production of phytotoxic metabolites by pseudomonas ... - CiteSeerX

Journal of Plant Pathology (2014), 96 (1), 169-176

Edizioni ETS Pisa, 2014

Andolfi et al. 169

PRODUCTION OF PHYTOTOXIC METABOLITES BY PSEUDOMONAS SYRINGAE PV. ACTINIDIAE, THE CAUSAL AGENT OF BACTERIAL CANKER OF KIWIFRUIT A. Andolfi1, P. Ferrante2, M.. Petriccione3, A. Cimmino1, A. Evidente1 and M. Scortichini2,3 1Dipartimento

di Scienze Chimiche, Complesso Universitario Monte Sant’Angelo, Via Cintia 4, 80126, Napoli, Italy per la Ricerca e la Sperimentazione in Agricultura, Centro di Ricerca per la Frutticoltura, Via di Fioranello 52, 00134, Roma, Italy 3Consiglio per la Ricerca e la Sperimentazione in Agricultura, Unità di ricerca per la Frutticoltura, Via Torrino 3, 81100, Caserta, Italy 2Consiglio

SUMMARY

Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker of Actinidia chinensis and A. deliciosa, is currently causing severe economic losses worldwide. A study was conducted to verify if a highly virulent Psa strain, isolated during the current outbreaks of bacterial canker of kiwifruit in Italy, produces phytotoxic metabolites in vitro. Culture filtrate, obtained from 14-day-old cells grown in Pseudomonas minimal medium, induced an evident hypersensitivity-like reaction to both tobacco and kiwifruit leaves. From culture filtrates, extracts were obtained using different solvents and pH values. The extracts and their corresponding aqueous phases, were further tested for phytotoxicity. Basic, hydrophilic, lowmolecular weight and hydrophilic, high-molecular weight compounds belonging to exopolysaccharides were isolated and analyzed. These compunds proved highly phytotoxic to kiwifruit, tobacco leaves and lemon fruits. Gas-chromatography-mass-spectrometry analysis carried out on crude exopolysaccharides showed glucose as the main monosaccharide constituent. These results suggest that phytotoxic metabolites, other than the antimetabolite phaseolotoxin, could be involved in the virulence of the pathogen to kiwifruit species. Key words: toxins, exopolysaccharides, culture filtrate, GC-MS analysis, Actinidia INTRODUCTION

Pseudomonas syringae pv. actinidiae (Psa), the causal agent of bacterial canker of kiwifruit, is a destructive pathogen currently causing severe economic losses to kiwifruit production worldwide. A population of the pathogen has spread to all major areas of cultivation of Actinidia deliciosa, the green-fleshed, and A. chinensis, the yellowfleshed kiwifruit. All cultivated germplasm, pollinators included, is susceptible to the disease (Scortichini et al., Corresponding author: M. Scortichini Fax:+39.06.79340158 E-mail: [email protected]

2012). So far, the studies on the pathogen mainly regarded the population structure, its origin and adaptation to Actinidia species, the detection techniques and the disease cycle (Ferrante and Scortichini, 2010; Gallelli et al., 2011; Marcelletti et al., 2011; Ferrante et al., 2012; Mazzaglia et al., 2012; Butler et al., 2013). Main field symptoms include leaf spotting, shoot wilting, twig dieback, blossom necrosis, reddening of the lenticels, extensive cankers along leaders and trunks accompanied by exudates mainly oozing out during winter. An important phase of the disease cycle is the systemic migration of the bacterium from the leaves to the shoot through the xylem vessels (Serizawa and Ichikawa, 1993; Spinelli et al., 2011; Petriccione et al., 2013). The systemic colonization of the shoot is fundamental for the pathogen since it can start an endophytic phase that can result in subsequent canker formation that enables further dispersal in the environment through the exudates (Ferrante et al., 2012). During spring, it is possible to observe a sudden tip shoot wilting, one of the most striking Psa symptoms both Actinidia species. Subsequently, the whole shoot withers in a few days. The presence of this symptom can be very relevant in kiwifruit orchards. If a toxin(s) and/or an antimetabolite(s) produced by the pathogen are involved in the rapid collapse of the shoot is still unknown but this may be a possibility worth investigating. In fact, it is known that many P. syringae pathovars produce secondary metabolites of different chemical structure that can be effective at low concentrations and can cause or aggravate symptoms on infected plants (Arrebola et al., 2011). Strains of Pseudomas syringae pv. phaseolicola, the causal agent of bean (Phaseoluis vulgaris) blight, and Psa produce phaseolotoxin, an antimetabolite toxin, composed by a tripeptide consisting of ornithine, alanine and homoarginine. This antimetabolite induces chlorosis of the leaves by inhibiting the ornithine carbamoyl transferase (OCT), an enzyme involved in arginine biosynthesis (Mitchell et al., 1976; Patil et al., 1976; Tamura et al., 2002). Interestingly, it has been shown that other strains of these two P. syringae pathovars, including the Psa strains causing the current bacterial canker outbreaks worldwide, do not produce phaseolotoxin, even though they induce severe damages to their host plants (Rico et al., 2003; Ferrante and

