V N,CHUMAKOVA O A,SUBBOTIN S A,et al.Morphological and molecular characterization of foliar nematodes of the genus Aphelencholdes:A.fragariae and A.ritzemabosi(Nematoda:Aphelenchoididae)from the main botanical garden of the Russian Academy of Sciences[J].Russian journal of nematology,2006,14(2):179-184.
[12]SUBBOTIN S A,STURHAN D,CHIZHOV V N,et al.Phylogenetic analysis of Tylenchida Thorne,1949as inferred from D2and D3expansion fragments of the 28SrRNA gene sequences[J].Nematology,2006,8(3):455-474.
[13]VRAIN T C,WAKARCHUK D A,LEVESQUE A C,et al.Intraspecific rDNA restriction fragment length polymorphism in the Xiphinema americanum group[J].Fundamental and applied nematology,1992,15(6):563-573.
[14]王宏洪,申东,黄少彬,等.竹子根际螺旋线虫中国新记录种---尖尾螺旋线虫Helicotylenchus cuspicaudatus[J].植物保护,2018,44(2):75-80.
[15]SUBBOTIN S A,RAGSDALE E J,MULLENS T,et al.A phylogenetic framework for root lesion nematodes of the genus Pratylenchus(Nematoda):evidence from 18Sand D2-D3expansion segments of 28Sribosomal RNA genes and morphological characters[J].Molecular phylogenetics and evolution,2008,48(2):491-505.
[16]WAEYENBERGE L,VIAENE N,MOENS M.Species-specific duplex PCR for the detection of Pratylenchus penetrans[J].Nematology,2009,11(6):847-857.
[17]DE LUCA F,REYES A,TROCCOLI A,et al.Molecular variability and phylogenetic relationships among different species and populations of Pratylenchus(Nematoda:Pratylenchidae)as inferred from the analysis of the ITS rDNA[J].European journal of plant pathology,2011,130(3):415-426.
[18]TAMURA K,STECHER G,PETERSON D,et al.MEGA6:molecular evolutionary genetics analysis version 6.0[J].Molecular biology and evolution,2013,30(12):2725-2729.
[19]SWOFFORD D L.PAUP*:Phylogenetic analyses using parsimony(and other methods).Version 4b10[M].Sunderland:Sinauer Associates,2002.
[20]POSADA D,CRANDALL K A.Modeltest:testing the model of DNA substitution[J].Bioinformatics,1998,14:817-818.
[21]RONQUIST F,TESLENKO M,MARK P V D,et al.MrBayes3.2:efficient Bayesian phylogenetic inference and model choice across a large model space[J].Systematic biology,2012,61(3):539-542.
[22]PAGE R D.TreeView:an application to display phylogenetic trees on personal computers[J].Computer applications in the biosciences cabios,1996,12(4):357-358.
[23]周银丽,胡先奇,林丽飞,等.云南石榴根际刻痕短体线虫的鉴定[J].中国农学通报,2008,24(11):403-405.
[24]李惠霞,彭焕,彭德良,等.甘肃省高寒草原牧草孢囊线虫的鉴定[J].草业学报,2015,24(8):174-180.
[25]薛会英,罗大庆,于宝政.西藏色季拉山急尖长苞冷杉林土壤线虫群落特征[J].应用生态学报,2012,23(12):3402-3408.
[26]薛会英,罗大庆.藏东南冷杉林采伐迹地土壤线虫群落特征[J].林业科学,2013,49(6):107-114.
[27]陈德来,普布,巴桑,等.西藏拉鲁湿地夏季土壤线虫群落特征[J].动物学杂志,2014,49(5):744-753.
[28]薛会英.西藏北部高寒草甸土壤线虫群落特征研究[D].南京:南京农业大学,2015.
[29]赵文霞,杨宝君.中国植物线虫名录[M].北京:中国林业出版社,2006.
[30]金惺惺.西藏主要耕作区植物线虫的调查与鉴定[D].广州:华南农业大学,2008.
[31]李惠霞,柳永娥,魏庄,等.新疆和西藏发现禾谷孢囊线虫[J].中国线虫学研究,2012(4):164-165.
[32]段玉玺.中国短体科线虫分类的研究[D].沈阳:沈阳农业大学,1994.
[33]李建立,赵洪海,李锡宏,等.湖北省烟草上植物线虫种类鉴定[J].湖北农业科学,2012,51(21):4795-4798.
[34]朱艳.大豆根围线虫生物多样性研究[D].沈阳:沈阳农业大学,2007.
[35]于焦,金惺惺,秦萌,等.西藏农作物短体线虫种类的鉴定和描述[J].华中农业大学学报,2017,36(5):20-24.
[36]江丽辉,边勇,汪万春.短体线虫Pratylenchus spp.非中国种问题探讨[J].中国线虫学研究,2010(3):116-122.