The Development and Deployment of Rapid In-Field Phytoplasma Diagnostics Exploiting Isothermal Amplification DNA Detection Systems

  • Mattew Dickinson
  • Jennifer Hodgetts


Detection and diagnosis of phytoplasmas is an important requirement, both for the identification of their hosts, alternate hosts and potential insect vector species and for the management of these devastating diseases of plants. Being able to detect pathogen presence at pre-symptomatic stages allows the removal of infected plants before they provide a significant reservoir of the pathogen for spread of disease to other plants or dissemination via propagation material, whilst detection in weeds from which the disease might spread into crops allows appropriate weed control to be undertaken. Similarly, detection in potential insect vectors allows for management practices to be considered that might reduce the spread of the diseases. As new technologies have developed over the years since phytoplasmas were first discovered, the methods for detection have evolved, as have their sensitivity, reliability and ease of use. In-field or point-of-care diagnostics, which allows tests to be conducted rapidly on site with minimal equipment and costs and which also allows easy and unambiguous interpretation of the results, is a goal that has driven the implementation of diagnostic techniques. This chapter will detail how, with the advent of isothermal DNA amplification methods, particularly loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), this goal is now being attained.


DNA extraction In-field diagnostics Lateral flow devices Loop-mediated isothermal amplification Recombinase polymerase amplification 


