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Simultaneous detection and discrimination of Pratylenchus and Meloidogyne species in Japanese fields using group-specific primers and denaturing gradient gel electrophoresis

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Abstract

We developed a simultaneous detection and discrimination method for the main Pratylenchus and Meloidogyne species of nematodes inhabiting Japanese fields. The method consists of a single polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) process using group-specific primers (SNem 1–4) constructed from the 18S rDNA region with high specificity toward these nematodes. Optimal electrophoresis conditions to differentiate PCR products of each species were 60 °C at a constant voltage of 70 V for 17 h using DGGE gel, with a 6 % acrylamide concentration and a denaturant gradient ranging from 25 to 41 %. Differentiation of most species was achieved according to their main band positions. Only bands derived from the Pratylenchus and Meloidogyne species were present in the DGGE profiles of many nematode communities from various locations in Japan, so we concluded that these primers were sufficiently specific to the targeted nematodes. Detection sensitivity was so high that we could detect one second-stage juvenile of Pratylenchus species among 1,000 free-living nematodes using a single PCR step (35 cycles). Even when two or more target species coexisted in a community, we could usually detect and distinguish them accurately. Most target species formed some minor bands in addition to the primary band. Any interference of the minor bands with the main bands of other target species was overcome using optimal denaturing gradient conditions for the electrophoresis.

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References

  • Blaxter ML, De Ley PD, Garey JR, Liu LX, Scheldeman P, Vierstraete A, Vanfleteren JR, Mackey LY, Dorris M, Frisse LM, Vida JT, Thomas WK (1998) A molecular evolutionary framework for the phylum Nematoda. Nature 392:71–75

    Article  CAS  PubMed  Google Scholar 

  • Blok VC, Powers TO (2009) Biochemical and molecular identification. In: Perry RN, Moens M, Starr JL (eds) Root-knot nematodes. CAB International, Wallingford, pp 98–118

    Chapter  Google Scholar 

  • Blok VC, Phillips MS, Fargette M (1997) Comparison of sequences from the ribosomal DNA intergenic region of Meloidogyne mayaguensis and other major tropical root-knot nematodes. J Nematol 29:16–22

    PubMed Central  CAS  PubMed  Google Scholar 

  • Castillo P, Vovlas N (2007) Biochemical and molecular approaches in Pratylenchus diagnosis. Pratylenchus (Nematoda: Pratylenchidae): diagnosis, biology, pathogenicity and management. Brill Academic Publishers, Leiden, pp 295–303

    Chapter  Google Scholar 

  • Cook AA, Bhadury P, Debenham NJ, Meldal BHM, Blaxter ML, Smerdon GR, Austen MC, Lambshead PJD, Rogers AD (2005) Denaturing gradient gel electrophoresis (DGGE) as a tool for identification of marine nematodes. Mar Ecol Prog Ser 291:103–113

    Article  CAS  Google Scholar 

  • Dar SA, Kuenen JG, Muyzer G (2005) Nested PCR-denaturing gradient gel electrophoresis approach to determine the diversity of sulfate-reducing bacteria in complex microbial communities. Appl Environ Microbiol 71:2325–2330

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Foucher ALJL, Bongers T, Noble LR, Wilson MJ (2004) Assessment of nematode biodiversity using DGGE of 18S rDNA following extraction of nematodes from soil. Soil Biol Biochem 36:2027–2032

    Article  CAS  Google Scholar 

  • Hu MX, Zhuo K, Liao JL (2011) Multiplex PCR for the simultaneous identification and detection of Meloidogyne incognita, M. enterolobii, and M. javanica using DNA extracted directly from individual galls. Phytopathology 101:1270–1277

    Article  CAS  PubMed  Google Scholar 

  • Iwahori H (2010) Identification and geographical distribution of plant-parasitic nematodes in Kyushu-Okinawa region (in Japanese). Shokubutsu boeki (Plant Prot) 64:239–246

    Google Scholar 

  • Kushida A (2013) Design and evaluation of PCR primers for denaturing gradient gel electrophoresis analysis of plant parasitic and fungivorous nematode communities. Microbes Environ 28:269–274

    Article  PubMed Central  PubMed  Google Scholar 

  • Markoulatos P, Siafakas N, Moncany M (2002) Multiplex polymerase chain reaction: a practical approach. J Clin Lab Anal 16:47–51

    Article  CAS  PubMed  Google Scholar 

  • Mizukubo T (1992a) Morphological and statistical differentiation of Pratylenchus coffeae complex in Japan (Nematoda: Pratylenchidae). Appl Entomol Zool 27:213–224

    Google Scholar 

  • Mizukubo T (1992b) The classification, morphology and distribution of the genus Pratylenchus in Japan (in Japanese). In: Nakasono K (ed) Senchu kenkyu no ayumi (Progress in nematology). The Japanese Nematological Society, Tsukuba, pp 40–47

    Google Scholar 

  • Mizukubo T, Orui Y, Hanada K, Sano Z (2003) Microevolutionary trend in Pratylenchus coffeae sensu stricto (Nematoda: Pratylenchidae): the diversity in PCR-RFLP phenotype, compatibility on host plants and reproductive segregation. Jpn J Nematol 33:57–76

    Article  Google Scholar 

  • Mühling M, Woolven-Allen J, Murrell JC, Joint I (2008) Improved group-specific PCR primers for denaturing gradient gel electrophoresis analysis of the genetic diversity of complex microbial communities. ISME J 2:379–392

    Article  PubMed  Google Scholar 

  • Narabu T (2004) Identification of root-knot nematodes (in Japanese). Shokubutsu boeki (Plant Prot) 8:11–16 (an extra number issue)

