Multiplex real-time PCR for the detection of Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and pathogenic Xanthomonas species on tomato plants

Autoři: Eliška Peňázová aff001;  Miloň Dvořák aff002;  Lucia Ragasová aff003;  Tomáš Kiss aff001;  Jakub Pečenka aff001;  Jana Čechová aff001;  Aleš Eichmeier aff001
Působiště autorů: Mendeleum–Department of Genetics, Faculty of Horticulture, Mendel University in Brno, Lednice, Czech Republic aff001;  Department of Forest Protection and Wildlife Management, Faculty of Forestry and Wood Technology, Mendel University in Brno, Brno, Czech Republic aff002;  Department of Vegetable Sciences, Faculty of Horticulture, Mendel University in Brno, Lednice, Czech Republic aff003
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article


A multiplex real-time PCR method based on fluorescent TaqMan® probes was developed for the simultaneous detection of the tomato pathogenic bacteria Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and bacterial spot-causing xanthomonads. The specificity of the multiplex assay was validated on 44 bacterial strains, including 32 target pathogen strains as well as closely related species and nontarget tomato pathogenic bacteria. The designed multiplex real-time PCR showed high sensitivity when positive amplification was observed for one pg of bacterial DNA in the cases of Clavibacter michiganensis subsp. michiganensis and Pseudomonas syringae pv. tomato bacteria and 100 pg for bacterial spot-causing xanthomonads. The reliability of the developed multiplex real-time PCR assay for in planta detection was verified by recognition of the target pathogens in 18 tomato plants artificially inoculated by each of the target bacteria and tomato samples from production greenhouses.

Klíčová slova:

Bacterial pathogens – Tomatoes – Polymerase chain reaction – Leaves – Plant bacterial pathogens – Nucleotide sequencing – Pseudomonas syringae – Bacterial cultures


1. Özdemir Z. Development of a multiplex PCR assay for the simultaneous detection of Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and Xanthomonas axonopodis pv. vesicatoria using pure cultures. J Plant Pathol. 2009; 91: 495–497.

2. Kolomiets JV, Grygoryuk IP, Butsenko LM. Bacterial diseases of tomato plants in terms of open and covered growing of Ukraine. Annals of Agrarian Science. 2017; 15(2): 213–216.

3. Moretti C, Amatulli MT, Buonaurio R. PCR-based assay for the detection of Xanthomonas euvesicatoria causing pepper and tomato bacterial spot. Lett Appl Microbiol. 2009; 49(4): 466–471. doi: 10.1111/j.1472-765X.2009.02690.x 19674291

4. EPPO. EPPO A2 List of pests recommended for regulation as quarantine pests, version 2018–09. 2018 [cited 1 May 2019]. In: European and Mediterranean Plant Protection Organisation [Internet]. Available from:

5. Preston G. Pseudomonas syringae pv. tomato: the right pathogen, of the right plant, at the right time. Mol Plant Pathol. 2000; 1(5): 263–275. doi: 10.1046/j.1364-3703.2000.00036.x 20572973

6. Zaccardelli M, Parisi M, Giordano I. Susceptibility of tomato genotypes to Pseudomonas syringae pv. tomato in the field conditions. J Plant Pathol. 2002; 84: 200.

7. Carlton WM, Braun EJ, Gleason ML. Ingress of Clavibacter michiganensis subsp michiganensis into tomato leaves through hydathodes. Phytopathology. 1998; 88(6): 525–529 doi: 10.1094/PHYTO.1998.88.6.525 18944904

8. De León L, Siverio F, López MM, Rodríguez A. Clavibacter michiganensis subsp. michiganensis, a seedborne tomato pathogen: healthy seeds are still the goal. Plant Dis. 2011; 95: 1328–1338. doi: 10.1094/PDIS-02-11-0091 30731794

9. Chalupowicz L, Zellermann E-M, Fluegel M, Dror O, Eichenlaub R, Gartemann K-H, et al. Colonization and movement of GFP-labeled Clavibacter michiganensis subsp. michiganensis during tomato infection. Phytopathology. 2012; 102: 23–31. doi: 10.1094/PHYTO-05-11-0135 21879791

