Effects of plant antimicrobial phenolic compounds on virulence of the genus Pectobacterium

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Research in Microbiology xx (2015) 1e11 www.elsevier.com/locate/resmic

Original article

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Effects of plant antimicrobial phenolic compounds on virulence of the genus Pectobacterium Janak Raj Joshi a,b, Saul Burdman a, Alex Lipsky b, Iris Yedidia b,*

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Department of Plant Pathology and Microbiology and the Otto Warburg Minerva Center for Agricultural Biotechnology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel b Department of Plant Sciences, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel Received 3 March 2015; accepted 23 April 2015 Available online ▪ ▪ ▪

Abstract Pectobacterium spp. are among the most devastating necrotrophs, attacking more than 50% of angiosperm plant orders. Their virulence strategy is based mainly on the secretion of exoenzymes that degrade the cell walls of their hosts, providing nutrients to the bacteria, but conversely, exposing the bacteria to plant defense compounds. In the present study, we screened plant-derived antimicrobial compounds, mainly phenolic acids and polyphenols, for their ability to affect virulence determinants including motility, biofilm formation and extracellular enzyme activities of different Pectobacteria: Pectobacterium carotovorum, P. brasiliensis, Pectobacterium atrosepticum and Pectobacterium aroidearum. In addition, virulence assays were performed on three different plant hosts following exposure of the bacteria to selected phenolic compounds. These experiments showed that cinnamic, coumaric, syringic and salicylic acids and catechol can considerably reduce disease severity, ranging from 20 to 100%. The reduced disease severity was not only the result of reduced bacterial growth, but also of a direct effect of the compounds on important bacterial virulence determinants, including pectolytic and proteolytic exoenzyme activities, that were reduced by 50e100%. This is the first report revealing a direct effect of phenolic compounds on virulence factors in a wide range of Pectobacterium strains. © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Biofilm; Motility; Pectobacterium; Phenolics; Plant cell wall degrading enzymes; Virulence determinants

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1. Introduction Pectobacterium spp. are ubiquitous necrotrophic plant pathogens implicated in bacterial soft rot of many fruit, ornamental and vegetable crops produced in tropical and temperate regions, resulting in serious worldwide economic losses [32,34]. They are Gram-negative, facultative anaerobic, non-sporing, motile bacteria belonging to the Enterobacteriaceae family. Soft rot symptoms caused by Pectobacteria are mediated by the secretion of multiple exoenzymes, which act synergistically to degrade plant cell

* Corresponding author. E-mail addresses: [email protected] (J.R. Joshi), saul. [email protected] (S. Burdman), [email protected] (A. Lipsky), [email protected] (I. Yedidia).

walls. Pectin-degrading enzymes comprise the major group of these enzymes. Most of the exoenzymes, including pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), pectin lyase (Pnl) and proteases (Prt) are secreted by the type II secretion system (T2SS), under the control of quorum sensing, and collectively determine bacterial pathogenicity [6,20,28]. The Pectobacterium host range extends to more than 50% of angiosperm plant orders [32,34]. This fact, together with the ability of Pectobacterium to remain latent inside several hosts, has led pathogen species from this group to acquire a respectable place on the ‘top-ten’ list of plant pathogenic bacteria [34]. Pectobacterium are able to detect multiple types of small molecules to coordinate pathogenesis, modify the plant environment and attack competing microbes [4]. Of these molecules, plant-derived compounds have been studied mostly in

http://dx.doi.org/10.1016/j.resmic.2015.04.004 0923-2508/© 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Joshi JR, et al., Effects of plant antimicrobial phenolic compounds on virulence of the genus Pectobacterium, Research in Microbiology (2015), http://dx.doi.org/10.1016/j.resmic.2015.04.004

