Development of a Bacteriophage Cocktail against Pectobacterium carotovorum Subsp. carotovorum and Its Effects on Pectobacterium Virulence

Hyeongsoon Kim; Minsik Kim; Sam Nyu Jee; Sunggi Heu; Sangryeol Ryu

doi:10.1128/aem.00761-22

Development of a Bacteriophage Cocktail against Pectobacterium carotovorum Subsp. carotovorum and Its Effects on Pectobacterium Virulence

Hyeongsoon Kim, Minsik Kim, Sam Nyu Jee, Sunggi Heu, Sangryeol Ryu

Department of Food and Nutrition

Research output: Contribution to journal › Article › peer-review

1 Citation (Scopus)

Abstract

Pectobacterium carotovorum subsp. carotovorum is a necrotrophic plant pathogen that secretes plant cell wall-degrading enzymes (PCWDEs) that cause soft rot disease in various crops. Bacteriophages have been under consideration as harmless antibacterial agents to replace antibiotics and copper-based pesticides. However, the emergence of bacteriophage resistance is one of the main concerns that should be resolved for practical phage applications. In this study, we developed a phage cocktail with three lytic phages that recognize colanic acid (phage POP12) or flagella (phages POP15 and POP17) as phage receptors to minimize phage resistance. The phage cocktail effectively suppressed the emergence of phage-resistant P. carotovorum subsp. carotovorum compared with single phages in in vitro challenge assays. The application of the phage cocktail to napa cabbage (Brassica rapa subsp. pekinensis) resulted in significant growth retardation of P. carotovorum subsp. carotovorum (P, 0.05) and prevented the symptoms of soft rot disease. Furthermore, phage cocktail treatments of young napa cabbage leaves in a greenhouse environment indicated effective prevention of soft rot disease compared to that in the nonphage negative control. We isolated 15 phage-resistant mutants after a phage cocktail treatment to assess the virulence-associated phenotypes compared to those of wild-type (WT) strain Pcc27. All mutants showed reduced production of four different PCWDEs, leading to lower levels of tissue softening. Ten of the 15 phage-resistant mutants additionally exhibited decreased swimming motility. Taken together, these results show that the phage cocktail developed here, which targets two different types of phage receptors, provides an effective strategy for controlling P. carotovorum subsp. carotovorum in agricultural products, with a potential ability to attenuate P. carotovorum subsp. carotovorum virulence.

Original language	English
Journal	Applied and Environmental Microbiology
Volume	88
Issue number	19
DOIs	https://doi.org/10.1128/aem.00761-22
Publication status	Published - 2022 Oct

Bibliographical note

Funding Information:
This study received support from LG Chem and a National Research Foundation of Korea (NRF) grant (grant no. NRF-2020R1A2B5B03094303). This work was carried out with the support of the Cooperative Research Program for Agriculture Science and Technology Development (project no. PJ01574702), Rural Development Administration, Republic of Korea, with a Yonsei University Future-leading Research Initiative of 2019 grant (grant no. 2019-22-0083). H.K. was supported by the BK21 Plus Program of Department of Agricultural Biotechnology, Seoul National University, Seoul, South Korea.

Publisher Copyright:
© 2022 American Society for Microbiology. All Rights Reserved.

All Science Journal Classification (ASJC) codes

Biotechnology
Food Science
Ecology
Applied Microbiology and Biotechnology

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10.1128/aem.00761-22

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@article{2487212690934f5a8b687de349e732dc,

title = "Development of a Bacteriophage Cocktail against Pectobacterium carotovorum Subsp. carotovorum and Its Effects on Pectobacterium Virulence",

abstract = "Pectobacterium carotovorum subsp. carotovorum is a necrotrophic plant pathogen that secretes plant cell wall-degrading enzymes (PCWDEs) that cause soft rot disease in various crops. Bacteriophages have been under consideration as harmless antibacterial agents to replace antibiotics and copper-based pesticides. However, the emergence of bacteriophage resistance is one of the main concerns that should be resolved for practical phage applications. In this study, we developed a phage cocktail with three lytic phages that recognize colanic acid (phage POP12) or flagella (phages POP15 and POP17) as phage receptors to minimize phage resistance. The phage cocktail effectively suppressed the emergence of phage-resistant P. carotovorum subsp. carotovorum compared with single phages in in vitro challenge assays. The application of the phage cocktail to napa cabbage (Brassica rapa subsp. pekinensis) resulted in significant growth retardation of P. carotovorum subsp. carotovorum (P, 0.05) and prevented the symptoms of soft rot disease. Furthermore, phage cocktail treatments of young napa cabbage leaves in a greenhouse environment indicated effective prevention of soft rot disease compared to that in the nonphage negative control. We isolated 15 phage-resistant mutants after a phage cocktail treatment to assess the virulence-associated phenotypes compared to those of wild-type (WT) strain Pcc27. All mutants showed reduced production of four different PCWDEs, leading to lower levels of tissue softening. Ten of the 15 phage-resistant mutants additionally exhibited decreased swimming motility. Taken together, these results show that the phage cocktail developed here, which targets two different types of phage receptors, provides an effective strategy for controlling P. carotovorum subsp. carotovorum in agricultural products, with a potential ability to attenuate P. carotovorum subsp. carotovorum virulence.",

