Determination of differences in Ralstonia solanacearum phylotype II, sequevar 1 forms as related to their colony characteristics on Kelman medium and pathogenesis

  • Yasser E. IbrahimEmail author
  • Naglaa M. Balabel
  • Amgad A. Saleh
  • Nabil S. Farag
Original Article


Potato bacterial wilt caused by Ralstonia solanacearum is one of the most destructive diseases in potato production areas worldwide. One-hundred-six colonies were obtained from a single typical colony recovered from a potato tuber showing internal brown rot symptoms. Colonies’ identity was confirmed as R. solanacearum phylotype II sequevar 1 via PCR. Based on the morphological characterization and the average attenuation index (AI), colonies grouped into typical, intermediate, and atypical forms. However, the small variations in AI make it very difficult to make sure identification and this index cannot be a definitive identification system for R. solanacearum forms. The atypical colonies did not revert to typical or intermediate forms, whereas the both latter ones changed to the three forms with different percentages. For forthcoming experiments, two subcultures of each morphological form were selected. The pathogenicity experiments showed that these subcultures were also grouped into three forms: virulent, intermediate and avirulent. The source of peptone in the agar medium caused significant effects on R. solanacearum colonial morphological characteristics and its pathogenesis. Consequently, it is recommended to use one source of peptone during subculturing and maintaining R. solanacearum forms, as well as studying host-pathogen interaction. The subcultures of R. solanacearum produced a brown diffusible pigment from L-tyrosine on TZC media devoid of ascorbic acid but 1 mM ascorbic acid affected pigmentation. It is suggested that the efficacy of ascorbic acid as a plant-resistance inducer in controlling bacterial wilt disease should be carefully evaluated as it apparently favors R. solanacearum inside the plant tissues.


Phenotypic conversion Different sources of peptone Ascorbic acid Melanin production TZC medium 



The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through the research group no. RG-1440-001.


