Microbial Pathogenesis 78 (2015) 95e102
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Antibacterial activity of carob (Ceratonia siliqua L.) extracts against phytopathogenic bacteria Pectobacterium atrosepticum Saïda Meziani a, e, B.Dave Oomah b, Farid Zaidi c, Annabel Simon-Levert d, dric Bertrand e, Rachida Zaidi-Yahiaoui a, * Ce a
Laboratory of Applied Microbiology, Faculty of Nature and Life Sciences, A. Mira University, Bejaia 06000, Algeria Formerly with the National Bioproducts and Bioprocesses Program, Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, Summerland, BC V0H 1Z0, Canada c Department of Food Science, Faculty of Nature and Life Sciences, A. Mira University, Bejaia 06000, Algeria d AkiNaO SAS, Perpignan, France e Laboratory of Chemistry of Biomolecules and Environment (LCBE), University of Perpignan Via Domitia, Perpignan, France b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 September 2014 Received in revised form 1 December 2014 Accepted 5 December 2014 Available online 6 December 2014
Acetone and ethanol extracts of carob (Ceratonia siliqua L.) leaf and pods were evaluated for their in vitro inhibitory ability against the pectinolytic Gram negative Pectobacterium atrosepticum (Pca, CFBP-5384) bacteria, the causal agent of potato soft rot. Potato (Solanum tuberosum, var nicola) tuber rot tissues obtained after 5 day bacterial inoculation was analyzed by LCeMS and GCeMS to study Pca pathogenicity. Trans/cis N-feruloylputrescine was identified in potato tuber after 5-day inoculation with Pca in a dark moist chamber. Although glycoalkoloid (a-chaconine and a-solanine) production increased due to Pca soft rot infection, it was not a resistance-determining factor. Many secondary metabolites were identified including the phytoalexins solavetivone and fatty acids responsible for plant defence responses. Acetone extract of carob leaf (FCA) exhibited the strongest inhibitory effect (IC50 ¼ 1.5 mg/ml) and displayed synergistic antimicrobial effect in the presence of infected potato tuber extract (Pdt-Pca extract) against Pca. This synergy could be used in an integrated control program against potato soft rot pathogens, thereby reducing chemical treatments. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Antimicrobial activity Phytoalexins Pectobacterium atrosepticum Soft rot Carob Leaf extract
1. Introduction Pectobacterium atrosepticum, formely named Erwinia carotovora subs. atroseptica, almost exclusively infects potato, causing blackleg of the stem and tuber soft rot [1,2]. Consequently, it causes important losses within cool temperate regions, where potatoes have traditionally been grown [3]. There are currently no efficient curative methods to protect potato against Pectobacterium spp [4]. Although copper compounds reduce the spread of these pathogens [5], they may cause environmental damages and present a risk to human health [6]. Selection of blackleg-resistant potato cultivars was only partially successful and never resulted in completely resistant cultivars [7]. There is growing interest in developing new antibacterial agents that can control phytopathogenic bacteria without environmental
* Corresponding author. E-mail address: [email protected] (R. Zaidi-Yahiaoui). http://dx.doi.org/10.1016/j.micpath.2014.12.001 0882-4010/© 2014 Elsevier Ltd. All rights reserved.
effects. Different studies provide evidence that some medicinal plants produce many biologically active compounds representing a source of great importance in research of new antimicrobial agents [8]. These compounds, derived from several secondary metabolic pathways, include alkaloids, flavonoids, lignins, phenolic compounds and terpenoids [9,10]. Recently, the antimicrobial proprieties of various plant extracts against certain pathogen have been reported [11]. Phenolic compounds are often considered to play an important role in resistance to many plant pathogens including resistance of potato to erwinias [12]. Phenolics can directly inhibit bacterial growth, by inhibiting wall-degrading enzymes, or as precursors in the formation of physical barriers such as lignin. Carob (Ceratonia siliqua L.) has received considerable attention because its extracts exhibit very strong antimicrobial activity against various species of bacteria and fungi [13e16]. The present study aimed to: (1) examine the potential role of ethanol and acetone extracts of carob (C. siliqua) leaf and pods to control in vitro potato soft rot caused by P. atrosepticum; (2) determine their
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antibacterial property, and the interaction of carob leaves extracts in combination with potato tuber extract (Solanum tuberosum, var, nicola) against phytopathegenic bacteria (Pca) and (3) to identify and characterize the active compounds present in infected potato tuber extract by LCeMS and GCeMS analysis. 2. Materials and methods 2.1. Plant material C. siliqua leaves and pods were collected in Aftis, Wilaya of Bejaia Algeria. The pods were randomly collected from selected plants and then pooled. The leaves and pods (without seeds) were separated, after being washed with tap water to remove all impurities and then with distilled water. The samples were dried at room temperature and ground to a fine powder in an electric blender (Super Blender National®, Japan). The samples were stored under vacuum in a desiccator prior to analysis. 2.2. Extraction procedure Dried samples (2 g) were sequentially extracted by maceration in 50 ml of 80% (v/v) acetone and 80% (v/v) ethanol according to the method described by Ranalli et al. [17]. The extraction was carried out at room temperature for 1 h under magnetic stirring. The extracts were filtered through filter paper (10 mm) and the extraction process repeated three times by adding another 50 ml of solvents to the sample residue. The combined filtrate from each extraction was concentrated under vacuum on a rotary evaporator (Büchi, R-114) at 40 C, and completely dried in a freeze drier. Finally, the obtained extracts were kept at 4 C in the dark until analysis. 2.3. Determination of total phenolic content Total phenolic content was determined according to the FolinCiocalteu colorimetric assay following the method described by Singleton et al. [18]. An aliquot of diluted sample extract (0.5 ml) was added to 0.9 ml of diluted Folin-Ciocalteu reagent and 3.6 ml sodium carbonate solution (75 g/l). The test tubes were allowed to stand in the dark at room temperature for 30 min. Absorbance at 765 nm was read versus the prepared blank with spectrophotometer (Shi-madzu Uvi-mini1240, Suzhou Jiangsu, China). Total phenolic content of samples were expressed as milligrams gallic acid equivalents per mg of dry weight (mg GAE mg1 DW) through the calibration curve with gallic acid. All samples were analyzed in three replications. 2.4. Screening for antibacterial activity 2.4.1. Bacterial strain Antimicrobial activity was tested in vitro against P. atrosepticum (strain CFBP-5384) provided by the Stock Culture Collections of Phytopathogenic Bacteria (CFBP, INRA Angers, France). Bacteria were maintained for extended periods as deep-frozen cultures (20 C) and cultivated on King B Agar Medium at 25 C for 48 h. Strain was grown in peptone water liquid nutritive medium (WPN) at 25 C overnight before use for antibacterial activity test. 2.4.2. Antibacterial activity The microplate bioassay (micro-dilution) was used to study the antimicrobial activities of P. atrosepticum. Plant extracts dissolved in aqueous dimethylsulfoxide (DMSO) at 50, 100, 250, 500 and 1000 mg ml1 concentration were pippeted in sterile 96-well plates (250 mL volume, Fisher Scientific) based on a National Committee for Clinical Laboratory Standard method (NCCLS) [19].
