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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Antibacterial activity of essential oils mixture against PSA a
a
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Elisabetta Vavala , Claudio Passariello , Federico Pepi , Marisa c
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Colone , Stefania Garzoli , Rino Ragno , Adele Pirolli , Annarita c
Stringaro & Letizia Angiolella
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Department of Public Health and Infectious Diseases, University of Rome ‘Sapienza’, Piazzale Aldo Moro, 5, 00161 Rome, Italy b
Department of Drugs Chemistry and Technology, University of Rome ‘Sapienza’, Piazzale Aldo Moro, 5, 00161 Rome, Italy c
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Department of Technology and Health, Italian National Institute of Health, Viale Regina Elena 299, 00161 Rome, Italy d
Department of Drugs Chemistry and Technology, Rome Center for Molecular Design, University of Rome ‘Sapienza’, Rome, Italy Published online: 18 Mar 2015.
To cite this article: Elisabetta Vavala, Claudio Passariello, Federico Pepi, Marisa Colone, Stefania Garzoli, Rino Ragno, Adele Pirolli, Annarita Stringaro & Letizia Angiolella (2015): Antibacterial activity of essential oils mixture against PSA, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1022543 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1022543
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Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1022543
Antibacterial activity of essential oils mixture against PSA Elisabetta Vavalaa, Claudio Passarielloa, Federico Pepib, Marisa Colonec, Stefania Garzolib, Rino Ragnod, Adele Pirollid, Annarita Stringaroc and Letizia Angiolellaa*
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a Department of Public Health and Infectious Diseases, University of Rome ‘Sapienza’, Piazzale Aldo Moro, 5, 00161 Rome, Italy; bDepartment of Drugs Chemistry and Technology, University of Rome ‘Sapienza’, Piazzale Aldo Moro, 5, 00161 Rome, Italy; cDepartment of Technology and Health, Italian National Institute of Health, Viale Regina Elena 299, 00161 Rome, Italy; dDepartment of Drugs Chemistry and Technology, Rome Center for Molecular Design, University of Rome ‘Sapienza’, Rome, Italy
(Received 16 December 2014; final version received 19 February 2015)
Pseudomonas syringae pv. actinidiae (PSA) is the causal agent of bacterial canker of kiwifruit. It is very difficult to treat pandemic disease. The prolonged treatment with antibiotics, has resulted in failure and resistance and alternatives to conventional antimicrobial therapy are needed. The aim of our study was to analyse the phenotypic characteristics of PSA, identify new substances from natural source i.e. essential oils (EOs) able to contain the kiwifruit canker and investigate their potential use when utilised in combination. Specially, we investigated the morphological differences of PSA isolates by scanning electron microscope, and the synergic action of different EOs by time-kill and checkerboard methods. Our results demonstrated that PSA was able to produce extracellular polysaccharides when it was isolated from trunk, and, for the first time, that it was possible to kill PSA with a mixture of EOs after 1 h of exposition. We hypothesise on its potential use in agriculture. Keywords: Pseudomonas syringae pv. actinidiae; kiwifruit canker; exopolymeric material; SEM; essential oils; synergism
1. Introduction Agriculture production records heavy yearly losses due to plant diseases. Many product used as control for plant pathogenic bacteria contain antibiotics (mostly streptomycin) or heavy metals (mostly copper). These treatments do have limitations to either phytotoxicity or authorised restriction in some countries (e.g. antibiotics in Europe) (Reglinski et al. 2013).