  1. Abeysinghe S, Abeysinghe PD, Kanatiwela-de Silva C, Udagama P, Warawichanee K, Aljafar N, Kawicha P, Dickinson M (2016) Refinement of the taxonomic structure of 16SrXI and 16SrXIV phytoplasmas of gramineous plants using multilocus sequence typing. Plant Disease 100, 2001-2010.PubMedCrossRefPubMedCentralGoogle Scholar
  2. Al-Jaf B (2016) Development of improved methods for phytoplasma diagnostics. PhD Thesis, University of Nottingham, UK.Google Scholar
  3. Angelini E, Bianchi GL, Filippin L, Morassutti C, Borgo M (2007) A new TaqMan method for the identification of phytoplasmas associated with grapevine yellows by real-time PCR assay. Journal of Microbiological Methods 68, 613-622.PubMedCrossRefPubMedCentralGoogle Scholar
  4. Baric S, Dalla-Via J (2004) A new approach to apple proliferation detection: a highly sensitive real-time PCR assay. Journal of Microbiological Methods 57, 135-145.PubMedCrossRefPubMedCentralGoogle Scholar
  5. Bekele B, Hodgetts J, Tomlinson J, Boonham N, Nikolic P, Swarbrick P, Dickinson M (2011) Use of a real-time LAMP isothermal assay for detecting 16SrII and XII phytoplasmas in fruit and weeds of the Ethiopian Rift Valley. Plant Pathology 60, 345-355.CrossRefGoogle Scholar
  6. Braun-Kiewnick A, Altenbach D, Oberhansli T, Bitterlin W, Duffy B (2011) A rapid lateral-flow immunoassay for phytosanitary detection of Erwinia amylovora and on-site fire blight diagnosis. Journal of Microbiological Methods 87, 1-9.PubMedCrossRefPubMedCentralGoogle Scholar
  7. Chomczynski P, Rymaszewski M (2006) Alkaline polyethylene glycol-based method for direct PCR from bacteria, eukaryotic tissue samples, and whole blood. BioTechniques 40, 454-458.PubMedCrossRefPubMedCentralGoogle Scholar
  8. Christensen NM, Nicolaisen M, Hansen M, Schulz A (2004) Distribution of phytoplasmas in infected plants as revealed by real-time PCR and bioimaging. Molecular Plant-Microbe Interactions 17, 1175-1184.PubMedCrossRefPubMedCentralGoogle Scholar
  9. Contaldo N, Satta E, Zambon Y, Paltrinieri S, Bertaccini A (2016) Development and evaluation of different complex media for phytoplasma isolation and growth. Journal of Microbiological Methods 127, 105-110.PubMedPubMedCentralCrossRefGoogle Scholar
  10. Contaldo N, D’Amico G, Paltrinieri S, Diallo HA, Bertaccini A, Arocha Rosete Y (2019) Molecular and biological characterization of phytoplasmas from coconut palms affected by the lethal yellowing disease in Africa. Microbiological Research 223–225: 51-57.PubMedPubMedCentralCrossRefGoogle Scholar
  11. Crosslin JM, Vandemark GJ, Munyaneza JE (2006) Development of a real-time, quantitative PCR for detection of the Columbia basin potato purple top phytoplasma in plants and beet leafhoppers. Plant Disease 90, 663-667.PubMedCrossRefPubMedCentralGoogle Scholar
  12. Danks C, Barker I (2000) On-site detection of plant pathogens using lateral-flow devices. Bulletin OEPP/EPPO Bulletin 30, 421-426.CrossRefGoogle Scholar
  13. de Jonghe K, De Roo I, Maes M (2017) Fast and sensitive on-site isothermal assay (LAMP) for diagnosis and detection of three fruit tree phytoplasmas. European Journal of Plant Pathology 147, 749-759.CrossRefGoogle Scholar
  14. Dickinson M (2015) Loop Mediated Isothermal amplification (LAMP) for detection of phytoplasmas in the field. In: Plant Pathology: Techniques and Protocols. Methods in Molecular Biology, ed Lacomme C, Springer Science + Business Media, New York, United States of America, 99-111 pp.CrossRefGoogle Scholar
  15. Donoso A, Valenzuela S (2018) In-field molecular diagnosis of plant pathogens: recent trends and future perspectives. Plant Pathology 67, 1451-1461.CrossRefGoogle Scholar
  16. Fire A, Xu SQ (1995) Rolling replication of short DNA circles. Proceedings of the National Academy of Science United States of America 92, 4641-4645.CrossRefGoogle Scholar
  17. Galetto L, Bosco B, Marzachì C (2005) Universal and group-specific real-time PCR diagnosis of “flavescence dorée” (16SrV), “bois noir” (16SrXII) and apple proliferation (16SrX) phytoplasmas from field-collected plant hosts and insect vectors. Annals of Applied Biology 147, 191-201.CrossRefGoogle Scholar
  18. Gundersen DE, Lee I-M (1996) Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs. Phytopathologia Mediterranea 35, 144-151.Google Scholar