The bacterial suspension was adjusted with sterile distilled water to 2.105 cfu/ml concentration. Briefly, 96-well plates were prepared by dispensing into each well 198 ml of microorganism suspension. Then, 2 mL from their serial dilutions of extracts was transferred into five consecutive wells. The last well containing 198 mL of WPN medium and 2 mL of the serial dilutions of extracts were used as negative control. The final volume in each well was 200 mL. The plate was covered with a sterile plate sealer and then incubated at 25 C for 48 h. After agitation, microorganism growth was estimated by reading absorbance in microplate wells at 600 nm with a Microplate Reader (Aviso, Sirius HT, Ebersberg, Germany). The MIC50 was defined as the lowest concentration of plant extracts able to inhibit 50% of the microorganism growth. 2.5. Pathogenicity tests on potato tubers Potato tubers (S. tuberosum, var. nicola) were selected free of wounds, rots and homogeneous in size. They were stored at 4 C until use. Tubers were washed, surface rinsed in sterile distilled water and air-dried under a laminar flow hood. Before inoculation, a bacterial suspension was prepared at 2.108 cfu/ml concentrations in liquid nutritive media (WPN) from cultures on King's B medium and incubated at 25 C for 24 h. The half-tuber method of Ibrahim et al. [20] was followed for the study of the pathogenic variability of P. atrosepticum. Each tuber was cut from the rose end to the heel end in two roughly equal parts with a sterile knife. A hole (5 mm diameter 5 mm depth) was drilled with a cork borer in the center of each half-tuber. Ten half-tubers were inoculated by depositing 50 ml of the bacterial suspensions prepared previously. Control tubers received 50 ml of sterile distilled water (SDW). After 5 days incubation at 25 C in a dark and water-saturated environment, symptoms were assessed visually; the rotted tissue was collected with a spatula, weighed and used for extraction. 2.6. Extraction from potato tubers The collected tissues were extracted according to the method described by Andreu et al. [21]. These tissues were blended in a mixture of dichloromethane and methanol (50:50, v/v) using an Ultra-Turax® homogenizer. The homogenate was allowed to stand overnight, filtered and evaporated to dryness. Solid-phase extraction, the method of choice for fast and effective purification, was performed using C18 extraction SPE cartridges (Strata C18-E, phenomenex®, 55 mm, 70A; 2g/12 ml Giga tubes, 8B-S001-KDG). The C18 cartridges were conditioned and activated. Briefly, the cartridges were preconditioned using 10 ml of methanol (MeOH) and equilibrated with 10 ml of distilled water. The MeOH/water (5:95, v/v) reconstituted sample extract was loaded onto the cartridges and allowed to flow by gravity. The columns were washed with 5 ml of distilled water. The analyte of interest was then eluted with 15 ml of MeOH into a tube, and the eluates were dried under nitrogen at 40 C. The residue was immediately dissolved in 1 ml of HPLC methanol solution, vortexed and sonicated for 2 min. 2.7. HPLC-MS analysis Phenolic acids were analyzed by high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS). The extracts were dissolved in HPLC grade methanol at 5 mg/mL, filtered (0.22 mm) and two microliters of the sample solutions were injected. LCeMS analysis was conducted using an Accela 600 system (Thermo® Scientific, France) connected with a quaternary pump, refrigerated autosampler, a diode array detector (Accela PDA
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detector) and a 3DLCQ-Fleet ion trap mass spectrometer (Thermo® Scientific, Inc, Bremen, Germany). A reversed phase column (Kinetex C-18 reverse phase column, 100 3.00 mm Phenomenex®) was used at a flow rate of 500 mL/min. Mobile phase was water containing 1‰ formic acid (solvent A) and acetonitrile containing 1‰ formic acid (solvent B). The elution gradient was from 95% (A)5% (B) 0e5 min, 40% (B) 5e30 min, 100% (B) 30e35 min and return to 95% (A) 35e45 min. UV analysis was performed at 254, 280 and 340 nm and UV spectra were recorded from 200 to 600 nm. The MS experiments were carried out using an electrospray interface (ESI) operating in positive [MþH]þ and negative [MH] ionization mode. The molecular ions were scanned from 100.0 to 2000.0 (m/z) and multiple MSn spectra were used to provide comprehensive structural information of the compounds. The MS data were collected and processed on an Xcalibur version 2.0 data system.
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antibacterial activity was measured using a well diffusion method according to the National Committee for Clinical Laboratory Standard (NCCLS) [19]. The bacterial suspension was adjusted with sterile distilled water to a concentration of 2.105 cfu/ml. Stock solutions of extract from carob leaf; infected potato tubers and the control were prepared for evaluation of their combined effect on P. atrosepticum. Plant extracts were dissolved in aqueous dimethylsulfoxide (DMSO) at concentration corresponding to MIC50/8, MIC50/4, MIC50/2, MIC50 and MIC50X2. The antibacterial activity was determined by the micro-dilution method described above. The first antagonist, additive or synergistic effect of the extracts in combination was determined with calculation of fractional inhibitory concentration indices (FICI). The experiment was done in triplicate to ensure reproducibility of results. The fractional effects (FIC) of two compounds (or extracts) X and Y for antibacterial studies, in terms of their MIC for bacterial growth, can be expressed as follows [23]. The FIC index was calculated as follows:
2.8. Gas chromatography/mass spectrometry (GC/MS) analysis 2.8.1. Derivatization for GC/MS analysis The derivatization is crucial in GCeMS analysis to increase metabolite volatilities. Silylation is the most prevalent derivatization method as it readily volatizes the sample and therefore very suitable for non-volatile samples for GC analysis. The mechanism involves the replacement of the active hydrogen with a trimethylsilyl group. An aliquot of the extracts was evaporated to dryness using a centrifugal evaporator. For this purpose, 10 mg of the extract dried with N2 gas, then 100 ml of derivatization agent N,O-Bis(thylsilyl) trifluoroacetamide (BSTFA) with 1% of trimethyltrime chlorosilane (TMCS) BSTFA/TMCS and 100 ml of pyridine was added, mixed and heated 15 min at 105 C [22]. The resulting solution containing the TMS derivatives was filtered and analyzed by GCeMS. 2.8.2. GCeMS analysis Extracts were analyzed by GCeMS, each sample was injected twice and the results were averaged. GCeMS analysis was carried out using a Trace Gas Chromatograph Focus GC (Thermo® Scientifique) 10904116 series equipped with a Trace MS mass spectrometer (DSQ II), and with a ZB-5MS (phenomenex®) column (30 m 0.25 mm i.d., 0.25 mm film thickness), using helium as carrier gas (35 cm/s). The chromatographic conditions were as follows: initial temperature: 150 C for 2 min; final temperature 325 C for 20 min; injector temperature 320 C; transfer-line temperature was held at 320 C; split ratio 10. Briefly, 2 ml of the derivative solution was injected. The injection conditions were: split mode with 1 min duration pulse, and 1 ml/ min He column flow. MS data were collected and processed on an Xcalibur™ version 1.4.1. For Mass spectrometry conditions, the EI ionization was 70 eV, the transfer line was at 320 C, mass range acquisition was from m/z 50 to m/z 650 and carried in Full Scan mode. Qualitative analysis of compounds was based on the comparison of their spectral mass and their relative Retention time with those of NIST mass spectra database and Kovats RI on the chromatograms recorded in Full Scan or in SIM mode using the characteristic ions. 2.9. Determination of the combined activity For the combination assays, bacterial suspension of P. atrosepticum was tested against acetone extract (FCA) and ethanol extract (FCE) of carob leaf combined with purified extract of infected potato tubers (Potatoes-Pca-extract, Pdt-Pca) in 96-well microplates (250 mL volume, Fisher Scientific). Combined effect of
FICindex ¼ FICX þ FICY
X
FIC ¼
FICY ¼
MICðX with YÞ MICðX aloneÞ MICðY with XÞ MICðY aloneÞ
! the fractional effect of X ! the fractional effect of Y
A FIC index 0.5 to 4.0 as additive or indifferent, and >4.0 as antagonistic [23,24]. The corresponding FIC values were used to construct isobolograms of the combinations [25]. The graph is represented with the ratio to the FIC of the infected potato tuber extract (Pdt-Pca) on the x-axis and the ratio of the FIC of the leaf of carob (FCA, FCE) on the yaxis. A line was drawn in the graph connecting the highest concentrations of each of the extract used to produce a reference line. Deviation from linearity relative to the reference line was used as the basis for interpretation of antimicrobic relationship between combined extracts against Pca. A curved deviation of the isobologram to the left of the reference line indicates synergy of the antimicrobial agents, while a right deviation would indicate antagonism. An additive relationship between antimicrobials was represented by an isobologram with points falling proximate to the reference line.
2.10. Data analysis Data were subjected to analysis of variance (ANOVA) by the general linear model procedure by means of comparison with Duncan's test and Pearson correlation according to R methods. Differences between means (P < 0.001) level were considered to be statistically significant.
Table 1 Total phenolic content of carob leaves and pods. Samples
Extracts
Total phenolic content (mg GAE/g)
Carob leaves
Acetone Ethanol Acetone Ethanol
21.41a 22.98a 2.03b 1.05b
Carob pods
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3. Results and discussion 3.1. Total phenolic content Acetone and ethanol extracts of C. siliqua L. leaves and pods grown in Algeria contained total phenolics ranging from 0.61 to 22.98 mg GAE/g. Carob leaf extracts had significantly higher (10) phenolic contents than those from pods, independent of solvent (Table 1). Phenolic content of ethyl acetate extract of carob leaves from Morocco was reported to be around 2.6 g/L GAE [26]. 3.2. Antibacterial activity The in vitro antibacterial activity of C. siliqua leaf and pod extracts against the Gram (e) bacteria P. atrosepticum (Pca, CFBP3584) was determined in this study. The leaf extracts showed strong inhibitory activity against Pca with MIC50 values between 1.5 and 2.4 mg/ml depending on concentration and extracting solvent. No antibacterial activity was observed from acetone and ethanol extracts of carob pods against Pca. The acetone leaf extract was more effective than ethanolin inhibiting Pca growth. Antibacterial activities were influenced by changes in leaf extracts induced by solvent choice [27]. Solvent also affected the phenolic distribution and concentration of olive leaf extracts resulting in varying antimicrobial efficiency against S. aureus, E. coli, S. enteritidis, and S. thypimurium [28]. The observed activity could be explained by the presence of biologically active compounds in these extracts. The extracts of leaves of C. siliqua have been known to contain a number of antimicrobial compounds. Phytochemical screening of this plant has shown the presence of gallic acid, quercetin -glucoside, Kaempferol-rhamnoside, Quercetin-rhamnoside, 1,2,6 tri-O-galloyl- glucopyranose, (e)-Epigallocatechin-3-O-gallate, kaempferol and quercetin [29]. Several studies attribute the inhibitory effect of plant extracts against bacterial pathogens to their phenol content [27,30e32]. The phenols and flavonoids contribute significantly to the antibacterial activity and can form complexes with cell wall and also disrupt bacterial envelopes [33]. In this study, the antibacterial effect may be attributed to the presence of different bioactive compounds such as tannins, phenolic acids, flavonoids and flavonoidal glycosids in carob leaf that impact growth and metabolism of microorganisms. It has been reported that the phenolics, tannins and propyle gallate were strong microbial inhibitors. C. siliqua leaves from Turkey had a significant effect on a broad range of microbial pathogens [14]. The toxicity of tannins to various bacteria has been demonstrated in several studies with hydrolysable tannins exerting potent antibacterial effects against B. subtilis and S. aureus [34,35]. This antibacterial effect may be largely due to the o-diphenol groups of tannins enabling them to act as iron chelators, thus depriving microorganisms of this essential element [36]. 3.3. Identification of chromatographic peaks 3.3.1. LC/MS analysis In the LC/MS analyses of constituents in the extracts of potato tuber, the peak area ratio of each compound was recorded by the UV detector. The chemical structures of these constituents were identified on the basis of their retention times and mass fragmentation patterns. Analysis of contents was performed after integration of single ion chromatograms of protonated molecular ions [MþH]þ. Pca infection induced differential metabolic changes demonstrated by increased peak intensities in potato tuber extract (Pdt-Pca extract) (Fig. 1).
