Detection of the Bacterial Potato Pathogens Pectobacterium and Dickeya spp. Using Conventional and Real-Time PCR

  • Sonia N. HumphrisEmail author
  • Greig Cahill
  • John G. Elphinstone
  • Rachel Kelly
  • Neil M. Parkinson
  • Leighton Pritchard
  • Ian K. Toth
  • Gerry S. Saddler
Part of the Methods in Molecular Biology book series (MIMB, volume 1302)


Blackleg and soft rot of potato, caused by Pectobacterium and Dickeya spp., are major production constraints in many potato-growing regions of the world. Despite advances in our understanding of the causative organisms, disease epidemiology, and control, blackleg remains the principal cause of down-grading and rejection of potato seed in classification schemes across Northern Europe and many other parts of the world. Although symptom recognition is relatively straightforward and is applied universally in seed classification schemes, attributing disease to a specific organism is problematic and can only be achieved through the use of diagnostics. Similarly as disease spread is largely through the movement of asymptomatically infected seed tubers and, possibly in the case of Dickeya spp., irrigation waters, accurate and sensitive diagnostics are a prerequisite for detection. This chapter describes the diagnostic pathway that can be applied to identify the principal potato pathogens within the genera Pectobacterium and Dickeya.

Key words

Pectobacterium Dickeya Real-time PCR Blackleg Soft rot 


  1. 1.
    Charkowski A, Blanco C, Condemine G, Expert D, Franza T, Hayes C, Hugouvieux-Cotte-Pattat N, López Solanilla E, Low D, Moleleki L, Pirhonen M, Pitman A, Perna N, Reverchon S, Rodríguez Palenzuela P, San Francisco M, Toth I, Tsuyumu S, van der Waals J, van der Wolf J, Van Gijsegem F, Yang CH, Yedidia I (2012) The role of secretion systems and small molecules in soft-rot enterobacteriaceae pathogenicity. Annu Rev Phytopathol 50:425–449PubMedCrossRefGoogle Scholar
  2. 2.
    Perombelon MCM (2002) Potato diseases caused by soft rot erwinias: an overview of pathogenesis. Plant Pathol 51:1–12CrossRefGoogle Scholar
  3. 3.
    Charkowski AO (2006) The soft rot Erwinia. In: Gnanamanickam SS (ed) Plant-associated bacteria. Springer, The Netherlands, pp 423–505Google Scholar
  4. 4.
    Hauben L, Moore ER, Vauterin L, Steenackers M, Mergaert J, Verdonck L, Swings J (1998) Phylogenetic position of phytopathogens within the Enterobacteriaceae. Syst Appl Microbiol 21:384–397Google Scholar
  5. 5.
    Waldee EL (1945) Comparative studies of some peritrichous phytopathogenic bacteria. Iowa State J Sci 19:435–484Google Scholar
  6. 6.
    Samson R, Legendre JB, Christen R, Fischer-Le Saux M, Achouak W, Gardan L (2005) Transfer of Pectobacterium chrysanthemi (Burkholder et al. 1953) Brenner et al. 1973 and Brenneria paradisiaca to the genus Dickeya gen. nov. as Dickeya chrysanthemi comb. nov. and Dickeya paradisiaca comb. nov. and delineation of four novel species, Dickeya dadantii sp. nov., Dickeya dianthicola sp. nov., Dickeya dieffenbachiae sp. nov. and Dickeya zeae sp. nov. Int J Syst Evol Microbiol 55:1415–1427PubMedCrossRefGoogle Scholar
  7. 7.
    Brady C, Cleenwerk I, Denman S, Venter S, Rodríguez-Palenzuela P, Coutinho TA, de Vos P (2012) Proposal to reclassify Brenneria quercina (Hildebrand & Schroth 1967) Hauben et al. 1999 into a new genus, Lonsdalea gen. nov., as Lonsdalea quercina comb. nov., descriptions of Lonsdalea quercina subsp. quercina comb. nov., Lonsdalea quercina subsp. iberica subsp. nov., and Lonsdalea quercina subsp. britannica subsp. nov., emendation of the description of the genus Brenneria, reclassification of Dickeya dieffenbachiae as Dickeya dadantii subsp. dieffenbachiae comb. nov., and emendation of the description of Dickeya dadantii. Int J Syst Evol Microbiol 62:1592–1602Google Scholar
  8. 8.