  19. Hatano B, Maki T, Obara T, Fukumoto H, Hagisawa K, Matsushita Y, Okutani A, Bazartseren B, Inoue S, Sata T, Katano H (2010) LAMP using a disposable pocket warmer for anthrax detection, a highly mobile and reliable method for anti-bioterrorism. Japanese Journal of Infectious Disease 63, 36-40.Google Scholar
  20. Hodgetts J, Boonham N, Mumford R, Dickinson M (2009) Panel of 23S rRNA gene-based real-time PCR assays for improved universal and group-specific detection of phytoplasmas. Applied and Environment Microbiology 75, 2945-2950.CrossRefGoogle Scholar
  21. Hodgetts J, Johnson G, Perkins K, Ostoja-Starzewska S, Boonham N, Mumford R, Dickinson MJ (2014) The development of monoclonal antibodies to the SecA protein of Cape St Paul wilt disease phytoplasma and their evaluation as a diagnostic tool. Molecular Biotechnology 56, 803-813.PubMedCrossRefPubMedCentralGoogle Scholar
  22. Howson ELA, Kurosaki Y, Yasuda J, Takahashi M, Goto H, Gray AR, Mioulet V, King DP and Fowler VL (2017) Defining the relative performance of isothermal assays that can be used for rapid and sensitive detection of foot-and-mouth virus. Journal of Virological Methods 249, 102-110.PubMedPubMedCentralCrossRefGoogle Scholar
  23. Kakizawa S, Oshima K, Kuboyama T, Nishigawa H, Jung H-Y, Sawayanagi T, Tsuchizaki T, Miyata S, Ugaki M, Namba S (2001) Cloning and expression analysis of phytoplasma protein translocation genes. Molecular Plant Microbe Interactions 14, 1043-1050.PubMedCrossRefPubMedCentralGoogle Scholar
  24. Karakkat BB, Hockemeyer K, Franchett M, Olson M, Mullenberg C, Koch PL (2018) Detection of root-infecting fungi on cool-season turfgrass using loop-mediated isothermal amplification and recombinase polymerase amplification. Journal of Microbiological Methods 151, 90-98.PubMedCrossRefPubMedCentralGoogle Scholar
  25. Kogovšek P, Hodgetts J, Hall J, Prezelj N, Nikolic P, Mehle N, Lenarcic R, Rotter A, Dickinson M, Boonham N, Dermastia M, Ravnikar M (2015) LAMP assay and rapid sample preparation method for on-site detection of “flavescence dorée” phytoplasma in grapevine. Plant Pathology 64, 286-296.PubMedCrossRefPubMedCentralGoogle Scholar
  26. Kogovšek P, Mehle N, Pugelj A, Jakomin T, Schroers H-J, Ravnikar M, Dermastia M (2017) Rapid loop-mediated isothermal amplification assays for grapevine yellows phytoplasmas on crude leaf-vein homogenate has the same performance as qPCR. European Journal of Plant Pathology 148, 75-84.CrossRefGoogle Scholar
  27. Lau HY, Botella JR (2017) Advanced DNA-based point-of-care diagnostic methods for plant diseases detection. Frontiers in Plant Science 8, 2016.Google Scholar
  28. Lau HY, Palanisamy R, Trau M, Botella JR (2014) Molecular inversion probe: a new tool for highly specific detection of plant pathogens. Plos One 9, e111182.PubMedPubMedCentralCrossRefGoogle Scholar
  29. Lu H, Wilson BAL, Ash GJ, Woruba SB, Fletcher MJ, You M, Yang G, Gurr GM (2016) Determining putative vectors of the Bogia coconut syndrome phytoplasma using loop-mediated isothermal amplification of single-insect feeding media. Scientific Reports 6, 35801.Google Scholar
  30. Mekuria TA, Zhang SL, Eastwell KC (2014) Rapid and sensitive detection of little cherry virus 22 using isothermal reverse transcription-recombinase polymerase amplification. Virological Methods 205, 24-30.CrossRefGoogle Scholar
  31. Mori Y, Nagamine K, Tomita N, Notomi T (2001) Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochemical and Biophysical Research Communications 289, 150-154.PubMedCrossRefPubMedCentralGoogle Scholar
  32. Nagamine K, Hase T, Notomi T (2002) Accelerated reaction by loop-mediated isothermal amplification using loop primers. Molecular and Cellular Probes 16, 223-229.PubMedCrossRefPubMedCentralGoogle Scholar
  33. Nair S, Manimekalai R, Raj PG, Hegde V (2016) Loop mediated isothermal amplification (LAMP) assay for detection of coconut root wilt disease and arecanut yellow leaf disease phytoplasma. World Journal of Microbiology and Biotechnology 32, 108.PubMedCrossRefPubMedCentralGoogle Scholar
  34. Notomi T, Okayama H, Masubuchi H, Yonekawa K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Research 28, e63.PubMedPubMedCentralCrossRefGoogle Scholar
  35. Obura E, Masiga D, Wachira F, Gurja B, Khan ZR (2011) Detection of phytoplasmas by loop-mediated isothermal amplification of DNA (LAMP). Journal of Microbiological Methods 84, 312-316.PubMedCrossRefPubMedCentralGoogle Scholar
