This study characterized 38 bacteriophage strains for their ability to lyse various bacterial plant pathogens. 24 phage strains were able to lyse at least one bacterial strain tested. One phage strain (KΦ1) isolated from diseased pepper tissue formed clear plaques on Xanthomonas euvesicatoria and had a titer of 1.26x1010 plaque forming units per ml. Six phage strains were specifically lytic against Xanthomonas species affecting peppers and tomatoes, while two additional phages lysed three bacterial species. Selected phages that lysed pathogens of vegetable crops could potentially be used to control these economically important diseases in vivo.

1. 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 strains
X. 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. PLP100 - - - - - - - - - - - - -
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.
Introduction
Bacterial 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.
Results
Phage 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.
Conclusion
Bacteriophages 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.