10. Medina-Mora C, Hausbeck MK, Fulbright DW. Bird’s eye lesions of tomato fruit produced by aerosol and direct application of Clavibacter michiganensis subsp. michiganensis. Plant Dis. 2001; 85: 88–91. doi: 10.1094/PDIS.2001.85.1.88 30832078

11. Gartemann K-H, Kirchner O, Engemann J, Gräfen I, Eichenlaub R, Burger A. Clavibacter michiganensis subsp. michiganensis: first steps in the understanding of virulence of a Gram-positive phytopathogenic bacterium. J Biotechnol. 2003; 106: 179–191. doi: 10.1016/j.jbiotec.2003.07.011 14651860

12. McCarter SM, Jones JB, Gitaitis RD, Smitky DR. Survival of Pseudomonas svringae pv. tomato in association with tomato seed, soil, host tissue and epiphytic weed hosts in Georgia. Phytopathology. 1983; 73: 1393–1398.

13. Bashan Y, Diab S, Okon Y. Survival of Xanthomonas campestris pv. vesicatoria in pepper seeds and roots in symptomless and dry leaves in non-host plants and in the soil. Plant Soil. 1982; 68(2): 161–170.

14. Goode MJ, Sasser M. Prevention–The key to controlling bacterial spot and bacterial speck of tomato. Plant Dis. 1980; 64: 831–834.

15. Jones JB, Lacy GH, Bouzar H, Stall RE, Schaad NW. Reclassification of the Xanthomonads associated with Bacterial spot disease of tomato and pepper. Syst Appl Microbiol. 2004; 27(6): 755–762. doi: 10.1078/0723202042369884 15612634

16. Young JM, Park DC, Shearman HM, Fargier F. A multilocus sequence analysis of the genus Xanthomonas. Syst Appl Microbiol. 2008; 31: 366–377. doi: 10.1016/j.syapm.2008.06.004 18783906

17. Hamza AA, Robène-Soustrade I, Jouen E, Gagnevin L, Lefeuvre P. Genetic and pathological diversity among Xanthomonas strains responsible for bacterial spot on tomato and pepper in the southwest Indian Ocean region. Plant Dis. 2010; 94: 993–999. doi: 10.1094/PDIS-94-8-0993 30743480

18. Schwartz AR, Potnis N, Timilsina S, Wilson M, Patane J, Martins J, et al. Phylogenomics of Xanthomonas field strains infecting pepper and tomato reveals diversity in effector repertoires and identifies determinants of host specificity. Front Microbiol. 2015; 6: 535. doi: 10.3389/fmicb.2015.00535 26089818

19. Blancard D. Tomato Diseases: A Colour Handbook. 2nd ed. London: Academic Press; 2012.

20. Jones JB, Pohronezny KL, Stall RE, Jones JP. Survival of Xanthomonas campestris pv. vesicatoria in Florida on tomato crop residue, weeds, seeds, and volunteer tomato plants. Phytopathology. 1986; 76(4): 430–434.

21. Dutta B, Gitaitis R, Sanders H, Booth C, Smith S, Langston DB. Role of blossom colonization in pepper seed infestation by Xanthomonas euvesicatoria. Phytopathology. 2014; 104: 232–239. doi: 10.1094/PHYTO-05-13-0138-R 24111576

22. Ritchie DF. Bacterial spot of pepper and tomato. The Plant Health Instructor. 2000 [cited 1 May 2019]. In: The American Phytopathological Society (APS) [Internet]. Available from:

23. Burokiene D. Early detection of Clavibacter michiganensis subsp. michiganensis in tomato seedlings. Agronomy Research. 2006; 4: 151–154.

24. Pastrik KH, Rainey FA. Identification and differentiation of Clavibacter michiganensis subspecies by polymerase chain reaction-based techniques. J Phytopathol. 1999; 147: 687–693.

25. Zaccardelli M, Spasiano A, Bazzi C, Merighi M. Identification and in planta detection of Pseudomonas syringae pv. tomato using PCR amplification of hrpZ(Pst). Eur J Plant Pathol. 2005; 111(1): 85–90.

26. Fath-Allah MM, Ali MH, Rasmi MR. Using hrpL gene specific primers to detect Pseudomonas syringae pv. tomato by polymerase chain reaction. Egyptian Journal of Experimental Biology (Agric.). 2006; 2: 7–13.

