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Research article

Environmental Microbiology and Engineering

Isolation and Characterization of Bacteriophages Against Pseudomonas syringae pv. actinidiae Causing Bacterial Canker Disease in Kiwifruit

Ji-Gang Yu 1, Jeong-A Lim 2, Yu-Rim Song 1, Sunggi Heu 3, Gyoung Hee Kim 4, Young Jin Koh 4 and Chang-Sik Oh 1*

1Department of Horticultural Biotechnology, Kyung Hee University, Yongin 17104, Republic of Korea, 2Division of Microbial Safety, National Academy of Agricultural Science, Rural Development Administration, Jeonju 55365, Republic of Korea, 3Crop Cultivation and Environment Research Division, National Institute of Crop Science, Rural Development Administration, Suwon 16429, Republic of Korea, 4Department of Plant Medicine, Sunchon National University, Suncheon 57922, Republic of Korea

Received: September 4, 2015; Accepted: December 1, 2015

J. Microbiol. Biotechnol. 2016; 26(2): 385-393

Published February 28, 2016 https://doi.org/10.4014/jmb.1509.09012

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

Pseudomonas syringae pv. actinidiae causes bacterial canker disease in kiwifruit. Owing to the prohibition of agricultural antibiotic use in major kiwifruit-cultivating countries, alternative methods need to be developed to manage this disease. Bacteriophages are viruses that specifically infect target bacteria and have recently been reconsidered as potential biological control agents for bacterial pathogens owing to their specificity in terms of host range. In this study, we isolated bacteriophages against P. syringae pv. actinidiae from soils collected from kiwifruit orchards in Korea and selected seven bacteriophages for further characterization based on restriction enzyme digestion patterns of genomic DNA. Among the studied bacteriophages, two belong to the Myoviridae family and three belong to the Podoviridae family, based on morphology observed by transmission electron microscopy. The host range of the selected bacteriophages was confirmed using 18 strains of P. syringae pv. actinidiae, including the Psa2 and Psa3 groups, and some were also effective against other P. syringae pathovars. Lytic activity of the selected bacteriophages was sustained in vitro until 80 h, and their activity remained stable up to 50oC, at pH 11, and under UV-B light. These results indicate that the isolated bacteriophages are specific to P. syringae species and are resistant to various environmental factors, implying their potential use in control of bacterial canker disease in kiwifruits.