  1. Ahmad S, Lee SY, Kong HG, Jo EJ, Choi HK (2016) Genetic determinants for pyomelanin production and its protective effect against oxidative stress in Ralstonia solanacearum. PLoS One 11:1–16Google Scholar
  2. Balabel NM, Eweda WE, Mostafa MI, Farag NS (2005) Some epidemiological aspects of Ralstonia solanacearum. Egypt J Agric Res 83:1547–1564Google Scholar
  3. Boucher CA, Van Gijsegem F, Barberis PA, Arlat M, Zischek C (1987) Pseudomonas solanacearum genes controlling both pathogenicity on tomato and hypersensitivity on tobacco are clustered. J Bacteriol 169:5626–5632CrossRefGoogle Scholar
  4. Brumbley SM, Denny T (1990) Cloning of wild-type Pseudomonas solanacearum phcA, a gene that when mutated alters expression of multiple traits that contribute to virulence. J Bacteriol 175:5477–5487CrossRefGoogle Scholar
  5. Buddenhagen I, Kelman A (1964) Biological and physiological aspects of bacterial wilt caused by Pseudomonas solanacearum. Annu Rev Phytopathol 2:203–230CrossRefGoogle Scholar
  6. Carney BF, Denny TP (1990) A cloned avirulent gene from Pseudomonas solanacearum determines incompatibility on Nicotiana tabacum at the host species level. J Bacteriol 172:4836–4843Google Scholar
  7. Caruso P, Palomo JL, Bertolini E, Alvarez B, Lo’pez MM, Biosca EG (2005) Seasonal variation of Ralstonia solanacearum biovar 2 populations in a Spanish river: recovery of stressed cells at low temperatures. Appl Environ Microbiol 71:140–148CrossRefGoogle Scholar
  8. Champoiseau PG, Jones JB, Allen C (2009) Ralstonia solanacearum race 3 biovar 2 causes tropical losses and temperate anxieties. Plant Health Prog 10:1–10CrossRefGoogle Scholar
  9. Denny TP, Makini FW, Brumbley SM (1988) Characterization of Pseudomonas solanacearum Tn5 mutants deficient in extracellular polysaccharide. Molecular Plant-Microbe Interactions 1:215–223Google Scholar
  10. Di Bscegliei DP, Saccardi A, Giosue S, Traversa F, Mazzucchi U (2005) Survival of Ralstonia solanacearum on wood, high density polyethylene and on jute fabric in cold storage. Journal of Plant Pathology 87:145–147Google Scholar
  11. Elphinstone JG (2005) The current bacterial wilt situation: a global overview. In: Allen C, Prior P, Hayward AC (eds) Bacterial wilt: the disease and the Ralstonia solanacearum species complex. APS Press, St Paul, MN, USA, pp 9–28Google Scholar
  12. Elphinstone JG, Stanford HM, Stead DE (1998) Detection of Ralstonia solanacearum in potato tubers, Solanum dulcamara and associated irrigated water. In: Prior P, Allen C, Elphinstone J (eds) Bacterial wilt disease: molecular and ecological aspects, Reports of the second international bacterial wilt symposium, 22–7 June 1997. Guadeloupe, France, pp 133–139CrossRefGoogle Scholar
  13. Elphinstone JG, Hennessy J, Wilson JK, Stead DE (1996) Sensitivity of different methods for the detection of Ralstonia solanacearum in potato tuber extracts. Bulletin OEEP/EPPO Bulletin 26:663–678CrossRefGoogle Scholar
  14. Engelbrecht MC (1994) Modification of a semi-selective medium for the isolation and quantification of Pseudomonas solanacearum. ACIAR Bacterial Wilt Newsletter 10:3–5Google Scholar
  15. Fava F, Gioia DD, Marchetti L (1993) Characterization of a pigment produced by Pseudomonas fluorescens during 3-chlorobenzoate co-metabolism. Chemosphere 27:825–835CrossRefGoogle Scholar
  16. Farag, NS (1976) Interaction Between Some Soil Microflora and Pseudomonas solanasearum (Ph.D). Department of Microbiology, Fac. of Agric., Ain Shams Univ ,118Google Scholar
  17. Farag NS, Fawzi FG, El-Said SIA, Mikhail MS (1986) Streptomycin in relation to potato brown rot control. Acta Phytopathological et Entomologica Hungarica 21:115–122Google Scholar
  18. Farag SMA, Elhalag KMA, Mohamed H, Hagag MH, Khairy ASM, Ibrahim HM, Saker MT, Messiha NAS (2017) Potato bacterial wilt suppression and plant health improvement after application of different antioxidants. J Phytopathol 165:522–537CrossRefGoogle Scholar