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relation to Dickeya spp. and less in relation to Pectobacterium spp. Two phenolic compounds, o-coumaric acid and t-cinnamic acid, have been shown to upregulate the expression of type III secretion (T3S) genes [26]. More specifically, in Dickeya dadantii, the two phenolic acids were shown to play a role in virulence via the GacAS two-component system, through activation of the expression of several virulence genes. Both compounds are intermediates in the plant phenylpropanoid biosynthesis pathway, which is involved in biosynthesis of several defense compounds such as phenolic acids, flavonoids, isoflavonoids, stilbenes and lignins [22]. These phenolic compounds have been reported to act as antimicrobial agents against a wide range of microorganisms, including Gram-positive (e. g., Staphylococcus aureus and Bacillus cereus) and Gram-negative bacteria (e. g., Escherichia coli, Salmonella spp., Pseudomonas aeruginosa and Xylella fastidiosa) [9,12]. Pectobacterium spp. are capable of avoiding plant defense to improve their ability to infect the host [16,37]. Following the initial infection, specific plant signals are released by the bacteria, which in many ways determine the outcome of the interaction [5]. Plants produce an enormous variety of small molecular compounds, many of which possess antimicrobial activity, and at least some of these antimicrobial compounds target the virulence mechanisms of the pathogens [25]. Based on the aforementioned background, we hypothesized that small plant molecules derived from the plant phenylpropanoid pathway may have crucial effects on Pectobacterium virulence. To verify this hypothesis, we investigated the effects of such small phenolic molecules on the growth and virulence-associated traits of Pectobacterium spp. Since many Pectobacterium strains are pathogens of potato, special emphasis was given to molecules that have been reported in the literature as constituents of potatoes or potato peel extracts [15]; however, additional compounds were considered. We performed experiments involving selected phenolic compounds to: (i) determine their minimal inhibitory concentrations (MICs) on pectolytic bacteria; (ii) examine the efficacy of potent compounds on growth; (iii) quantify their effect on several virulence factors, such as motility, biofilm formation and exoenzyme activity; and (iv) monitor their effects on bacterial virulence in the interaction with potato, as well as in two other Pectobacterium hosts, calla lily and cabbage (monocot and dicot hosts, respectively). 2. Materials and methods

Table 1 List of bacterial strains used in this study. Species

Strain

Pectobacterium aroidearum P. brasiliensis

PC1

Host

Location of isolation/source

Ornithogalum dubium Pcb3 Solanum tuberosum P. carotovorum PC11 Solanum lycopersicum P. carotovorum WPP14 S. tuberosum P. atrosepticum EcaSCRI1043 S. tuberosum P. brasiliensis Pcb1692 S. tuberosum Escherichia coli K12

Israel (2004)/Shulamit Manulis Israel (2004)/S. Manulis Israel (1998)/S. Manulis USA (2001) Scotland (2001) Brazil (1999) ATCC 10798

2.2. Determination of minimum inhibitory concentrations All plant-derived compounds used in this study are secondary metabolites and were selected based on their effects on microbes (see Table 2 for selected phenolic acids and polyphenolics). All compounds were purchased from SigmaeAldrich (SigmaeAldrich Co., MO, USA). Minimum inhibitory concentrations (MICs) were determined by the broth dilution method, as recommended by the CLSI [7] with minor modifications. Briefly, bacterial cultures of all strains were grown overnight in LB and normalized to 1  108 colony forming units (cfu) mL1 with fresh liquid LB. Ten microliters were then used to inoculate 190 mL of LB containing 2-fold serial dilutions of each of the tested compounds. The MIC was determined as the lowest concentration of the compound at which no measurable growth (increase in turbidity) occurred for a given strain. MIC values for the different compound/ strain combinations are shown in Table S1. Assessment of the effects of selected compounds on growth, motility, biofilm formation, exoenzyme activity and virulence of different strains was carried out using a non-lethal concentration of the compounds that lead to 50% growth inhibition relative to untreated bacteria (Table 3). Importantly, all phenolic acids were found to be unable to alter the pH of the growth medium at the used concentrations (data not shown). 2.3. Growth curves Bacterial strains were grown overnight at 28
C in 4 mL liquid LB medium under continuous shaking at 150 rpm in a TU-400 incubator shaker (MRC, Holon, Israel). The Table 2 Phenolic compounds used in the present study and their common concentrations in plant tissue as reported in the literature.

2.1. Bacterial strains, growth media and chemicals

Compound

Source

Conc.(mg 100 g1 fresh tissue)

References

Pectobacterium and E. coli strains used in this study are described in Table 1. All strains were cultivated at 28
C in LuriaeBertani (LB) medium (Difco Laboratories, MI, USA), unless stated otherwise. Murashiage and Skoog minimal medium (MS; Duchefa, Haarlem, The Netherlands) was used for the plant infection assays.