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
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Pseudomonas syringae is a worldwide spread phytopathogenic micro-organism mainly adapted to plant species (Gardan et al. 1999), either cultivated or grown in wild habitats. P. syringae pv. actinidiae (PSA) the causal agent of bacterial canker of kiwigreen (Actinidia deliciosa and Actinidia chinensis) was first reported in Japan (Takikawa et al. 1989), and subsequently isolated in South Korea (Koh et al. 1994) and Italy (Scortichini 1994; Balestra et al. 2009). During 2008– 2011, PSA suddenly incited the rapid spread of severe epidemics of bacterial canker in central Italy (Ferrante & Scortichini 2009, 2010). The symptoms of bacterial disease included brown leaf spots, necrosis of the withering flowers, redness of lenticels, extended cankers along the main trunk, bleeding of cankers on the trunk with exudates that go from white to orange (Marcelletti et al. 2011). The bacterium owes its virulence to the ability to acquire exogenous material that allows it to exceed the plant stress conditions, such as lack of nutrients (Arnold et al. 2007) and loss of mobile genetic elements (Johnson et al. 2011), and to the presence of an ATP binding cassette (ABC) transporter called Pseudomonas efflux system, involved in multiple resistance to drugs (Marcelletti et al. 2011). A further PSA virulence factor is represented by copA and copB genes, which play a key role in the copper resistance (Brotz & Sahl 2000). The bacterium propagates quickly within and between orchards due to bacterial exudates that oozes from cankers during late autumn – winter and early spring, and are spread by the wind (Serizawa et al. 1989; Serizawa & Ichikawa 1993). To worsen the scenario, some agronomic techniques, such as pruning cut, cause injury that greatly facilitates the pathogen penetration within the plant. PSA survive even in the pollen grains of Actinidia (Vanneste et al. 2011), contributing to pathogen spread. The research of new antimicrobial protocols which include natural products has recently increased in many fields and the essential oils (EOs) and/or some of their constituents have demonstrated their effectiveness against a large variety of organisms, including bacteria (Basile et al. 2006), viruses (Duschatzky et al. 2005; Civitelli et al. 2014), fungi (Pietrella et al. 2011; Stringaro et al. 2014), protozoa and parasites (Sarrou et al. 2013). Recent studies have highlighted a synergism between the various components of EOs and antibiotics or antifungals, which are able to penetrate into the cell through the membrane and produce different types of radical reactions, thus causing cellular death (Bakkali et al. 2008). EOs have no specific cellular target and are mainly composed of mono- and di-terpenes (Carson et al. 2002). The aim of this study was to investigate the morphological differences of PSA strains isolated from trunk and from exudate, as well as to identify new substances of natural origin able to kill or contain the bacterium infections. In the present study, we have demonstrated (i) the presence of exopolymeric material around the bacterium in particular growth conditions; (ii) the bactericidal activity of Mentha suaveolens, Rosmarinus officinalis and Melaleuca alternifolia EOs mixture against PSA. 2. Results and discussion 2.1. Isolation and molecular identification of PSA Strains of PSA were isolated from trunk and from exudate, in a case of bacterial canker of A. chinensis, and grown on NSA. As reported in Figure S1A, the colony of the strains isolated from trunk were small, opaque, irregular, and cream-white, while the colony isolated from exudates were large, mucoid and cream dark. Pseudomonas spp. often has a non-mucoid phenotype in standard laboratory media. Growth in the presence of 0.3 M sodium chloride or 3 –5% ethanol reportedly can lead to the generation of mucoid variants of non-mucoid strains of Pseudomonas (Singh et al. 1992). These morphological variations can be induced by exposing cells to stress such as high antibiotic concentration (Silva et al. 2013). Molecular analyses by polymerase chain reaction using the Rees-George protocol (Rees-George et al. 2010) were performed, to confirm that the strains isolated from trunk and exudate were PSA. Specific
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sequences of 16S and 23S ribosomal RNA have been used as control. Figure S1B shows a band of 280 Kb in both strains confirming, as reported by other authors (Chapman et al. 2012), that the isolated strains were PSA with different phenotypic characteristics.