  19. French EB, Gutarra L, Aley P, Elphinstone J (1995) Culture media for Ralstonia solanacearum isolation, identification, and maintenance. Fitopatologia 30:126–130Google Scholar
  20. Gomez KA, Gomez AA (1984) Statistical procedures for agricultural research, 2nd edn. John Wiley and Sons Inc., New YorkGoogle Scholar
  21. González ET, Brown DG, Swanson JK, Allen C (2007) Using the Ralstonia solanacearum T at Secretome to identify bacterial wilt virulence factors. Appl Environ Microbiol 73:3779–3786CrossRefGoogle Scholar
  22. Granada GA, Sequeira L. (1975) A hypersensitive reaction induced in tobacco leaves by a compatible isolate of Pseudomonas solanacearum. Phytopathology 65:731–733Google Scholar
  23. Granada GA, Sequeira L (1983) Survival of Pseudomonas solanacearum in soil, rhizosphere, and plant roots. Can J Microbiol 29:433–440Google Scholar
  24. Habashy WH, Fawzi FG, Neweigy NA, El-Huseiny TM (1991) Bacterial wilt of potatoes: 1. Enzymes secreted by fluidal virulent and butyrous avirulent mutants of Pseudomonas solanacearum Egypt J Agric Res 69:709–720Google Scholar
  25. Hanafy MS, El-Habbaa GM, Mohamed FG, Balabel NM, Ahmed GA (2018) Surveying and fast detection of Ralstonia solanacearum bacterium in some Egyptian governorates. Annals of Agric. Sci. Moshtohor 56:405–416Google Scholar
  26. Hayward AC (1964) Characteristics of Pseudomonas solanacearum. J Appl Bacteriol 27:265–277CrossRefGoogle Scholar
  27. Hayward AC (1991) Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum. Annu Rev Phytopathol 29:65–87CrossRefGoogle Scholar
  28. Hayward AC, Denny TP (2001) Ralstonia pp. In: Schaad NW, Jones JB, Chun W (eds) Laboratory guide for identification of plant pathogenic Bacteria, 5th edn. APS Press, St. Paul, Minnesota, MN, pp 151–173Google Scholar
  29. Henson JM, Butler MJ, Day AW (1999) The dark side of the mycelium: Melanins of phytopathogenic fungi. Annu Rev Phytopathol 37:447–471CrossRefGoogle Scholar
  30. Hudelson, BD, Williamson, L, Nakaho, K, Allen, C (2002) Ralstonia solanacearum race 3, biovar 2 strains isolated from geranium are pathogenic on potato. In: Proceedings 3rd International Symposium on Bacterial Wilt, White River 2002, 14Google Scholar
  31. Janse JD (1988) A detection method for Pseudomonas solanacearum in symptomless potato tubers and some data on its sensitivity and specificity. EPPO Bulletin 18:343–351CrossRefGoogle Scholar
  32. Janse JD, van den Beld HE, Elphinstone J, Simpkins S, Tjou-Tam-Sin NN, van Vaerenbergh J (2004) Introduction to Europe of Ralstonia solanacearum biovar 2, race 3 in pelargonium zonale cuttings. J Plant Pathol 86:147–155Google Scholar
  33. Kelman A (1954) The relationship of pathogenicity in Pseudomonas solanacearum to colony appearance on a tetrazolium medium. Phytopathology 44:693–695Google Scholar
  34. Kelman A, Hruschka J (1973) The role of motility and aerotaxis in the selective increase of virulent bacteria in still broth culture of Pseudomonas solanacearum. J. Gen. Microbiol. 76:177–188PCCrossRefGoogle Scholar
  35. Klement Z (1963) Rapid detection of the pathogenicity of phytopathogenic Pseudomonads. Nature 199(4890):299–300CrossRefGoogle Scholar
  36. Liu, B, Lin, YZ, Zhu, YJ, Ge, CB Cao, Y (2004) Attenuation characteristics of bacterial wilt disease biocontrol strain anti-8098A (Bacillus cereus) to Ralstonia solanacearum. J Agric Biotech, 12:322–329Google Scholar
  37. Mikhail MS, Abdel-Alim AI, Balabel NM, Abbas AM (2017) Virulence of Ralstonia solanacearum phylotype II sequevar 1, the causal pathogen of potato brown rot. J Biol Chem Environ Sci 21:219–235Google Scholar
  38. Pradhanang PM, Elphinstone JG, Fox RTV (2000) Sensitive detection of Ralstonia solanacearum in soil: a comparison of different detection techniques. Plant Pathology 49:414–422Google Scholar