Fig. 1. Total scan PDA and ESI Full MS chromatogram of potato tuber extracts: (a) infected potato tubers by Pca (Pdt-Pca) and (b) control, not infected potato tubers (PdtT) obtained by LC-ESI-MS.
Peak 2 (tR, 5.37 min; lmax, 316 nm) at [MþH]þ m/z 265 and fragments ions of m/z 248 [M þ HeNH3]þand 177 [M þ HeC4H12N2]þ (Fig. 1) was identified in potato tuber after five days of bacterial inoculation as trans/cis N-Feruloylputrescine as reported by Yan et al. [37]. Trans/cis N-Feruloylputrescine has been isolated from the zone of potato tuber (cv. Bintje) infected by Phoma exigua var. foveata, and may be produced in stress-exposed tuber, but not in healthy tuber [38]. Peaks1and 3 remains unidentified (Fig. 1). The mass spectrum of peak 1 (tR, 5.05 min; lmax, 279 nm) with a [MþH]þ ion at m/a 205 yielded MS ion at m/z 188. Peak 3(tR, 47.91 min; lmax, 249 nm) with a [MþH]þ ion at m/z 589 produced ions at m/z 571, 553 and 417. The comparison of LC-ESI-MS analysis of potato tubers extracts revealed the presence of glycoalkaloids, a class of natural toxicants, in infected tissues of potato tubers (Pdt-Pca extract) and present only in control potato tubers (Pdt-T extract). The peaks4 and 5 (tR, 13.02 min; lmax, 248 nm) and (tR, 13.26 min; lmax, 248 nm) (Fig. 1a), were two major glycoalkaloids, a-solanine and a-chaconine, respectively identified by comparison with pure commercial
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standards. Using the mass detector, it was possible to distinguish the two molecules based on their corresponding intense [MþH]þ ions at m/z 868 and 852 m/z for a-solanine and a-chaconine, respectively. Both are glycosylated (trisaccharide) derivatives of the aglycone solanidine, a steroid alkaloid, cleavage at the interglycosidic bonds is the dominant process in the fragmentation of a -chaconine (m/z 852) and a -solanine (m/z 868) by ESI-MS. From a-chaconine, fragments of m/z 706, 560 and 398 corresponding to [M þ H-Rham]þ, [M þ H-Rham-Rham]þ, and [M þ HRham-Rham-Glu]þ were produced in addition to the protonated molecular ion [MþH]þ at m/z 852. The fragments of m/z 722,706 and 398 corresponding to [M þ H-Rham]þ, [M þ H-Glu]þand [M þ H-Glu-Rham-Gal or M þ H-Rham-Glu-Gal]þ were produced from m/z 868 for a-solanine. The results of the present study showed significant differences in glycoalkaloid accumulation before and after inoculation, between the infected (Pdt-Pca) and the control (Pdt-T) uninfected potato tuber extracts. Fewell et Roddick [39] reported that the two most abundant potato glycoalkaloids, a-chaconine and a-solanine, synergistically inhibit in vitro growth and germination of several important pathogenic fungi, and according to Friedman [40] and Lachman et al. [41] potato glycoalkaloids serve as natural defenses and display antimicrobial activity. However, there are inconsistent reports about their antimicrobial effects against pathogenic fungi and bacteria [42]. Studies report variable result on possible relationships between glycoalkaloid levels of potato tuber and P. infestans infection. For example, Smith et al. [43] suggested that glycoalkaloid have a protective function, although glycoalkaloid accumulation was suppressed in tuber slices or potato leaves after fungal inoculations or treatments with P. infestans, P. citrophthora or H. carbonum [21]. Fewell and Roddick [39] showed that P. infestans affect glycoalkaloid production and induced production of phytoalexins. In addition, eicosapentaenoic and arachidonic acid extracts or elicitors of P. infestans caused inhibition in glycoalkaloid accumulation [44]. Infection of potato tubers by P. infestans induced increases in sesquiterpene cyclase and squalene synthase, enzymes of the isoprenoid pathways. These enzymes catalyze the synthesis of sesquiterpenoid phytoalexins and glycoalkaloids [45]. Pathogen infections are known to affect glycoalkaloid content.
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In this study, glycoalkaloid production increased due to bacterial soft rot infection caused by P. atrosepticum (CFBP-5384), in potato tubers, these results are in accordance with previous report [46]. Glycoalkaloid content in potato tuber extracts was not correlated with their resistance to soft rot caused by P. atrosepticum, suggesting that glycoalkaloids were not the factor determining resistance. 3.3.2. GCeMS analysis The components present in the extracts of Pca infected potato tubers (Pdt-Pca) and healthy uninfected control potato tubers (Pdt-T) were identified by GCeMS analysis. The sesquiterpenoid metabolite solavetivone (C15H22O, MW 218) accumulated in smaller amounts in potato tuber extracts inoculated with the pathogen (Pdt-Pca) (Fig. 2). The research on phytoalexins in tubers has focused on the major sesquiterpenoid metabolites rishitin, lubimin and solavetivone found in tubers [47]. Srikrishna and Ramasastry [48] reported that phytoalexin solavetivone was the major stress metabolite from potato tubers infected with the blight fungus P. infestans or with the soft-rot bacterium E. carotovora var. atroseptica. In addition to the phytoalexins, the GCeMS profile showed the presence of methyl esters of various types of fatty acid compounds in the rotted tissue (Pdt-Pca extracts). High concentrations were detected in rotted tissue, whereas in the wounded control tissue lower concentrations were present. Table 2 list the compounds including the relative content, retention time, mean relative molecular weight (MW), and formula indentified by GCeMS. The main components were: 9-12-otadecadienoic acid (Z,Z)-methyl ester (18:2, syn. linoleic acid methyl ester), 9-12-octadecadienoic acid (Z,Z)- trimethylsilyl ester (18:2, syn. linoleic acid), hexadecanoic acid trimethylsilyl ester (16:0, syn. Palmitic acid trimethylsilyl ester), octadecanoic acid (Z,Z)- trimethylsilyl ester (18:0, syn. stearic acid trimethylsilyl ester), n-pentadecanoic acid trimethylsilyl ester (15:0), palmitelaidic acid trimethylsilyl ester, linolenic acid trimethylsilyl (18:3), hexadecanoic acid,2,3-bis [(trimethylsilyl)oxy]propyl ester (syn. 1-monopalmitin trimethylsilyl ether), Octadecanoic acid,2,3-bis-[(trimethylsilyl)oxy]propyl ester (syn. Stearic acid,2,3 bis-(trimethylsiloxy)propyl ester).
Fig. 2. GCeMS analysis of potato tuber extracts (a) infected potato tubers by Pca (Pdt-Pca) and (b) control: not infected potato tubers (Pdt-T).