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2.2. Scanning electron microscope observations and exopolymeric production To evaluate the different PSA strains morphology, scanning electron microscope (SEM) analyses were performed. SEM observations of the trunk isolated PSA showed the bacteria with a typical shape, dimension and surface morphology (Figure 1(A)). Differently, exudate isolated PSA, showed evident exopolymeric material on the bacteria, as biofilm (Figure 1(B)), in agreement with the report by Renzi et al. (2012). Extracellular polysaccharides or exopolysaccharides (EPSs) are high-molecular weight sugar-based polymers that are synthesised and secreted by many micro-organisms (Ferreira et al. 2011). The importance of their production has been studied in many bacteria (Li et al. 2014; Shi et al. 2014). Furthermore, it is known that the physiological role of polysaccharides is to defend the cell from environmental stress and from the action of toxic substances or antibiotics. EPSs production represents a main virulence factor for the survival of the micro-organism both inside and outside the plant. To confirm EPSs production, trunk isolated PSA was grown in aerobic or anaerobic conditions for 72 h, in aerobic conditions the colonies were non-mucoid (Figure 1(C)), while in anaerobic conditions the colonies turned to be mucoid (Figure 1(D)) similarly to exudate isolated PSA (Figure 1(E)). The EPS production could explain their survival in low presence of oxygen. In particular, EPS is typically correlated with bacterial resistance and protection against different stress conditions such as desiccation, salt stress and UV radiations (Ferreira et al. 2011).
Figure 1. SEM observations of PSA strains isolated from trunk (A) and from exudate (B) and production of exopolymeric material. (A) Bacteria with their typical shape, dimension and surface morphology. (B) PSA cells isolated from exudate with evident exopolymeric material (as biofilm) on their surfaces. Images were examined at magnifications (A: 9500 £; B: 3000 £). PSA isolated from trunk grown (C) aerobic conditions; (D) anaerobic conditions. (E) PSA isolated from exudate grown in aerobic conditions.
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2.3. Antibacterial activity minimal inhibitory concentration, MBC The major relevance of PSA is its high resistance to antimicrobial drugs and its adaptability to environment. During the last decade, a variety of EOs have been screened for antimicrobial activity to assess their use as both potential sources of new antimicrobial compounds and alternatives drugs to treat infectious diseases and/or to promote food preservation. As a matter of fact, EOs were extensively tested against a broad spectrum of bacteria, yeasts and fungi (Reichling et al. 2009). Nevertheless only few studies on EOs antimicrobial activity towards phytopathogenic microorganisms were performed. In particular, two monoterpenes, as geraniol and citronellol have been used as alternative compounds against PSA (Ferrante & Scortichini 2010), but at a deeper inspection the antibacterial activity assays were performed by no standard methods and data are not comparable. In this study, the determination of EOs antimicrobial activity against PSA strains isolated from trunk and exudate was performed in vitro by standardised CLSI methods (CLSI 2009), with some modification for EOs. Minimal inhibitory concentration (MIC) values for either PSA, were 3.12, 12.5 and 12.5 g/L for Mentha suaveolens essential oil (EOMS), Rosmarinus essential oil (REO) and Tea Tree oil (TTO) respectively. MIC .12.5 g/L were reported for Salvia essential oil (SEO) and Lauro essential oil (LEO). The minimal bactericidal concentration (MBC) values ranged from 3.12 to 6.25 g/L for EOMS, 12.5 and 6.25 g/L for REO and TTO respectively. No bactericidal effect was observed for SEO and LEO. REO and TTO were less efficient than EOMS, and also MBC values for REO and TTO were higher than EOMS for both strains tested (Table S1). Unfortunately, all MIC and MBC values reported could be too higher for a safe application. 2.4. EOs synergism To resolve the problem of the high concentrations at which EOs must be used to be effective, one suggestion is to utilise these oils together drugs so they can act synergistically. The concept of antimicrobial synergy is based on the principle that, in combination with other drugs, the formulation may enhance efficacy, reduce toxicity, decrease adverse side effects, increase bioavailability, lower the dose and reduce the development of antimicrobial resistance (Li et al. 1993). The fact that many agents engage in surface disruption and energy balance disruption would advocate their potential for augmenting each other’s antibacterial effects. Synergism was generally studied in the combination of EO and synthetic drugs (White et al. 1996). In this case, due to lack of any synthetic drugs for the first time, it was evaluated the potential synergism in a combination of different EOs, and as reported by other authors, we used a mixture of three components (Rey-Jurado et al. 2013). Since, chemical constituents of the three active EOs were no overlapping (see Table S2) thus suggesting that a mixture could exert some synergistic effect. To explore the possibility of developing a more powerful combination therapy of EOMS with REO and TTO, the checkerboard microtiter test was performed. Table S2 shows the results obtained in terms of MIC. EOMS inhibition of PSA growth was achieved at 3.12 g/L. In comparison, the MIC of REO and TTO was 12.5 and 6.5 g/L, respectively. The fractional inhibitory concentration indexes (FICIs) calculated from the results of the checkerboard titer assays (Table 1) revealed the following: treating PSA with REO and TTO in Table 1. Fractional inhibitory concentrations (FICs) and indices (FICI) of EOMS, REO and TTO alone or combined together against Pseudomonas syringae pv. actinidiae (PSA).