Cinnamic acid Coumaric acid

potato red cabbage, radish, pepper, potato, parsley potato soybean, potato, rye olive potato, soybean

0.05 3e9.6

[44] [35]

0.4 0.5e25 0.14 0.5e25

[8] [35,36] [42] [35]

Salicylic acid Syringic acid Tyrosol Vanillin

Please cite this article in press as: Joshi JR, et al., Effects of plant antimicrobial phenolic compounds on virulence of the genus Pectobacterium, Research in Microbiology (2015), http://dx.doi.org/10.1016/j.resmic.2015.04.004

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Table 3 Non-lethal concentration (mM) of phenolic compounds used in the experimenta. Compounds

Pectobacterium strains

E. coli

PC1 Pcb3 PC11 WPP14 EcaSCRI1043 Pcb1692 K12 Caffeic acid Catechin Catechol Chlorogenic acid Cinnamic acid Coumaric acid Epicatechin Ferulic acid Gallic acid Hydroquinone Salicylic acid Syringic acid Tyrosol Vanillin

10 7.5 7.5 5 3 10 15 10 10 10 3 5 10 6

5 3.75 3.75 5 3 5 15 5 10 5 3 5 5 3

5 7.5 7.5 5 3 10 15 5 10 5 3 5 10 6

10 7.5 7.5 5 3 10 15 10 10 5 3 5 10 6

5 7.5 7.5 5 3 10 7.5 10 10 5 3 5 10 6

5 3.75 3.75 5 3 5 7.5 5 10 5 3 5 5 3

10 15 15 10 6 10 15 10 20 10 3 10 20 12

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Non-lethal concentrations that caused 50% growth inhibition of a given strain relative to untreated controls.

suspensions were then diluted to a final concentration of 1  106 cfu in 200 mL fresh LB containing non-lethal concentrations (causing 50% growth inhibition) of the phenolic compounds, or without any compound in controls, in Bradford 96-well microtiter plates (Bar-Naor, Ramat Gan, Israel). Plates were incubated at 28
C with continuous shaking, and growth was determined by measuring optical density at 600 nm (OD600) at 1 h intervals for 14 h, using a micro-plate reader (Spectra MR, Dynext Technologies, VA, USA). Each experiment was performed at least twice with five replicates for each strain/compound combination. 2.4. Motility assays We examined the effects of the phenolic compounds on the motility of the strains under study. Motility was tested on plates containing semi-solid LB medium (0.3% agar) with 0.005% tetrazolium chloride to detect the presence of bacteria by deep pink staining (Northeast Laboratory Services, Maine, England). Two hundred microliters of normalized bacterial suspensions of 1  106 cfu (prepared from overnight culture) were exposed to the non-lethal concentrations of the compounds under study for 2 h. Then, bacteria were collected by dipping a sterile needle into the suspensions and transferred to the center of the plates. Plates were incubated at 28
C, and motility was assessed after 24 h by measuring the pink-colored area (which is the swimming area) using ImageJ (National Institute of Health, MD, USA; http://imagej.nih.gov/ij/). 2.5. Biofilm formation Biofilm formation was evaluated using the microtiter dish assay with crystal violet (CV) for biofilm staining as described by O'Toole [40]. Briefly, bacterial suspensions containing 1  106 cfu in 200 mL LB were incubated at 28
C in the presence of phenolic compounds at 50% inhibitory

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concentrations in Bradford 96-well microtiter plate for 72 h without shaking. Then, the suspensions were carefully discarded and the plates were washed gently with double-distilled water (DDW) to remove any unattached cells. Two hundred microliters of 0.1% crystal violet were added to each well. After 20 min of incubation at 25
C and three washings with deionized water, 200 mL of 30% acetic acid were added to each well to solubilize the crystal violet, and the plates were incubated for 15 min at 25
C. The formation of biofilms was quantified by measuring absorbance at 550 nm in a microplate reader. 2.6. Activities of hydrolytic enzymes The activities of pectate lyase (Pel), polygalacturonase (Peh) and proteolytic enzymes (Prt) of Pectobacterium strains were tested following overnight exposure of bacteria grown in LB medium at 28
C to non-lethal concentrations (50% inhibition) of the different phenolic compounds under study. Semiquantitative assays for Peh, Pel and Prt activities were assessed using a plate assay as described by Chatterjee [6]. The plates were prepared as described and then poked to form 4 mm holes, which were filled with the supernatants from overnight grown cultures and incubated at 28
C for 24 h. Activity of the enzymes were expressed as the size of the observed haloes. Two independent experiments were carried out, each with four replicates of each strain/compound combination. A quantitative assay available for Pel activity was also performed as described by Lee et al. [24]. Also, the activity of hydrolytic enzymes was evaluated in presence ciprofloxacin, at non-lethal concentration (6 ng mL1) that would contribute 50% growth inhibition. Ciprofloxacin is an antibiotic that belongs to the group of DNA gyrase inhibitors, known to specifically inhibit cell division and growth [23]. 2.7. Virulence assays Virulence was evaluated by assessing symptom severity in three plants-Zantedeschia aethiopica (calla lily), Brassica oleracea 'Proctor' and Solanum tuberosum 'Lady Rosetta'- as previously described [31,47]. Fully expanded young leaves of calla lily, young cabbage leaves or small (about 100 g) potato tubers were externally disinfected by soaking in 0.7% sodium hypochlorite for 20 min, and then washed twice with sterilized distilled water. Discs (20 mm in diameter) were excised from disinfected leaves of calla lily and cabbage and transferred to Petri dishes containing Murashige and Skoog (MS) medium. Whole disinfected potato tubers were used for inoculation. Bacterial strains were grown overnight in LB liquid medium at 28
C with continuous shaking and diluted to 1  108 cfu mL1 (OD600 ¼ 0.1) in sterile DDW containing non-lethal concentrations of the phenolic compounds. The bacterial suspensions were shaken at 150 rpm in a TU-400 incubator shaker for 2 h at 28
C before inoculation. At this time point, bacterial samples of each treatment and control were assessed by dilution plating assay, revealing no significant difference in bacterial counts before host inoculation

