Skip to main content
SpringerLink
Log in
Book cover

Plant Pathology pp 113–122Cite as

Diagnosis of Phytoplasmas by Real-Time PCR Using Locked Nucleic Acid (LNA) Probes

  • Protocol

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1302))

Abstract

Phytoplasma infections are regularly reported worldwide, and concerns about their threats on agricultural production, especially in relation to global climate change, are increasing. Sensitive and reliable detection methods are important to ensure that propagation material is free of phytoplasma infection and for epidemiological studies that may provide information to limit the extent of phytoplasma diseases and to prevent large-scale crop losses. The detection method described here uses LNA chemistry in real-time PCR. It has been developed and validated for use on potatoes, and its sensitivity and specificity make it suitable for use in postentry potato quarantine and initiation of potato nuclear stocks to ensure that material is phytoplasma-free.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Doi YM, Teranaka M, Yora K et al (1967) Mycoplasma or PLT-group-like microorganisms found in the phloem elements of plants infected with mulberry dwarf, potato witches’ broom, aster yellows, or paulownia witches’ broom. Ann Phytopathol Soc Jpn 33:259–266

    Article  Google Scholar 

  2. McCoy RE, Caudwell A, Chang CJ et al (1989) Plant diseases associated with mycoplasma like organisms. In: Whitcomb RF, Tully JG (eds) The mycoplasmas. Academic, New York, NY, pp 545–560

    Chapter  Google Scholar 

  3. Bertaccini A (2007) Phytoplasmas: diversity, taxonomy, and epidemiology. Front Biosci 12:673–689

    Article  CAS  PubMed  Google Scholar 

  4. The IRPCM Phytoplasma/Spiroplasma Working Team—Phytoplasma taxonomy group (2004) ‘Candidatus Phytoplasma’, a taxon for the wall-less, non-helical prokaryotes that colonize plant phloem and insects. Int J Syst Evol Microbiol 54:1243–1255

    Article  Google Scholar 

  5. Kube M, Mitrovic J, Duduk B et al (2012) Current view on phytoplasma genomes and encoded metabolism. Sci World J 2012:185942

    Article  Google Scholar 

  6. Lee I-M, Gundersen DE, Davis RE (1998) Revised classification scheme of phytoplasmas based on RFLP analysis of 16S rRNA and ribosomal protein gene sequences. Int J Syst Bacteriol 48:1153–1169

    Article  CAS  Google Scholar 

  7. Bertaccini A, Duduk B (2009) Phytoplasma and phytoplasma diseases: a review of recent research. Phytopathol Mediterr 48:355–378

    CAS  Google Scholar 

  8. Musetti R (2010) Biochemical changes in plants infected by phytoplasmas. In: Weintraub PG, Jones P (eds) Phytoplasmas: genomes, plant hosts and vectors. CABI, Cambridge, UK, pp 132–146

    Google Scholar 

  9. Bertamini M, Nedunchezhian N (2001) Effects of phytoplasma stolbur subgroup (Bois noir-BN) on photosynthetic pigments, saccharides, ribulose 1,5-bisphosphate carboxylase, nitrate and nitrite reductases, and photosynthetic activities in field-grown grapevine (Vitis vinifera L. cv. Chardonnay) leaves. Photosynthetica 39:119–122

    Article  CAS  Google Scholar 

  10. Choi YH, Tapias EC, Kim HK et al (2004) Metabolic discrimination of Catharanthus roseus leaves infected by phytoplasma using H-1-NMR spectroscopy and multivariate data analysis. Plant Physiol 135:2398–2410

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Maust BE, Espadas F, Talavera C et al (2003) Changes in carbohydrate metabolism in coconut palms infected with the lethal yellowing phytoplasma. Phytopathology 93:976–981

    Article  CAS  PubMed  Google Scholar 

  12. Junqueira A, Bedendo I, Pascholati S (2004) Biochemical changes in corn plants infected by the maize bushy stunt phytoplasma. Physio Mol Plant Pathol 65:181–185

    Article  CAS  Google Scholar 

  13. Margaria P, Ferrandino A, Caciagli P et al (2014) Metabolic and transcript analysis of the flavonoid pathway in diseased and recovered Nebbiolo and Barbera grapevines (Vitis vinifera L.) following infection by Flavescence dorée phytoplasma. Plant Cell Environ 37: 2183–2200

    Google Scholar 

  14. Musetti R, Buxa SV, De Marco F et al (2013) Phytoplasma-triggered Ca2+ influx is involved in sieve-tube blockage. Mol Plant Microbe Interact 26:379–386