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Table 2 GCeMS profile of extracts of infected potato tuber (Pdt-Pca). Peak RT MW Prob. Formula (min)
Peak name
1 2 3 4
9.55 11.14 11.47 11.97
5
13.37 352 79.87 C27H40O4Si
6 7 8
13.46 350 41.47 C21H38O2Si 13.66 356 94.43 C21H44O2Si 15.32 352 5.72 C21H40O2Si
9
17.04 474 94.70 C25H54O4Si2
10
18.80 502 77.32 C27H58O4Si2
n-pentadecanoic acid trimethylsilyl ester palmitelaidic trimethylsilyl ester hexadecanoic acid trimethylsilyl ester 9,12-Octadecadienoic acid, methyl ester (Z,Z)9,12-Octadecadienoic acid, trimethylsilyl ester linolenic acid trimethylsilyl ester octadecadienoic trimethylsilyl ester 9,12-Octadecadienoic acid, trimethylsilyl ester octadecanoic acid, 2,3 bis[(trimethylsilyl) oxy]propyl ester octadecanoic acid, 2,3 bis[(trimethylsilyl) oxy]propyl ester
314 326 328 294
35.93 89.95 95.96 21.52
C18H38O2Si C19H38O2Si C19H40O2Si C19H34O2
Vernenghi et al. [49] report that linoleic and linolenic acids derivatives accumulate in tomato (Lycopersicum esculentum Mill.) resistant to Phytophtora sp. and to Verticillium alboatrum; these acids are involved in the defense mechanisms against fungal pathogen by producing hydroxylated and peroxidized fatty acids [50]. The a-linolenic acid is the first potential precursor for the production of jasmonic acid and its derivatives, which are key signaling molecules known to activate different plant defense responses. Jasmonic acid induces phenylalanine ammonia-lyase (PAL) and several pathogenesis related proteins [51]. Fatty acids were phytotoxic to potato leaves, and elicited systemic induced resistance efficiently against P. infestans [52]. Linoleic acid found to elicit induced systemic resistance (ISR) of tobacco against the bacterial soft rot pathogen, Pectobacterium carotovorum subsp. carotovorum (Pcc) [53]. Furthermore, linolenic and linoleic acids were phytotoxic and effective in inducing systemic resistance, whereas oleic acid was the least phytotoxic and induced no systemic resistance [52]. According to Blechert et al. [54], the octadecanoic acid derivative, octadecatrienoic acid, (Z,Z)-, methyl ester plays an important role in plant defense mechanism. The hexadecanoic acid ethyl ester acts as antioxidant, nematicide and pesticide and may contribute to the antimicrobial and antioxidant activities [55]. Farmer and Ryan [56,57] proposed a model according to which primary wound signals like oligogalacturonides and system in trigger the activation of the octadecanoid pathway resulting in a burst of jasmonic acid production which ultimately leads to the activation of defense genes. The model proposed that wounding by insects or microbial
pathogen attack led to an interaction of elicitors with receptors, thus initiating the octadecanoic-based pathway from the C18 linolenic acid fatty acid to jasmonic acid.
3.4. Evaluation of the combined activity Representative isobolograms of combined carob leaf extracts and infected potato tuber extract against P. atrosepticum are shown graphically in Fig. 3. A synergistic effect was observed for the combination of carob leaf acetone extract (FCA) with infected potato tuber extract (PdtPca) with FIC indices ( 0.5e1.0) as indicated by isobologram with MIC points falling very proximate to a reference additive line (Fig. 3b). According to the effect obtained for each extract, the combination was qualified as acting additively and synergistically. Extracts of carob leaf and potato tuber present a combined antimicrobial effect potentialized against P. atrosepticum. This antimicrobial effect is even more evident in the presence of PdtPca extracts and acetone extract of carob leaf (FCA). Liu [58] opined that the additive and synergistic effects of phytochemicals in vegetables are attributed to the complexes mixture of phytochemicals present. The strong antibacterial activity of acetone extract of leaf of C. siliqua and synergistic effect with combination with infected potato tuber extract may be attributed to its content of flavonoids and tannins. Naringin, apigenin and rutin are carob flavonoids had reported antibacterial activity [59]. Potato tuber infected by P. atrosepticum accumulates phenolics and metabolites. Phenolics play an important role in many plant-pathogen interactions, both as constitutive defense metabolites and as induced phytoalexins [60]. Andreu et al. [21] found that beside phytoalexins and glycoalkaloids, phenolics are also involved in potato resistance to late blight. Since the amount of phenolics induced after infection was lower in a susceptible potato cultivar compared to a resistant cultivar, they suggested a role for phenolics in resistance of potato to P. infestans. Finally, the bioactive phytocompounds in carob leaf inhibit the growth of Pca and their joint activity with potato tubers extracts lead to an enhanced antibacterial effect. The mechanism governing the joint action of plant extract compounds is still unknown. Some authors suggest that phytocompounds disturb cell wall or increase permeability of the cytoplasmic membrane [61e63].
Fig. 3. Isobolograms of antimicrobial combinations of C. Siliqua leaf extract and infected potato tuber extract (Pdt-Pca) against P. atrosepticum: (a) acetone (FCA) and (b) ethanol (FCE) leaf extract of C. Siliqua (FCA) and Pdt-Pca extract.
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4. Conclusion This study describes the antimicrobial activity from leaves and pods extract of C. siliqua (Carob) against P. atrosepticum. The leaf extracts exhibited potent antimicrobial activity against P. atrosepticum that may be related to the presence of phenolic compounds, flavonoids and tannins in considerable quantities in carob leaf. The results obtained from the present study suggested that C. siliqua leaf extracts possess significant antibacterial property. This work confirms the antibacterial activity of C. siliqua extract and shows their potential use as microbial agents. The results revealed the importance of combining plant extracts to control bacteria. The study suggests the possibility of concurrent use of plant extracts in combinations in treating soft rot caused by P. atrosepticum. Acknowledgments We are grateful to the Algerian Ministry of Higher Education and Scientific Research (MESRS) for providing financial assistance and scholarship (BAF) to Ms. Saida Maziani. References rombelon, Potato diseases caused by soft rot erwinias: an overview [1] M.C.M. Pe of pathogenesis, Plant Pathol. 51 (2002) 1e12. rombelon, J.A. van Veen, J.M. van der Wolf, Control of [2] R. Czajkowski, M.C.M. Pe blackleg and tuber soft rot of potato caused by Pectobacterium and Dickeya species: a review, Plant Pathol. 60 (2011) 999e1013. [3] B. Smadja, X. Latour, S. Trigui, J.-F. Burini, S. Chevalier, N. Orange, Thermodependence of growth and enzymatic activities implicated in pathogenicity of two Erwinia carotovora subspecies (Pectobacterium spp.), Can. J. Microbiol. 50 (2004) 19e27. [4] X. Latour, D. Faure, S. Diallo, A. Cirou, B. Smadjia, Y. Dessaux, N. Orange, Control of bacterial diseases of potato caused by Pectobacterium spp. (E. carotovora), Cah. Agric. 17 (2008) 355e360. [5] J.A. Gracia-Garza, T.J. Blom, W. Brown, W. Allen, Pre- and post plant applications of copper-based compounds to control Erwinia soft rot of calla lilies, Can. J. Plant Pathol. 24 (2002) 274e280. [6] M. Gulzar, T. Croguennec, J. Julien, P. Michel, B. Saı€d, Copper modulates the heat-induced sulfhydryl/disulfide interchange reactions of b-Lactoglobulin, Food Chem. 2009 (116) (2009) 884e891. [7] D.H. Lapwood, P.J. Reed, The susceptibility of stems of different potato cultivars to blackleg caused by Erwinia carotovora subsp. atroseptica, Ann. Appl. Biol. 109 (1986) 555e560. [8] D.J. Newman, G.M. Cragg, Natural products as sources of new drugs over the 30 days from 1981 to 2010, J. Nat. Prod. 75 (2012) 311e335. [9] M. Saleem, M. Nazir, M. Shaiq Ali, H. Hussain, Y.S. Lee, N. Riaz, A. Jabbar, Antimicrobial natural products: an update on future antibiotic drug candidates, Nat. Prod. Rep. 27 (2009) 238e254. [10] K. Lewis, F.M. Ausubel, Prospects of plant derived antibacterials, Nat. Biotechnol. 24 (2006) 1504e1507. [11] C. Papadoupoulo, K. Soulti, I.G. Roussis, Potential antimicrobial activity of red and white wine phenolic extracts against strains of Staphyloccocus aureus, Escherichia coli and Candida albicans, F. Tech-Biotech. 43 (2005) 41e46. [12] L. Lovrekovitch, H. Lovrekovitch, M.A. Stahman, Inhibition of phenol oxidation by Erwinia carotovora in potato tuber tissue and its significance in disease resistance, Phytopathology 57 (1967) 737e742 in Lyon GD, Heilbronn J, Forrest RS, Johnston DJ. The biochemical basis of resistance of potato to soft rot bacteria. Neth J P1 Path 1992; 98. Supplement 2:127-133. [13] Y. Henis, H. Tagari, R. Volcani, Effect of water extracts of carob pods, tannic acid, and their derivatives on the morphology and growth of microorganisms, J. Appl. Microbiol. 12 (1964) 204e209. [14] B. Kivçak, T. Mert, H.T. Ozturk, Antimicrobial and cytotoxic activity of Ceratonia siliqua L. Extracts, Turk J. Biol. 26 (2002) 197e200. [15] A. Ben Hsouna, M. Trigui, R. Ben Mansour, R.M. Jarraya, M. Damak, S. Jaoua, Chemical composition, cytotoxicity effect and antimicrobial activity of Ceratonia siliqua essential oil with preservative effects against Listeria inoculated in minced beef meat, Int. J. Food Microbiol. 148 (2011) 66e72. [16] A.H. Ibrahim, M.A.E.B. Rehab, Y.D. Samar, A.L. Ahmed, S.K. Mohamed, Bacterial growth inhibitory effect of Ceratonia siliqua L. plant extracts alone and in combination with some antimicrobial agents, J. Adv. Biotechnol. Bioeng. 1 (2013) 3e13. [17] A. Ranalli, S. Contento, L. Lucera, M. Di Febo, D. Marchegiani, V. Di Fonzo, Factors affecting the contents of iridoid oleuropein in olive leaves (Olea europaea L.), J. Agric. Food Chem. 54 (2006) 434e440.