EOMS REO TTO
MICa (g/L)
MICc (g/L)
FIC
FICI
3.12 12.5 6.5
0.78 1.56 0.78
0.25 0.125 0.125
0.5
Note: MICa, MIC of the sample alone; MICc, MIC of the sample of the most effective combination; FIC of oil, MIC of oil combined together/MIC of oil alone; FICI, summa of three FICs oils.
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combination with EOMS caused a significant decrease in the MIC, compared with their individual MIC values. For example, the MIC of REO alone against PSA was dropped from 12.5 to 1.56 g/L in the presence of EOMS and TTO. The MIC of EOMS alone decreased from 3.12 to 0.78 g/L and also the MIC of TTO alone decreased from 6.25 to 0.78 g/L. Synergistic effects were obtained using combinations of 0.78, 1.56 and 0.78 g/L of EOMS, REO and TTO respectively with a FICI of 0.50. Nevertheless, the results indicate a significant synergistic value of three EOs all together. Checkerboard assay was used to measure the inhibition activity while time – kill study was used to assess bactericidal activity, which was dependent on time instead of being concentration dependent.
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2.5. Time –kill kinetic To confirm the synergistic effect of combined EOs and assess the kinetics of killing effect, in vitro time – kill kinetic experiment was carried out with PSA (Figure S2) at EOs concentrations equivalent to 1/2;, 1, 2 and 4 times their synergic values (s.v). As reported in Figure S2, at a concentration of 1/2; s.v. PSA micro-organism was not killed, while at a concentration of 1 £ s.v. (0.78, 1.56 and 0.78 g/L of EOMS, REO and TTO respectively), the number of colonies (colonies forming unit) was significantly reduced from 4.6 to 1.5 log after 30 min of incubation and the total bactericidal effect was observed within 1 h of contact. At a concentration of 2 £ s.v. (1.56, 3.12 and 1.56 g/L of EOMS, REO and TTO respectively) the total bactericidal effect was observed within only 30 min of contact. The results demonstrated that PSA was highly susceptible to synergic combination of sub-inhibitory concentration of EOMS, REO and TTO. This suggests that the combination treatments exerted a stronger bactericidal action. 3. Conclusion In this article, we demonstrated that the exudate or trunk isolated PSA strains shared the same for phenotypic switching and that the production of EPS is correlated with bacterial resistance and protection against different environmental conditions. We have established that the EOs have activity against PSA, indeed, our results clearly demonstrated that a mixture of three low active EOs, EOMS, REO and TTO, is able to kill PSA at a concentration about sixteen times lower than the corresponding MIC values of single EO utilised alone, after 1 h of exposition. Further studies are in due course to evaluate the toxicity of the above substances towards plants and set the appropriate formulation useful for the purpose. Finally, the significant antibacterial activity of EOs towards the bacteria pathogens of kiwifruit suggests the possibility to use the substances also in this crop. However, also in this case, further studies are necessary to evaluate the toxic effects of the EOs on the kiwifruit production. In vivo studies are in due course to assess the EOs mixture utility in PSA affected plant. Supplementary material Experimental details relating to this paper are available online, alongside Tables S1 and S2 and Figures S1 and S2. Acknowledgements We are grateful to Cristina Buffone for editorial assistance. Universita` Agraria di Tarquinia for the access to their territory for gathering fresh wild M. suaveolens plants and Claudio Quacquarelli of Talia s.r.l for providing TTO sample.
Funding The authors acknowledge Sapienza University [Ateneo grant number C26A122JAW].
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Conflict of Interests The authors declare that they have no conflict of interests.
