This article was downloaded by: [UQ Library] On: 27 July 2015, At: 23:46 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: 5 Howick Place, London, SW1P 1WG
Bacteriophage Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kbac20
Protection of Erwinia amylovora bacteriophage Y2 from UV-induced damage by natural compounds Yannick Born
abc
c
bd
c
ac
, Lars Bosshard , Brion Duffy , Martin J. Loessner & Lars Fieseler
a
Institute of Food and Beverage Innovation, Zurich University of Applied Sciences, CH-8820 Wädenswil, Switzerland b
Agroscope Changins-Wädenswil ACW, Swiss National Competence Center for Fire Blight, CH-8820 Wädenswil, Switzerland c
Institute of Food, Nutrition and Health, ETH Zurich, CH-8092 Zürich, Switzerland
d
Click for updates
Institute of Natural Resource Sciences, Zurich University of Applied Sciences, CH-8820 Wädenswil, Switzerland Accepted author version posted online: 24 Jul 2015.
To cite this article: Yannick Born, Lars Bosshard, Brion Duffy, Martin J. Loessner & Lars Fieseler (2015): Protection of Erwinia amylovora bacteriophage Y2 from UV-induced damage by natural compounds, Bacteriophage, DOI: 10.1080/21597081.2015.1074330 To link to this article: http://dx.doi.org/10.1080/21597081.2015.1074330
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Protection of Erwinia amylovora bacteriophage Y2 from UV-induced damage by natural compounds
Yannick Born1,2,3, Lars Bosshard3, Brion Duffy2,4, Martin J. Loessner3, and Lars Fieseler1,3*
Downloaded by [UQ Library] at 23:46 27 July 2015
1
Institute of Food and Beverage Innovation, Zurich University of Applied Sciences, CH-8820 Wädenswil, Switzerland
2
Agroscope Changins-Wädenswil ACW, Swiss National Competence Center for Fire Blight, CH8820 Wädenswil, Switzerland 3
4
Institute of Food, Nutrition and Health, ETH Zurich, CH-8092 Zürich, Switzerland
Institute of Natural Resource Sciences, Zurich University of Applied Sciences, CH-8820 Wädenswil, Switzerland
*
Correspondence: Lars Fieseler; E-mail: [email protected]
Keywords Phage, Erwinia amylovora, fire blight, stability, UV damage, carotenoids, betalaines
1
Abbreviations
Downloaded by [UQ Library] at 23:46 27 July 2015
DMSO Dimethyl sulfoxide DNA
Deoxyribonucleic acid
PFU
Plaque forming units
Phe
Phenylalanine
SM
Phage buffer containing sodium and magnesium
Trp
Tryptophan
Tyr
Tyrosine
UV
Ultraviolet
Abstract Bacteriophages have regained much attention as biocontrol agents against bacterial pathogens.
However, with respect to stability, phages are biomolecules and are therefore sensitive to a number of environmental influences. UV-irradiation can readily inactivate phage infectivity, which impedes their potential application in the plant phyllosphere. Therefore, phages for control of Erwinia amylovora, the causative agent of fire blight, need to be protected from UVdamage by adequate measures. We investigated the protective effect of different lightabsorbing substances on phage particles exposed to UV-light. For this, natural extracts from carrot, red pepper, and beetroot, casein and soy peptone in solution, and purified substances 2
such as astaxanthin, aromatic amino acids, and Tween 80 were prepared and tested as natural sunscreens for phage. All compounds were found to significantly increase half-life of UVirradiated phage particles and they did not negatively affect phage viability or infectivity. Altogether, a range of readily available, natural substances are suitable as UV-protectants to
Downloaded by [UQ Library] at 23:46 27 July 2015
prevent phage particles from UV-light damage.