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Antibacterial activity of carob (Ceratonia siliqua L.) extracts against phytopathogenic bacteria Pectobacterium atrosepticum Saïda Meziani a, e, B.Dave Oomah b, Farid Zaidi c, Annabel Simon-Levert d, dric Bertrand e, Rachida Zaidi-Yahiaoui a, * Ce a
Laboratory of Applied Microbiology, Faculty of Nature and Life Sciences, A. Mira University, Bejaia 06000, Algeria Formerly with the National Bioproducts and Bioprocesses Program, Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, Summerland, BC V0H 1Z0, Canada c Department of Food Science, Faculty of Nature and Life Sciences, A. Mira University, Bejaia 06000, Algeria d AkiNaO SAS, Perpignan, France e Laboratory of Chemistry of Biomolecules and Environment (LCBE), University of Perpignan Via Domitia, Perpignan, France b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 September 2014 Received in revised form 1 December 2014 Accepted 5 December 2014 Available online 6 December 2014
Acetone and ethanol extracts of carob (Ceratonia siliqua L.) leaf and pods were evaluated for their in vitro inhibitory ability against the pectinolytic Gram negative Pectobacterium atrosepticum (Pca, CFBP-5384) bacteria, the causal agent of potato soft rot. Potato (Solanum tuberosum, var nicola) tuber rot tissues obtained after 5 day bacterial inoculation was analyzed by LCeMS and GCeMS to study Pca pathogenicity. Trans/cis N-feruloylputrescine was identified in potato tuber after 5-day inoculation with Pca in a dark moist chamber. Although glycoalkoloid (a-chaconine and a-solanine) production increased due to Pca soft rot infection, it was not a resistance-determining factor. Many secondary metabolites were identified including the phytoalexins solavetivone and fatty acids responsible for plant defence responses. Acetone extract of carob leaf (FCA) exhibited the strongest inhibitory effect (IC50 ¼ 1.5 mg/ml) and displayed synergistic antimicrobial effect in the presence of infected potato tuber extract (Pdt-Pca extract) against Pca. This synergy could be used in an integrated control program against potato soft rot pathogens, thereby reducing chemical treatments. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Antimicrobial activity Phytoalexins Pectobacterium atrosepticum Soft rot Carob Leaf extract
1. Introduction Pectobacterium atrosepticum, formely named Erwinia carotovora subs. atroseptica, almost exclusively infects potato, causing blackleg of the stem and tuber soft rot [1,2]. Consequently, it causes important losses within cool temperate regions, where potatoes have traditionally been grown [3]. There are currently no efficient curative methods to protect potato against Pectobacterium spp [4]. Although copper compounds reduce the spread of these pathogens [5], they may cause environmental damages and present a risk to human health [6]. Selection of blackleg-resistant potato cultivars was only partially successful and never resulted in completely resistant cultivars [7]. There is growing interest in developing new antibacterial agents that can control phytopathogenic bacteria without environmental
* Corresponding author. E-mail address: [email protected] (R. Zaidi-Yahiaoui). http://dx.doi.org/10.1016/j.micpath.2014.12.001 0882-4010/© 2014 Elsevier Ltd. All rights reserved.
effects. Different studies provide evidence that some medicinal plants produce many biologically active compounds representing a source of great importance in research of new antimicrobial agents [8]. These compounds, derived from several secondary metabolic pathways, include alkaloids, flavonoids, lignins, phenolic compounds and terpenoids [9,10]. Recently, the antimicrobial proprieties of various plant extracts against certain pathogen have been reported [11]. Phenolic compounds are often considered to play an important role in resistance to many plant pathogens including resistance of potato to erwinias [12]. Phenolics can directly inhibit bacterial growth, by inhibiting wall-degrading enzymes, or as precursors in the formation of physical barriers such as lignin. Carob (Ceratonia siliqua L.) has received considerable attention because its extracts exhibit very strong antimicrobial activity against various species of bacteria and fungi [13e16]. The present study aimed to: (1) examine the potential role of ethanol and acetone extracts of carob (C. siliqua) leaf and pods to control in vitro potato soft rot caused by P. atrosepticum; (2) determine their
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antibacterial property, and the interaction of carob leaves extracts in combination with potato tuber extract (Solanum tuberosum, var, nicola) against phytopathegenic bacteria (Pca) and (3) to identify and characterize the active compounds present in infected potato tuber extract by LCeMS and GCeMS analysis. 2. Materials and methods 2.1. Plant material C. siliqua leaves and pods were collected in Aftis, Wilaya of Bejaia Algeria. The pods were randomly collected from selected plants and then pooled. The leaves and pods (without seeds) were separated, after being washed with tap water to remove all impurities and then with distilled water. The samples were dried at room temperature and ground to a fine powder in an electric blender (Super Blender National®, Japan). The samples were stored under vacuum in a desiccator prior to analysis. 2.2. Extraction procedure Dried samples (2 g) were sequentially extracted by maceration in 50 ml of 80% (v/v) acetone and 80% (v/v) ethanol according to the method described by Ranalli et al. [17]. The extraction was carried out at room temperature for 1 h under magnetic stirring. The extracts were filtered through filter paper (10 mm) and the extraction process repeated three times by adding another 50 ml of solvents to the sample residue. The combined filtrate from each extraction was concentrated under vacuum on a rotary evaporator (Büchi, R-114) at 40 C, and completely dried in a freeze drier. Finally, the obtained extracts were kept at 4 C in the dark until analysis. 2.3. Determination of total phenolic content Total phenolic content was determined according to the FolinCiocalteu colorimetric assay following the method described by Singleton et al. [18]. An aliquot of diluted sample extract (0.5 ml) was added to 0.9 ml of diluted Folin-Ciocalteu reagent and 3.6 ml sodium carbonate solution (75 g/l). The test tubes were allowed to stand in the dark at room temperature for 30 min. Absorbance at 765 nm was read versus the prepared blank with spectrophotometer (Shi-madzu Uvi-mini1240, Suzhou Jiangsu, China). Total phenolic content of samples were expressed as milligrams gallic acid equivalents per mg of dry weight (mg GAE mg1 DW) through the calibration curve with gallic acid. All samples were analyzed in three replications. 2.4. Screening for antibacterial activity 2.4.1. Bacterial strain Antimicrobial activity was tested in vitro against P. atrosepticum (strain CFBP-5384) provided by the Stock Culture Collections of Phytopathogenic Bacteria (CFBP, INRA Angers, France). Bacteria were maintained for extended periods as deep-frozen cultures (20 C) and cultivated on King B Agar Medium at 25 C for 48 h. Strain was grown in peptone water liquid nutritive medium (WPN) at 25 C overnight before use for antibacterial activity test. 2.4.2. Antibacterial activity The microplate bioassay (micro-dilution) was used to study the antimicrobial activities of P. atrosepticum. Plant extracts dissolved in aqueous dimethylsulfoxide (DMSO) at 50, 100, 250, 500 and 1000 mg ml1 concentration were pippeted in sterile 96-well plates (250 mL volume, Fisher Scientific) based on a National Committee for Clinical Laboratory Standard method (NCCLS) [19].
The bacterial suspension was adjusted with sterile distilled water to 2.105 cfu/ml concentration. Briefly, 96-well plates were prepared by dispensing into each well 198 ml of microorganism suspension. Then, 2 mL from their serial dilutions of extracts was transferred into five consecutive wells. The last well containing 198 mL of WPN medium and 2 mL of the serial dilutions of extracts were used as negative control. The final volume in each well was 200 mL. The plate was covered with a sterile plate sealer and then incubated at 25 C for 48 h. After agitation, microorganism growth was estimated by reading absorbance in microplate wells at 600 nm with a Microplate Reader (Aviso, Sirius HT, Ebersberg, Germany). The MIC50 was defined as the lowest concentration of plant extracts able to inhibit 50% of the microorganism growth. 2.5. Pathogenicity tests on potato tubers Potato tubers (S. tuberosum, var. nicola) were selected free of wounds, rots and homogeneous in size. They were stored at 4 C until use. Tubers were washed, surface rinsed in sterile distilled water and air-dried under a laminar flow hood. Before inoculation, a bacterial suspension was prepared at 2.108 cfu/ml concentrations in liquid nutritive media (WPN) from cultures on King's B medium and incubated at 25 C for 24 h. The half-tuber method of Ibrahim et al. [20] was followed for the study of the pathogenic variability of P. atrosepticum. Each tuber was cut from the rose end to the heel end in two roughly equal parts with a sterile knife. A hole (5 mm diameter 5 mm depth) was drilled with a cork borer in the center of each half-tuber. Ten half-tubers were inoculated by depositing 50 ml of the bacterial suspensions prepared previously. Control tubers received 50 ml of sterile distilled water (SDW). After 5 days incubation at 25 C in a dark and water-saturated environment, symptoms were assessed visually; the rotted tissue was collected with a spatula, weighed and used for extraction. 2.6. Extraction from potato tubers The collected tissues were extracted according to the method described by Andreu et al. [21]. These tissues were blended in a mixture of dichloromethane and methanol (50:50, v/v) using an Ultra-Turax® homogenizer. The homogenate was allowed to stand overnight, filtered and evaporated to dryness. Solid-phase extraction, the method of choice for fast and effective purification, was performed using C18 extraction SPE cartridges (Strata C18-E, phenomenex®, 55 mm, 70A; 2g/12 ml Giga tubes, 8B-S001-KDG). The C18 cartridges were conditioned and activated. Briefly, the cartridges were preconditioned using 10 ml of methanol (MeOH) and equilibrated with 10 ml of distilled water. The MeOH/water (5:95, v/v) reconstituted sample extract was loaded onto the cartridges and allowed to flow by gravity. The columns were washed with 5 ml of distilled water. The analyte of interest was then eluted with 15 ml of MeOH into a tube, and the eluates were dried under nitrogen at 40 C. The residue was immediately dissolved in 1 ml of HPLC methanol solution, vortexed and sonicated for 2 min. 2.7. HPLC-MS analysis Phenolic acids were analyzed by high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS). The extracts were dissolved in HPLC grade methanol at 5 mg/mL, filtered (0.22 mm) and two microliters of the sample solutions were injected. LCeMS analysis was conducted using an Accela 600 system (Thermo® Scientific, France) connected with a quaternary pump, refrigerated autosampler, a diode array detector (Accela PDA
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detector) and a 3DLCQ-Fleet ion trap mass spectrometer (Thermo® Scientific, Inc, Bremen, Germany). A reversed phase column (Kinetex C-18 reverse phase column, 100 3.00 mm Phenomenex®) was used at a flow rate of 500 mL/min. Mobile phase was water containing 1‰ formic acid (solvent A) and acetonitrile containing 1‰ formic acid (solvent B). The elution gradient was from 95% (A)5% (B) 0e5 min, 40% (B) 5e30 min, 100% (B) 30e35 min and return to 95% (A) 35e45 min. UV analysis was performed at 254, 280 and 340 nm and UV spectra were recorded from 200 to 600 nm. The MS experiments were carried out using an electrospray interface (ESI) operating in positive [MþH]þ and negative [MH] ionization mode. The molecular ions were scanned from 100.0 to 2000.0 (m/z) and multiple MSn spectra were used to provide comprehensive structural information of the compounds. The MS data were collected and processed on an Xcalibur version 2.0 data system.
