较小拟毛刺线虫不同群体的形态及分子特征比较研究
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摘要
毛刺类线虫系一类重要的植物外寄生线虫,其内包括一些可传播植物病毒的种类。本研究基于形态学和rDNA分子特征从我国海南儋州、云南呈贡和昆明、福建厦门等植物根围土壤样品中分离鉴定出8个较小拟毛刺线虫(Paratrichodorus minor)群体;通过对种群数量较大的海南儋州群体与福建厦门群体的形态及测量值比较,发现不同地理群体间主要形态测量值存在一定差异;各群体18S RNA区、r DNA-ITS1区、r DNA-ITS2区和28S RNA基因中D2D3区序列与Gen Bank已登录的较小拟毛刺线虫不同群体相应序列(AJ438053、AJ438054及AJ438056;KJ934126;JN123380和JN123381;JN123396和KJ513001)相似度分别介于99.1%~99.9%、95.8%~96.3%、99.5%~99.8%和99.4%~99.9%。对上述较小拟毛刺线虫群体r DNA-ITS1区、r DNA-ITS2区和28S RNA基因中D2D3区序列比较和酶切分析显示,该3段序列在较小拟毛刺线虫种内群体间都较为保守。其中28S RNA基因中D2D3区序列在种内群体稳定,种间群体有明显的差异,系毛刺类线虫鉴定一较好的分子靶标。有关结果对较小拟毛刺线虫的鉴定及鉴别有直接的指导意义。
Trichodorids constitute an important group of ectoparasitic nematodes of plant,in which some of the species can transmit various viruses to economically important crops. In this study,8 Paratrichodorus populations from the rhizosphere of eggplant,chinaberry,lettuce,peach,pear,walnut and longan in Hainan( Danzhou),Yunnan( Chenggong and Kunming),and Fujian( Xiamen) regions of China were identified as P. minor based on the morphological and rDNA characteristics. Morphological and morphometrical comparisons of Hainan( Danzhou) and Fujian( Xiamen) populations showed certain intra-species morphometric variations.The sequence similarity of 18 S RNA gene,rDNA-ITS1,rDNA-ITS2 and D2 D3 region of 28 S RNA gene among P. minor populations used in this study and those submitted in GenBank( AJ438053,AJ438054 and AJ438056;KJ934126; N123380 and JN123381; JN123396 and KJ513001) ranged in 99. 1%-99. 9%,95. 8%-96. 3%,99.5%-99.8% and 99.4%-99.9%,respectively. PCR-RFLP of ITS1,ITS2 region of rDNA and D2 D3 region of28 S RNA gene revealed the consistency among these intra-species populations. D2 D3 region of 28 S RNA gene sequences are stable within the intra-species populations and have clear differences among inter-species of trichodorid nematodes which is a good molecular marker for identification of trichodorid nematodes. These results indicate that rDNA sequences can provide useful information for molecular identification of P. minor.
Trichodorids constitute an important group of ectoparasitic nematodes of plant,in which some of the species can transmit various viruses to economically important crops. In this study,8 Paratrichodorus populations from the rhizosphere of eggplant,chinaberry,lettuce,peach,pear,walnut and longan in Hainan( Danzhou),Yunnan( Chenggong and Kunming),and Fujian( Xiamen) regions of China were identified as P. minor based on the morphological and rDNA characteristics. Morphological and morphometrical comparisons of Hainan( Danzhou) and Fujian( Xiamen) populations showed certain intra-species morphometric variations.The sequence similarity of 18 S RNA gene,rDNA-ITS1,rDNA-ITS2 and D2 D3 region of 28 S RNA gene among P. minor populations used in this study and those submitted in GenBank( AJ438053,AJ438054 and AJ438056;KJ934126; N123380 and JN123381; JN123396 and KJ513001) ranged in 99. 1%-99. 9%,95. 8%-96. 3%,99.5%-99.8% and 99.4%-99.9%,respectively. PCR-RFLP of ITS1,ITS2 region of rDNA and D2 D3 region of28 S RNA gene revealed the consistency among these intra-species populations. D2 D3 region of 28 S RNA gene sequences are stable within the intra-species populations and have clear differences among inter-species of trichodorid nematodes which is a good molecular marker for identification of trichodorid nematodes. These results indicate that rDNA sequences can provide useful information for molecular identification of P. minor.
