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Preparation and characterization of polyclonal antibody against resting spores of Olpidium virulentus, fungal vector of lettuce big-vein disease

  • Viral and Viroid Diseases
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Abstract

Research on Olpidium virulentus, the vector of the serious lettuce big-vein disease, is difficult because its resting spores persist in soil and retain virus for years and the fungus is not culturable. We developed protocols to obtain purified, viable resting spores from infected roots using an enzymatic treatment and two-step density gradient centrifugation and to generate a polyclonal antibody against these spores. The antibody was species-specific in a direct immunostaining assay and in western blots produced two specific bands (ca. 30.5, 29.0 kDa) against resting spores. The detection limit for resting spores in an indirect ELISA was 200 spores/100 μL.

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References

  • Arie T, Namba S, Yamashita S, Doi Y (1988) Detection of resting spores of Plasmodiophora brassicae Woron. from soil and root by fluorescent-antibody technique (in Japanese with English abstract). Ann Phytopathol Soc Jpn 54:242–245

    Article  Google Scholar 

  • Campbell RN (1962) Relationship between the Lettuce big-vein virus and its vector, Olpidium brassicae. Nature 195:675–677

    Article  PubMed  CAS  Google Scholar 

  • Campbell RN (1985) Longevity of Olpidium brassicae in air-dry soil and the persistence of the lettuce big-vein agent. Can J Bot 63:2288–2289

    Article  Google Scholar 

  • Campbell RN, Grogan RG (1964) Acquisition and transmission of Lettuce big-vein virus by Olpidium brassicae. Phytopathology 54:681–690

    Google Scholar 

  • Delfosse P, Reddy AS, Legrève A, Thirumala Devi K, Abdurahman MD, Maraite H, Reddy DVR (2000) Serological methods for detection of Polymyxa graminis, an obligate root parasite and vector of plant viruses. Phytopathology 90:537–545

    Article  PubMed  CAS  Google Scholar 

  • Grogan RG, Zink FW, Hewitt WB, Kimble KA (1958) The association of Olpidium with the big-vein disease of lettuce. Phytopathology 48:292–297

    Google Scholar 

  • Gugerli P (1984) Isopycnic centrifugation of plant viruses in Nycodemz® density gradients. J Virol Methods 9:249–258

    Article  PubMed  CAS  Google Scholar 

  • Harrison JG, Rees EA, Barker H, Lowe R (1993) Detection of spore balls of Spongospora subterranea on potato tubers by enzyme-linked immunosorbent assay. Plant Pathol 42:181–186

    Article  CAS  Google Scholar 

  • Iwaki M, Nakano A, Iemura H, Tochihara H (1978) The occurrence of lettuce big vein disease and its soil transmission in Japan (in Japanese with English abstract). Ann Phytopath Soc Jpn 44:578–584

    Article  Google Scholar 

  • Kageyama K, Komatsu T, Suga H (2003) Refined PCR protocol for detection plant pathogens in soil. J Gen Plant Pathol 69:153–160

    CAS  Google Scholar 

  • Kassanis B, MacFarlane I (1965) Interaction of virus strain, fungus isolate, and host species in the transmission of tobacco necrosis virus. Virology 26:603–612

    Article  PubMed  CAS  Google Scholar 

  • Koganezawa H, Sasaya T, Nomiyama K, Morikawa T (2003) Evaluation of host specificity of a single-sporangial isolate of Olpidium brassicae based on zoospore release in a sea-sand culture (in Japanese). Proc Assoc Pl Protec Shikoku 38:23–28

    Google Scholar 

  • Koganezawa H, Takayama T, Sasaya T (2004) Difference in resting-spore formation between crucifer and noncrucifer strains of Olpidium brassicae sensu lato (in Japanese with English abstract). Jpn J Phytopathol 70:307–313

    Article  Google Scholar 

  • Lot H, Campbell RN, Souche S, Milne RG, Roggero P (2002) Transmission by Olpidium brassicae of Mirafiori lettuce virus and Lettuce big-vein virus, and their roles in lettuce big-vein etiology. Phytopathology 92:288–293

    Article  PubMed  Google Scholar 

  • Morikawa T (2005) Handling techniques in preparation of Olpidium culture (in Japanese). Shokubutsu Boeki [Plant Protection] 59:78–81

    Google Scholar 

  • Morikawa T, Chikuo Y, Natsuaki T (1997) Transmission of tulip mild mottle mosaic by Olpidium brassicae (abstract in Japanese). Ann Phytopathl Soc Jpn 63:504