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(data not shown). Leaf discs and potato tubers were pierced at the center with a sterile tip and inoculated with 10 mL of the bacterial suspensions. The inoculated plant material was incubated at 28
C. For calla lily and cabbage, disease severity was expressed as the percentage of decayed tissue relative to the total area of the disc, and was recorded 15 h after inoculation. For potato tubers, disease severity was expressed as the percentage of rotten tissue, which was determined by weighing the whole tuber and then subtracting the corresponding tuber weight after the decayed portion was scraped away, 48 h after inoculation. As an additional control, calla lily leaf discs were also inoculated with Pectobacterium strains PC1 and Pcb1692 that were treated with a non-lethal concentration (50% growth inhibition) of ciprofloxacin. Two independent experiments were carried out, with 10 and 4 replicates per strain  compound combination for the leaf-disc and tuber assays, respectively. 2.8. Data analyses We performed a one-way analysis of variance (ANOVA) using the JMP-Version 5 software, (SAS Institute Inc., NC, USA). When the ANOVA indicated a significant difference (P < 0.05), student's t test was performed. Graphs were generated with Sigma Plot Version 10.0 (Systat Software Inc., CA, USA). 3. Results and discussion 3.1. Growth of bacteria in response to phenolic compounds Polyphenols and phenolic acids are secondary metabolites that are ubiquitous in higher plants and have important roles in plant defense against pathogens, herbivores and abiotic stress [2]. Moreover, salicylic acid (a phenolic compound) induced resistance against Dickeya solani and Pectobacterium carotovirum subsp. carotovorum in potato and tobacco [41,11]. Besides induced resistance, plant phenolic compounds were found to have antimicrobial activity against bacterial pathogens [4,43,46]. In order to study the effect of plant phenolic compounds on bacterial growth and virulence traits, MIC as well as the non-lethal concentrations of each compound (causing 50% growth inhibition) were determined for each of the tested Pectobacterium strains and E. coli (Table S1 and 3, respectively). These experiments revealed that, among the tested compounds, salicylic acid was the most effective compound, with the lowest MIC and 50% growth inhibition values for all tested Pectobacterium strains (6 and 3 mM, respectively). Relatively low 50% growth inhibition values were also obtained for cinnamic acid, syringic acid and catechol (Table S1). These values were also determined for E. coli K12, a nonplant-pathogenic bacterium belonging to the Enterobacteriaceae family. A consistent trend was observed for E. coli K12 which was more tolerant than the Pectobacterium strains for most tested compounds as reflected in the highest MIC and 50% growth inhibition values observed for this strain