引文
[1]Decraemer W.The family Trichodoridae:stubby root and virus vector nematodes[M].Dordrecht,Boston&London:Kluw er Academic Publishers,1995.
[2]Marais M,Botha-Greeff M S.A new species of Paratrichodorus Siddiqi(Nemata:Trichodoridae)from Namibia,w ith a note on males of P.minor(Colbran)Siddiqi from South Africa[J].African Plant Protection,1997,3(1):17-22.
[3]Almeida M T M de,Santos M S N de A,Abrantes I M de O,et al.Paratrichodorus divergens sp.n.,a new potential virus vector of tobacco rattle virus and additional observations on P.hispanus Roca&Arias,1986from Portugal(Nematoda:Trichodoridae)[J].Nematology,2005,7(3):343-361.
[4]Colbran R C.Studies of plant and soil nematodes.1.Tw o new species from Queensland[J].Queensland Journal of Agricultural Science,1956,13:123-126.
[5]Siddiqi M R.Systematics of the genus Trichodorus Cobb,1913(Nematoda:Dorylaimida),w ith descriptions of three new species[J].Nematologica,1974,19(3):259-278.
[6]Allen M W.A review of the nematode genus Trichodorus w ith descriptions of ten new species[J].Nematologica,1957,2(1):32-62.
[7]Heyns J.Paratrichodorus christiei[J].C.I.H.Descriptions of Plant-parasitic Nematodes,1975,5:69-72.
[8]Mamiya Y.Descriptive notes on three species of Trichodorus(Dorylaimida:Trichodoridae)from forest Nurseries in Japan[J].Applied Entomology and Zoology,1967,2(2):61-68.
[9]Hooper D J.Paratrichodorus(Nanidorus)minor[J].C.I.H.Descriptions of Plant-Parasitic Nematodes,1977,7:103-105.
[10]Walkinshaw C H,Griffin G D,Larson R H.Trichodorus christiei as a vector of potato corky ringspot(tobacco rattle)virus[J].Phytopathology,1961,51(11):806-807.
[11]Ayala A,Allen M.Transmission of the Californian Tobacco rattle virus by three species of the nematode genus Trichodorus[J].Journal of Agriculture of the University of Puerto Rico,1968,52(2):101-125.
[12]Komuro Y,Yoshino M,Ichinohe M.Tobacco rattle virus isolated from aster show ing ringspot syndrome and its transmission by Trichodorus minor Colbran(in Japanese)[C].Japan:Annals of the Phytopathological Society of Japan,1970:17-36.
[13]Salomao T A.Soil transmission of artichoke yellow band virus[C].Italy,2nd Congresso Internazionale di Studi sul Carciofo,1973:831-854.
[14]Xie H,Feng Z X.Identification of the species of genus Paratrichodorus Siddiqi,1974(Nemata:Trichodoridae)from Hongkong(in Chinese)[J].Journal of South China Agricultural(华南农业大学学报),1996,17(2):70-73.
[15]Decraemer W,Baujard P.A polytomous key the identification of species of the family Trichodoridae Thorne,1935(Nematoda:Triplonchida)[J].Fundamental and Applied Nematology,1998,21(1):37-62.
[16]Decraemer W,Baujard P.Additions and corrections to:Decraemer:the family Trichodoridae[J].Fundamental and Applied Nematology,1998,21(2):207-212.
[17]Zhang S S,Chen Y B.Studies on nematodes of bayberry(Myrica rubra)(in Chinese)[J].Journal of Fujian Agricultural University(Natural Sciences Edition)(福建农林大学学报:自然科学版),1994,23(2):172-177.
[18]Lin L F,Li W F,Hu X Q,et al.Identification of the nematodes causing the stubby root disease in rhizospheric soils of Hydrangea macrophylla(in Chinese)[J].Plant Protection(植物保护),2009,35(2):131-133.
[19]Du Y,Jiang X L,Li S G,et al.Description on three Paratrichodorids nematodes in Yunnan(in Chinese)[J].Plant Quarantine(植物检疫),2010,24(5):42-45.
[20]Brown D F J,Boag B.An examibation of methods used to extract virus-vector nematodes(Nematoda:Longidoriae and Trichodoridae)from soil samples[J].Nematologia Mediterranea,1988,16(1):93-99.