    van der Wolf JM, Nijhuis EH, Kowalewska MJ, Saddler GS, Parkinson N, Elphinstone JG, Pritchard L, Toth IK, Lojkowska E, Potrykus M, Waleron M, de Vos P, Cleenwerck I, Pirhonen M, Garlant L, Hélias V, Pothier JF, Pflüger V, Duffy B, Tsror L, Manulis S (2014) Dickeya solani sp. nov., a pectinolytic plant pathogenic bacterium isolated from potato (Solanum tuberosum). Int J Syst Evol Microbiol 64:768–774PubMedCrossRefGoogle Scholar
  9. 9.
    Parkinson N, De Vos P, Pirhonen M, Elphinstone J (2014) Dickeya aquatica sp. nov., isolated from waterways. Int J Syst Evol Microbiol 64:2264–2266PubMedCrossRefGoogle Scholar
  10. 10.
    Gardan L, Gouy C, Christen R, Samson R (2003) Elevation of three subspecies of Pectobacterium carotovorum to species level: Pectobacterium atrosepticum sp. nov., Pectobacterium betavasculorum sp. nov. and Pectobacterium wasabiae sp. nov. Int J Syst Evol Microbiol 53:381–391PubMedCrossRefGoogle Scholar
  11. 11.
    Duarte V, de Boer SH, Ward TL, de Oliveora AMR (2004) Characterisation of atypical Erwinia carotovora strains causing blackleg of potato in Brazil. J App Microbiol 96:535–545CrossRefGoogle Scholar
  12. 12.
    Laurila J, Ahola V, Lehtinen A, Joutsjoki T, Hannukkala A, Rahkonen A, Pirhonen M (2008) Characterization of Dickeya strains isolated from potato and river water samples in Finland. Eur J Plant Pathol 122:213–225CrossRefGoogle Scholar
  13. 13.
    Sławiak M, van Beckhoven JRCM, Speksnijder AGCL, Czajkowski R, Grabe G, van der Wolf JM (2009) Biochemical and genetical analysis reveal a new clade of biovar 3 Dickeya spp. strains isolated from potato in Europe. Eur J Plant Pathol 125:245–261CrossRefGoogle Scholar
  14. 14.
    Pitman AR, Harrow SA, Visnovsky SB (2010) Genetic characterisation of Pectobacterium wasabiae causing soft rot disease of potato in New Zealand. Eur J Plant Pathol 126:423–435CrossRefGoogle Scholar
  15. 15.
    Baghaee-Ravari S, Rahimian H, Shams-Bakhsh M, Lopez-Solanilla E, Antunez-Lamaz M, Rodriguez-Penzuela P (2011) Characterization of Pectobacterium species from Iran using biochemical and molecular methods. Eur J Plant Pathol 129:413–425CrossRefGoogle Scholar
  16. 16.
    Moleleki LN, Onkendi EM, Mongae A, Kubheka GC (2013) Characterisation of Pectobacterium wasabiae causing blackleg and soft rot diseases in South Africa. Eur J Plant Pathol 135:279–288CrossRefGoogle Scholar
  17. 17.
    Pasanen M, Laurila J, Brader G, Palva ET, Ahola V, van der Wolf J, Hannukkala A, Pirhonen M (2013) Characterisation of Pectobacterium wasabiae and Pectobacterium carotovorum subsp. carotovorum isolates from diseased potato plants in Finland. Ann Appl Biol 163:403–419Google Scholar
  18. 18.
    Onkendi EM, Moleleki LN (2014) Characterization of Pectobacterium carotovorum subsp. carotovorum and brasiliense from diseased potatoes in Kenya. Eur J Plant Pathol 139:557–566CrossRefGoogle Scholar
  19. 19.
    Czajkowski R, Pe´rombelon MCM, van Veen JA, van der Wolf JM (2011) Control of blackleg and tuber soft rot of potato caused by Pectobacterium and Dickeya species: a review. Plant Pathol 60:999–1013CrossRefGoogle Scholar
  20. 20.
    Maas Geesteranus HP (1972) Natrot en zwartbenigheid bij aardappelen. Bedrijfsontwikkeling 3:941–945Google Scholar
  21. 21.
    Parkinson N, Stead D, Bew J, Heeney J, Tsror L, Elphinstone J (2009) Dickeya species relatedness and clade structure determined by comparison of recA sequences International. J Syst Evol Microbiol 59:2388–2393Google Scholar
  22. 22.
    Toth IK, van der Wolf JM, Saddler G, Lojkowska E, Hélias E, Pirhonen M, Tsror L, Elphinstone JG (2011) Dickeya species: an emerging problem for potato production in Europe. Plant Pathol 60:385–399CrossRefGoogle Scholar
  23. 23.
    Ngadze E, Coutinho TA, van der Waals JE (2010) First report of soft rot of potatoes caused by Dickeya dadantii in Zimbabwe. Plant Dis 94:1263CrossRefGoogle Scholar
  24. 24.
    Perombelon MCM, Kelman A (1980) Ecology of the soft rot erwinias. Annu Rev Phytopathol 18:361–387CrossRefGoogle Scholar