  36. Pérez-López E, Rodriguez-Martinez D, Olivier CY, Luna-Rodriguez M, Dumonceaux TJ (2017) Molecular diagnostic assays based on cpn60 UT sequences reveal the geographic distribution of subgroup 16SrXIII-(A/I)I phytoplasma in Mexico. Scientific Reports 7, 950.PubMedPubMedCentralCrossRefGoogle Scholar
  37. Piepenburg O, Williams CH, Stemple DL, Armes NA (2006) DNA detection using recombination proteins. Plos Biology 4, e204.PubMedPubMedCentralCrossRefGoogle Scholar
  38. Smart CD, Schneider B, Blomquist CL, Guerra LJ, Harrison NA, Ahrens U, Lorenz K-H, Seemuller E, Kirkpatrick B (1996) Phytoplasma-specific PCR primers based on sequence of the 16S-23S rRNA spacer region. Applied and Environmental Microbiology 62, 2988-2993.PubMedPubMedCentralGoogle Scholar
  39. Stainton K, Hall J, Budge GE, Boonham N, Hodgetts J (2018) Rapid molecular methods for in-field and laboratory identification of the yellow-legged Asian hornet (Vespa velutina nigrithorax). Journal of Applied Entomology 142, 610-616.CrossRefGoogle Scholar
  40. Sugawara K, Himeno M, Keima T, Kitazawa Y, Maejima K, Oshima K, Namba S (2012) Rapid and reliable detection of phytoplasmas by loop-mediated isothermal amplification targeting a housekeeping gene. Journal of General Plant Pathology 78, 389-397.CrossRefGoogle Scholar
  41. Thornton CR, Groenhof AC, Forrest R, Lamotte R (2004) A one-step, immunochromatographic lateral flow device specific to Rhizoctonia solani and certain related species, and its use to detect and quantify R. solani in soil. Phytopathology 94, 280-288.PubMedCrossRefPubMedCentralGoogle Scholar
  42. Tomlinson JA, Dickinson M, Boonham N (2010a) Rapid method for detection of Phytophthora ramorum and P. kernoviae by a two-minute DNA extraction method followed by isothermal amplification, and amplicon detection by generic lateral flow device. Phytopathology 100, 143-149.PubMedCrossRefPubMedCentralGoogle Scholar
  43. Tomlinson JA, Boonham N, Dickinson M (2010b) Development and evaluation of a one-hour DNA extraction and loop-mediated isothermal amplification assay for rapid detection of phytoplasmas. Plant Pathology 59, 465-471.CrossRefGoogle Scholar
  44. Valasevich N, Schneider B (2017) Rapid detection of ‘Candidatus Phytoplasma mali’ by recombinase polymerase amplification assays. Journal of Phytopathology 165, 762-770.CrossRefGoogle Scholar
  45. Villalobos W, Montero-Astua M, Coto T, Sandoval I, Moreira L (2018) Genipa Americana and Ageratina anisochroma, two new hosts of ‘Candidatus Phytoplasma asteris’ in Costa Rica. Australasian Plant Disease Notes 18, 31.CrossRefGoogle Scholar
  46. Villamor DEV, Eastwell K (2019) Multilocus characterization, gene expression analysis of putative immunodominant protein coding regions, and development of recombinase polymerase amplification assay for detection of ‘Candidatus Phytoplasma pruni’ in Prunus avium. Phytopathology 109, 983-992.PubMedCrossRefPubMedCentralGoogle Scholar
  47. Vincent M, Xu Y, Kong HM (2004) Helicase-dependant isothermal DNA amplification. EMBO Reports 5, 795-800.PubMedPubMedCentralCrossRefGoogle Scholar
  48. Vu NT, Pardo JM, Alvarez E, Le HH, Wyckhuys K, Nguyen K-L, Le DT (2016). Establishment of a loop-mediated isothermal amplification (LAMP) assay for the detection of phytoplasma-associated cassava witches’ broom disease. Applied Biological Chemistry 59, 151-156.CrossRefGoogle Scholar
  49. Wamalwa NIE, Midega CAO, Ajanga S, Omukunda NE, Muyekho FN, Asudi GO, Mulaa M, Khan ZR (2017) Screening napier grass accessions for resistance to napier grass stunt disease using the loop-mediated isothermal amplification of DNA. Crop Protection 98, 61-69.CrossRefGoogle Scholar
  50. Wambua L, Schneider B, Okwaro A, Wanga JO, Imali O, Wambua PN, Agutu L, Olds C, Jones CS, Masiga D, Midega C, Khan Z, Jones J, Fischer A (2017) Development of field-applicable tests for rapid and sensitive detection of ‘Candidatus Phytoplasma oryzae’. Molecular and Cellular Probes 35, 44-56.PubMedCrossRefPubMedCentralGoogle Scholar
  51. Zou Y, Mason MG, Wang Y, Wee E, Turni C, Blakall P, Trau M, Botella JR (2017) Nucleic acid purification from plants, animals and microbes in under 30 seconds. Plos Biology 15, e2003916.PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2019

Authors and Affiliations

  • Mattew Dickinson
    • 1
  • Jennifer Hodgetts
    • 2
  1. 1.School of BiosciencesUniversity of NottinghamLoughboroughUK
  2. 2.Elsoms Seeds LtdSpaldingUK

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