27. Koenraadt H, van Betteray B, Germain R, Hiddink G, Jones JB, Oosterhof J, et al. Development of specific primers for the molecular detection of bacterial spot of pepper and tomato. Acta Hortic. 2009; 808: 99–102.

28. Kokošková B, Mráz I, Fousek J. Comparison of specificity and sensitivity of immunochemical and molecular techniques for determination of Clavibacter michiganensis subsp. michiganensis. Folia Microbiol (Praha). 2010; 55 (3): 239–244.

29. Bach HJ, Jessen I, Schloter M, Munch JC. A TaqMan-PCR protocol for quantification and differentiation of the phytopathogenic Clavibacter michiganensis subspecies. J Microbiol Methods. 2003; 52: 85–91. doi: 10.1016/s0167-7012(02)00152-5 12401230

30. Fanelli V, Cariddi C, Finetti-Sialer M. Selective detection of Pseudomonas syringae pv. tomato using dot blot hybridization and real-time PCR. Plant Pathol. 2007; 56(4): 683–691.

31. Oosterhof J, Berendsen S. The development of a specific Real‐Time TaqMan for the detection of Clavibacter michiganensis supsp. michiganensis (Abstr.). Phytopathology. 2011; 101: S133.

32. Araujo ER, Costa JR, Ferreira MASV, Quezado-Duval AM. Simultaneous detection and identification of the Xanthomonas species complex associated with tomato bacterial spot using species-specific primers and multiplex PCR. J Appl Microbiol. 2012; 113: 1479–1490. doi: 10.1111/j.1365-2672.2012.05431.x 22900936

33. Dreier J, Bermpohl A, Eichenlaub R. Southern hybridization and PCR for specific detection of phytopathogenic Clavibacter michiganensis subsp. michiganensis. Phytopathology. 1995; 85: 462–468.

34. Ftayeh R, von Tiedemann A, Koopmann B, Rudolph K, Abu-Ghorrah M. First record of Clavibacter michiganensis subsp. michiganensis causing canker of tomato plants in Syria. Plant Dis. 2008; 92(4), 649.

35. Kozik EU, Sobiczewski P. Assessment of inoculation techniques suitability for determination of tomato plants resistance to bacterial speck (Pseudomonas syringae pv. tomato). Phytopathol Polonica. 2007; 44: 17–25.

36. Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics. 2012; 13: 134. doi: 10.1186/1471-2105-13-134 22708584

37. Evans RN, Blaha G, Bailey S, Steitz TA. The structure of LepA, the ribosomal back translocase. Proc Natl Acad Sci USA. 2008; 105(12): 4673–4678. doi: 10.1073/pnas.0801308105 18362332

38. Youngman EM, Green R. Ribosomal translocation: lepA does it backwards. Curr Biol. 2007; 17(4): R136–R139. doi: 10.1016/j.cub.2006.12.029 17307049

39. He L-S, Zhang P-W, Huang J-M, Zhu F-C, Danchin A, Wang Y. The enigmatic genome of an obligate ancient Spiroplasma symbiont in a hadal holothurian. Appl Environ Microbiol. 2018; 84: e01965–17. doi: 10.1128/AEM.01965-17 29054873

40. Bereswill S, Bugert P, Volksch B, Ullrich M, Bender CL, Geider K. Identification and relatedness of coronatine-producing Pseudomonas syringae pathovars by PCR analysis and sequence determination of the amplification products. Appl Environ Microbiol. 1994; 60(8): 2924–2930. 7916181

41. Leite RP, Minsavage GV, Bonas U, Stall RE. Detection and identification of phytopathogenic Xanthomonas strains by amplification of DNA sequences related to the hrp genes of Xanthomonas campestris pv. vesicatoria. Appl Environ Microbiol. 1994; 60(4): 1068–1077. 8017904

42. Zhao W-J, Chen H-Y, Zhu S-F, Xia M-X, Tan T-W. One-step detection of Clavibacter michiganensis subsp. michiganensis in symptomless tomato seeds using a TaqMan probe. J Plant Pathol. 2007; 89(3): 349–351.