    Google Scholar 

  • Oba H, Okada H (2008) Methods for analysis of soil communities by PCR-DGGE (2) nematode community (in Japanese). Tsuchi to biseibutsu (Soil Microorg) 62:69–73

    Google Scholar 

  • Okada H, Oba H (2008) Comparison of nematode community similarities assessed by polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) and by morphological identification. Nematology 10:689–700

    Article  CAS  Google Scholar 

  • Orui Y, Mizukubo T (1999) Discrimination of seven Pratylenchus species (Nematoda: Pratylenchidae) in Japan by PCR-RFLP analysis. Appl Entomol Zool 34:205–211

    CAS  Google Scholar 

  • Powers TO, Todd TC, Burnell AM, Murray PCB, Fleming CC, Szalanski AL, Adams BA, Harris TS (1997) The rDNA internal transcribed spacer region as a taxonomic marker for nematodes. J Nematol 29:441–450

    PubMed Central  CAS  PubMed  Google Scholar 

  • Saeki Y, Kawano E, Yamashita C, Akao S, Nagatomo Y (2003) Detection of plant parasitic nematodes, Meloidogyne incognita and Pratylenchus coffeae by multiplex PCR using specific primers. Soil Sci Plant Nutr 49:291–295

    Article  CAS  Google Scholar 

  • Sato E, Toyota K (2006) Application of PCR-DGGE into community structure analysis of soil nematodes (in Japanese with English summary). Jpn J Soil Sci Plant Nutr 77:157–163

    CAS  Google Scholar 

  • Subbotin SA, Waeyenberge L, Moens M (2013) Molecular systematics. In: Perry RN, Moens M (eds) Plant nematology, 2nd edn. CAB International, Wallingford, pp 40–72

    Google Scholar 

  • Uehara T, Mizukubo T, Kushida A, Momota Y (1998) Identification of Pratylenchus coffeae and P. loosi using specific primers for PCR amplification of ribosomal DNA. Nematologica 44:357–368

    Article  Google Scholar 

  • Uehara T, Kushida A, Momota Y (1999) Rapid and sensitive identification of Pratylenchus spp. using reverse dot blot hybridization. Nematology 1:549–555

    Article  CAS  Google Scholar 

  • Uehara T, Kushida A, Itou K, Narabu T, Momota Y (2005) Discrimination of three cyst-forming nematodes of the genus Globodera (Nematode: Heteroderidae) from Japan based on PCR-RFLP of ribosomal DNA. Appl Entomol Zool 40:537–543

    Article  CAS  Google Scholar 

  • Uesugi K, Iwahori H, Tateishi Y (2009) Distribution of three Pratylenchus species in chrysanthemum fields in the Kyushu-Okinawa region of southern Japan, with notes on their identification based on PCR-RFLP analysis. Nematol Res 39:17–22

    Article  Google Scholar 

  • Wada S, Toyota K, Takada A (2011) Effects of the nematicide imicyafos on soil nematode community structure and damage to radish caused by Pratylenchus penetrans. J Nematol 43:1–6

    PubMed Central  PubMed  Google Scholar 

  • Yamada E, Takakura S (1995) Species and distribution of the root-lesion nematodes Pratylenchus spp. (Nematoda: Tylenchida) found from the cultivated fields in Hokkaido, Japan 2. Geographical distribution and densities in the soil at harvest time of crop (in Japanese with English summary). Bull Hokkaido Pref Agric Exp Stn 69:19–29

    Google Scholar 

Download references

Acknowledgments

The authors are grateful to Drs. H. Iwahori (NARO/KARC), T. Mizukubo (NARO/ARC), T. Narabu (NARO/HARC), T. Uehara (NARO/ARC), and K. Uesugi (NARO/KARC) for providing nematode isolates and to Drs. H. Iwahori, M. Maeda (Niigata Agricultural Research Institute), T. Mizukoshi (HRO/Donan Agricultural Experiment Station), H. Sekiguchi (NARO/WARC), T. Suzuki (NARO/KARC), and N. Yamagishi (Nagano Vegetable and Ornamental Crops Experiment Station) for providing field soil samples.

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Authors and Affiliations

  1. Upland Farming Research Division, NARO Hokkaido Agricultural Research Center, Shinsei-minami 9-4, Memuro-cho, Kasai-gun, Hokkaido, 082-0071, Japan

    Atsuhiko Kushida

  2. Graduate School of Agriculture, Hokkaido University, Kita 9 Nishi 9, Kita-ku, Sapporo, Hokkaido, 060-8589, Japan

    Atsuhiko Kushida & Norio Kondo

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  1. Atsuhiko Kushida
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  2. Norio Kondo
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Correspondence to Atsuhiko Kushida.

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10327_2014_572_MOESM1_ESM.jpg

Supplementary Fig. 1 Denaturing gradient gel electrophoresis (DGGE) profiles of nematode communities from various districts (community numbers 23–36) using 25–41% denaturant gradient. Sample locations and results of microscopic examination of each community are given in Table 2. M: DGGE markers of the main species (JPEG 274 kb)

10327_2014_572_MOESM2_ESM.jpg

Supplementary Fig. 2 Denaturing gradient gel electrophoresis (DGGE) profile of Helicotylenchus dihystera using 25–41% denaturant gradient. M: DGGE markers of the main species (JPEG 46 kb)

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Kushida, A., Kondo, N. Simultaneous detection and discrimination of Pratylenchus and Meloidogyne species in Japanese fields using group-specific primers and denaturing gradient gel electrophoresis. J Gen Plant Pathol 81, 151–161 (2015). https://doi.org/10.1007/s10327-014-0572-9

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  • DOI: https://doi.org/10.1007/s10327-014-0572-9

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