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Antibacterial activity of essential oils mixture against PSA a
a
b
Elisabetta Vavala , Claudio Passariello , Federico Pepi , Marisa c
b
d
d
Colone , Stefania Garzoli , Rino Ragno , Adele Pirolli , Annarita c
Stringaro & Letizia Angiolella
a
a
Department of Public Health and Infectious Diseases, University of Rome ‘Sapienza’, Piazzale Aldo Moro, 5, 00161 Rome, Italy b
Department of Drugs Chemistry and Technology, University of Rome ‘Sapienza’, Piazzale Aldo Moro, 5, 00161 Rome, Italy c
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Department of Technology and Health, Italian National Institute of Health, Viale Regina Elena 299, 00161 Rome, Italy d
Department of Drugs Chemistry and Technology, Rome Center for Molecular Design, University of Rome ‘Sapienza’, Rome, Italy Published online: 18 Mar 2015.
To cite this article: Elisabetta Vavala, Claudio Passariello, Federico Pepi, Marisa Colone, Stefania Garzoli, Rino Ragno, Adele Pirolli, Annarita Stringaro & Letizia Angiolella (2015): Antibacterial activity of essential oils mixture against PSA, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1022543 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1022543
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Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1022543
Antibacterial activity of essential oils mixture against PSA Elisabetta Vavalaa, Claudio Passarielloa, Federico Pepib, Marisa Colonec, Stefania Garzolib, Rino Ragnod, Adele Pirollid, Annarita Stringaroc and Letizia Angiolellaa*
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a Department of Public Health and Infectious Diseases, University of Rome ‘Sapienza’, Piazzale Aldo Moro, 5, 00161 Rome, Italy; bDepartment of Drugs Chemistry and Technology, University of Rome ‘Sapienza’, Piazzale Aldo Moro, 5, 00161 Rome, Italy; cDepartment of Technology and Health, Italian National Institute of Health, Viale Regina Elena 299, 00161 Rome, Italy; dDepartment of Drugs Chemistry and Technology, Rome Center for Molecular Design, University of Rome ‘Sapienza’, Rome, Italy
(Received 16 December 2014; final version received 19 February 2015)
Pseudomonas syringae pv. actinidiae (PSA) is the causal agent of bacterial canker of kiwifruit. It is very difficult to treat pandemic disease. The prolonged treatment with antibiotics, has resulted in failure and resistance and alternatives to conventional antimicrobial therapy are needed. The aim of our study was to analyse the phenotypic characteristics of PSA, identify new substances from natural source i.e. essential oils (EOs) able to contain the kiwifruit canker and investigate their potential use when utilised in combination. Specially, we investigated the morphological differences of PSA isolates by scanning electron microscope, and the synergic action of different EOs by time-kill and checkerboard methods. Our results demonstrated that PSA was able to produce extracellular polysaccharides when it was isolated from trunk, and, for the first time, that it was possible to kill PSA with a mixture of EOs after 1 h of exposition. We hypothesise on its potential use in agriculture. Keywords: Pseudomonas syringae pv. actinidiae; kiwifruit canker; exopolymeric material; SEM; essential oils; synergism
1. Introduction Agriculture production records heavy yearly losses due to plant diseases. Many product used as control for plant pathogenic bacteria contain antibiotics (mostly streptomycin) or heavy metals (mostly copper). These treatments do have limitations to either phytotoxicity or authorised restriction in some countries (e.g. antibiotics in Europe) (Reglinski et al. 2013).