(Table S1 and 3). Surprisingly, this strain also displayed more rapid recovery after exposure to all of the tested compounds (Fig. 1), suggesting that its natural environment, the gastrointestinal tract exposes the bacterium to compounds of the same nature. To explore the effects of selected phenolic compounds on virulence traits of Pectobacterium strains, we chose to use non-lethal concentrations, which allow considerable bacterial growth, thus permitting to explore the effect of the different compounds in a comparative manner. Growth curves in the presence of the non-lethal concentrations (50% inhibition) of the compounds were determined for the Pectobacterium strains as well as for E. coli K12 (shown in Fig. 1 for compounds that were selected for further studies and Fig. S1 for the other compounds). As expected, the presence of phenolic compounds at non-lethal concentrations in the medium affected the growth of all strains relative to the control. In agreement with the MIC values (Table S1), some strains were more sensitive to certain phenolics than others. For example, P. brasiliensis strains Pcb1692 and Pcb3 displayed higher sensitivity to cinnamic, salicylic and coumaric acids (Fig. 1). Alternatively, the Pectobacterium carotovorum strains PC11 and WPP14, as well as the Pectobacterium aroidearum strain PC1 were more tolerant. The strain PC1 was once considered to belong to P. carotovorum, but was recently re-classified as P. aroidearum, a monocot specializing species [39,47]. This specialization could be at least in part attributed to the high tolerance of this Pectobacterium strain to most of the phenolic compounds. The effect of the compounds on Pectobacterium growth revealed potent compounds belonging mainly to the group of non-flavonoid phenolic acids. These compounds may be subdivided into benzoic acid derivatives (gallic, syringic, salicylic and vanillic acids) and cinnamic acid derivatives (coumaric, ferulic and caffeic acids) [11]. Both classes of phenolic acids were found active with no definite advantage to any of the groups. Based on that, we chose salicylic, cinnamic, coumaric and syringic acids as well as vanillin, catechol and tyrosol for further experiments to assess their effects on virulence traits. Importantly, most of these compounds were reported to be naturally produced in potato (Table 2), an economically important host of Pectobacterium strains. 3.2. Effect of phenolic acids and other phenolics on motility Motility is an important virulence factor of many plantpathogenic bacteria, including Pectobacterium [14,19,45]. Here, we assessed the effects of phenolic compounds on the motility of the different strains. In controls (untreated bacteria), the highest motility was observed for the strain PC1 followed by Pcb3, WPP14 and Pcb1692. Interestingly, an increased motility was observed in most strains following exposure to non-lethal concentrations of the tested compounds (Fig. 2). This effect was particularly clear in strains that showed relatively high motility under control conditions, and following exposure to cinnamic, coumaric and salicylic acid. The

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Fig. 1. Effect of treatments with non-lethal concentrations (causing 50% inhibition) of different phenolic compounds on the growth curves of Pectobacterium strains (P. aroidearum PC1; P. carotovorum PC11 and WPP14; P. brasiliensis Pcb3 and Pcb1692; and P. atrosepticum EcaSCRI1043) and E. coli K12. Bacteria were grown at 28
C for 14 h and growth was assessed by measuring absorbance at 600 nm every hour (bar ¼ SE; n ¼ 14).

combination of PC1 and cinnamic acid was an exception to that. A similar pattern of effect, though less pronounced, was observed for syringic acid. PC11, that displayed least motility, was not affected by any of the compounds tested. Similarly, catechol increased the motility of strains WPP14 and K12 but not of the other strains. Additionally, tyrosol and vanillin did not have a marked influence on the motility of most strains with the exception of strain PC1 and K12 for which motility was reduced and increased respectively (Fig. 2). Overall, with few exceptions, the tested phenolic compounds were found to either increase significantly the motility of most strains, or at cases

not affect motility. More specifically, coumaric and salicylic acids along with cinnamic and syringic acid, which appreciably reduced bacterial growth, also consistently increased the motility of most strains. Since non-lethal concentration still allowed considerable growth, it is reasonable to assume, that their toxic effects, may induce higher motility in many strain/ compound combinations. Similar observations were made in P. aeruginosa on exposure to non-lethal concentration of common antibiotics [27]. Collectively, it appeared that compounds that had a stronger inhibitory effect on growth were more potent in enhancing bacterial motility.

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Fig. 2. Effect of exposure with phenolic compounds at non-lethal concentrations on the motility of Pectobacterium strains (P. aroidearum PC1; P. carotovorum PC11 and WPP14; P. brasiliensis Pcb3 and Pcb1692; and P. atrosepticum EcaSCRI1043) and E. coli K12. Bacteria were grown at 28
C in 0.3% agar medium. The motility area was measured after 24 h. Bars with * sign are significantly higher than their respective control with the exception of PC1/tyrosol, where it is significantly lower (P < 0.05; bar ¼ SE; n ¼ 4).

3.3. Effect of phenolic compounds on biofilm formation Biofilm formation ability is another recognized trait in terms of its importance for successful infection of any bacteria including Pectobacterium [18]. It is represented as the absorbance of crystal violet dye bound to biofilm cells. Therefore, a large absorbance indicates more biofilm and vice versa. Under the tested conditions, application of phenolic compounds significantly reduced the biofilm formation ability of all the tested isolates by 50e90% (Fig. 3). The P. brasiliensis strains Pcb3 and Pcb1692 were shown to possess higher biofilm formation ability than the other strains under control condition; therefore the effect of the phenolic compounds on their biofilm ability was more pronounced. In contrast, strains EcaSCRI1043 and PC1, as well as E. coli K12, showed relatively low levels of biofilm formation capacity, however, reduction of biofilm formation following exposure to the different compounds could also be observed in all tested strains (Fig. 3). Similar to our results, Lagonenko et al., observed reduction in biofilm formation in plant pathogenic bacteria in the presence of salicylic acid. The authors suggested that this might be the result of salicylic acid interference with communication between bacteria [21]. Also, natural phenolic compounds were shown to reduce biofilm formation ability of P. aeruginosa, which in that case were related to their redox potential or antioxidant capacity [17]. Based on these studies, the reduced biofilm that was demonstrated here for all Pectobacterium strains might be the result of phenolic compounds interference with