author = "Hyeongsoon Kim and Minsik Kim and Jee, {Sam Nyu} and Sunggi Heu and Sangryeol Ryu",

note = "Funding Information: This study received support from LG Chem and a National Research Foundation of Korea (NRF) grant (grant no. NRF-2020R1A2B5B03094303). This work was carried out with the support of the Cooperative Research Program for Agriculture Science and Technology Development (project no. PJ01574702), Rural Development Administration, Republic of Korea, with a Yonsei University Future-leading Research Initiative of 2019 grant (grant no. 2019-22-0083). H.K. was supported by the BK21 Plus Program of Department of Agricultural Biotechnology, Seoul National University, Seoul, South Korea. Publisher Copyright: {\textcopyright} 2022 American Society for Microbiology. All Rights Reserved.",

year = "2022",

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doi = "10.1128/aem.00761-22",

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AU - Kim, Hyeongsoon

AU - Kim, Minsik

AU - Jee, Sam Nyu

AU - Heu, Sunggi

AU - Ryu, Sangryeol

N1 - Funding Information: This study received support from LG Chem and a National Research Foundation of Korea (NRF) grant (grant no. NRF-2020R1A2B5B03094303). This work was carried out with the support of the Cooperative Research Program for Agriculture Science and Technology Development (project no. PJ01574702), Rural Development Administration, Republic of Korea, with a Yonsei University Future-leading Research Initiative of 2019 grant (grant no. 2019-22-0083). H.K. was supported by the BK21 Plus Program of Department of Agricultural Biotechnology, Seoul National University, Seoul, South Korea. Publisher Copyright: © 2022 American Society for Microbiology. All Rights Reserved.

PY - 2022/10

Y1 - 2022/10

N2 - Pectobacterium carotovorum subsp. carotovorum is a necrotrophic plant pathogen that secretes plant cell wall-degrading enzymes (PCWDEs) that cause soft rot disease in various crops. Bacteriophages have been under consideration as harmless antibacterial agents to replace antibiotics and copper-based pesticides. However, the emergence of bacteriophage resistance is one of the main concerns that should be resolved for practical phage applications. In this study, we developed a phage cocktail with three lytic phages that recognize colanic acid (phage POP12) or flagella (phages POP15 and POP17) as phage receptors to minimize phage resistance. The phage cocktail effectively suppressed the emergence of phage-resistant P. carotovorum subsp. carotovorum compared with single phages in in vitro challenge assays. The application of the phage cocktail to napa cabbage (Brassica rapa subsp. pekinensis) resulted in significant growth retardation of P. carotovorum subsp. carotovorum (P, 0.05) and prevented the symptoms of soft rot disease. Furthermore, phage cocktail treatments of young napa cabbage leaves in a greenhouse environment indicated effective prevention of soft rot disease compared to that in the nonphage negative control. We isolated 15 phage-resistant mutants after a phage cocktail treatment to assess the virulence-associated phenotypes compared to those of wild-type (WT) strain Pcc27. All mutants showed reduced production of four different PCWDEs, leading to lower levels of tissue softening. Ten of the 15 phage-resistant mutants additionally exhibited decreased swimming motility. Taken together, these results show that the phage cocktail developed here, which targets two different types of phage receptors, provides an effective strategy for controlling P. carotovorum subsp. carotovorum in agricultural products, with a potential ability to attenuate P. carotovorum subsp. carotovorum virulence.

AB - Pectobacterium carotovorum subsp. carotovorum is a necrotrophic plant pathogen that secretes plant cell wall-degrading enzymes (PCWDEs) that cause soft rot disease in various crops. Bacteriophages have been under consideration as harmless antibacterial agents to replace antibiotics and copper-based pesticides. However, the emergence of bacteriophage resistance is one of the main concerns that should be resolved for practical phage applications. In this study, we developed a phage cocktail with three lytic phages that recognize colanic acid (phage POP12) or flagella (phages POP15 and POP17) as phage receptors to minimize phage resistance. The phage cocktail effectively suppressed the emergence of phage-resistant P. carotovorum subsp. carotovorum compared with single phages in in vitro challenge assays. The application of the phage cocktail to napa cabbage (Brassica rapa subsp. pekinensis) resulted in significant growth retardation of P. carotovorum subsp. carotovorum (P, 0.05) and prevented the symptoms of soft rot disease. Furthermore, phage cocktail treatments of young napa cabbage leaves in a greenhouse environment indicated effective prevention of soft rot disease compared to that in the nonphage negative control. We isolated 15 phage-resistant mutants after a phage cocktail treatment to assess the virulence-associated phenotypes compared to those of wild-type (WT) strain Pcc27. All mutants showed reduced production of four different PCWDEs, leading to lower levels of tissue softening. Ten of the 15 phage-resistant mutants additionally exhibited decreased swimming motility. Taken together, these results show that the phage cocktail developed here, which targets two different types of phage receptors, provides an effective strategy for controlling P. carotovorum subsp. carotovorum in agricultural products, with a potential ability to attenuate P. carotovorum subsp. carotovorum virulence.

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Development of a Bacteriophage Cocktail against Pectobacterium carotovorum Subsp. carotovorum and Its Effects on Pectobacterium Virulence

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