Keywords

biocontrol agents, host range, phage therapy, Psa, specificity

References

  1. Balestra GM, Renzi M, Mazzaglia A. 2010. First report of bacterial canker of Actinidia deliciosa caused by Pseudomonas syringae pv. actinidiae in Portugal. New Dis. Rep. 22: 20440588.2010.
  2. Balestra GM, Renzi M, Mazzaglia A. 2011. First report of Pseudomonas syringae pv. actinidiae on kiwifruit plants in Spain. New Dis. Rep. 24: 2044-0588.2011.
  3. Balestra GM, Taratufolo MC, Vinatzer BA, Mazzaglia A. 2013. A multiplex PCR assay for detection of Pseudomonas syringae pv. actinidiae and differentiation of populations with different geographic origin. Plant Dis. 97: 472-478.
    CrossRef
  4. Boulé J, Sholberg PL, Lehman SM, O’Gorman DT, Svircev AM. 2011. Isolation and characterization of eight bacteriophages infecting Erwinia amylovora and their potential as biological control agents in British Columbia, Canada. Can. J. Plant Pathol. 33: 308-317.
    CrossRef
  5. Cameron A, Sarojini V. 2014. Pseudomonas syringae pv. actinidiae: chemical control, resistance mechanisms and possible alternatives. Plant Pathol. 63: 1-11.
    CrossRef
  6. Chapman JR, Taylor RK, Weir BS, Romberg MK, Vanneste JL, Luck J, Alexander BJR. 2012. Phylogenetic relationships among global populations of Pseudomonas syringae pv. actinidiae. Phytopathology 102: 1034-1044.
    Pubmed CrossRef
  7. Czajkowski R, Ozymko Z, Lojkowska E. 2014. Isolation and characterization of novel soilborne lytic bacteriophages infecting Dickeya spp. biovar 3 (‘D. solani’). Plant Pathol. 63:758-772.
    CrossRef
  8. De Paepe M, Taddei F. 2006. Viruses' life history: towards a mechanistic basis of a trade-off between survival and reproduction among phages. PLoS Biol. 4: e193.
    Pubmed PMC CrossRef
  9. Deveau H, Labrie SJ, Chopin M-C, Moineau S. 2006. Biodiversity and classification of lactococcal phages. Appl. Environ. Microbiol. 72: 4338-4346.
    Pubmed PMC CrossRef
  10. Di Lallo G, Evangelisti M, Mancuso F, Ferrante P, Marcelletti S, Tinari A, et al. 2014. Isolation and partial characterization of bacteriophages infecting Pseudomonas syringae pv. actinidiae, causal agent of kiwifruit bacterial canker. J. Basic Microbiol. 54: 1210-1221.
    Pubmed CrossRef
  11. Donati I, Buriani G, Cellini A, Mauri S, Costa G, Spinelli F. 2014. New insights on the bacterial canker of kiwifruit (Pseudomonas syringae pv. actinidiae). J. Berry Res. 4: 53-67.
  12. Everett KR, Taylor RK, Romberg MK, Rees-George J, Fullerton RA, Vanneste JL, Manning MA. 2011. First report of Pseudomonas syringae pv. actinidiae causing kiwifruit bacterial canker in New Zealand. Australasian Plant Dis. Notes 6: 67-71.
    CrossRef
  13. Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA. 2005. Virus Taxonomy: VIIIth Report of the International Committee on Taxonomy of Viruses. Academic Press.
  14. Frampton RA, Pitman AR, Fineran PC. 2012. Advances in bacteriophage-mediated control of plant pathogens. Int. J. Microbiol. 2012: 1-11.
    Pubmed PMC CrossRef
  15. Frampton RA, Taylor C, Holguin Moreno AV, Visnovsky SB, Petty NK, Pitman AR, Fineran PC. 2014. Identification of bacteriophages for the biocontrol of the kiwifruit canker phytopathogen Pseudomonas syringae pv. actinidiae. Appl. Environ. Microbiol. 80: 2216-2228.
    Pubmed PMC CrossRef
  16. Fujiwara A, Fujisawa M, Hamasaki R, Kawasaki T, Fujie M, Yamada T. 2011. Biocontrol of Ralstonia solanacearum by treatment with lytic bacteriophages. Appl. Environ. Microbiol. 77: 4155-4162.
    Pubmed PMC CrossRef
  17. Garden L, Shafik H, Belouin S, Broch R, Grimont F, Grimont P. 1999. DNA relatedness among the pathovars of Pseudomonas syringae and description of Pseudomonas tremae sp. nov. and Pseudomonas cannabina sp. nov. (ex Sutic and Dowson 1959). Int. J. Syst. Bacteriol. 49: 469-478.
    Pubmed CrossRef
  18. Han HS, Koh YJ, Hur J-S, Jung JS. 2004. Occurrence of the strA-strB streptomycin resistance genes in Pseudomonas species isolated from kiwifruit plants. J. Microbiol. 42: 365-368.
    Pubmed
  19. Kim J, Park SS, Cho N, Kim W, Cho HK. 2011. Recent variations of UV irradiance at Seoul 2004-2010. Atmosphere 21: 429-438.
  20. Kim M, Ryu S. 2011. Characterization of a T5-like coliphage, SPC35, and differential development of resistance to SPC35 in Salmonella enterica serovar Typhimurium and Escherichia coli. Appl. Environ. Microbiol. 77: 2042-2050.
    Pubmed PMC CrossRef
  21. Koh HS, Lee YS, Jung JS. 2014. Molecular characteristics of Pseudomonas syringae pv. actinidiae strains isolated in Korea and a multiplex PCR assay for haplotype differentiation. Plant Pathol. J. 30: 96-101.
    Pubmed PMC CrossRef
  22. Koh YJ, Chung HJ, Cha BJ, Lee DH. 1994. Outbreak and spread of bacterial canker in kiwifruit. Korean J. Plant Pathol. 10.
  23. Koh YJ, Kim GH, Jung JS, Lee YS, Hur JS. 2010. Outbreak of bacterial canker on Hort16A (Actinidia chinensis Planchon) caused by Pseudomonas syringae pv. actinidiae in Korea. N. Z. J. Crop Hort. Sci. 38: 275-282.
    CrossRef
  24. Kropinski AM, Mazzocco A, Waddell TE, Lingohr E, Johnson RP. 2009. Enumeration of bacteriophages by double agar overlay plaque assay, p. 69-76. Bacteriophages. Springer.
  25. Kutter E, Sulakvelidze A. 2004. Bacteriophages: Biology and Applications. CRC Press.
    CrossRef
  26. Lim JA, Lee DH, Jung KS, Heu S. 2013. Biocontrol of Pectobacterium carotovorum subsp. carotovorum using bacteriophage PP1. J. Microbiol. Biotechnol. 23: 1147-1153.
    Pubmed CrossRef
  27. Maniloff J, Ackermann H-W. 1998. Taxonomy of bacterial viruses: establishment of tailed virus genera and the other Caudovirales. Arch. Virol. 143: 2051-2063.
    Pubmed CrossRef
  28. Nakajima M, Tadaaki H. 2002. Similarity between copper resistance genes from Pseudomonas syringae pv. actinidiae and P. syringae pv. tomato. J. Gen. Plant Pathol. 68: 68-74.
    CrossRef
  29. Scortichini M. 1994. Occurrence of Pseudomonas syringae pv. actinidiae on kiwifruit in Italy. Plant Pathol. 43: 1035-1038.
    CrossRef
  30. Scortichini M, Marcelletti S, Ferrante P, Petriccione M, Firrao G. 2012. Pseudomonas syringae pv. actinidiae: a reemerging, multi-faceted, pandemic pathogen. Mol. Plant Pathol. 13: 631-640.
    Pubmed CrossRef
  31. Snelgar WP, Hall AJ, Ferguson AR, Blattmann P. 2005. Temperature influences growth and maturation of fruit on 'Hayward' kiwifruit vines. Funct. Plant Biol. 32: 631-642.
    CrossRef
  32. Takikawa Y, Serizawa S, Ichikawa T, Tsuyumu S, Goto M. 1989. Pseudomonas syringae pv. actinidiae pv. nov.: the causal bacterium of canker of kiwifruit in Japan. Ann. Phytopathol. Soc. Jap. 55: 437-444.
    CrossRef
  33. Vanneste JL, Poliakoff F, Audusseau C, Cornish DA, Paillard S, Rivoal C, Yu J. 2012. First report of Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker of kiwifruit in France. Mol. Plant Pathol. 13: 631-640.
  34. Young BJ, Lee HJ, Murugaiyan S, Lee SW. 2012. Biocontrol potential of a lytic bacteriophage PE204 against bacterial wilt of tomato. J. Microbiol. Biotechnol. 22: 1613-1620.
    CrossRef