communication between bacterial cells, which is the basis for biofilm formation. Moreover, dissecting motility and biofilm together, displayed an inverse trend between these two virulence determinants. Treating the bacteria with phenolic compounds increased motility in most cases (in accord with the strain's innate motility) while biofilm formation was significantly reduced in all pectobacteria, regardless of the strain's basic capacity to form biofilm. The inverse correlation is well suited with a mechanism in which the bacteria increase motility in order to escape the toxic effects of the compounds; an activity which is opposite to biofilm formation, an act that is largely dependent on cell attachment, communication and bacterial ability to anchor onto a surface. This result is in agreement with Caiazza et al. and Merritt et al., who proposed the existence of inverse correlation between motility and biofilm formation in P. aeruginosa, that was guided either by SadBdependent chemotaxis like regulatory pathway or SadC and phosphodiesterase BifA [3,38]. 3.4. Effect of phenolic compounds on exoenzyme activity Exoenzyme activity is another virulence determinant that is crucial for necrotrophic pathogens, including Pectobacterium, and is mediated by the secretion and activity of enzymes that promote cell wall degradation and induction of soft rot decay. These enzymes are secreted to the extracellular space through the type II secretion system [6,10,13]. Accordingly, assessment of exoenzyme activities like pectate lyases (Pel),

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Fig. 3. Effect of exposure to phenolic compounds on biofilm formation ability of Pectobacterium strains (P. aroidearum PC1; P. carotovorum PC11 and WPP14; P. brasiliensis Pcb3 and Pcb1692; and P. atrosepticum EcaSCRI1043) and E. coli K12. Biofilm formation was measured after 72 h of growth in LB medium (with and without phenolic compounds) at 28
C. Each bar represents the absorbance of crystal violet dye (bound to the biofilm cells) measured at 570 nm. Bars with * sign are significantly lower than their respective control (P < 0.05; bar ¼ SE; n ¼ 5).

polygalacturonases (Peh) and proteases (Prt) in response to phenolic compounds is expected at least in part, to illuminate the mechanism by which these compounds may affect the virulence of pectobacteria toward their hosts. Strains PC1, WPP14 and Pcb1692, each representing three different Pectobacterium species (P. aroidearum, P. carotovorum and P. brasiliensis, respectively), were exposed to non-lethal concentrations of the selected phenolic compounds and assessed in semi-quantitative assays for their pectate lyase (Pel), polygalacturonase (Peh) and protease (Prt) activities. The experiments revealed that all compounds reduced the exoenzyme activities of the strains by 50e100% (Fig. 4). Specifically, Pel was most strongly inhibited, with no detected activity for strains WPP14 and Pcb1692 following exposure to all compounds (Fig. 4A). Pel activity was also strongly inhibited by the phenolic compounds in strain PC1, with the exception of low levels of Pel activity detected after exposure to syringic acid and tyrosol (Fig. 4A). Peh and Prt activities were also reduced by 50e80% in all strains following exposure to the compounds; however, in contrast to Pel, activities of these enzymes could still be detected for all strain/compound combinations under the tested conditions (Fig. 4B and C). To test whether the Pel inhibition also occurs at higher bacterial growth rates, a quantitative assay for Pel-specific activity was performed at lower concentration of phenolics (corresponding to 25% growth inhibition). In these quantitative experiments, despite the lower concentrations, Pel-specific activity was significantly reduced for strains WPP14 and Pcb1692, with up to 90% inhibition following exposure to some