    Article  CAS  PubMed  Google Scholar 

  15. Weintraub PG, Beanland L (2006) Insect vectors of phytoplasmas. Annu Rev Entomol 51:91–111

    Article  CAS  PubMed  Google Scholar 

  16. Ahrens U, Seemüller E (1992) Detection of DNA of plant pathogenic mycoplasma-like organisms by a polymerase chain reaction that amplifies a sequence of the 16S rRNA gene. Phytopathology 82:828–832

    Article  CAS  Google Scholar 

  17. Berges R, Rott M, Seemüller E (2000) Range of phytoplasma concentrations in various plant hosts as determined by competitive polymerase chain reaction. Phytopathology 90:1145–1152

    Article  CAS  PubMed  Google Scholar 

  18. Demeke T, Adams RP (1992) The effects of plant polysaccharides and buffer additives on PCR. Biotechniques 12:332–334

    CAS  PubMed  Google Scholar 

  19. Lee IM, Gundersen DE, Hammond RD et al (1994) Use of mycoplasma like organism (MLOs) group specific oligonucleotide primers for nested-PCR assays to detect mixed-MLO infections in a single host plant. Phytopathology 84:559–566

    Article  CAS  Google Scholar 

  20. Gundersen DE, Lee IM (1996) Ultrasensitive detection of phytoplasmas by nested PCR assays using two universal primer pairs. Phytopathol Mediterr 35:114–151

    Google Scholar 

  21. Christensen NM, Nicolaisen M, Hansen M et al (2004) Distribution of phytoplasmas in infected plants as revealed by real-time PCR and bioimaging. Mol Plant Microbe Interact 17:1175–1184

    Article  CAS  PubMed  Google Scholar 

  22. Baric S, Dalla-Via J (2004) A new approach to apple proliferation detection: a highly sensitive real-time PCR assay. J Microbiol Meth 57:135–145

    Article  CAS  Google Scholar 

  23. Galetto L, Marzachì C, Bosco D (2005) Universal and group specific real-time PCR diagnosis of Flavescence dorée (16Sr-V), Bois noir (16Sr-XII) and apple proliferation (16Sr-X) phytoplasmas from field-collected plant hosts and insect vectors. Ann Appl Biol 147:191–201

    Article  CAS  Google Scholar 

  24. Hren M, Boben J, Rotter A et al (2007) Real-time PCR detection systems for Flavescence doree and Bois noir phytoplasmas in grapevine: comparison with conventional PCR detection and application in diagnostics. Plant Pathol 56:785–796

    Article  CAS  Google Scholar 

  25. Margaria P, Turina M, Palmano S (2009) Detection of Flavescence dorée and Bois noir phytoplasmas, Grapevine leafroll associated virus-1 and -3 and Grapevine virus A from the same crude extract by reverse transcription-RealTime Taqman assays. Plant Pathol 58:838–845

    Article  Google Scholar 

  26. Holland PM, Abramson RD, Watson R et al (1991) Detection of specific polymerase chain reaction product by utilizing the 5′-3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A 88:7276–7280

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Reynisson E, Josefsen MH, Krause A et al (2006) Evaluation of probe chemistries and platforms to improve the detection limit of real-time PCR. J Microbiol Methods 66:206–216

    Article  CAS  PubMed  Google Scholar 

  28. Kumar R, Singh SK, Koshkin AA et al (1998) The first analogues of LNA (locked nucleic acids): phosphorothioate-LNA and 2V-thio-LNA. Bioorg Med Chem Lett 8:2219–2222

    Article  CAS  PubMed  Google Scholar 

  29. Costa JM, Ernault P, Olivi M et al (2004) Chimeric LNA/DNA probes as a detection system for real-time PCR. Clin Biochem 37:930–932

    Article  CAS  PubMed  Google Scholar 

  30. Jepsen JS, Sørensen MD, Wengel J (2004) Locked nucleic acid: a potent nucleic acid analog in therapeutics and biotechnology. Oligonucleotides 14:130–146

    Article  CAS  PubMed  Google Scholar 

  31. Montone KT (2009) Differentiation of fusarium from aspergillus species by colorimetric in situ hybridization in formalin-fixed, paraffin-embedded tissue sections using dual fluorogenic-labeled LNA probes. Am J Clin Pathol 132:866–870

    Article  CAS  PubMed  Google Scholar 

  32. Ruiz-Ruiz S, Moreno P, Guerri J et al (2009) Discrimination between mild and severe citrus tristeza virus isolates with a rapid and highly specific real-time reverse transcription-polymerase chain reaction method using TaqMan LNA probes. Phytopathology 99:307–315