    Google Scholar 

  • Nomiyama K, Sasaya T, Ishikawa K (2004) Thermal effect of infested soil on lettuce big-vein disease (abstract in Japanese). Jpn J Phytopathol 70:239

    Google Scholar 

  • Okubara PA, Schroeder KL, Paulitz TC (2005) Real-time polymerase chain reaction: applications to studies on soilborne pathogens. Can J Plant Pathol 27:300–313

    Article  CAS  Google Scholar 

  • Orihara S, Yamamoto T (1998) Detection of resting spores of Plasmodiophora brassicae from soil and plant tissues by enzyme immunoassay. Ann Phytopathol Soc Jpn 64:569–573

    Article  Google Scholar 

  • Roggero P, Ciuffo M, Vaira AM, Accotto GP, Masenga V, Milne RG (2000) An Ophiovirus isolated from lettuce with big-vein symptoms. Arch Virol 145:2629–2642

    Article  PubMed  CAS  Google Scholar 

  • Sasaya T, Fujii H, Ishikawa K, Koganezawa H (2008) Further evidence of Mirafiori lettuce big-vein virus but not of Lettuce big-vein associated virus with big-vein disease in lettuce. Phytopathology 98:464–468

    Article  PubMed  CAS  Google Scholar 

  • Sasaya T, Kognezawa H (2006) Molecular analysis and virus transmission tests place Olpidium virulentus, a vector of Mirafiori lettuce big-vein virus and tobacco stunt virus, as a distinct species rather than a strain of Olpidium brassicae. J Gen Plant Pathol 72:20–25

    Article  CAS  Google Scholar 

  • Takeuchi S, Hikichi Y, Kawada Y, Okuno T (1999) Direct immunostaining assay, a new simplified technique for detection tobamoviruses from seeds of green pepper (Capsicum annuum L.). Ann Phytopathol Soc Jpn 65:189–191

    Article  Google Scholar 

  • Teakle DS (1962) Transmission of tobacco necrosis virus by a fungus, Olpidium brassicae. Virology 18:224–231

    Article  PubMed  CAS  Google Scholar 

  • Teakle DS, Hiruki C (1964) Vector specificity in Olpidium. Virology 24:539–544

    Article  Google Scholar 

  • Verbeek M, Dullemans AM, van Bekkum PJ, van der Vlugt RAA (2012) Evidence for Lettuce big-vein associated virus as the causal agent of a syndrome of necrotic rings and spots in lettuce. Plant Pathol. doi:10.1111/j.1365-3059.2012.02645.x

  • Wakeham AJ, White JG (1996) Serological detection in soil of Plasmodiophora brassicae resting spores. Physiol Mol Plant Pathol 48:289–303

    Article  Google Scholar 

  • Walsh JA, Merz U, Harrison JG (1996) Serological detection of spore balls of Spongospora subterranea and quantification in soil. Plant Pathol 45:884–895

    Article  Google Scholar 

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Acknowledgments

We thank Dr. Kanda and Dr. Tsuda (National Agricultural Research Center) for providing isolate NA of O. bornovanus. We also thank Dr. Takehara and Dr. Sekiguchi (Western Region Agricultural Research Center) for critical reading of the manuscript.

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Author notes

  1. Takahide Sasaya

    Present address: Kyushu Okinawa Agricultural Research Center, National Agriculture and Food Research Organization, Koshi, Kumamoto, 861-1192, Japan

Authors and Affiliations

  1. Western Region Agricultural Research Center, National Agriculture and Food Research Organization, Fukuyama, Hiroshima, 721-8514, Japan

    Koji Nomiyama & Hideki Osaki

  2. National Agricultural Research Center, Tsukuba, Ibaraki, 305-8666, Japan

    Takahide Sasaya

  3. Western Region Agricultural Research Center, National Agriculture and Food Research Organization, Zentsuji, Kagawa, 765-8508, Japan

    Koichi Ishikawa

Authors
  1. Koji Nomiyama
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  2. Hideki Osaki
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  3. Takahide Sasaya
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  4. Koichi Ishikawa
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Correspondence to Koji Nomiyama.

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Nomiyama, K., Osaki, H., Sasaya, T. et al. Preparation and characterization of polyclonal antibody against resting spores of Olpidium virulentus, fungal vector of lettuce big-vein disease. J Gen Plant Pathol 79, 64–68 (2013). https://doi.org/10.1007/s10327-012-0424-4

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  • DOI: https://doi.org/10.1007/s10327-012-0424-4

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