compounds. Weaker inhibition of Pel specific activity was observed for strain PC1, yet significant inhibition was still detected following exposure to salicylic, cinnamic and coumaric acids as well as vanillin (Fig. S2). Similar evidence was reported by Zaidi-Yahiaouli et al. [48], who claimed that gallic acid and tannic acid inactivate or precipitate protease and pectate lyase of Pectobacterium chrysanthemi by binding to their active sites. Also, in this regard, some studies have suggested that plant phenolic compounds affect bacterial metabolism, including synthesis of host cell-wall-degrading enzymes, by inducing oxygen-dependent inhibitors of polygalacturonase [33,29]. Moreover, cinnamic and coumaric acids were already reported to regulate the virulence of Dickeya dadanti, a well-studied soft rot bacterium, through regulatory RNA for exoenzyme secretion which eventually affected virulence [46]. Based on these studies, phenolic compounds appear to inhibit synthesis of exoenzymes which might have resulted in lower levels of Pel, Peh and Prt in the tested Pectobacterium strains upon exposure to the phenolic compounds. 3.5. Virulence response to different phenolic compounds Disease development following host tissue infection is the most prominent expression of virulence. This ability of strains PC1, WPP14 and Pcb1692 in the presence of selected phenolic compounds was tested in three unrelated hosts (calla lily, cabbage and potato), in two tissues, the leaves and the tuber. The strains were exposed to non-lethal concentrations of the compounds and used to inoculate leaf discs of calla lily and

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Fig. 4. Effect of phenolic compounds on the activities of exoenzymes of Pectobacterium strains: P. aroidearum PC1, P. carotovorum WPP14 and P. brasiliensis Pcb1692. Enzymatic activity was estimated after overnight exposure of bacterial suspensions to non-lethal concentrations (50% inhibition) of the tested compounds. Pectate lyase (Pel, A), polygalacturonase (Peh, B), and proteolytic (Prt, C) activities were determined based on the size of substrate degradation haloes. Results are expressed as the percentage of activity relative to controls (bacteria that were not exposed to the compounds). Bars not connected by same letter are significantly different from each other (P < 0.05; bar ¼ SE; n ¼ 4).

cabbage, as well as potato tubers, as described in the Methods section. Before plant tissue inoculation, bacterial cells of all treatments were enumerated and found to be similar. The results demonstrated that exposure of Pectobacteria to non-lethal concentrations of phenolic compounds severely affected the ability of the strains to induce disease symptoms in most treatments (Fig. 5). Significant reductions in the decay of calla lily leaf tissue were observed for all strain/compound combinations with the exception of strain Pcb1692 exposed to vanillin (Fig. 5A). Of the three tested strains, WPP14 was most affected by the phenolic compounds in the calla lily infection assays. In addition, salicylic and syringic acid as well as catechol showed a particularly strong inhibitory effect on virulence in all tested strains in the calla lily (Fig. 5A). Similarly, significant reduction in the decay of cabbage leaves was observed after inoculation with all strains following exposure to the compounds, with the exception of WPP14/vanillin and PC1/tyrosol combinations (Fig. 5B). Salicylic acid consistently inhibited symptominducing ability of the three strains in these assays. In potato tuber assays, again, salicylic acid potently inhibited disease severity for all three strains, while syringic acid, catechol and cinnamic acid showed strong inhibition of symptom-inducing ability in some of the strains (Fig. 5C). In this regard, salicylic acid was already demonstrated to be a compound that may affect growth as well as virulence of P. aeruginosa [1]. Here, being a well-recognized defense elicitor in plants, it is

important to note, that salicylic acid does not play a critical role in the plant's defense against necrotrophic pathogens, unlike jasmonic acid and ethylene [16,31]. More specifically, previous studies have shown that at least in the monocot host Z. aethiopica, defense against P. carotovorum, involved the jasmonic acid signaling pathway rather than the salicylic acid pathway [30,16]. Moreover, the concentration of salicylic acid that was used in the plant inoculation assays was negligible in terms of defense induction, and the time frame was too short to allow defense development. Thus, it is possible to claim that the negative effects of salicylic acid and the other phenolic compounds on disease development were the result of a direct negative effect on pathogen growth and/or virulence, rather than indirect induction of the plant's defense response. Our findings demonstrated a direct effect of phenolic compounds on bacterial growth. Thus, we considered the option that it was the effect on growth, and not necessarily the effect on virulence-associated traits, that was responsible for the impaired ability of the bacterium to cause disease. The use of non-lethal concentrations (50% growth inhibition) throughout the study was one way of dealing with the problem. It enabled studying the effect of the compounds on several virulence determinants in a comparative way. For instance, some specific compounds such as salicylic and syringic acids inhibited bacterial growth to a level of 50%, but repressed disease-inducing ability of the bacteria by nearly 100%.