3
Introduction Erwinia amylovora is the causative agent of fire blight, a devastating plant disease affecting Rosaceae species, and a major threat to pome fruit production causing high economic losses every year.1 To date, the most effective control option is the prophylactic application of antibiotics, e.g., streptomycin, during the flowering period. The use is limited due to regulatory restrictions, pathogen resistance development, and consumers’ demands.2
Downloaded by [UQ Library] at 23:46 27 July 2015
Biocontrol of bacterial plant pathogens using bacteriophages (phages) has gained increased attention in agriculture as an alternative treatment,3,4 and several studies have demonstrated the potential of phages to control fire blight. Phage-treatment significantly reduced the number of viable E. amylovora and symptom development in vitro,5,6 on immature pear fruit,7,8 on detached flowers,8,9 and on apple trees.9,10 Furthermore, “Erwiphage”, a bacteriophage cocktail consisting of two phages, was recently introduced and is commercially available.11 However, unfavorable conditions, which prevail in the phyllosphere, can inactivate the phage, i.e., variation of temperature and pH, rain, desiccation, and sunlight irradiation, with the latter being the most detrimental factor for phage persistence.12–14 Light of a wide spectrum, i.e., 254 nm, UVB (280-320 nm), and > 320 nm, was shown to have a negative impact on phage DNA.15–21 Sunlight-mediated damage of phage particles is characterized by the formation of pyrimidine (mostly thymine) dimers in DNA caused by direct absorption of UV-light.22 The decay of infective phage is in direct correlation with the received irradiation dose.15,17 Given that the number of infective phage is proportional to the outcome of the treatment, phage persistence must be guaranteed by adequate measures.13 A feasible option is the avoidance of exposure to 4
sunlight by applying the phage in the evening, as shown in experiments on tomato leaves.14,23 Another option to improve phage persistence in the phyllosphere is the application of an avirulent bacterial strain, which supports phage replication. This strategy was successfully applied with phages of E. amylovora (carrier: Pantoea agglomerans) on apple trees9 and on tomato leaves using an attenuated Xanthomonas perforans strain as host bacterium.24 Phages can also be protected from inactivation by the addition of substances, which absorb and prevent phages from UV-light. Balogh et al.23 developed three different formulations including
Downloaded by [UQ Library] at 23:46 27 July 2015
(i) pregelatinized corn flour (PCF) and sucrose; (ii) casecrete, sucrose, and PCF; and (iii) skim milk and sucrose (M+S). Phage-longevity was significantly increased and a reduction of disease severity was achieved. In a subsequent study, the formulation with M+S was found to successfully prevent the decline of infective phages caused by temperature, desiccation, fluorescent light, and copper.14 In general, any substance absorbing detrimental light offers potential as a protectant. Plant compounds such as pigments (e.g., carotenoids or betalaines) or polyphenols absorb light of a wide spectral range and could reduce the inactivation of phages by sunlight irradiation. They are readily available, natural, and safe substances demonstrated by their use as colorants and antioxidants in the food-industry.25–27 Alternatively, peptides or aromatic amino acids could have a similar effect. As a proof of concept study, we investigated the efficacy of juices from vegetables (carrot, red pepper, and beetroot), astaxanthin, a pure carotenoid from the freshwater chlorophyte algae Haematococcus pluvialis,28 aromatic amino acids, casein, peptone, and Tween 80 to prevent E. amylovora phage Y2 (vB_EamM-Y2), a virulent myovirus5, from UVdamage. 5
Materials and Methods
Preparation of plant extracts and UV adsorbing substances A series of substances were tested for their abilities to protect Y2 from UV-inactivation. These included juice of beetroot, red pepper, and carrot, casein from bovine milk, soy peptone,
Downloaded by [UQ Library] at 23:46 27 July 2015
astaxanthin, aromatic amino acids (Phe, Trp, Tyr), and Tween 80. Carrot juice, red peppers and beetroots were bought in a local grocery store. The juice from the peppers was extracted using a commercial juice squeezer. Beetroots were cut into pieces and homogenized using an UltraTurrax high speed blender (IKA®-Werke GmbH & Co. KG) for 5 min. Solid particles were removed from all juices by centrifugation (1000 ×g, 5 min) and serial passages through filters. The final filter had a pore size of 0.2 μm. The juices were diluted in SM buffer (50 mM Tris, 100 mM NaCl, 8 mM MgSO4, pH 7.4) to a stock-concentration of 20 % ([vol/vol], carrot and beetroot) or 10 % ([vol/vol], red pepper) and stored at 4 °C. Casein from bovine milk (Sigma-Aldrich) was dissolved in 1 M NaOH (final concentration: 20 mg/ml), soy peptone in ddH2O (100 mg/ml), astaxanthin (Sigma-Aldrich) in DMSO (6 mg/ml), aromatic amino acids (Sigma-Aldrich) in ddH2O (100 mM each), and Tween 80 (Sigma-Aldrich) in ddH2O (10 %, [vol/vol]).
