Identification and differentiation of soft rot and blackleg bacteria from potato using nested and multiplex PCR

  • Nader A. Ashmawy
  • Ahmed F. El-Bebany
  • Amany H. M. Shams
  • Alia A. ShoeibEmail author
Original Article


The bacterial genera Pectobacterium, Dickeya and Enterobacter are plant pathogens that cause soft rot and blackleg of potato. In the present study, 25 soft rot bacteria were isolated from potato tubers and stems showing typical tuber soft rot and blackleg symptoms. The samples were obtained from different locations in Egypt. Initial identification was based on morphological, physiological and biochemical characteristics. Pathogenicity of the identified isolates was assessed on potato tuber slices. Molecular techniques (16S rRNA sequencing and PCR analysis) revealed the identification of new clades of D. solani and E. cloacae. The primers (Eca1f/Eca2r) selected for the detection of P. atrosepticum specifically amplified a 690-bp region in only two out of 25 soft rot isolates. A single 550-bp band was produced from 14 isolates of P. carotovorum subsp. carotovorum using the species-specific primer EXPCC. Out of 23 isolates, 12 isolates gave a 666-bp band using primers to detect pmrA. Moreover, D. solani isolates were confirmed by the PCR amplification of a 420-bp region using specific primers (ADE1/ADE2). Nested PCR using the primer set EXPCCF/EXPCCR followed by INPCCF/INPCCR produced a 400-bp band from all P. c. subsp. carotovorum isolates tested. Nested PCR and multiplex PCR assays provided fast and reliable detection of the major soft rot and blackleg potato pathogens using three pairs of primers (Df/Dr, Y45/46 and EXPCCF/EXPCCR) and enabled the simultaneous detection of four pectinolytic bacteria found in potato: P. atrosepticum, P. c. subsp. carotovorum, D. solani and E. cloacae.


Pectobacterium Dickeya Enterobacter Nested PCR Multiplex PCR 



We would like to thank Prof. Kamal A. M. Abo-Elyouser, Plant Pathology Department, Faculty of Agriculture, Assiut University, Egypt, for providing us P. atrosepticum isolates.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.