43. Luo LX, Walters C, Bolkan H, Liu XL, Li JQ. Quantification of viable cells of Clavibacter michiganensis subsp. Michiganensis using a DNA binding dye and a real-time PCR assay. Plant Pathol. 2008; 57: 332–337.

44. Hadas R, Kritzman G, Kleitman F, Gefen T, Manulis S. Comparison of extraction procedures and determination of the detection threshold for Clavibacter michiganensis ssp. michiganensis in tomato seeds. Plant Pathol. 2005; 54: 643–649.

45. Kleitman F, Barash I, Burger A, Iraki N, Falah Y, Sessa G, et al. Characterization of a Clavibacter michiganensis subsp. michiganensis population in Israel. Eur J Plant Pathol. 2008; 121: 463–475.

46. Caraguel ChGB, Stryhn H, Gagné N, Dohoo IR, Hammell KL. Selection of a cutoff value for real-time polymerase chain reaction results to fit a diagnostic purpose: analytical and epidemiologic approaches. J Veg Diagn Invest. 2011; 23: 2–15.

47. Oosterhof J, Berendsen S. The development of a specific Real-Time TaqMan for the detection of Clavibacter michiganensis supsp. michiganensis (Abstr.). Phytopathology. 2011; 101: S133.

48. EPPO. PM 7/42 (3) Clavibacter michiganensis subsp. michiganensis. EPPO Bulletin. 2016; 46(2): 202–225.

49. Lindgren PB, Peet RC, Panapoulos NJ. Gene cluster of Pseudomonas syringae pv. phaseolicola controls pathogenicity of bean plants and hypersensitivity of nonhost plant. J Bacteriol. 1986; 168(2): 515–522.

50. Lonetto MA, Brown KL, Rudd KE, Buttner M. Analysis of the Streptomyces coelicolor sig E gene reveals the existence of a sub-family of eubacterial RNA polymerase sigma factors involved in the regulation of extracytoplasmatic functions. Proc Natl Acad Sci USA. 1994; 91: 7573–7577. doi: 10.1073/pnas.91.16.7573 8052622

51. Huang HC, Lin RH, Chang CJ, Collmer A, Deng WL. The complete hrp gene cluster of Pseudomonas syringae pv. syringae 61 includes two blocks of genes required for harpin Pss secretion that are arranged colinearly with Yersinia ysc homologs. Mol Plant Microbe Interact. 1995; 8: 733–746 doi: 10.1094/mpmi-8-0733 7579617

52. Alfano JR, Collmer A. The type III (Hrp) secretion pathway of plant pathogenic bacteria: trafficking harpins, Avr proteins, and death. J Bacteriol. 1997; 179(18): 5655–62. doi: 10.1128/jb.179.18.5655-5662.1997 9294418

53. Strayer A, Jeyaprakash A, Minsavage GV, Timilsina S, Vallad GE, Jones JB, et al. A multiplex real-time PCR assay differentiates four Xanthomonas species associated with bacterial spot of tomato. Plant Dis. 2016; 100: 1660–1668. doi: 10.1094/PDIS-09-15-1085-RE 30686244

54. Potnis N, Timilsina S, Strayer A, Shantharaj D, Barak JD, Paret ML, et al. Bacterial spot of tomato and pepper: diverse Xanthomonas species with a wide variety of virulence factors posing a worldwide challenge. Mol Plant Pathol. 2015; 16: 907–20. doi: 10.1111/mpp.12244 25649754

55. Timilsina S, Jibrin MO, Potnis N, Minsavage GV, Kebede M, Schwartz A, et al. Multilocus sequence analysis of xanthomonads causing bacterial spot of tomato and pepper reveals strains generated by recombination among species and recent global spread of Xanthomonas gardneri. Appl Environ Microbiol. 2015; 81(4): 1520–1529. doi: 10.1128/AEM.03000-14 25527544

Článok vyšiel v časopise


2020 Číslo 1
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle

Zvýšte si kvalifikáciu online z pohodlia domova

Eozinofilní granulomatóza s polyangiitidou
nový kurz
Autori: doc. MUDr. Martina Doubková, Ph.D.

Všetky kurzy
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.


Nemáte účet?  Registrujte sa