UV sensitivity assays In wells of a 96 multiwell plate (Nunc GmbH & Co. KG), 100 μl aliquots containing phage Y2 (2 × 108 PFU/ml) in SM buffer were mixed with 100 μl of the UV-absorbing substance or a dilution thereof. A UV lamp (λ=254 nm; Camag 29200), switched on 15 min prior to use, was placed 6 cm 6
above the plate assuring equal radiation of every well (an estimated 1 mW/cm2 was applied). The wavelength of 254 nm was used in the present work since it is near the peak absorption of DNA and it was the wavelength of choice for a vast number of studies on UV-inactivation due to its detrimental impact on DNA.29 After 0 (no irradiation), 30, 60, 120, and 300 s of exposure to UV-light, the remainder of infective phage particles was determined by the soft agar overlay method30 using E. amylovora strain 4/82 as host as described previously.5 Each experiment was carried out in triplicates and independently repeated twice.
Downloaded by [UQ Library] at 23:46 27 July 2015
In addition, phages were mixed with the UV-absorbing substances without being irradiated to investigate if the UV-protectants influence the number of infective phage. The concentrations of phage were determined with the same soft agar overlay method at the same time points as above.
Statistical analysis Statistical significance of the effects observed after 5 min of irradiation was evaluated by analysis of variance (ANOVA).
Results The inactivation of phage Y2 by UV-irradiation could be minimized by addition of different substances to the phage suspension. Yet, the UV-protective effects differed depending on the substance and the concentration applied. Importantly, phage Y2 was not adversely affected by the UV-protectants, i.e., the number of PFU mixed with any substance remained constant over 5 min (data not shown). In contrast, the number of infective particles not supplemented with a 7
UV-protectant was rapidly reduced by UV-light. This demonstrates the instability of phages exposed to UV-irradiation. In average, only 0.21 % of the initial number of PFU remained infective after being irradiated with UV-light for 5 min. When treated with carrot (10 %) or red pepper juice (5 %), 28 % and 34 %, respectively, of the original number of PFU remained infective after 5 min of UV-irradiation. The number of infective particles was approximately 2 logs higher compared to the untreated controls (Figure 1). These protective effects were found to be highly significant (P
Bacteriophage Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kbac20
Protection of Erwinia amylovora bacteriophage Y2 from UV-induced damage by natural compounds Yannick Born
abc
c
bd
c
ac
, Lars Bosshard , Brion Duffy , Martin J. Loessner & Lars Fieseler
a
Institute of Food and Beverage Innovation, Zurich University of Applied Sciences, CH-8820 Wädenswil, Switzerland b
Agroscope Changins-Wädenswil ACW, Swiss National Competence Center for Fire Blight, CH-8820 Wädenswil, Switzerland c
Institute of Food, Nutrition and Health, ETH Zurich, CH-8092 Zürich, Switzerland
d
Click for updates
Institute of Natural Resource Sciences, Zurich University of Applied Sciences, CH-8820 Wädenswil, Switzerland Accepted author version posted online: 24 Jul 2015.
To cite this article: Yannick Born, Lars Bosshard, Brion Duffy, Martin J. Loessner & Lars Fieseler (2015): Protection of Erwinia amylovora bacteriophage Y2 from UV-induced damage by natural compounds, Bacteriophage, DOI: 10.1080/21597081.2015.1074330 To link to this article: http://dx.doi.org/10.1080/21597081.2015.1074330
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Protection of Erwinia amylovora bacteriophage Y2 from UV-induced damage by natural compounds
Yannick Born1,2,3, Lars Bosshard3, Brion Duffy2,4, Martin J. Loessner3, and Lars Fieseler1,3*
Downloaded by [UQ Library] at 23:46 27 July 2015
1
Institute of Food and Beverage Innovation, Zurich University of Applied Sciences, CH-8820 Wädenswil, Switzerland
2
Agroscope Changins-Wädenswil ACW, Swiss National Competence Center for Fire Blight, CH8820 Wädenswil, Switzerland 3
4
Institute of Food, Nutrition and Health, ETH Zurich, CH-8092 Zürich, Switzerland
Institute of Natural Resource Sciences, Zurich University of Applied Sciences, CH-8820 Wädenswil, Switzerland
*
Correspondence: Lars Fieseler; E-mail: [email protected]
Keywords Phage, Erwinia amylovora, fire blight, stability, UV damage, carotenoids, betalaines
1
Abbreviations
Downloaded by [UQ Library] at 23:46 27 July 2015
DMSO Dimethyl sulfoxide DNA
Deoxyribonucleic acid
PFU
Plaque forming units
Phe
Phenylalanine
SM
Phage buffer containing sodium and magnesium
Trp
Tryptophan
Tyr
Tyrosine
UV
Ultraviolet
Abstract Bacteriophages have regained much attention as biocontrol agents against bacterial pathogens.