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antibacterial activity was measured using a well diffusion method according to the National Committee for Clinical Laboratory Standard (NCCLS) [19]. The bacterial suspension was adjusted with sterile distilled water to a concentration of 2.105 cfu/ml. Stock solutions of extract from carob leaf; infected potato tubers and the control were prepared for evaluation of their combined effect on P. atrosepticum. Plant extracts were dissolved in aqueous dimethylsulfoxide (DMSO) at concentration corresponding to MIC50/8, MIC50/4, MIC50/2, MIC50 and MIC50X2. The antibacterial activity was determined by the micro-dilution method described above. The first antagonist, additive or synergistic effect of the extracts in combination was determined with calculation of fractional inhibitory concentration indices (FICI). The experiment was done in triplicate to ensure reproducibility of results. The fractional effects (FIC) of two compounds (or extracts) X and Y for antibacterial studies, in terms of their MIC for bacterial growth, can be expressed as follows [23]. The FIC index was calculated as follows:
2.8. Gas chromatography/mass spectrometry (GC/MS) analysis 2.8.1. Derivatization for GC/MS analysis The derivatization is crucial in GCeMS analysis to increase metabolite volatilities. Silylation is the most prevalent derivatization method as it readily volatizes the sample and therefore very suitable for non-volatile samples for GC analysis. The mechanism involves the replacement of the active hydrogen with a trimethylsilyl group. An aliquot of the extracts was evaporated to dryness using a centrifugal evaporator. For this purpose, 10 mg of the extract dried with N2 gas, then 100 ml of derivatization agent N,O-Bis(thylsilyl) trifluoroacetamide (BSTFA) with 1% of trimethyltrime chlorosilane (TMCS) BSTFA/TMCS and 100 ml of pyridine was added, mixed and heated 15 min at 105 C [22]. The resulting solution containing the TMS derivatives was filtered and analyzed by GCeMS. 2.8.2. GCeMS analysis Extracts were analyzed by GCeMS, each sample was injected twice and the results were averaged. GCeMS analysis was carried out using a Trace Gas Chromatograph Focus GC (Thermo® Scientifique) 10904116 series equipped with a Trace MS mass spectrometer (DSQ II), and with a ZB-5MS (phenomenex®) column (30 m 0.25 mm i.d., 0.25 mm film thickness), using helium as carrier gas (35 cm/s). The chromatographic conditions were as follows: initial temperature: 150 C for 2 min; final temperature 325 C for 20 min; injector temperature 320 C; transfer-line temperature was held at 320 C; split ratio 10. Briefly, 2 ml of the derivative solution was injected. The injection conditions were: split mode with 1 min duration pulse, and 1 ml/ min He column flow. MS data were collected and processed on an Xcalibur™ version 1.4.1. For Mass spectrometry conditions, the EI ionization was 70 eV, the transfer line was at 320 C, mass range acquisition was from m/z 50 to m/z 650 and carried in Full Scan mode. Qualitative analysis of compounds was based on the comparison of their spectral mass and their relative Retention time with those of NIST mass spectra database and Kovats RI on the chromatograms recorded in Full Scan or in SIM mode using the characteristic ions. 2.9. Determination of the combined activity For the combination assays, bacterial suspension of P. atrosepticum was tested against acetone extract (FCA) and ethanol extract (FCE) of carob leaf combined with purified extract of infected potato tubers (Potatoes-Pca-extract, Pdt-Pca) in 96-well microplates (250 mL volume, Fisher Scientific). Combined effect of
FICindex ¼ FICX þ FICY
X
FIC ¼
FICY ¼
MICðX with YÞ MICðX aloneÞ MICðY with XÞ MICðY aloneÞ
! the fractional effect of X ! the fractional effect of Y
A FIC index 0.5 to 4.0 as additive or indifferent, and >4.0 as antagonistic [23,24]. The corresponding FIC values were used to construct isobolograms of the combinations [25]. The graph is represented with the ratio to the FIC of the infected potato tuber extract (Pdt-Pca) on the x-axis and the ratio of the FIC of the leaf of carob (FCA, FCE) on the yaxis. A line was drawn in the graph connecting the highest concentrations of each of the extract used to produce a reference line. Deviation from linearity relative to the reference line was used as the basis for interpretation of antimicrobic relationship between combined extracts against Pca. A curved deviation of the isobologram to the left of the reference line indicates synergy of the antimicrobial agents, while a right deviation would indicate antagonism. An additive relationship between antimicrobials was represented by an isobologram with points falling proximate to the reference line.
2.10. Data analysis Data were subjected to analysis of variance (ANOVA) by the general linear model procedure by means of comparison with Duncan's test and Pearson correlation according to R methods. Differences between means (P < 0.001) level were considered to be statistically significant.
Table 1 Total phenolic content of carob leaves and pods. Samples
Extracts
Total phenolic content (mg GAE/g)
Carob leaves
Acetone Ethanol Acetone Ethanol
21.41a 22.98a 2.03b 1.05b
Carob pods
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3. Results and discussion 3.1. Total phenolic content Acetone and ethanol extracts of C. siliqua L. leaves and pods grown in Algeria contained total phenolics ranging from 0.61 to 22.98 mg GAE/g. Carob leaf extracts had significantly higher (10) phenolic contents than those from pods, independent of solvent (Table 1). Phenolic content of ethyl acetate extract of carob leaves from Morocco was reported to be around 2.6 g/L GAE [26]. 3.2. Antibacterial activity The in vitro antibacterial activity of C. siliqua leaf and pod extracts against the Gram (e) bacteria P. atrosepticum (Pca, CFBP3584) was determined in this study. The leaf extracts showed strong inhibitory activity against Pca with MIC50 values between 1.5 and 2.4 mg/ml depending on concentration and extracting solvent. No antibacterial activity was observed from acetone and ethanol extracts of carob pods against Pca. The acetone leaf extract was more effective than ethanolin inhibiting Pca growth. Antibacterial activities were influenced by changes in leaf extracts induced by solvent choice [27]. Solvent also affected the phenolic distribution and concentration of olive leaf extracts resulting in varying antimicrobial efficiency against S. aureus, E. coli, S. enteritidis, and S. thypimurium [28]. The observed activity could be explained by the presence of biologically active compounds in these extracts. The extracts of leaves of C. siliqua have been known to contain a number of antimicrobial compounds. Phytochemical screening of this plant has shown the presence of gallic acid, quercetin -glucoside, Kaempferol-rhamnoside, Quercetin-rhamnoside, 1,2,6 tri-O-galloyl- glucopyranose, (e)-Epigallocatechin-3-O-gallate, kaempferol and quercetin [29]. Several studies attribute the inhibitory effect of plant extracts against bacterial pathogens to their phenol content [27,30e32]. The phenols and flavonoids contribute significantly to the antibacterial activity and can form complexes with cell wall and also disrupt bacterial envelopes [33]. In this study, the antibacterial effect may be attributed to the presence of different bioactive compounds such as tannins, phenolic acids, flavonoids and flavonoidal glycosids in carob leaf that impact growth and metabolism of microorganisms. It has been reported that the phenolics, tannins and propyle gallate were strong microbial inhibitors. C. siliqua leaves from Turkey had a significant effect on a broad range of microbial pathogens [14]. The toxicity of tannins to various bacteria has been demonstrated in several studies with hydrolysable tannins exerting potent antibacterial effects against B. subtilis and S. aureus [34,35]. This antibacterial effect may be largely due to the o-diphenol groups of tannins enabling them to act as iron chelators, thus depriving microorganisms of this essential element [36]. 3.3. Identification of chromatographic peaks 3.3.1. LC/MS analysis In the LC/MS analyses of constituents in the extracts of potato tuber, the peak area ratio of each compound was recorded by the UV detector. The chemical structures of these constituents were identified on the basis of their retention times and mass fragmentation patterns. Analysis of contents was performed after integration of single ion chromatograms of protonated molecular ions [MþH]þ. Pca infection induced differential metabolic changes demonstrated by increased peak intensities in potato tuber extract (Pdt-Pca extract) (Fig. 1).