  1. Abd-Elhafeez E, AlKhazindar M, Sayed ETA (2018) Isolation and characterization of Enterobacter strains caused Potato soft rot disease in Egypt. Minia Sci Bull Bot Sect 29(1):1–13Google Scholar
  2. Abo-El-Dahab MK, El-Goorani MA (1969) Antagonism among strains of Pseudomonas solanacearum. Phytopathology 59:1005–1007PubMedGoogle Scholar
  3. Abo-El-Dahab MK, El-Goorani MA, Shoeib AA (1982) New corn and pseudostem diseases of banana in Egypt. In: Proceedings of the second Egyptian–Hungarian conference of plant protection, Sept 21–24, Alexandria, Egypt, pp 133–143Google Scholar
  4. Abu-Obeid I, Khlaif H, Salem N (2017) Detection and identification of bacterial soft rot of potato Pectobacterium carotovorum subsp. carotovorum using specific PCR primers in Jordan. Afr J Agric Res 12(39):2910–2918CrossRefGoogle Scholar
  5. Ahmed Asia RE (2009) Pathological studies on potato soft rot disease caused by Erwinia carotovora subsp. carotovora. M.Sc. thesis, Alexandria University, Faculty of Agriculture, Damnhour Branch, EgyptGoogle Scholar
  6. Al-Enazi MA, Shoeib AA (2017) Molecular characterization of vancomycin resistance Enterococcus faecium clinical isolates and efficacy of ozone on pattern of antibiotic(s) resistance bacterial cell wall using SEM. IOSR J Dent Med Sci 16(2):38–45CrossRefGoogle Scholar
  7. Anbazhagan D, Kathirvalu GG, Mansor M, Yan GO, Yusof MY, Sekaran SD (2010) Multiplex polymerase chain reaction (PCR) assays for the detection of Enterobacteriaceae in clinical samples. Afr J Microbiol Res 4:1186–1191Google Scholar
  8. Ashmawy NA, Jadalla NM, Shoeib AA, El-Bebany AF (2015) Identification and genetic characterization of Pectobacterium spp. and related Enterobacteriaceae causing potato soft rot diseases in Egypt. J Pure Appl Microbiol 9:1847–1858Google Scholar
  9. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1995) Preparation of genomic DNA from bacteria. In: Ausubel FA, Brent RE, Kingston DD, Moore JG, Seidman JA (eds) Current protocols in molecular biology. Wiley, New York, pp 630–634Google Scholar
  10. Behiry SI (2013) Molecular and pathological studies on potato bacterial soft rot disease. Ph.D. thesis, Alex University, Agricultural Botany Department, Faculty of Agriculture, Saba Basha, EgyptGoogle Scholar
  11. Behiry SI, Ashmawy NA, Abdelkhalek AA, Younes HA, Khaled AE, Hafez EE (2018) Compatible and incompatible type interactions related to defense genes in potato elucidation by Pectobacterium carotovorum. J Plant Dis Prot 125(3):197–204. CrossRefGoogle Scholar
  12. Chao YC, Feng CT, Ho WC (2006) First report of Aglaonema bacterial blight caused by Erwinia chrysanthemi in Taiwan. Plant Dis 10:1358–1358CrossRefGoogle Scholar
  13. Czajkowski R, Pérombelon MCM, Jafra S, Łojkowska E, Potrykus M, van der Wolf JM, Śledź W (2015) Detection, identification and differentiation of Pectobacterium and Dickeya species causing potato blackleg and tuber soft rot: a review. Ann Appl Biol 166:18–38PubMedCrossRefGoogle Scholar
  14. De Boer SH, Ward LJ (1995) PCR detection of Erwinia carotovorora subsp. atroseptica associated with potato tissue. Phytopathology 85:854–858CrossRefGoogle Scholar
  15. Duarte V, De Boer SH, Ward LJ, De Oliveira AMR (2004) Characterization of atypical Erwinia carotovora strains causing blackleg of potato in Brazil. J Appl Microbiol 96:535–545PubMedCrossRefGoogle Scholar
  16. Fahy PC, Hayward AC (1983) Plant bacterial disease (a diagnostic guide). Academic Press, New YorkGoogle Scholar
  17. FAOSTAT (2017) Agriculture data source. Accessed 18 Jan 2019
  18. Frechon D, Exbrayat P, Helias V, Hyman LJ, Jouan B, Llop P, Lopez MM, Payet N, Perombelon MCM, Toth IK, van Backhoven JRCM, van der Wolf JM, Bertheau Y (1998) Evaluation of a PCR kit for the detection of Erwinia carotovora subsp. atroseptica on potato tubers. Potato Res 41:163–173CrossRefGoogle Scholar
  19. García-González T, Sáenz-Hidalgo HK, Silva-Rojas HV, Morales-Nieto C, Vancheva T, Koebnik R, Ávila-Quezada GD (2018) Enterobacter cloacae, an emerging plant-pathogenic bacterium affecting chili pepper seedlings. Plant Pathol J 34(1):1–10PubMedPubMedCentralGoogle Scholar
  20. 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
  21. Kang HW, Kwon SW, Go SJ (2003) PCR-based specific and sensitive detection of Pectobacterium carotovorum spp. carotovorum by primers generated from a URP-PCR fingerprinting-derived polymorphic band. Plant Pathol 52:127–133CrossRefGoogle Scholar
  22. Kettani-Halabi M, Terta M, Amdan M, El Fahime E, Bouteau F, Ennaji MM (2013) An easy, simple inexpensive test for the specific detection of Pectobacterium carotovorum subsp. carotovorum based on sequence analysis of the pmrAgene. BMC Microbiol 13:176–183PubMedPubMedCentralCrossRefGoogle Scholar