However, with respect to stability, phages are biomolecules and are therefore sensitive to a number of environmental influences. UV-irradiation can readily inactivate phage infectivity, which impedes their potential application in the plant phyllosphere. Therefore, phages for control of Erwinia amylovora, the causative agent of fire blight, need to be protected from UVdamage by adequate measures. We investigated the protective effect of different lightabsorbing substances on phage particles exposed to UV-light. For this, natural extracts from carrot, red pepper, and beetroot, casein and soy peptone in solution, and purified substances 2
such as astaxanthin, aromatic amino acids, and Tween 80 were prepared and tested as natural sunscreens for phage. All compounds were found to significantly increase half-life of UVirradiated phage particles and they did not negatively affect phage viability or infectivity. Altogether, a range of readily available, natural substances are suitable as UV-protectants to
Downloaded by [UQ Library] at 23:46 27 July 2015
prevent phage particles from UV-light damage.
3
Introduction Erwinia amylovora is the causative agent of fire blight, a devastating plant disease affecting Rosaceae species, and a major threat to pome fruit production causing high economic losses every year.1 To date, the most effective control option is the prophylactic application of antibiotics, e.g., streptomycin, during the flowering period. The use is limited due to regulatory restrictions, pathogen resistance development, and consumers’ demands.2
Downloaded by [UQ Library] at 23:46 27 July 2015
Biocontrol of bacterial plant pathogens using bacteriophages (phages) has gained increased attention in agriculture as an alternative treatment,3,4 and several studies have demonstrated the potential of phages to control fire blight. Phage-treatment significantly reduced the number of viable E. amylovora and symptom development in vitro,5,6 on immature pear fruit,7,8 on detached flowers,8,9 and on apple trees.9,10 Furthermore, “Erwiphage”, a bacteriophage cocktail consisting of two phages, was recently introduced and is commercially available.11 However, unfavorable conditions, which prevail in the phyllosphere, can inactivate the phage, i.e., variation of temperature and pH, rain, desiccation, and sunlight irradiation, with the latter being the most detrimental factor for phage persistence.12–14 Light of a wide spectrum, i.e., 254 nm, UVB (280-320 nm), and > 320 nm, was shown to have a negative impact on phage DNA.15–21 Sunlight-mediated damage of phage particles is characterized by the formation of pyrimidine (mostly thymine) dimers in DNA caused by direct absorption of UV-light.22 The decay of infective phage is in direct correlation with the received irradiation dose.15,17 Given that the number of infective phage is proportional to the outcome of the treatment, phage persistence must be guaranteed by adequate measures.13 A feasible option is the avoidance of exposure to 4
sunlight by applying the phage in the evening, as shown in experiments on tomato leaves.14,23 Another option to improve phage persistence in the phyllosphere is the application of an avirulent bacterial strain, which supports phage replication. This strategy was successfully applied with phages of E. amylovora (carrier: Pantoea agglomerans) on apple trees9 and on tomato leaves using an attenuated Xanthomonas perforans strain as host bacterium.24 Phages can also be protected from inactivation by the addition of substances, which absorb and prevent phages from UV-light. Balogh et al.23 developed three different formulations including
Downloaded by [UQ Library] at 23:46 27 July 2015
(i) pregelatinized corn flour (PCF) and sucrose; (ii) casecrete, sucrose, and PCF; and (iii) skim milk and sucrose (M+S). Phage-longevity was significantly increased and a reduction of disease severity was achieved. In a subsequent study, the formulation with M+S was found to successfully prevent the decline of infective phages caused by temperature, desiccation, fluorescent light, and copper.14 In general, any substance absorbing detrimental light offers potential as a protectant. Plant compounds such as pigments (e.g., carotenoids or betalaines) or polyphenols absorb light of a wide spectral range and could reduce the inactivation of phages by sunlight irradiation. They are readily available, natural, and safe substances demonstrated by their use as colorants and antioxidants in the food-industry.25–27 Alternatively, peptides or aromatic amino acids could have a similar effect. As a proof of concept study, we investigated the efficacy of juices from vegetables (carrot, red pepper, and beetroot), astaxanthin, a pure carotenoid from the freshwater chlorophyte algae Haematococcus pluvialis,28 aromatic amino acids, casein, peptone, and Tween 80 to prevent E. amylovora phage Y2 (vB_EamM-Y2), a virulent myovirus5, from UVdamage. 5