Fig. 1. Total scan PDA and ESI Full MS chromatogram of potato tuber extracts: (a) infected potato tubers by Pca (Pdt-Pca) and (b) control, not infected potato tubers (PdtT) obtained by LC-ESI-MS.
Peak 2 (tR, 5.37 min; lmax, 316 nm) at [MþH]þ m/z 265 and fragments ions of m/z 248 [M þ HeNH3]þand 177 [M þ HeC4H12N2]þ (Fig. 1) was identified in potato tuber after five days of bacterial inoculation as trans/cis N-Feruloylputrescine as reported by Yan et al. [37]. Trans/cis N-Feruloylputrescine has been isolated from the zone of potato tuber (cv. Bintje) infected by Phoma exigua var. foveata, and may be produced in stress-exposed tuber, but not in healthy tuber [38]. Peaks1and 3 remains unidentified (Fig. 1). The mass spectrum of peak 1 (tR, 5.05 min; lmax, 279 nm) with a [MþH]þ ion at m/a 205 yielded MS ion at m/z 188. Peak 3(tR, 47.91 min; lmax, 249 nm) with a [MþH]þ ion at m/z 589 produced ions at m/z 571, 553 and 417. The comparison of LC-ESI-MS analysis of potato tubers extracts revealed the presence of glycoalkaloids, a class of natural toxicants, in infected tissues of potato tubers (Pdt-Pca extract) and present only in control potato tubers (Pdt-T extract). The peaks4 and 5 (tR, 13.02 min; lmax, 248 nm) and (tR, 13.26 min; lmax, 248 nm) (Fig. 1a), were two major glycoalkaloids, a-solanine and a-chaconine, respectively identified by comparison with pure commercial
S. Meziani et al. / Microbial Pathogenesis 78 (2015) 95e102
standards. Using the mass detector, it was possible to distinguish the two molecules based on their corresponding intense [MþH]þ ions at m/z 868 and 852 m/z for a-solanine and a-chaconine, respectively. Both are glycosylated (trisaccharide) derivatives of the aglycone solanidine, a steroid alkaloid, cleavage at the interglycosidic bonds is the dominant process in the fragmentation of a -chaconine (m/z 852) and a -solanine (m/z 868) by ESI-MS. From a-chaconine, fragments of m/z 706, 560 and 398 corresponding to [M þ H-Rham]þ, [M þ H-Rham-Rham]þ, and [M þ HRham-Rham-Glu]þ were produced in addition to the protonated molecular ion [MþH]þ at m/z 852. The fragments of m/z 722,706 and 398 corresponding to [M þ H-Rham]þ, [M þ H-Glu]þand [M þ H-Glu-Rham-Gal or M þ H-Rham-Glu-Gal]þ were produced from m/z 868 for a-solanine. The results of the present study showed significant differences in glycoalkaloid accumulation before and after inoculation, between the infected (Pdt-Pca) and the control (Pdt-T) uninfected potato tuber extracts. Fewell et Roddick [39] reported that the two most abundant potato glycoalkaloids, a-chaconine and a-solanine, synergistically inhibit in vitro growth and germination of several important pathogenic fungi, and according to Friedman [40] and Lachman et al. [41] potato glycoalkaloids serve as natural defenses and display antimicrobial activity. However, there are inconsistent reports about their antimicrobial effects against pathogenic fungi and bacteria [42]. Studies report variable result on possible relationships between glycoalkaloid levels of potato tuber and P. infestans infection. For example, Smith et al. [43] suggested that glycoalkaloid have a protective function, although glycoalkaloid accumulation was suppressed in tuber slices or potato leaves after fungal inoculations or treatments with P. infestans, P. citrophthora or H. carbonum [21]. Fewell and Roddick [39] showed that P. infestans affect glycoalkaloid production and induced production of phytoalexins. In addition, eicosapentaenoic and arachidonic acid extracts or elicitors of P. infestans caused inhibition in glycoalkaloid accumulation [44]. Infection of potato tubers by P. infestans induced increases in sesquiterpene cyclase and squalene synthase, enzymes of the isoprenoid pathways. These enzymes catalyze the synthesis of sesquiterpenoid phytoalexins and glycoalkaloids [45]. Pathogen infections are known to affect glycoalkaloid content.
99
In this study, glycoalkaloid production increased due to bacterial soft rot infection caused by P. atrosepticum (CFBP-5384), in potato tubers, these results are in accordance with previous report [46]. Glycoalkaloid content in potato tuber extracts was not correlated with their resistance to soft rot caused by P. atrosepticum, suggesting that glycoalkaloids were not the factor determining resistance. 3.3.2. GCeMS analysis The components present in the extracts of Pca infected potato tubers (Pdt-Pca) and healthy uninfected control potato tubers (Pdt-T) were identified by GCeMS analysis. The sesquiterpenoid metabolite solavetivone (C15H22O, MW 218) accumulated in smaller amounts in potato tuber extracts inoculated with the pathogen (Pdt-Pca) (Fig. 2). The research on phytoalexins in tubers has focused on the major sesquiterpenoid metabolites rishitin, lubimin and solavetivone found in tubers [47]. Srikrishna and Ramasastry [48] reported that phytoalexin solavetivone was the major stress metabolite from potato tubers infected with the blight fungus P. infestans or with the soft-rot bacterium E. carotovora var. atroseptica. In addition to the phytoalexins, the GCeMS profile showed the presence of methyl esters of various types of fatty acid compounds in the rotted tissue (Pdt-Pca extracts). High concentrations were detected in rotted tissue, whereas in the wounded control tissue lower concentrations were present. Table 2 list the compounds including the relative content, retention time, mean relative molecular weight (MW), and formula indentified by GCeMS. The main components were: 9-12-otadecadienoic acid (Z,Z)-methyl ester (18:2, syn. linoleic acid methyl ester), 9-12-octadecadienoic acid (Z,Z)- trimethylsilyl ester (18:2, syn. linoleic acid), hexadecanoic acid trimethylsilyl ester (16:0, syn. Palmitic acid trimethylsilyl ester), octadecanoic acid (Z,Z)- trimethylsilyl ester (18:0, syn. stearic acid trimethylsilyl ester), n-pentadecanoic acid trimethylsilyl ester (15:0), palmitelaidic acid trimethylsilyl ester, linolenic acid trimethylsilyl (18:3), hexadecanoic acid,2,3-bis [(trimethylsilyl)oxy]propyl ester (syn. 1-monopalmitin trimethylsilyl ether), Octadecanoic acid,2,3-bis-[(trimethylsilyl)oxy]propyl ester (syn. Stearic acid,2,3 bis-(trimethylsiloxy)propyl ester).
Fig. 2. GCeMS analysis of potato tuber extracts (a) infected potato tubers by Pca (Pdt-Pca) and (b) control: not infected potato tubers (Pdt-T).