[21]Jiang L,Li X,Zheng J.First record of Bursaphelenchua rainulfi on pine trees from eastern China and its phylogenetic relationship w ith intro-genus species[J].Journal of Zhejiang University:Science B,2007,8(5):345-351.
[22]Boutsika K,Blok V C,Phillips M S,et al.Confirmation of the synonymy of Paratrichodorus christiei(Allen,1957)Siddiqi,1974 w ith P.minor(Colbran,1956)Siddiqi,1974(Nematoda:Triplonchida)based on sequence data obtained for the ribosomal DNA 18S gene[J].Nematology,2004,6(1):145-151.
[23]Waite I S,O’Donnell A G,Harrison A,et al.Design and evaluation of nematode 18S r DNA primers for PCR and denaturing gradient gel electrophoresis(DGGE)of soil community DNA[J].Soil Biology and Biochemistry,2003,35(9):1165-1173.
[24]Vrain T C,Wakarchuk D A,Levesque A C,et al.Intraspecific r DNA restriction fragment length polymorphism in the Xiphinema americanum group[J].Fundamental and Applied Nematology,1992,15(6):563-573.
[25]Boutsika K,Brown D J F,Phillips M S,et al.Molecular characterisation of the ribosomal DNA of Paratrichodorus macrostylus,P.pachydermus,Trichodorus primitivus and T.similis(Nematoda:Trichodoridae)[J].Nematology,2004,6(5):641-654.
[26]De Ley P,Félix M A,Frisse L M,et al.Molecular and morphological characterization of tw o reproductively isolated species w ith mirror-image anatomy(Nematoda:Cephalobidae)[J].Nematology,1999,1(6):591-612.
[27]Hooper D J.Three new species of Trichodorus(Nematoda:Dorylaimoidea)and observations on T.minor Colbran,1956[J].Nematologica,1962,7(4):273-280.
[28]Bird G W,Mai W F.Morphometric and allometric variations of Trichodorus christiei[J].Nematologica,1967,13(4):617-632.
[29]Chaves E J.Observation on plant parasitic nematodes from Argentina[D].Gent:Rijkuniverisiteit Gent,1984.
[30]Zheng J W,Li D B.Recent progress on molecular taxonomy and identification of plant nematodes(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),1998,28(1):1-4.
[2]Marais M,Botha-Greeff M S.A new species of Paratrichodorus Siddiqi(Nemata:Trichodoridae)from Namibia,w ith a note on males of P.minor(Colbran)Siddiqi from South Africa[J].African Plant Protection,1997,3(1):17-22.
[3]Almeida M T M de,Santos M S N de A,Abrantes I M de O,et al.Paratrichodorus divergens sp.n.,a new potential virus vector of tobacco rattle virus and additional observations on P.hispanus Roca&Arias,1986from Portugal(Nematoda:Trichodoridae)[J].Nematology,2005,7(3):343-361.
[4]Colbran R C.Studies of plant and soil nematodes.1.Tw o new species from Queensland[J].Queensland Journal of Agricultural Science,1956,13:123-126.
[5]Siddiqi M R.Systematics of the genus Trichodorus Cobb,1913(Nematoda:Dorylaimida),w ith descriptions of three new species[J].Nematologica,1974,19(3):259-278.
[6]Allen M W.A review of the nematode genus Trichodorus w ith descriptions of ten new species[J].Nematologica,1957,2(1):32-62.
[7]Heyns J.Paratrichodorus christiei[J].C.I.H.Descriptions of Plant-parasitic Nematodes,1975,5:69-72.
[8]Mamiya Y.Descriptive notes on three species of Trichodorus(Dorylaimida:Trichodoridae)from forest Nurseries in Japan[J].Applied Entomology and Zoology,1967,2(2):61-68.
[9]Hooper D J.Paratrichodorus(Nanidorus)minor[J].C.I.H.Descriptions of Plant-Parasitic Nematodes,1977,7:103-105.
[10]Walkinshaw C H,Griffin G D,Larson R H.Trichodorus christiei as a vector of potato corky ringspot(tobacco rattle)virus[J].Phytopathology,1961,51(11):806-807.
[11]Ayala A,Allen M.Transmission of the Californian Tobacco rattle virus by three species of the nematode genus Trichodorus[J].Journal of Agriculture of the University of Puerto Rico,1968,52(2):101-125.