Materials and Methods
Preparation of plant extracts and UV adsorbing substances A series of substances were tested for their abilities to protect Y2 from UV-inactivation. These included juice of beetroot, red pepper, and carrot, casein from bovine milk, soy peptone,
Downloaded by [UQ Library] at 23:46 27 July 2015
astaxanthin, aromatic amino acids (Phe, Trp, Tyr), and Tween 80. Carrot juice, red peppers and beetroots were bought in a local grocery store. The juice from the peppers was extracted using a commercial juice squeezer. Beetroots were cut into pieces and homogenized using an UltraTurrax high speed blender (IKA®-Werke GmbH & Co. KG) for 5 min. Solid particles were removed from all juices by centrifugation (1000 ×g, 5 min) and serial passages through filters. The final filter had a pore size of 0.2 μm. The juices were diluted in SM buffer (50 mM Tris, 100 mM NaCl, 8 mM MgSO4, pH 7.4) to a stock-concentration of 20 % ([vol/vol], carrot and beetroot) or 10 % ([vol/vol], red pepper) and stored at 4 °C. Casein from bovine milk (Sigma-Aldrich) was dissolved in 1 M NaOH (final concentration: 20 mg/ml), soy peptone in ddH2O (100 mg/ml), astaxanthin (Sigma-Aldrich) in DMSO (6 mg/ml), aromatic amino acids (Sigma-Aldrich) in ddH2O (100 mM each), and Tween 80 (Sigma-Aldrich) in ddH2O (10 %, [vol/vol]).
UV sensitivity assays In wells of a 96 multiwell plate (Nunc GmbH & Co. KG), 100 μl aliquots containing phage Y2 (2 × 108 PFU/ml) in SM buffer were mixed with 100 μl of the UV-absorbing substance or a dilution thereof. A UV lamp (λ=254 nm; Camag 29200), switched on 15 min prior to use, was placed 6 cm 6
above the plate assuring equal radiation of every well (an estimated 1 mW/cm2 was applied). The wavelength of 254 nm was used in the present work since it is near the peak absorption of DNA and it was the wavelength of choice for a vast number of studies on UV-inactivation due to its detrimental impact on DNA.29 After 0 (no irradiation), 30, 60, 120, and 300 s of exposure to UV-light, the remainder of infective phage particles was determined by the soft agar overlay method30 using E. amylovora strain 4/82 as host as described previously.5 Each experiment was carried out in triplicates and independently repeated twice.
Downloaded by [UQ Library] at 23:46 27 July 2015
In addition, phages were mixed with the UV-absorbing substances without being irradiated to investigate if the UV-protectants influence the number of infective phage. The concentrations of phage were determined with the same soft agar overlay method at the same time points as above.
Statistical analysis Statistical significance of the effects observed after 5 min of irradiation was evaluated by analysis of variance (ANOVA).
Results The inactivation of phage Y2 by UV-irradiation could be minimized by addition of different substances to the phage suspension. Yet, the UV-protective effects differed depending on the substance and the concentration applied. Importantly, phage Y2 was not adversely affected by the UV-protectants, i.e., the number of PFU mixed with any substance remained constant over 5 min (data not shown). In contrast, the number of infective particles not supplemented with a 7
UV-protectant was rapidly reduced by UV-light. This demonstrates the instability of phages exposed to UV-irradiation. In average, only 0.21 % of the initial number of PFU remained infective after being irradiated with UV-light for 5 min. When treated with carrot (10 %) or red pepper juice (5 %), 28 % and 34 %, respectively, of the original number of PFU remained infective after 5 min of UV-irradiation. The number of infective particles was approximately 2 logs higher compared to the untreated controls (Figure 1). These protective effects were found to be highly significant (P