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Fig. 5. Effect of selected phenolic compounds on the virulence of three Pectobacterium strains (P. aroidearum PC1, P. carotovorum WPP14 and P. brasiliensis Pcb1692) on three hosts: A, calla lily; B, cabbage; and C, potato. Strains were exposed to non-lethal (50% inhibition) concentrations of the selected compounds and bacterial concentrations were normalized to 1  108 cfu mL1 upon inoculation. Virulence was determined as the percentage of decayed tissue 15 h after inoculation of lily and cabbage leaves, and 48 h after inoculation of potato tubers relative to the decay induced by untreated bacterial cultures (controls). Data represent means ± SE of two independent experiments with similar results, with 10 replicates (discs) for calla lily and cabbage, and 4 replicates (tubers) for potato, in each experiment. Treatments that are not labeled with the same letter in each panel are significantly different (P < 0.05). Representative pictures are shown for infected discs of calla lily (bottom left) and cabbage (bottom right).

Nonetheless, to dissect the direct contribution of growth inhibition to the reduced virulence, we conducted two additional experiments under similar conditions: an exoenzyme activity assay and a virulence assay, with non-lethal concentrations of a known growth inhibiting antibiotic e ciprofloxacin. Two strains of Pectobacterium, P. aroidearum PC1 and P. brasiliensis Pcb1692, were exposed to equivalent concentrations of ciprofloxacin (corresponding to 50% growth inhibition): 6 ng mL1 [23]. Ciprofloxacin is an antibiotic that belongs to the group of DNA gyrase inhibitors, mostly recognized for inhibition of cell division and growth. DNA gyrase is a vital enzyme for almost all cellular processes involving double-stranded DNA, namely, replication, recombination and transcription. Therefore, if the observed inhibiting effect of phenolic compounds on Pectobacterium virulence traits is a direct result of growth inhibition, exposure to a corresponding non-lethal concentration of ciprofloxacin is expected to inhibit Pectobacterium exoenzyme activity and disease-inducing ability in a similar manner as observed for the phenolic compounds.

Experiments with ciprofloxacin clearly revealed that despite ciprofloxacin's effect on cell growth, pre-exposure of bacteria to a non-lethal concentration of this antibiotic did not affect either exoenzyme activity (Fig. 6B) or development of disease symptoms in calla lily leaf discs following infection (Fig. 6C). This lack of an effect observed upon ciprofloxacin treatment of two strains of Pectobacterium, PC1 and Pcb1692 was in contrast to the effects exerted by almost all phenolic compounds on these traits at comparable non-lethal concentrations and under the same experimental conditions (Figs. 4 and 5). This observation supports the notion that the negative effects of phenolic compounds on the disease-initiation ability of Pectobacterium are largely due to a direct effect of the compounds on specific virulence determinants, including exoenzyme activity, biofilm formation and motility. In conclusion, based on accumulated knowledge from the literature, we determined the effects of common phenolic acids and other phenolics on the growth and virulenceassociated traits (motility, biofilm, exoenzyme activity) of several Pectobacterium strains representing different species

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Fig. 6. Effect of ciprofloxacin at non-lethal concentration on growth and virulence of Pectobacterium strains PC1 and Pcb1692. (A) Effect of ciprofloxacin on bacterial growth. Bacteria were exposed to non-lethal concentrations of ciprofloxacin (6 ng mL1) before incubating them at 28
C with continuous shaking. Growth was assessed by measuring absorbance at 600 nm and expressed in terms of percentage relative to untreated bacteria. Results are means ± SE of 5 replicates per treatment. (B) Effect of ciprofloxacin on the activity of the cell-wall-degrading enzymes pectate lyase (Pel), polygalacturonase (Peh) and proteases (Prt). Supernatant from overnight grown cultures (control and ciprofloxacin) were used to evaluate their activity. Results are expressed as the percentage of activity relative to controls. Bars not connected by the same letter are significantly different from each other (P < 0.05; bar ¼ SE; n ¼ 4). (C) Effect of ciprofloxacin on the virulence of Pectobacterium strains on the calla lily. Virulence was determined as the percentage of decayed tissue 15 h after inoculation of calla lily leaf discs relative to the decay induced by untreated bacteria (control) (*P < 0.05; bar ¼ SE; n ¼ 10). Representative pictures of infected calla lily discs are shown.

of this genus. Specifically, phenolic compounds affected the virulence determinants of Pectobacterium, which can be seen by reduced exoenzyme secretion and ultimately less or no infection in the hosts tested. Moreover, the data presented in this study support a role for plant phenolic compounds in virulence inhibition in the genus Pectobacterium and probably in other soft rot bacteria as well. Further work is required to understand the mechanism of action of plant-derived phenolic compounds, which holds great potential for the development of stable and ecologically sound measures to minimize the damage that soft rot bacteria cause to agricultural crops.

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