[12]Komuro Y,Yoshino M,Ichinohe M.Tobacco rattle virus isolated from aster show ing ringspot syndrome and its transmission by Trichodorus minor Colbran(in Japanese)[C].Japan:Annals of the Phytopathological Society of Japan,1970:17-36.
[13]Salomao T A.Soil transmission of artichoke yellow band virus[C].Italy,2nd Congresso Internazionale di Studi sul Carciofo,1973:831-854.
[14]Xie H,Feng Z X.Identification of the species of genus Paratrichodorus Siddiqi,1974(Nemata:Trichodoridae)from Hongkong(in Chinese)[J].Journal of South China Agricultural(华南农业大学学报),1996,17(2):70-73.
[15]Decraemer W,Baujard P.A polytomous key the identification of species of the family Trichodoridae Thorne,1935(Nematoda:Triplonchida)[J].Fundamental and Applied Nematology,1998,21(1):37-62.
[16]Decraemer W,Baujard P.Additions and corrections to:Decraemer:the family Trichodoridae[J].Fundamental and Applied Nematology,1998,21(2):207-212.
[17]Zhang S S,Chen Y B.Studies on nematodes of bayberry(Myrica rubra)(in Chinese)[J].Journal of Fujian Agricultural University(Natural Sciences Edition)(福建农林大学学报:自然科学版),1994,23(2):172-177.
[18]Lin L F,Li W F,Hu X Q,et al.Identification of the nematodes causing the stubby root disease in rhizospheric soils of Hydrangea macrophylla(in Chinese)[J].Plant Protection(植物保护),2009,35(2):131-133.
[19]Du Y,Jiang X L,Li S G,et al.Description on three Paratrichodorids nematodes in Yunnan(in Chinese)[J].Plant Quarantine(植物检疫),2010,24(5):42-45.
[20]Brown D F J,Boag B.An examibation of methods used to extract virus-vector nematodes(Nematoda:Longidoriae and Trichodoridae)from soil samples[J].Nematologia Mediterranea,1988,16(1):93-99.
[21]Jiang L,Li X,Zheng J.First record of Bursaphelenchua rainulfi on pine trees from eastern China and its phylogenetic relationship w ith intro-genus species[J].Journal of Zhejiang University:Science B,2007,8(5):345-351.
[22]Boutsika K,Blok V C,Phillips M S,et al.Confirmation of the synonymy of Paratrichodorus christiei(Allen,1957)Siddiqi,1974 w ith P.minor(Colbran,1956)Siddiqi,1974(Nematoda:Triplonchida)based on sequence data obtained for the ribosomal DNA 18S gene[J].Nematology,2004,6(1):145-151.
[23]Waite I S,O’Donnell A G,Harrison A,et al.Design and evaluation of nematode 18S r DNA primers for PCR and denaturing gradient gel electrophoresis(DGGE)of soil community DNA[J].Soil Biology and Biochemistry,2003,35(9):1165-1173.
[24]Vrain T C,Wakarchuk D A,Levesque A C,et al.Intraspecific r DNA restriction fragment length polymorphism in the Xiphinema americanum group[J].Fundamental and Applied Nematology,1992,15(6):563-573.
[25]Boutsika K,Brown D J F,Phillips M S,et al.Molecular characterisation of the ribosomal DNA of Paratrichodorus macrostylus,P.pachydermus,Trichodorus primitivus and T.similis(Nematoda:Trichodoridae)[J].Nematology,2004,6(5):641-654.
[26]De Ley P,Félix M A,Frisse L M,et al.Molecular and morphological characterization of tw o reproductively isolated species w ith mirror-image anatomy(Nematoda:Cephalobidae)[J].Nematology,1999,1(6):591-612.
[27]Hooper D J.Three new species of Trichodorus(Nematoda:Dorylaimoidea)and observations on T.minor Colbran,1956[J].Nematologica,1962,7(4):273-280.
[28]Bird G W,Mai W F.Morphometric and allometric variations of Trichodorus christiei[J].Nematologica,1967,13(4):617-632.
[29]Chaves E J.Observation on plant parasitic nematodes from Argentina[D].Gent:Rijkuniverisiteit Gent,1984.
[30]Zheng J W,Li D B.Recent progress on molecular taxonomy and identification of plant nematodes(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),1998,28(1):1-4.