  23. Klement Z, Rudolph K, Sands DC (1990) Methods in phytobacteriology. Akademiai Kiado, BudapestGoogle Scholar
  24. Lee YA, Yu CP (2005) A differential medium for the isolation and rapid identification of a plant soft rot pathogen, Erwinia chrysanthemi. J Microbiol Methods 64:200–206PubMedCrossRefGoogle Scholar
  25. Li PQ, Lin BR, Shen HF, Pu XM (2011) Species-specific detection of Dickeya sp. (Pectobacterium chrysanthemi) in infected banana tissues, soil and water. Afr J Biotechnol 10:16774–16780Google Scholar
  26. Lin Y-H, Lee P-J, Shie W-T, Chern L-L, Chao Y-C (2015) Pectobacterium chrysanthemi as the dominant causal agent of bacterial soft rot in Oncidium “Grower Ramsey”. Eur J Plant Pathol 142:331–343CrossRefGoogle Scholar
  27. Liu S, Tang Y, Wang D, Lin N, Zhou J (2016) Identification and characterization of a new Enterobacter onion bulb decay caused by Lelliottia amnigena in China. Appl Microbiol Open Access 2:2. CrossRefGoogle Scholar
  28. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  29. Martín A, Pérez-Ayala A, Chaves FF, Lora F, ÁngelesOrellana M (2018) Evaluation of the multiplex PCR Allplex-GI assay in the detection of bacterial pathogens in diarrheic stool samples. J Microbiol Methods 144:33–36PubMedCrossRefGoogle Scholar
  30. McManus PS, Jones AL (1995) Detection of Erwinia amylovora by nested PCR and PCR-dot-blot and reverse-blot hybridization. Phytopathology 85:618–623CrossRefGoogle Scholar
  31. Nassar A, Darrasse A, Lemattre M, Kotoujansky A, Dervin C, Vedel R, Bertheau Y (1996) Characterization of Erwinia chrysanthemi by pectinolytic isozyme polymorphism and restriction fragment length polymorphism analysis of PCR-amplified fragments of pel genes. Appl Environ Microbiol 62:2228–2235PubMedPubMedCentralGoogle Scholar
  32. Ngadze E, Brady CL, Coutinho T, Van Der Waals JE (2012) Pectinolytic bacteria associated with potato soft rot and blackleg in South Africa and Zimbabwe. Eur J Plant Pathol 134:533–549CrossRefGoogle Scholar
  33. Ozturk M, Aksoy HM (2017) First report on Dickeya solani associated with potato blackleg and soft rot in Turkey. J Plant Pathol 99(1):287–304Google Scholar
  34. Ozturk M, Aksoy HM, Potrykus M, Lojkowska E (2018) Genotypic and phenotypic variability of Pectobacterium strains causing blackleg and soft rot on potato in Turkey. Eur J Plant Pathol. CrossRefGoogle Scholar
  35. Potrykus M, Śledź W, Golanowska M, Sławiak M, Binek A, Motyka A, Żołędowska S, Czajkowski R, Łojkowska E (2014) Simultaneous detection of major blackleg and soft rot bacterial pathogens in potato by multiplex polymerase chain reaction. Ann Appl Biol 165:474–487PubMedPubMedCentralCrossRefGoogle Scholar
  36. Saettler AW, Schaad NW, Roth DA (1989) Detection of bacteria in seed and other planting material. APS Press, St PaulGoogle Scholar
  37. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold SpringGoogle Scholar
  38. Shams AHM, Ashmawy NA, El-Bebany AF, Shoeib AA (2016) Identification and pathogenicity of phytopathogenic bacteria associated with soft rot disease on some potato cultivars. Alex J Agric Sci 61:541–550Google Scholar
  39. Staley JT, Boone DR, Garrity GM, Devos P, Fellow MG, Rainey FA, Schlifer KH, Brenner DJ, Castenholz RW, Holt JG, Krieg NR, Liston J, Moulder JW, Murray RGE, Niven CF Jr, Pfenning N, Sneath PHA, Jully JG, Williams S (2005) Bergey’s manual of systematic bacteriology, vol 2. Williams and Wilking Company, Baltimore, MDGoogle Scholar
  40. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.1. Mol Biol Evol 30:2725–2729PubMedPubMedCentralCrossRefGoogle Scholar
  41. Thompson IJD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting position specific gap penalties and weight matrix choice. Nucleic Acis Res 22:4673–4680CrossRefGoogle Scholar
  42. Van der Merwe JJ, Coutinho TA, Korsten L, van der Waals JE (2010) Pectobacterium carotovorum subsp. brasiliensis causing blackleg on potatoes in South Africa. Eur J Plant Pathol 126:175–185CrossRefGoogle Scholar
  43. Van der Wolf JM, De Boer SH (2007) Bacterial pathogens of potato. In: Bradshaw DV, Gebhardt C, Govers F, Mackerron DKL, Taylor MA, Ross HA (eds) Potato biology and biotechnology. Elsevier, Amsterdam, pp 595–617CrossRefGoogle Scholar
  44. Waleron M, Waleron K, Podhajska AJ, Łojkowska E (2002) Genotyping of bacteria belonging to the former Erwinia genus by PCR-RFLP analysis of a recA gene treatment. Microbiology 148:583–595PubMedCrossRefGoogle Scholar

Copyright information

© Deutsche Phytomedizinische Gesellschaft 2019

Authors and Affiliations

  1. 1.Department of Plant Pathology, Faculty of AgricultureAlexandria UniversityAlexandriaEgypt

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