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Article

Research article

J. Microbiol. Biotechnol. 2016; 26(2): 385-393

Published online February 28, 2016 https://doi.org/10.4014/jmb.1509.09012

Copyright © The Korean Society for Microbiology and Biotechnology.

Isolation and Characterization of Bacteriophages Against Pseudomonas syringae pv. actinidiae Causing Bacterial Canker Disease in Kiwifruit

Ji-Gang Yu 1, Jeong-A Lim 2, Yu-Rim Song 1, Sunggi Heu 3, Gyoung Hee Kim 4, Young Jin Koh 4 and Chang-Sik Oh 1*

1Department of Horticultural Biotechnology, Kyung Hee University, Yongin 17104, Republic of Korea, 2Division of Microbial Safety, National Academy of Agricultural Science, Rural Development Administration, Jeonju 55365, Republic of Korea, 3Crop Cultivation and Environment Research Division, National Institute of Crop Science, Rural Development Administration, Suwon 16429, Republic of Korea, 4Department of Plant Medicine, Sunchon National University, Suncheon 57922, Republic of Korea

Received: September 4, 2015; Accepted: December 1, 2015

Abstract

Pseudomonas syringae pv. actinidiae causes bacterial canker disease in kiwifruit. Owing to the
prohibition of agricultural antibiotic use in major kiwifruit-cultivating countries, alternative
methods need to be developed to manage this disease. Bacteriophages are viruses that
specifically infect target bacteria and have recently been reconsidered as potential biological
control agents for bacterial pathogens owing to their specificity in terms of host range. In this
study, we isolated bacteriophages against P. syringae pv. actinidiae from soils collected from
kiwifruit orchards in Korea and selected seven bacteriophages for further characterization
based on restriction enzyme digestion patterns of genomic DNA. Among the studied
bacteriophages, two belong to the Myoviridae family and three belong to the Podoviridae family,
based on morphology observed by transmission electron microscopy. The host range of the
selected bacteriophages was confirmed using 18 strains of P. syringae pv. actinidiae, including
the Psa2 and Psa3 groups, and some were also effective against other P. syringae pathovars.
Lytic activity of the selected bacteriophages was sustained in vitro until 80 h, and their activity
remained stable up to 50oC, at pH 11, and under UV-B light. These results indicate that the
isolated bacteriophages are specific to P. syringae species and are resistant to various
environmental factors, implying their potential use in control of bacterial canker disease in
kiwifruits.