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Table 2 GCeMS profile of extracts of infected potato tuber (Pdt-Pca). Peak RT MW Prob. Formula (min)
Peak name
1 2 3 4
9.55 11.14 11.47 11.97
5
13.37 352 79.87 C27H40O4Si
6 7 8
13.46 350 41.47 C21H38O2Si 13.66 356 94.43 C21H44O2Si 15.32 352 5.72 C21H40O2Si
9
17.04 474 94.70 C25H54O4Si2
10
18.80 502 77.32 C27H58O4Si2
n-pentadecanoic acid trimethylsilyl ester palmitelaidic trimethylsilyl ester hexadecanoic acid trimethylsilyl ester 9,12-Octadecadienoic acid, methyl ester (Z,Z)9,12-Octadecadienoic acid, trimethylsilyl ester linolenic acid trimethylsilyl ester octadecadienoic trimethylsilyl ester 9,12-Octadecadienoic acid, trimethylsilyl ester octadecanoic acid, 2,3 bis[(trimethylsilyl) oxy]propyl ester octadecanoic acid, 2,3 bis[(trimethylsilyl) oxy]propyl ester
314 326 328 294
35.93 89.95 95.96 21.52
C18H38O2Si C19H38O2Si C19H40O2Si C19H34O2
Vernenghi et al. [49] report that linoleic and linolenic acids derivatives accumulate in tomato (Lycopersicum esculentum Mill.) resistant to Phytophtora sp. and to Verticillium alboatrum; these acids are involved in the defense mechanisms against fungal pathogen by producing hydroxylated and peroxidized fatty acids [50]. The a-linolenic acid is the first potential precursor for the production of jasmonic acid and its derivatives, which are key signaling molecules known to activate different plant defense responses. Jasmonic acid induces phenylalanine ammonia-lyase (PAL) and several pathogenesis related proteins [51]. Fatty acids were phytotoxic to potato leaves, and elicited systemic induced resistance efficiently against P. infestans [52]. Linoleic acid found to elicit induced systemic resistance (ISR) of tobacco against the bacterial soft rot pathogen, Pectobacterium carotovorum subsp. carotovorum (Pcc) [53]. Furthermore, linolenic and linoleic acids were phytotoxic and effective in inducing systemic resistance, whereas oleic acid was the least phytotoxic and induced no systemic resistance [52]. According to Blechert et al. [54], the octadecanoic acid derivative, octadecatrienoic acid, (Z,Z)-, methyl ester plays an important role in plant defense mechanism. The hexadecanoic acid ethyl ester acts as antioxidant, nematicide and pesticide and may contribute to the antimicrobial and antioxidant activities [55]. Farmer and Ryan [56,57] proposed a model according to which primary wound signals like oligogalacturonides and system in trigger the activation of the octadecanoid pathway resulting in a burst of jasmonic acid production which ultimately leads to the activation of defense genes. The model proposed that wounding by insects or microbial
pathogen attack led to an interaction of elicitors with receptors, thus initiating the octadecanoic-based pathway from the C18 linolenic acid fatty acid to jasmonic acid.
3.4. Evaluation of the combined activity Representative isobolograms of combined carob leaf extracts and infected potato tuber extract against P. atrosepticum are shown graphically in Fig. 3. A synergistic effect was observed for the combination of carob leaf acetone extract (FCA) with infected potato tuber extract (PdtPca) with FIC indices ( 0.5e1.0) as indicated by isobologram with MIC points falling very proximate to a reference additive line (Fig. 3b). According to the effect obtained for each extract, the combination was qualified as acting additively and synergistically. Extracts of carob leaf and potato tuber present a combined antimicrobial effect potentialized against P. atrosepticum. This antimicrobial effect is even more evident in the presence of PdtPca extracts and acetone extract of carob leaf (FCA). Liu [58] opined that the additive and synergistic effects of phytochemicals in vegetables are attributed to the complexes mixture of phytochemicals present. The strong antibacterial activity of acetone extract of leaf of C. siliqua and synergistic effect with combination with infected potato tuber extract may be attributed to its content of flavonoids and tannins. Naringin, apigenin and rutin are carob flavonoids had reported antibacterial activity [59]. Potato tuber infected by P. atrosepticum accumulates phenolics and metabolites. Phenolics play an important role in many plant-pathogen interactions, both as constitutive defense metabolites and as induced phytoalexins [60]. Andreu et al. [21] found that beside phytoalexins and glycoalkaloids, phenolics are also involved in potato resistance to late blight. Since the amount of phenolics induced after infection was lower in a susceptible potato cultivar compared to a resistant cultivar, they suggested a role for phenolics in resistance of potato to P. infestans. Finally, the bioactive phytocompounds in carob leaf inhibit the growth of Pca and their joint activity with potato tubers extracts lead to an enhanced antibacterial effect. The mechanism governing the joint action of plant extract compounds is still unknown. Some authors suggest that phytocompounds disturb cell wall or increase permeability of the cytoplasmic membrane [61e63].
Fig. 3. Isobolograms of antimicrobial combinations of C. Siliqua leaf extract and infected potato tuber extract (Pdt-Pca) against P. atrosepticum: (a) acetone (FCA) and (b) ethanol (FCE) leaf extract of C. Siliqua (FCA) and Pdt-Pca extract.
S. Meziani et al. / Microbial Pathogenesis 78 (2015) 95e102
4. Conclusion This study describes the antimicrobial activity from leaves and pods extract of C. siliqua (Carob) against P. atrosepticum. The leaf extracts exhibited potent antimicrobial activity against P. atrosepticum that may be related to the presence of phenolic compounds, flavonoids and tannins in considerable quantities in carob leaf. The results obtained from the present study suggested that C. siliqua leaf extracts possess significant antibacterial property. This work confirms the antibacterial activity of C. siliqua extract and shows their potential use as microbial agents. The results revealed the importance of combining plant extracts to control bacteria. The study suggests the possibility of concurrent use of plant extracts in combinations in treating soft rot caused by P. atrosepticum. Acknowledgments We are grateful to the Algerian Ministry of Higher Education and Scientific Research (MESRS) for providing financial assistance and scholarship (BAF) to Ms. Saida Maziani. References rombelon, Potato diseases caused by soft rot erwinias: an overview [1] M.C.M. Pe of pathogenesis, Plant Pathol. 51 (2002) 1e12. rombelon, J.A. van Veen, J.M. van der Wolf, Control of [2] R. Czajkowski, M.C.M. Pe blackleg and tuber soft rot of potato caused by Pectobacterium and Dickeya species: a review, Plant Pathol. 60 (2011) 999e1013. [3] B. Smadja, X. Latour, S. Trigui, J.-F. Burini, S. Chevalier, N. Orange, Thermodependence of growth and enzymatic activities implicated in pathogenicity of two Erwinia carotovora subspecies (Pectobacterium spp.), Can. J. Microbiol. 50 (2004) 19e27. [4] X. Latour, D. Faure, S. Diallo, A. Cirou, B. Smadjia, Y. Dessaux, N. Orange, Control of bacterial diseases of potato caused by Pectobacterium spp. (E. carotovora), Cah. Agric. 17 (2008) 355e360. [5] J.A. Gracia-Garza, T.J. Blom, W. Brown, W. Allen, Pre- and post plant applications of copper-based compounds to control Erwinia soft rot of calla lilies, Can. J. Plant Pathol. 24 (2002) 274e280. [6] M. Gulzar, T. Croguennec, J. Julien, P. Michel, B. Saı€d, Copper modulates the heat-induced sulfhydryl/disulfide interchange reactions of b-Lactoglobulin, Food Chem. 2009 (116) (2009) 884e891. [7] D.H. Lapwood, P.J. Reed, The susceptibility of stems of different potato cultivars to blackleg caused by Erwinia carotovora subsp. atroseptica, Ann. Appl. Biol. 109 (1986) 555e560. [8] D.J. Newman, G.M. Cragg, Natural products as sources of new drugs over the 30 days from 1981 to 2010, J. Nat. Prod. 75 (2012) 311e335. [9] M. Saleem, M. Nazir, M. Shaiq Ali, H. Hussain, Y.S. Lee, N. Riaz, A. Jabbar, Antimicrobial natural products: an update on future antibiotic drug candidates, Nat. Prod. Rep. 27 (2009) 238e254. [10] K. Lewis, F.M. Ausubel, Prospects of plant derived antibacterials, Nat. Biotechnol. 24 (2006) 1504e1507. [11] C. Papadoupoulo, K. Soulti, I.G. Roussis, Potential antimicrobial activity of red and white wine phenolic extracts against strains of Staphyloccocus aureus, Escherichia coli and Candida albicans, F. Tech-Biotech. 43 (2005) 41e46. [12] L. Lovrekovitch, H. Lovrekovitch, M.A. Stahman, Inhibition of phenol oxidation by Erwinia carotovora in potato tuber tissue and its significance in disease resistance, Phytopathology 57 (1967) 737e742 in Lyon GD, Heilbronn J, Forrest RS, Johnston DJ. The biochemical basis of resistance of potato to soft rot bacteria. Neth J P1 Path 1992; 98. Supplement 2:127-133. [13] Y. Henis, H. Tagari, R. Volcani, Effect of water extracts of carob pods, tannic acid, and their derivatives on the morphology and growth of microorganisms, J. Appl. Microbiol. 12 (1964) 204e209. [14] B. Kivçak, T. Mert, H.T. Ozturk, Antimicrobial and cytotoxic activity of Ceratonia siliqua L. Extracts, Turk J. Biol. 26 (2002) 197e200. [15] A. Ben Hsouna, M. Trigui, R. Ben Mansour, R.M. Jarraya, M. Damak, S. Jaoua, Chemical composition, cytotoxicity effect and antimicrobial activity of Ceratonia siliqua essential oil with preservative effects against Listeria inoculated in minced beef meat, Int. J. Food Microbiol. 148 (2011) 66e72. [16] A.H. Ibrahim, M.A.E.B. Rehab, Y.D. Samar, A.L. Ahmed, S.K. Mohamed, Bacterial growth inhibitory effect of Ceratonia siliqua L. plant extracts alone and in combination with some antimicrobial agents, J. Adv. Biotechnol. Bioeng. 1 (2013) 3e13. [17] A. Ranalli, S. Contento, L. Lucera, M. Di Febo, D. Marchegiani, V. Di Fonzo, Factors affecting the contents of iridoid oleuropein in olive leaves (Olea europaea L.), J. Agric. Food Chem. 54 (2006) 434e440.
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![[Galactomannan breakdown in germinating carob seeds (Ceratonia siliqua L.)].](/assets/img/hold.png)