  39. Priou S, Gutarra L, De Mendiburu F, Llique R. (2010) Detection of Ralstonia solanacearum(Biovar 2A) in stems of symptomless plants before harvest of the potato crop using post-enrichment DAS-ELISA. Plant Pathol 59:59–67.Google Scholar
  40. Safni I, Cleenwerck I, De Vos P, Fegan M, Sly L, Kappler U (2014) Polyphasic taxonomic revision of the Ralstonia solanacearum species complex: proposal to emend the descriptions of Ralstonia solanacearum and Ralstonia syzygii and reclassify current R. syzygii strains as Ralstonia syzygii subsp. syzygii subsp. nov., R. solanacearum phylotype IV strains as Ralstonia syzygii subsp. indonesiensis subsp. nov., banana blood disease bacterium strains as Ralstonia syzygii subsp. celebesensis subsp. nov. and R. solanacearum phylotype I and III strains as Ralstonia pseudosolanacearum sp. nov. Int. J. Syst. Evol. Microbiol 64:3087–3103CrossRefGoogle Scholar
  41. Sajjan S, Kulkarni G, Yaligara V, Kyoung L, Karegoudar TB (2010) Purification and physiochemical characterization of melanin pigment from Klebsiella sp. GSK Journal of Microbiology and Biotechnology 20:1513–1520CrossRefGoogle Scholar
  42. SAS (2003) Statistical Analysis System. SAS Release 9.1 for windows, SAS Institute Inc. Cary, NC, USAGoogle Scholar
  43. Seal SE, Jackson LA, Young JPW, Daniels MJ (1993) Differentiation of Pseudomonas solanacearum, Pseudomonas syzygii, Pseudomonas pickettii and the blood disease bacterium by partial 16S rRNA sequencing: construction of oligonucleotide primers for sensitive detection by polymerase chain reaction. J Gen Microbiol 139:1587–1594CrossRefGoogle Scholar
  44. She X, Yu L, Lan G, Tang Y, He Z (2017) Identification and genetic characterization of Ralstonia solanacearum species complex isolates from Cucurbita maxima in China. Front Plant Sci 8:1794CrossRefGoogle Scholar
  45. Thomas P, Upreti R (2014) Significant effects due to peptone in Kelman medium on colony characteristics and virulence of Ralstonia solanacearum in tomato. Open J Microbiol 8:87–105CrossRefGoogle Scholar
  46. Van der Wolf JM, Vriend SGC, Kastelein P, Nijhuis EH, van Bekkum PJ, van Vuurde JWL. (2000). Immunofluorescence colony-staining (IFC) for detection and quantification of Ralstonia(Pseudomonas) solanacearum biovar 2 (race 3) in soil and verification of positive results by PCR and dilution plating. Eur J Plant Pathol. 106:123–133Google Scholar
  47. Velupillai M, Stall RE (1984) Variation among strains of Pseudomonas solanacearum from Florida. Proc Fla State Hort Soc 97:209–213Google Scholar
  48. Whatley MH, Hunter N, Cantrell MA, Hendrick, C, Keegstra, K, Sequeira, L. (1980) Lipopolysaccharide composition of the wilt pathogen, Pseudomonas solanacearum: Correlation with the hypersensitive response in tobacco. Plant Physiol 65:557–559Google Scholar
  49. Zheng XF, Zhu YJ, Liu B, Zhou Y, Che JM, Lin NQ (2014) Relationship between Ralstonia solanacearum diversity and severity of bacterial wilt disease in tomato fields in China. J Phytopathol 162:607–616CrossRefGoogle Scholar
  50. Zhu Y, Zhou H, Liu B, Zhang S (2004) Study on the growing competition relationship between virulent and avirulent strains Ralstonia solanacearum isolated from tomatoes. Xiamen Univ. Nat Sci 43:97–101Google Scholar

Copyright information

© Società Italiana di Patologia Vegetale (S.I.Pa.V.) 2019

Authors and Affiliations

  • Yasser E. Ibrahim
    • 1
    • 2
    Email author
  • Naglaa M. Balabel
    • 2
  • Amgad A. Saleh
    • 1
    • 3
  • Nabil S. Farag
    • 2
  1. 1.Plant Protection Department, College of Food and Agriculture SciencesKing Saud UniversityRiyadhSaudi Arabia
  2. 2.Bacterial Diseases Research Department, Agricultural Research Center (ARC)Plant Pathology Research InstituteGizaEgypt
  3. 3.Agricultural Research Center (ARC)Agricultural Genetic Engineering Research InstituteGizaEgypt

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