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Scortichini, 2010; Marcelletti et al., 2011). This, however, does not rule out the possibility that some other phytotoxic compound(s) might be produced by such Psa strains that could elicit some of the symptoms observed in kiwifruit orchards. Since no data are available on the possible production of bioactive phytotoxic metabolites by the current Psa population, a preliminary study was carried out for evaluating the phytotoxic activity of metabolites produced in vitro by a highly virulent Psa strain and for the preliminary characterization of the chemical and biological features of these metabolites. MATERIALS AND METHODS

Bacterial strain and culture condition. The strain Psa CRA-FRU 8.43, recovered in central Italy in 2008 from A. chinensis cv. Hort16A, was utilized in the study, due to its high virulence to both A. chinensis and A. deliciosa (Marcelletti et al., 2011). This strain had previously been phenotypically and genetically characterized (Ferrante and Scortichini, 2010; Marcelletti et al., 2011). It was routinely maintained on nutrient agar additioned with 3% sucrose (NSA) at 25-27°C. For media inoculation, an aliquot of 100 µl suspension, at 1-2 × 108 CFU/ml, prepared in sterile water from a 48-hour-old single colony grown on NSA was utilized. For metabolite production, the Psa strain was grown in Erlenmeyer flasks, containing 1 litre of Pseudomonas minimal medium (ammonium dihydrogen orthophosphate 1.0 g/l; potassium chloride 0.2 g/l; magnesium sulphate 0.2 g/l) (Gasson, 1980), additioned with peptone 1.0 g/l (0,45% tryptophan; Difco, USA) and glucose 10.0 g/l, for 14 days at 25-27°C, with gentle shaking (80 rpm). The experiment was repeated three times. For the chemical analysis of metabolites, 10 litres of bacterial cultures were prepared in the same way. Phytotoxic activity of the Psa culture filtrate. After six days of incubation, and at each day until the 14th day from inoculation, an aliquot of 1 ml of the bacterial suspension concentrated at 1-2 × 109 CFU/ml, was aseptically collected and filtered through a 0.2 µm nitrocellulose membrane (Millipore Corporation, USA) into a sterile Eppendorf tube. The filtrate was then injected with a sterile 1 ml syringe, into the lower surface of tobacco (Nicotiana tabacum, cv. White Burley) and kiwifruit (i.e. A. deliciosa cv. Hayward) young leaves. Three different leaves were infiltrated in spring, at room temperature and the appearance of visible symptoms (i.e., necrotic areas in the infiltrated area) was checked two days after infiltration. The assay was repeated three times. General procedures for chemical analyses. 1H nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz in D2O on a spectrometer AVANCE 400 (Bruker,