  25. 25.
    Tsror L, Erlich O, Lebiush S, Hazanovsky M, Zig U, Slawiak M, Grabe G, van der Wolf JM, van de Haar JJ (2009) Assessment of recent outbreaks of Dickeya sp. (syn. Erwinia chrysanthemi) slow wilt in potato crops in Israel. Eur J Plant Pathol 123:311–320CrossRefGoogle Scholar
  26. 26.
    Cahill G, Fraser K, Kowalewska MJ, Kenyon DM, Saddler GS (2010) Recent findings from the Dickeya survey and monitoring programme. Proceedings crop protection in Northern Britain, pp 171–176Google Scholar
  27. 27.
    Cuppels D, Kelman A (1974) Evaluation of selective media for isolation of soft-rot bacteria from soil and plant tissue. Phytopathology 64:468–475CrossRefGoogle Scholar
  28. 28.
    Hélias V, Hamon P, Huchet E, van der Wolf JM, Adrivon D (2012) Two new effective semi selective crystal violet pectate media for isolation of Pectobacterium and Dickeya. Plant Pathol 61:339–345CrossRefGoogle Scholar
  29. 29.
    Brierley J, Lees A, Hilton A, Wale S, Peters J, Elphinstone J, Boonham N (2008) Improving decision making for the management of potato diseases using real-time diagnostics. Potato Council Final Report R253 2008/6Google Scholar
  30. 30.
    Kim MH, Cho MS, Kim BK, Choi HJ, Hahn JH, Kim C, Kang MJ, Kim SH, Park DS (2012) Quantitative real-time polymerase chain reaction assay for detection of Pectobacterium wasabiae using YD repeat protein gene-based primers. Plant Dis 96:253–257CrossRefGoogle Scholar
  31. 31.
    Pritchard L, Humphris S, Saddler GS, Parkinson NM, Bertrand V, Elphinstone JG, Toth IK (2013) Detection of phytopathogens of the genus Dickeya using a PCR primer prediction pipeline for draft bacterial genome sequences. Plant Pathol 62:587–596. doi: 10.1111/j.1365-3059.2012.02678.x CrossRefGoogle Scholar
  32. 32.
    Meneley JC, Stanghellini M (1976) Isolation of soft rot Erwinia spp. from agricultural soils using an enrichment technique. Phytopathology 66:367–370CrossRefGoogle Scholar
  33. 33.
    Toth IK, Hyman LJ, Wood JR (1999) A one step PCR-based method for the detection of economically important soft rot Erwinia species on micropropagated potato plants. J Appl Microbiol 87:158–166CrossRefGoogle Scholar
  34. 34.
    De Boer SH, Ward LJ (1995) PCR detection of Erwinia carotovora subsp. atroseptica associated with potato tissue. Phytopathology 85:854–858CrossRefGoogle Scholar
  35. 35.
    Kang HW, Kwon SW, Go SJ (2003) PCR based and sensitive detection of Pectobacterium carotovorum by primers generated from URP-PCR fingerprinting-derived polymorphic band. Plant Pathol 52:127–133CrossRefGoogle Scholar
  36. 36.
    Nassar A, Darrasse A, Lemattre M, Kotoujansky A, Dervin C, Vedel R, Bertheau Y (1996) Characterization of Erwinia chrysanthemi by pectolytic isozyme polymorphism and restriction fragment length polymorphism analysis of PCR amplified fragment of pel genes. Appl Environ Microbiol 62:2228–2235PubMedCentralPubMedGoogle Scholar
  37. 37.
    Kelly RM, Cahill G, Elphinstone JG, Mitchell WJ, Mulholland V, Parkinson NM, Pritchard L, Toth IK, Saddler GS (2012) Development of a real-time PCR assay for the detection of “Dickeya solani.” Proceedings Crop Protection in Northern Britain 2012, pp 201–206Google Scholar
  38. 38.
    Weller SA, Elphinstone JG, Smith NC, Boonham N, Stead DE (2000) Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (Taq-Man) assay. Appl Environ Microbiol 66:2853–2858PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Sonia N. Humphris
    • 1
    Email author
  • Greig Cahill
    • 2
  • John G. Elphinstone
    • 3
  • Rachel Kelly
    • 2
  • Neil M. Parkinson
    • 3
  • Leighton Pritchard
    • 1
  • Ian K. Toth
    • 1
  • Gerry S. Saddler
    • 2
  1. 1.The James Hutton InstituteDundeeUK
  2. 2.Science and Advice for Scottish AgricultureRoddinglaw RoadEdinburghUK
  3. 3.Food and Environment Research AgencyYorkUK

Personalised recommendations