Keywords: biocontrol agents, host range, phage therapy, Psa, specificity

References

  1. Balestra GM, Renzi M, Mazzaglia A. 2010. First report of bacterial canker of Actinidia deliciosa caused by Pseudomonas syringae pv. actinidiae in Portugal. New Dis. Rep. 22: 20440588.2010.
  2. Balestra GM, Renzi M, Mazzaglia A. 2011. First report of Pseudomonas syringae pv. actinidiae on kiwifruit plants in Spain. New Dis. Rep. 24: 2044-0588.2011.
  3. Balestra GM, Taratufolo MC, Vinatzer BA, Mazzaglia A. 2013. A multiplex PCR assay for detection of Pseudomonas syringae pv. actinidiae and differentiation of populations with different geographic origin. Plant Dis. 97: 472-478.
    CrossRef
  4. Boulé J, Sholberg PL, Lehman SM, O’Gorman DT, Svircev AM. 2011. Isolation and characterization of eight bacteriophages infecting Erwinia amylovora and their potential as biological control agents in British Columbia, Canada. Can. J. Plant Pathol. 33: 308-317.
    CrossRef
  5. Cameron A, Sarojini V. 2014. Pseudomonas syringae pv. actinidiae: chemical control, resistance mechanisms and possible alternatives. Plant Pathol. 63: 1-11.
    CrossRef
  6. Chapman JR, Taylor RK, Weir BS, Romberg MK, Vanneste JL, Luck J, Alexander BJR. 2012. Phylogenetic relationships among global populations of Pseudomonas syringae pv. actinidiae. Phytopathology 102: 1034-1044.
    Pubmed CrossRef
  7. Czajkowski R, Ozymko Z, Lojkowska E. 2014. Isolation and characterization of novel soilborne lytic bacteriophages infecting Dickeya spp. biovar 3 (‘D. solani’). Plant Pathol. 63:758-772.
    CrossRef
  8. De Paepe M, Taddei F. 2006. Viruses' life history: towards a mechanistic basis of a trade-off between survival and reproduction among phages. PLoS Biol. 4: e193.
    Pubmed KoreaMed CrossRef
  9. Deveau H, Labrie SJ, Chopin M-C, Moineau S. 2006. Biodiversity and classification of lactococcal phages. Appl. Environ. Microbiol. 72: 4338-4346.
    Pubmed KoreaMed CrossRef
  10. Di Lallo G, Evangelisti M, Mancuso F, Ferrante P, Marcelletti S, Tinari A, et al. 2014. Isolation and partial characterization of bacteriophages infecting Pseudomonas syringae pv. actinidiae, causal agent of kiwifruit bacterial canker. J. Basic Microbiol. 54: 1210-1221.
    Pubmed CrossRef
  11. Donati I, Buriani G, Cellini A, Mauri S, Costa G, Spinelli F. 2014. New insights on the bacterial canker of kiwifruit (Pseudomonas syringae pv. actinidiae). J. Berry Res. 4: 53-67.
  12. Everett KR, Taylor RK, Romberg MK, Rees-George J, Fullerton RA, Vanneste JL, Manning MA. 2011. First report of Pseudomonas syringae pv. actinidiae causing kiwifruit bacterial canker in New Zealand. Australasian Plant Dis. Notes 6: 67-71.
    CrossRef
  13. Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA. 2005. Virus Taxonomy: VIIIth Report of the International Committee on Taxonomy of Viruses. Academic Press.
  14. Frampton RA, Pitman AR, Fineran PC. 2012. Advances in bacteriophage-mediated control of plant pathogens. Int. J. Microbiol. 2012: 1-11.
    Pubmed KoreaMed CrossRef
  15. Frampton RA, Taylor C, Holguin Moreno AV, Visnovsky SB, Petty NK, Pitman AR, Fineran PC. 2014. Identification of bacteriophages for the biocontrol of the kiwifruit canker phytopathogen Pseudomonas syringae pv. actinidiae. Appl. Environ. Microbiol. 80: 2216-2228.
    Pubmed KoreaMed CrossRef
  16. Fujiwara A, Fujisawa M, Hamasaki R, Kawasaki T, Fujie M, Yamada T. 2011. Biocontrol of Ralstonia solanacearum by treatment with lytic bacteriophages. Appl. Environ. Microbiol. 77: 4155-4162.