Germany). Analytical thin layer chromatography (TLC) was performed on Kieselgel 60 F254, 0.25 mm silica gel plates (Merck, Germany). The spots on TLC plates were visualized using different techniques: (i) exposing plates to UV light (254 nm); (ii) spraying the plates with 10% H 2SO4 in methanol, followed by 5% phosphomolybdic acid in ethanol, then heating at 110°C for 10 min; (iii) spraying the plates with 0.5% ninhydrin in acetone, followed by heating at 110°C for 10 min; (iv) spraying the plates with chromic mixture, followed by heating at 110°C for 10 min. Dialyses were carried out using the molecular porous membrane tubing Spectra/Por, with a 3.500 Da cut-off (Spectrum Medical Industries, USA). Cation exchange chromatography was performed using Dowex-50 resin (H+ form, Fluka, Switzerland). Ultrapure H 2O was obtained with a Milli Q system (Millipore, USA). Extraction of metabolites from Psa culture filtrate. Extractions were carried out at three different pH: (i) non-modified pH of culture filtrates (pH 5.5); (ii) pH 2, by acidification with 1 M formic acid, and (iii) pH 10, by alkalinization with 1 M NH4OH. Three aliquots of 20 ml were taken from the culture filtrate, their pH was modified according to the above described procedures then they were extracted three times with the same volume of ethyl acetate (EtOAc). Organic phases corresponding to the same pH value were combined, dried over Na 2SO4, filtered, and evaporated under reduced pressure. Residual aqueous phases were fluxed under a N2 stream to remove NH4OH and formic acid and lyophilized. n-butanol (BuOH) extracts, at three different pH values, were obtained with the same procedure described above. The extract was additioned with a same volume of water and the azeotropic mixture evaporated under reduced pressure. The extracts obtained from each solvent and pH value, and their corresponding aqueous phases, were tested for phytotoxicity as reported below. Dialysis of Psa culture filtrate. Five mls of Psa culture filtrate were dialysed. The tube was immersed in 10 ml of distilled water for 48 h at 10°C. Water was renewed every 6 h. The remaining tube content (IN) and the outer aqueous phases (OUT) were collected and lyophilized for phytotoxicity assays. Hydrophilic, low molecular weight and basic metabolites. The residue of organic extract obtained from BuOH (100 mg) was dissolved in 1 M formic acid (5 ml), then loaded on a Dowex-50 resin, previously packed in a chromatography column (15 × 3 cm i.d.). The column was washed with distilled H 2O (30 ml) and eluted with 1 M NH4OH (50 ml). The basic eluate was fluxed by a N2 stream, then lyophilized to give a yellow solid residue (28.7 mg), which was analyzed by TLC on silica gel eluted with BuOH/AcOH/H 2O 3:1:1 (v/v/v) and iPrOH:H 2O 4:1 (v/v). The chromatograms showed two


main ninhydrin-sensitive spots at R f = 0.40 and 0.2, R f = 0.43 and 0.11, respectively. Chemical analysis of extracellular polysaccharides. The Psa culture filtrate was fractionated with cold ethanol to recover the extracellular polysaccharide fraction (EPS) as follows: 80 ml of culture filtrate were cooled at 4°C, mixed with five volumes of absolute cold ethanol (400 ml) and left overnight at −20°C. The precipitate was separated by centrifugation at 7.000 g for 45 min at 4°C. The ethanolic phase was evaporated under reduced pressure, yielding a homogenous viscous oil. The precipitate was dissolved in 70 ml ultrapure Milli-Q water, and re-precipitated with 350 ml cold absolute ethanol as described above. The resulting precipitate was dissolved in 50 ml ultrapure Milli-Q water and dialysed as described. The IN crude EPS fractions together with the ethanolic fractions were tested for phytotoxicity. An EPS fraction (1 mg) was utilised for analysis of monosaccharides. The sample was hydrolyzed with CF3COOH 2 M, at 120°C, for 2 h. Sugar residues were converted to corresponding alditol-acetate derivatives and analyzed by GC-MS as described above (Leontein et al., 1978; Gerwig et al., 1979). GC-MS analysis was performed using a Perkin-Elmer Autosystem XL with a RTX-5MS WCOT capillary column, (Restek, 30 m × 0.25 mm; film thickness, 0.25 mm) that was coupled, through a heated transfer line (250°C), to a PE Turbomass-Gold quadrupole mass spectrometer. Chromatographic separation was achieved with the following temperature program: 100°C (3 min isothermal), rate 4°C/min to 300°C. Helium was used as carrier gas at 1.60 ml/min, the injector temperature was 250°C, the split-injection mode had a 30 ml/min of split flow. Mass spectra were obtained in EI mode (70 eV), scanning in the range 45-650 m/z, with a cycle time of 1 sec. Compound identification was based on the comparison of mass spectra data with the National Institute of Standards and Technology (NIST) library database, published spectra, and real standards. Phytotoxic assays. Organic extracts and relative aqueous phases, the chromatographic fraction, the crude EPS the IN and OUT fractions obtained from Psa culture filtrate were assayed on lemon (Citrus lemon) fruits, and kiwifruit and tobacco leaves The EtOAc extracts (2.0 mg) were diluted in DMSO (final concentration 4%) and the volume was later adjusted to 1 ml. Other samples were diluted in distilled water, BuOH extracts and basic fraction were assayed at a final concentration of 2 mg/ml, while other samples were assayed at final concentrations like those of the starting culture filtrate. Solutions were infiltrated into tobacco and kiwifruit leaves as described. Lemon fruits were surface-disinfected with sodium hypochlorite (50 µg/ml), followed by three rinses of sterile distilled water. Lemon fruits were chosen for phytotoxic activity assays, since they were effective in revealing the virulence of P. syringae isolates when inoculated on the