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
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Pseudomonas syringae is a worldwide spread phytopathogenic micro-organism mainly adapted to plant species (Gardan et al. 1999), either cultivated or grown in wild habitats. P. syringae pv. actinidiae (PSA) the causal agent of bacterial canker of kiwigreen (Actinidia deliciosa and Actinidia chinensis) was first reported in Japan (Takikawa et al. 1989), and subsequently isolated in South Korea (Koh et al. 1994) and Italy (Scortichini 1994; Balestra et al. 2009). During 2008– 2011, PSA suddenly incited the rapid spread of severe epidemics of bacterial canker in central Italy (Ferrante & Scortichini 2009, 2010). The symptoms of bacterial disease included brown leaf spots, necrosis of the withering flowers, redness of lenticels, extended cankers along the main trunk, bleeding of cankers on the trunk with exudates that go from white to orange (Marcelletti et al. 2011). The bacterium owes its virulence to the ability to acquire exogenous material that allows it to exceed the plant stress conditions, such as lack of nutrients (Arnold et al. 2007) and loss of mobile genetic elements (Johnson et al. 2011), and to the presence of an ATP binding cassette (ABC) transporter called Pseudomonas efflux system, involved in multiple resistance to drugs (Marcelletti et al. 2011). A further PSA virulence factor is represented by copA and copB genes, which play a key role in the copper resistance (Brotz & Sahl 2000). The bacterium propagates quickly within and between orchards due to bacterial exudates that oozes from cankers during late autumn – winter and early spring, and are spread by the wind (Serizawa et al. 1989; Serizawa & Ichikawa 1993). To worsen the scenario, some agronomic techniques, such as pruning cut, cause injury that greatly facilitates the pathogen penetration within the plant. PSA survive even in the pollen grains of Actinidia (Vanneste et al. 2011), contributing to pathogen spread. The research of new antimicrobial protocols which include natural products has recently increased in many fields and the essential oils (EOs) and/or some of their constituents have demonstrated their effectiveness against a large variety of organisms, including bacteria (Basile et al. 2006), viruses (Duschatzky et al. 2005; Civitelli et al. 2014), fungi (Pietrella et al. 2011; Stringaro et al. 2014), protozoa and parasites (Sarrou et al. 2013). Recent studies have highlighted a synergism between the various components of EOs and antibiotics or antifungals, which are able to penetrate into the cell through the membrane and produce different types of radical reactions, thus causing cellular death (Bakkali et al. 2008). EOs have no specific cellular target and are mainly composed of mono- and di-terpenes (Carson et al. 2002). The aim of this study was to investigate the morphological differences of PSA strains isolated from trunk and from exudate, as well as to identify new substances of natural origin able to kill or contain the bacterium infections. In the present study, we have demonstrated (i) the presence of exopolymeric material around the bacterium in particular growth conditions; (ii) the bactericidal activity of Mentha suaveolens, Rosmarinus officinalis and Melaleuca alternifolia EOs mixture against PSA. 2. Results and discussion 2.1. Isolation and molecular identification of PSA Strains of PSA were isolated from trunk and from exudate, in a case of bacterial canker of A. chinensis, and grown on NSA. As reported in Figure S1A, the colony of the strains isolated from trunk were small, opaque, irregular, and cream-white, while the colony isolated from exudates were large, mucoid and cream dark. Pseudomonas spp. often has a non-mucoid phenotype in standard laboratory media. Growth in the presence of 0.3 M sodium chloride or 3 –5% ethanol reportedly can lead to the generation of mucoid variants of non-mucoid strains of Pseudomonas (Singh et al. 1992). These morphological variations can be induced by exposing cells to stress such as high antibiotic concentration (Silva et al. 2013). Molecular analyses by polymerase chain reaction using the Rees-George protocol (Rees-George et al. 2010) were performed, to confirm that the strains isolated from trunk and exudate were PSA. Specific
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sequences of 16S and 23S ribosomal RNA have been used as control. Figure S1B shows a band of 280 Kb in both strains confirming, as reported by other authors (Chapman et al. 2012), that the isolated strains were PSA with different phenotypic characteristics.
