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European Journal of Plant Pathology

, Volume 112, Issue 4, pp 337–347 | Cite as

A PCR-based assay for the detection and identification of Pyrenochaeta lycopersici

  • Alessandro InfantinoEmail author
  • Nicoletta Pucci
Article

Abstract

The isolation of Pyrenochaeta lycopersici, causal agent of corky root of tomato, is difficult because of its slow growth and poor sporulation. Identification is complicated due the existence of two morphologically similar forms, Types 1 and 2, that differ in several physiological and molecular features. For the rapid and unambiguous identification of isolates, two oligonucleotide primer pairs were designed using ITS region sequences. Specific PCR products of 147 and 209 bp were obtained for isolates of Type 1 and Type 2, respectively. Specificity of both primer pairs was verified using several fungal and bacterial species. As little as 0.7 pg of target DNA could be detected with the protocol. A nested PCR procedure was necessary for the detection of the fungus in plant tissue. This technique will be of use in epidemiological studies and in the implementation of control strategies.

Key words

corky-root molecular diagnosis ribosomal DNA sequence analysis tomato 

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References

  1. Altschul, SF, Madden, TL, Schaffer, AA, Zhang, J, Zhang, Z, Miller, W, Lipman, DJ 1997Gapped BLAST and PSI-BLAST: A new generation of protein database search programsNucleic Acids Research2533893402CrossRefPubMedGoogle Scholar
  2. Bridge, PD, Arora, DK 1998Interpretation of PCR methods for species definitionBridge, PDArora, DKReddy, CAElander, RP eds. Application of PCR in Mycology CAB InternationalWallingfordUK6380Google Scholar
  3. Cascone, G, D’Emilio, A, Polizzi, G, Grillo, R 2000Effectiveness of greenhouse soil solarization with different plastic mulches in controlling corky root and root-knot on tomato plantsActa Horticulturae532145150Google Scholar
  4. Cenis, JL 1992Rapid extraction of fungal DNA for PCR amplificationNucleic Acids Research202380PubMedGoogle Scholar
  5. Chen, W, Gray, LE, Kurle, JE, Grau, CR 1999Specific detection of Phialophora gregata and Plectosporium tabacinum in infected soybean plants using polymerase chain reactionMolecular Ecology8871877CrossRefGoogle Scholar
  6. Cullen, DW, Lees, AK, Toth, IK, Duncan, JM 2002Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCRPlant Pathology51281292CrossRefGoogle Scholar
  7. Faggian, R, Bulman, SR, Lawrie, AC, Porter, IJ 1999Specific Polymerase Chain Reaction primers for the detection of Plasmodiophora brassicae in soil and waterPhytopathology89392397Google Scholar
  8. Ghignone, S, Tamietti, G, Girlanda, M 2003Development of specific PCR primers for identification and detection of Rhizopycnis vagumEuropean Journal of Plant Pathology109861870CrossRefGoogle Scholar
  9. Grove, GG, Campbell, RN 1987Host range and survival in soil of Pyrenochaeta lycopersiciPlant Disease71806809Google Scholar
  10. Higgins, D, Thompson, J, Gibson, T, Thompson, JD, Higgins, DG, Gibson, TJ 1994CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choiceNucleic Acids Research2246734680PubMedGoogle Scholar
  11. Hockey, AG, Jeves, TM 1984Isolation and identification of Pyrenochaeta lycopersici, causal agent of tomato brown root rotTransactions of the British Mycological Society82151152Google Scholar
  12. Infantino, A, Di Giambattista, G, Porta-Puglia, A 2000First report of Pyrenochaeta lycopersici on melon in ItalyPetria10195198Google Scholar