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surface (Scortichini et al., 2003). Six 10 µl drops of the samples were released onto the surface of the fruits which was immediately pricked with the needle of a sterile 1 ml syringe. Infiltrations were performed in spring, at room temperature. The appearance of leaf and lemon fruit necrosis was checked two days after infiltration. Leaves and fruits were checked for symptom severity using a 0 to 3 arbitrary scale: −: no symptoms; +: slight necrosis; ++: presence of necrotic areas; +++: extensive necrotic areas. Ophiobolin A (Evidente et al., 2006), at 1.5 mg/l, was used as positive control, whereas DMSO at 4% and sterile distilled water were used as negative controls. The experiments were repeated three times. RESULTS

Phytotoxic activity. Filtrates from Psa cultures grown for up to 13 days in Pseudomonas minimal medium did not induce any apparent phytotoxic effect to tobacco and kiwifruit leaves (i.e., no symptoms developed following infiltration). However, the filtrate from a 14-days-old Psa CRA-FRU 8.43 culture induced a clear-cut hypersensitivity-like reaction on tobacco and kiwifruit leaves (Fig. 1A). A comparable reaction was obtained with filtrated from cultures older than 14 days. The phytotoxic activity of the organic extracts and relative aqueous phases at various pH levels is reported in Table 1, whereas Table 2 reports the phytotoxic activity of the IN and OUT residues of dialysis experiments and of the crude EPS and relative ethanol phases and cationic exchange chromatography basic fraction. A representative image of the phytotoxic effect induced on lemon fruit by the BuOH extract at pH 10 is shown in Fig. 1B. Metabolite extraction and phytotoxicity. Comparison of the weight of residues obtained by EtOAc extraction and those of the corresponding aqueous phases showed that no lipophilic, low molecular weight phytotoxic metabolites were present in the culture filtrates. In fact, the ethyl acetate extracts at three different pH did not show any phytotoxic activity on tobacco and kiwifruit leaves and on lemon fruit (Table 1). In contrast, the aqueous phases induced large necrotic areas on tobacco and kiwifruit leaves as well as on lemon fruit. In addition, the weight of the residues obtained by BuOH extraction were similar to those of the corresponding aqueous phases (Table 1). This, probably, could be attributed to the presence of glucose in the organic extract, as checked by TLC on silica gel eluted with iso-PrOH:H 2O 8:2 (v:v) and developed with phosphomolybdic acid and chromic mixture. The chromatogram showed a main sensitive spot at R f = 0.52, which was identified as glucose by comparison with a standard sample. Both the organic and aqueous phases extracts obtained by BuOH showed phytotoxic activity. Apparently, the

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A

!

B

Fig. 1. A. Phytotoxic reaction shown by Actinidia deliciosa leaves following infiltration with a filtrate from a 14-day-old culture of Pseudomonas syringae pv. actinidiae CRAFRU 8.43 grown in Pseudomonas minimal medium. The reaction was observed two days after infiltration. B. Phytotoxic activity of the n-butanol (BuOH) organic extract of Pseudomonas syringae pv. actinidiae CRAFRU 8.43 culture filtrate at pH 10, shown by a lemon fruit two days after infiltration.

BuOH extract at pH 10 was the most active in the bioassays by inducing severe necrotic areas. However, similar results were observed by testing all the aqueous phase residues (Table 1). Ophiobolin, used as positive control in the bioassays caused necrotic areas similar to those of the aqueous phase and BuOH extracts. No symptoms were observed upon infiltration with DMSO and distilled water. Partial purification and characterization of metabolites. Dialysis experiments of culture filtrate showed a higher amount of low-molecular weight compounds collected outsite the tube with respect to the quantity of highmolecular weight compounds that had remained inside the tube. Both low and high molecular weight compounds showed a remarkable phytotoxic activity by inducing necrosis on leaves and fruits (Table 2). A crude EPS fraction obtained by precipitation of the culture filtrate with cold ethanol was active, as shown by bioassays, whereas the residues of the ethanolic phases had a low or no phytotoxic activity (Table 2). The GC MS analysis carried out on crude EPS showed glucose as the main monosaccharide constituent. The corresponding 1H NMR spectrum confirmed this result. In fact, it showed the presence of two doublets (J = 3.8 and 7.9 Hz) at ä 5.16 and 4.57, respectively. This feature is typical of two anomeric protons of α- and β-glucosides, respectively (Moran et al., 2000). In addition, taking into account the activity of the BuOH basic extract, the presence in the culture filtrate of low molecular weight basic compounds was also hypothesized. The BuOH basic extract was, subsequently, fractionated by cationic

exchange chromatography to remove the glucose. When the residue obtained by ammonia elution (basic fraction) was assayed, it was clearly phytotoxic to kiwifruit and tobacco leaves and lemon fruit (Table 2). A subsequent TLC analysis of this residue with two different eluents revealed the presence of two metabolites reacting with ninhydrin. The 1H NMR spectrum of this mixture showed different signals in the range δ 7.4-6.8, 4.0-3.0, and 1.8-0.7 that could be attributed to the presence of an α-amino acid and monosaccharide residues. In particular, multiplets at δ 7.36-7.24 and the two doublets (J = 8.3 Hz) at δ 7.12 and 6.82 suggested the presence of phenylalanine and tyrosine residues, while the doublet (J = 7.36 Hz) at δ 1.40 could putatively be attributed to the presence of a methyl group of an alanine residue. The monosaccharide residues could be attributed to the presence of glucose. DISCUSSION

The results obtained in this study clearly indicate that the Psa strain CRA-FRU 8.43, isolated during the current outbreak of bacterial canker of kiwifruit in central Italy, produces phytotoxic metabolites when grown, for at least 14 days, in the Pseudomonas minimal medium additioned with peptone and glucose. In fact, relevant phytotoxic effects induced by the culture filtrates to kiwifruit and tobacco leaves and lemon fruit were observed after this period of incubation. Taking into consideration that this strain does not produce phaseolotoxin (Ferrante and Scortichini, 2010;


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Table 1. Yield (mg) and phytotoxic activity on tobacco and kiwifruit leaf, and lemon fruit of ethyl acetate (AcOEt) and n-butanol (BuOH) extracts at three different pHs and residual aqueous phases, as obtained from 20 ml of Pseudomonas syringae pv. actinidiae CRAFRU 8.43 culture filtrate (CF). The symptoms were evaluated two days after the infiltration. Sample a