    Pubmed KoreaMed CrossRef
  17. Garden L, Shafik H, Belouin S, Broch R, Grimont F, Grimont P. 1999. DNA relatedness among the pathovars of Pseudomonas syringae and description of Pseudomonas tremae sp. nov. and Pseudomonas cannabina sp. nov. (ex Sutic and Dowson 1959). Int. J. Syst. Bacteriol. 49: 469-478.
    Pubmed CrossRef
  18. Han HS, Koh YJ, Hur J-S, Jung JS. 2004. Occurrence of the strA-strB streptomycin resistance genes in Pseudomonas species isolated from kiwifruit plants. J. Microbiol. 42: 365-368.
    Pubmed
  19. Kim J, Park SS, Cho N, Kim W, Cho HK. 2011. Recent variations of UV irradiance at Seoul 2004-2010. Atmosphere 21: 429-438.
  20. Kim M, Ryu S. 2011. Characterization of a T5-like coliphage, SPC35, and differential development of resistance to SPC35 in Salmonella enterica serovar Typhimurium and Escherichia coli. Appl. Environ. Microbiol. 77: 2042-2050.
    Pubmed KoreaMed CrossRef
  21. Koh HS, Lee YS, Jung JS. 2014. Molecular characteristics of Pseudomonas syringae pv. actinidiae strains isolated in Korea and a multiplex PCR assay for haplotype differentiation. Plant Pathol. J. 30: 96-101.
    Pubmed KoreaMed CrossRef
  22. Koh YJ, Chung HJ, Cha BJ, Lee DH. 1994. Outbreak and spread of bacterial canker in kiwifruit. Korean J. Plant Pathol. 10.
  23. Koh YJ, Kim GH, Jung JS, Lee YS, Hur JS. 2010. Outbreak of bacterial canker on Hort16A (Actinidia chinensis Planchon) caused by Pseudomonas syringae pv. actinidiae in Korea. N. Z. J. Crop Hort. Sci. 38: 275-282.
    CrossRef
  24. Kropinski AM, Mazzocco A, Waddell TE, Lingohr E, Johnson RP. 2009. Enumeration of bacteriophages by double agar overlay plaque assay, p. 69-76. Bacteriophages. Springer.
  25. Kutter E, Sulakvelidze A. 2004. Bacteriophages: Biology and Applications. CRC Press.
    CrossRef
  26. Lim JA, Lee DH, Jung KS, Heu S. 2013. Biocontrol of Pectobacterium carotovorum subsp. carotovorum using bacteriophage PP1. J. Microbiol. Biotechnol. 23: 1147-1153.
    Pubmed CrossRef
  27. Maniloff J, Ackermann H-W. 1998. Taxonomy of bacterial viruses: establishment of tailed virus genera and the other Caudovirales. Arch. Virol. 143: 2051-2063.
    Pubmed CrossRef
  28. Nakajima M, Tadaaki H. 2002. Similarity between copper resistance genes from Pseudomonas syringae pv. actinidiae and P. syringae pv. tomato. J. Gen. Plant Pathol. 68: 68-74.
    CrossRef
  29. Scortichini M. 1994. Occurrence of Pseudomonas syringae pv. actinidiae on kiwifruit in Italy. Plant Pathol. 43: 1035-1038.
    CrossRef
  30. Scortichini M, Marcelletti S, Ferrante P, Petriccione M, Firrao G. 2012. Pseudomonas syringae pv. actinidiae: a reemerging, multi-faceted, pandemic pathogen. Mol. Plant Pathol. 13: 631-640.
    Pubmed CrossRef
  31. Snelgar WP, Hall AJ, Ferguson AR, Blattmann P. 2005. Temperature influences growth and maturation of fruit on 'Hayward' kiwifruit vines. Funct. Plant Biol. 32: 631-642.
    CrossRef
  32. Takikawa Y, Serizawa S, Ichikawa T, Tsuyumu S, Goto M. 1989. Pseudomonas syringae pv. actinidiae pv. nov.: the causal bacterium of canker of kiwifruit in Japan. Ann. Phytopathol. Soc. Jap. 55: 437-444.
    CrossRef
  33. Vanneste JL, Poliakoff F, Audusseau C, Cornish DA, Paillard S, Rivoal C, Yu J. 2012. First report of Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker of kiwifruit in France. Mol. Plant Pathol. 13: 631-640.
  34. Young BJ, Lee HJ, Murugaiyan S, Lee SW. 2012. Biocontrol potential of a lytic bacteriophage PE204 against bacterial wilt of tomato. J. Microbiol. Biotechnol. 22: 1613-1620.
    CrossRef