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2.2. Scanning electron microscope observations and exopolymeric production To evaluate the different PSA strains morphology, scanning electron microscope (SEM) analyses were performed. SEM observations of the trunk isolated PSA showed the bacteria with a typical shape, dimension and surface morphology (Figure 1(A)). Differently, exudate isolated PSA, showed evident exopolymeric material on the bacteria, as biofilm (Figure 1(B)), in agreement with the report by Renzi et al. (2012). Extracellular polysaccharides or exopolysaccharides (EPSs) are high-molecular weight sugar-based polymers that are synthesised and secreted by many micro-organisms (Ferreira et al. 2011). The importance of their production has been studied in many bacteria (Li et al. 2014; Shi et al. 2014). Furthermore, it is known that the physiological role of polysaccharides is to defend the cell from environmental stress and from the action of toxic substances or antibiotics. EPSs production represents a main virulence factor for the survival of the micro-organism both inside and outside the plant. To confirm EPSs production, trunk isolated PSA was grown in aerobic or anaerobic conditions for 72 h, in aerobic conditions the colonies were non-mucoid (Figure 1(C)), while in anaerobic conditions the colonies turned to be mucoid (Figure 1(D)) similarly to exudate isolated PSA (Figure 1(E)). The EPS production could explain their survival in low presence of oxygen. In particular, EPS is typically correlated with bacterial resistance and protection against different stress conditions such as desiccation, salt stress and UV radiations (Ferreira et al. 2011).
Figure 1. SEM observations of PSA strains isolated from trunk (A) and from exudate (B) and production of exopolymeric material. (A) Bacteria with their typical shape, dimension and surface morphology. (B) PSA cells isolated from exudate with evident exopolymeric material (as biofilm) on their surfaces. Images were examined at magnifications (A: 9500 £; B: 3000 £). PSA isolated from trunk grown (C) aerobic conditions; (D) anaerobic conditions. (E) PSA isolated from exudate grown in aerobic conditions.
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2.3. Antibacterial activity minimal inhibitory concentration, MBC The major relevance of PSA is its high resistance to antimicrobial drugs and its adaptability to environment. During the last decade, a variety of EOs have been screened for antimicrobial activity to assess their use as both potential sources of new antimicrobial compounds and alternatives drugs to treat infectious diseases and/or to promote food preservation. As a matter of fact, EOs were extensively tested against a broad spectrum of bacteria, yeasts and fungi (Reichling et al. 2009). Nevertheless only few studies on EOs antimicrobial activity towards phytopathogenic microorganisms were performed. In particular, two monoterpenes, as geraniol and citronellol have been used as alternative compounds against PSA (Ferrante & Scortichini 2010), but at a deeper inspection the antibacterial activity assays were performed by no standard methods and data are not comparable. In this study, the determination of EOs antimicrobial activity against PSA strains isolated from trunk and exudate was performed in vitro by standardised CLSI methods (CLSI 2009), with some modification for EOs. Minimal inhibitory concentration (MIC) values for either PSA, were 3.12, 12.5 and 12.5 g/L for Mentha suaveolens essential oil (EOMS), Rosmarinus essential oil (REO) and Tea Tree oil (TTO) respectively. MIC .12.5 g/L were reported for Salvia essential oil (SEO) and Lauro essential oil (LEO). The minimal bactericidal concentration (MBC) values ranged from 3.12 to 6.25 g/L for EOMS, 12.5 and 6.25 g/L for REO and TTO respectively. No bactericidal effect was observed for SEO and LEO. REO and TTO were less efficient than EOMS, and also MBC values for REO and TTO were higher than EOMS for both strains tested (Table S1). Unfortunately, all MIC and MBC values reported could be too higher for a safe application. 2.4. EOs synergism To resolve the problem of the high concentrations at which EOs must be used to be effective, one suggestion is to utilise these oils together drugs so they can act synergistically. The concept of antimicrobial synergy is based on the principle that, in combination with other drugs, the formulation may enhance efficacy, reduce toxicity, decrease adverse side effects, increase bioavailability, lower the dose and reduce the development of antimicrobial resistance (Li et al. 1993). The fact that many agents engage in surface disruption and energy balance disruption would advocate their potential for augmenting each other’s antibacterial effects. Synergism was generally studied in the combination of EO and synthetic drugs (White et al. 1996). In this case, due to lack of any synthetic drugs for the first time, it was evaluated the potential synergism in a combination of different EOs, and as reported by other authors, we used a mixture of three components (Rey-Jurado et al. 2013). Since, chemical constituents of the three active EOs were no overlapping (see Table S2) thus suggesting that a mixture could exert some synergistic effect. To explore the possibility of developing a more powerful combination therapy of EOMS with REO and TTO, the checkerboard microtiter test was performed. Table S2 shows the results obtained in terms of MIC. EOMS inhibition of PSA growth was achieved at 3.12 g/L. In comparison, the MIC of REO and TTO was 12.5 and 6.5 g/L, respectively. The fractional inhibitory concentration indexes (FICIs) calculated from the results of the checkerboard titer assays (Table 1) revealed the following: treating PSA with REO and TTO in Table 1. Fractional inhibitory concentrations (FICs) and indices (FICI) of EOMS, REO and TTO alone or combined together against Pseudomonas syringae pv. actinidiae (PSA).