    Article  CAS  PubMed  Google Scholar 

  33. EPPO (2006) PM 3/21(2). Phytosanitary procedures: post-entry quarantine for potato http://archives.eppo.int/EPPOStandards/PM3_PROCEDURES/pm3-21-2-e%20web.pdf

  34. Nasir MM, Mughal SM, Khan SM (2007) Occurrence, distribution and detection of potato purple top phytoplasma disease in the Punjab (Pakistan). Bull Insectology 60:377–378

    Google Scholar 

  35. Lee I-M, Bottner KD, Sun M (2009) An emerging potato purple top disease associated with a new 16SrIII group phytoplasma in Montana. Plant Dis 93:970

    Article  Google Scholar 

  36. Hodgetts J, Chuquillangui C, Muller G et al (2009) Surveys reveal the occurrence of phytoplasmas in plants at different geographical locations in Peru. Ann Appl Biol 155:15–27

    Article  CAS  Google Scholar 

  37. Santos-Cervantes ME, Chavez-Medina JA, Acosta-Pardini J et al (2010) Genetic diversity and geographical distribution of phytoplasmas associated with potato purple top disease in Mexico. Plant Dis 94:388–395

    Article  Google Scholar 

  38. Eroglu S, Ozbek H, Sahin F (2010) First report of group 16SrXII phytoplasmas causing stolbur disease in potato plants in the Eastern and Southern Anatolia Region of Turkey. Plant Dis 94:1374

    Article  Google Scholar 

  39. Hosseini P, Bahar M, Madani G et al (2011) Molecular characterization of phytoplasmas associated with potato purple top disease in Iran. J Phytopathol 159:241–246

    Article  Google Scholar 

  40. Iftikhar S, Fahmeed F (2011) Detection of phytoplasma from diseased potato sample. Pakistan J Botany 43:1085–1090

    Google Scholar 

  41. Mejia JF, Contaldo N, Paltrinieri S et al (2011) Molecular detection and identification of group 16SrV and 16SrXII phytoplasmas associated with potatoes in Colombia. Bull Insectology 64:S97–S98

    Google Scholar 

  42. Gutierrez-Ibanez AT, Laguna-Cerda A, Rojas-Martinez R et al (2012) Molecular detection and classification of the phytoplasma that causes purple top in potatoes (Solanum tuberosum) in the State of Mexico. Cien Inv Agr 39:339–346

    Article  Google Scholar 

  43. Lee I-M, Bottner KD, Secor G et al (2006) ‘Candidatus Phytoplasma americanum’ a phytoplasma associated with a potato purple top wilt disease complex. Int J System Evol Microbiol 56:1593–1597

    Article  CAS  Google Scholar 

  44. Liefting LW, Veerakone S, Ward LI et al (2009) First report of ‘Candidatus Phytoplasma australiense’ in potato. Plant Dis 93:969

    Article  Google Scholar 

  45. Gardener BBM (2004) Ecology of Bacillus and Paenibacillus spp. in agricultural systems. Phytopathology 94:1252–1258

    Article  Google Scholar 

  46. Palmano S (2001) A comparison of different phytoplasma DNA extraction methods using competitive PCR. Phytopathol Mediterr 40:99–107

    CAS  Google Scholar 

Download references

Acknowledgments

The corresponding author would like to thank colleagues in the Diagnostic and Molecular Biology Section and Plant Health Section (the UK Potato Quarantine Unit) for their help and especially Gerry S. Saddler for giving the opportunity to be hosted at SASA for a fruitful collaboration. This work was supported by the Scottish Government.

Author information

Authors and Affiliations

  1. Istituto per la Protezione Sostenibile delle Piante (IPSP) C.N.R., Strada delle Cacce 73, 10135, Torino, Italy

    Sabrina Palmano

  2. Science and Advice for Scottish Agriculture (SASA), Roddinglaw Road, Edinburgh, EH12 9FJ, UK

    Vincent Mulholland, David Kenyon, Gerry S. Saddler & Colin Jeffries

Authors
  1. Sabrina Palmano
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vincent Mulholland
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David Kenyon
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gerry S. Saddler
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Colin Jeffries
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sabrina Palmano .

Editor information

Editors and Affiliations

  1. Virology & Zoology, SASA, Edinburgh, United Kingdom

    Christophe Lacomme

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Palmano, S., Mulholland, V., Kenyon, D., Saddler, G.S., Jeffries, C. (2015). Diagnosis of Phytoplasmas by Real-Time PCR Using Locked Nucleic Acid (LNA) Probes. In: Lacomme, C. (eds) Plant Pathology. Methods in Molecular Biology, vol 1302. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2620-6_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2620-6_9

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2619-0

  • Online ISBN: 978-1-4939-2620-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation