Marina Lazarević, Katarina Gašić, Aleksa Obradović

University of Belgrade, Faculty of Agriculture, Plant Pathology Department, Belgrade, Serbia

DETERMINATION OF BACTERIOPHAGE TITER AND HOST RANGE

Figure 1. X. euvesicatoria. Bacterial spot on pepper leaves (A) and pepper fruit (B). Natural infection.

A) B)

No. Phage strain

Bacterial strainsX.

euvesicatoria(KFB 189)

X. perforans(KFB 061)

X. perforans(KFB 0109)

X. gardneri(KFB 0110)

X. gardneri(KFB 0116)

X. vesicatoria(KFB 0108)

E. amylovora(KFB 182)

P. c. subsp. carotovorum

(KFB 68)

P. c. subsp. carotovorum

(KFB 85)

P. lachrymans(KFB 214)

P. lachrymans(KFB 0117)

P. fluorescens(B 130)

R.solanacearum

(KFB 025)

1. 1 - - - - - - - - - - - - -2. 2 + - - - - - - - - - - - -3. 3 - - - - - - - - - - - - -4. 44 - + - - - + - - - - - - -5. 45 - + - - - + - - - + - - -6. 46 - - - - - + - - - - - - -7. 47 - (+) - - - - - - - - - - -8. 48 - (+) - - - - - - - - - - -9. 49 - + - - - + - - - - - - -10. 50 - + - - - + - - - - - - -11. 51 - + - - - + - - - + - - -12. 52 - + - - - - - - - - - - -13. 53 - - - - - + - - - - - - -14. 54 - - - - - - - - - + - - -15. 55 - - - - - + - - - + - - -16. 56 - - - - - - - - - - - - -17. 57 + - - - - - - - - - - - -18. 58 - - - - - - - - - - - - -19. 59 + - - - - - - - - - - - -20. 60 + - - - - - - - - - - - -21. 61 - - - - - - - - - - - - -22. 62 - - - - - - - - - - - - -23. 63 + - - - - - - - - - - - -24. 64 + - - - - + - - - - - - -25. 65 - - - - - - - - - - - - -26. 66 + - - - - - - - - - - - -27. 67 - - - - - - - - - - - - -28. 68 - - - - - - - - - - - - -29. 69 + - - - - - - - - - - - -30. 70 - - - - - - - - - - - - -31. 71 - - - - - - - - - - - - -32. 72 - - - - - - - - - - - - -33. 73 + - - - - - - - - - - - -34. 74 - - - - - - - - - - - - -35. 75 + - - - - (+) - - - - - - -36. PLP10

0- - - - - - - - - - - - -

37. RS5M - - - - - - - - - - - - +38. KФ1 + - - - - - - - - - - - -

A) B) C)

Figure 2. X. euvesicatoria specific bacteriophage, strain KΦ1, Single plaque formation in NYA medium using different dilutions of phage suspension: A )10ˉ7, B)10ˉ8, C)10ˉ9.

IntroductionBacterial spot of pepper, caused by Xanthomonas euvesicatoria, is one of the major

pepper diseases in Serbia (Fig. 1). Copper-based treatments, frequently used for control of this disease, were ineffective especially in conditions favorable for the infection. Therefore, alternative disease control strategies are needed.

One of possible alternatives could be use of biocontrol agents in plant protection. Investigation of bacteriophages, viruses that attack bacteria, is a fast-expanding area of research. Being widespread natural bacterial enemies, simple for cultivation and management, host-specific, suitable for integration with other control practices, human and environment friendly, bacteriophages have a great advantage over other bactericides.

ResultsPhage strain KΦ1 formed clear plaques, 4 to 5 mm in diameter, with

sharp edges, on lawns of X. euvesicatoria strain KFB 189, after 48 h of incubation at 27°C (Fig. 2B and 2C). Based on the number of single plaques formed in NYA medium using three consecutive dilutions (Fig. 2), concentration of phage KΦ1 was calculated as 1.26x1010 PFU/ml.

Out of 38 tested phages, 24 strains lysed at least one bacterial strain (Tab. 1). Six phage strains were specific to the two Xanthomonas species, while two phages showed lytic activity to three different species. Fourteen strains showed no activity, which can be explained by lack of specificity to the tested bacteria or inactivation during the long term storage.

ConclusionBacteriophages that lysed strains of pepper- and tomato-associated

xanthomonads (X. euvesicatoria, X. vesicatoria, X. perforans) as well as strains of R. solanacearum or P. lachrymans were determinated. Since these bacteria have been identified as economically important pathogens of vegetable crops in Serbia, selected phages can be used for their control in vivo.

Material and methods X. euvesicatoria phage strain KΦ1, used in this study, was isolated from diseased pepper tissue showing symptoms of bacterial spot, in 2005. Phages were

purified by three subsequent single plaque isolations. Concentration was determined by dilution-plating-plaque count assay on NYA plates and calculated from the plaque number and specific dilution (Klement et al., 1990). Result was expressed as plaque forming units per ml (PFU/ml) of phage suspension in Nutrient Broth.

In order to determine host range of 38 phage strains stored in our collection at 4°C, we studied their lytic activity in cultures of pepper- and tomato-associated xanthomonads (X. euvesicatoria, X. vesicatoria, X. perforans and X. gardneri) as well as in culture of Pseudomonas fluorescens, Ralstonia solanacearum, Pseudomonas lachrymans and Pectobacterium carotovorum subsp. carotovorum (Gašić et al., 2011).

References:Gašić, K., Ivanović, M.M., Ignjatov, M., Ćalić, A., Obradović, A. (2011): Isolation and characterization of Xanthomonas euvesicatoria bacteriophages. Journal of Plant Pathology, 93 (2): 415-423.Klement, Z., Rudolf, K., Sands, D.C. (1990): Methods in Phytobacteriology. Akadémiai Kiadó, Budapest.

Table 1. Bacteriphage specificity to different bacterial species.

Legend: + clear plaque formation; (+) turbid plaque formation; - no plaque formation

This research is result of project: III46008 Development of integrated management of harmful organisms in plant production in order to overcome resistance and to improve food quality and safety, supported by Ministry of education and science, Republic of Serbia.