EOMS REO TTO
MICa (g/L)
MICc (g/L)
FIC
FICI
3.12 12.5 6.5
0.78 1.56 0.78
0.25 0.125 0.125
0.5
Note: MICa, MIC of the sample alone; MICc, MIC of the sample of the most effective combination; FIC of oil, MIC of oil combined together/MIC of oil alone; FICI, summa of three FICs oils.
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combination with EOMS caused a significant decrease in the MIC, compared with their individual MIC values. For example, the MIC of REO alone against PSA was dropped from 12.5 to 1.56 g/L in the presence of EOMS and TTO. The MIC of EOMS alone decreased from 3.12 to 0.78 g/L and also the MIC of TTO alone decreased from 6.25 to 0.78 g/L. Synergistic effects were obtained using combinations of 0.78, 1.56 and 0.78 g/L of EOMS, REO and TTO respectively with a FICI of 0.50. Nevertheless, the results indicate a significant synergistic value of three EOs all together. Checkerboard assay was used to measure the inhibition activity while time – kill study was used to assess bactericidal activity, which was dependent on time instead of being concentration dependent.
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2.5. Time –kill kinetic To confirm the synergistic effect of combined EOs and assess the kinetics of killing effect, in vitro time – kill kinetic experiment was carried out with PSA (Figure S2) at EOs concentrations equivalent to 1/2;, 1, 2 and 4 times their synergic values (s.v). As reported in Figure S2, at a concentration of 1/2; s.v. PSA micro-organism was not killed, while at a concentration of 1 £ s.v. (0.78, 1.56 and 0.78 g/L of EOMS, REO and TTO respectively), the number of colonies (colonies forming unit) was significantly reduced from 4.6 to 1.5 log after 30 min of incubation and the total bactericidal effect was observed within 1 h of contact. At a concentration of 2 £ s.v. (1.56, 3.12 and 1.56 g/L of EOMS, REO and TTO respectively) the total bactericidal effect was observed within only 30 min of contact. The results demonstrated that PSA was highly susceptible to synergic combination of sub-inhibitory concentration of EOMS, REO and TTO. This suggests that the combination treatments exerted a stronger bactericidal action. 3. Conclusion In this article, we demonstrated that the exudate or trunk isolated PSA strains shared the same for phenotypic switching and that the production of EPS is correlated with bacterial resistance and protection against different environmental conditions. We have established that the EOs have activity against PSA, indeed, our results clearly demonstrated that a mixture of three low active EOs, EOMS, REO and TTO, is able to kill PSA at a concentration about sixteen times lower than the corresponding MIC values of single EO utilised alone, after 1 h of exposition. Further studies are in due course to evaluate the toxicity of the above substances towards plants and set the appropriate formulation useful for the purpose. Finally, the significant antibacterial activity of EOs towards the bacteria pathogens of kiwifruit suggests the possibility to use the substances also in this crop. However, also in this case, further studies are necessary to evaluate the toxic effects of the EOs on the kiwifruit production. In vivo studies are in due course to assess the EOs mixture utility in PSA affected plant. Supplementary material Experimental details relating to this paper are available online, alongside Tables S1 and S2 and Figures S1 and S2. Acknowledgements We are grateful to Cristina Buffone for editorial assistance. Universita` Agraria di Tarquinia for the access to their territory for gathering fresh wild M. suaveolens plants and Claudio Quacquarelli of Talia s.r.l for providing TTO sample.
Funding The authors acknowledge Sapienza University [Ateneo grant number C26A122JAW].
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Conflict of Interests The authors declare that they have no conflict of interests.
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