  13. Infantino, A, Aragona, M, Brunetti, A, Lahoz, E, Oliva, A, Porta-Puglia, A 2003Molecular and physiological characterization of Italian isolates of Pyrenochaeta lycopersiciMycological Research107707716CrossRefPubMedGoogle Scholar
  14. Innis, MA, Gelfand, DH 1990Optimization of PCRsInnis, MAGelfand, DHSninsky, JJWhite, TJ. eds. PCR Protocols. A Guide to Methods and ApplicationsAcademic PressSan Diego, USA312Google Scholar
  15. Johansson, A, Turner, HC, McKay, GJ, Brown, AE 1998A PCR-based method to distinguish fungi of the rice sheath-blight complex, Rhizoctonia solani, R. oryzae and R. oryzae-sativaeFEMS Microbiology Letters162289294CrossRefPubMedGoogle Scholar
  16. Konstantinova, P, Bonants, PJM, Gent-Pelzer, MPE, Zouwen, P, Bulk, R 2002Development of specific primers for detection and identification of Alternaria spp. in carrot material by PCR and comparison with blotter and plating assaysMycological Research1062333CrossRefGoogle Scholar
  17. Kumar, S, Tamura, K, Nei, M 2004MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignmentBriefings in Bioinformatics5150163PubMedGoogle Scholar
  18. Lovic, BR, Martin, RD, Miller, ME 1995Sequence analysis of the ITS region of rDNA in Monosporascus spp. to evaluate its potential for PCR-mediated detectionPhytopathology85655661Google Scholar
  19. Martin, RR, James, D, Lévesque, CA 2000Impacts of molecular diagnostic technologies on plant disease managementAnnual Review of Phytopathology38207239CrossRefPubMedGoogle Scholar
  20. Milgroom, MG 1997Genetic variation and the application of genetic markers for studying plant pathogen populationsJournal of Plant Pathology78113Google Scholar
  21. Pohronezny, KL, Volin, RB 1991Corky root rotJones, JBJones, JPStall, REZitter, TA eds. Compendium of Tomato DiseasesAPS PressSt. Paul, Minnesota, USA1213Google Scholar
  22. Porta-Puglia, A, Pucci, N, Di Giambattista, G, Infantino, A 2001First report of Rhizopycnis vagum associated with tomato roots in ItalyPlant Disease851210Google Scholar
  23. Punithalingam, E, Holiday, P 1973Pyrenochaeta lycopersici. CMI Description of Pathogenic Fungi and BacteriaNo. 398. Commonwealth Agricultural BureauKew, UK.Google Scholar
  24. Rozen, S, Skaletsky, HJ 2000Primer3 on the WWW for general users and for biologist programmersKrawetz, SMisener, S eds. Bioinformatics Methods and Protocols: Methods in Molecular BiologyHumana PressTotowa, NJ365386Google Scholar
  25. Salazar, O, Julian, MCA, Rubio, V 2000Primers based on specific rDNA-ITS sequences for PCR detection of Rhizoctonia solani, R. solani AG2 subgroups and ecological types, and binucleate RhizoctoniaMycological Research104281285CrossRefGoogle Scholar
  26. Schneider, R, Gerlach, W 1966Pyrenochaeta lycopersici nov. spec., der Erreger der Korkwurzelkrankheit der TomatePhytopathologische Zeitschrift56117122Google Scholar
  27. Sugiura T, Horinouchi H, Taguchi Y and Hyahamachi M, (2003). Two types of Pyrenochaeta lycopersici, causal pathogen of corky root of tomato. Proceedings of the 8th International Congress of Plant Pathology, Christchurch, New Zealand 2–7 February 2003, p. 346Google Scholar
  28. Tamietti, G, Valentino, D 1990A semi-selective medium for the isolation of Pyrenochaeta lycoperici from soilPhytopatologia Mediterranea299094Google Scholar
  29. White, TJ, Bruns, T, Lee, S, Taylor, J 1990Amplification and direct sequencing of fungal ribosomal RNA genes for phylogeneticsInnis, MAGelfand, DHSninsky, JJWhite, TJ eds. PCR Protocols, a Guide to Methods and ApplicationsAcademic PressSan Diego, USA315322Google Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  1. 1.Istituto Sperimentale per la Patologia Vegetale (I.S.Pa.Ve.)RomeItaly

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