EtOAc extract at pH CF EtOAc extract at pH 2 EtOAc extract at pH 10 a Aqueous phase from extraction with AcOEt at pH CF b Aqueous phase from extraction with AcOEt at pH 2 Aqueous phase from extraction with AcOEt at pH 10b a BuOH extract at pH CF BuOH extract at pH 2 BuOH extract at pH 10 a Aqueous phase from extraction with BuOH at pH CF Aqueous phase from extraction with BuOH at pH 2b Aqueous phase from extraction with BuOH at pH 10b Positive control ophiobolin (1.5 mg/ml) Negative control DMSO at 4% in distilled water Negative control (distilled water)

c

Kiwifruit leaf

c

Yield (mg/20 ml of CF)

Tobacco leaf

Lemon fruit

3.4

−

−

−

3.8 3.2 320.3 327.6 332.0 134.8 125.2 207.2 178.0 182.0 148.2 − − −

− − + ++ ++ + + +++ ++ ++ ++ +++ − −

− − ++ +++ +++ + + +++ +++ +++ +++ ++ − −

− − ++ +++ +++ + + +++ ++ +++ +++ +++ − −

c

a pH

of culture filtrate: 5.5. aqueous phases were fluxed under N2 to remove NH 3 or formic acid c The organic extracts were assayed at concentration of 2 mg/ml and the aqueous phases at the correspondence of CF. Scale: −, no symptoms; +, slight necrosis; ++, presence of necrotic areas; +++, extensive necrotic areas. b The

Table 2. Yield (mg) and phytotoxic activity on tobacco and kiwifruit leaf, and lemon fruit, as recorded two days after the infiltration of low molecular weight basic compounds, dialysed and crude exopolysaccharides (EPS), and ethanolic fractions as obtained from 20 ml of Pseudomonas syringae pv. actinidiae CRAFRU 8.43 culture filtrate (CF).

Sample

Yield (mg/20 ml of CF)

Tobacco leaf

Kiwifruit leaf

Lemon fruit

Basic fraction Dialysed CF 3500 D (IN) Dialysed CF 3500 D (OUT) Crude EPS Ethanol residue 1 Ethanol residue 2 Control (distilled water)

14.6 32.4 293.2 6.2 233.4 78.7 -

++ ++ + ++ + -

++ ++ + ++ + -

++ ++ + ++ + -

Scale: −, no symptoms; +, slight necrosis; ++, presence of necrotic areas; +++, extensive necrotic areas.

Marcelletti et al., 2011), a preliminary assessment of the chemical features of the phytotoxic metabolites was made. The filtrate extraction, carried out with solvents of different polarity and three different pH and their corresponding aqueous phases, showed that the Psa strain produced hydrophilic, basic, low-molecular weight, together with hydrophilic, high-molecular weight phytotoxic metabolites. These findings were confirmed also by dialysis tests. A basic, hydrophilic, low-molecular weight metabolite has been partially purified by cationic exchange chromatography and its preliminary spectroscopic investigation (1H NMR) showed an α-amino acid and a monosaccharide residues. In addition, the hydrophilic, high-molecular weight metabolite was putatively identified as an exopolysaccaride. In fact, its GC-MS analysis revealed that it is mainly composed by glucose. To the best of our knowledge, the present study deals for the first time with the production of phytotoxic metabolites by Psa strains of the current outbreaks of bacterial canker of kiwifruit. The involvement of extracellular polysaccharides, namely levan and alginate, in bacterial diseases is well

known (Evidente and Motta, 2002). For example, they play several roles during the plant-microbe interaction. In particular, it is assumed that extracellular polysaccharides surround the bacterial cell in planta to retain water so, by reducing water evaporation, they protect the cell from desiccation (Denny, 1995). It has also been observed that in P. syringae-host interaction, alginate genes are expressed during the formation of water-soaked lesions (Keith et al., 2003). These compounds, by forming a hydrogenated matrix, can also prevent the recognition by the host and can function as detoxifying barriers for plant defense molecules (Kiraly et al., 1997). In addition, alginate would contribute to the P. syringae epiphytic fitness on the leaves (Yu et al., 1999). Recently, it has also been shown that both levan and alginate contribute to biofilm formation in P. syringae (Laue et al., 2006). In other pathosystems, exopolysaccharides play also a relevant role as pathogenicity or virulence factors. In fact, Erwinia amylovora, the causal agent of fire blight of Rosaceous plants, produces amylovoran, an acidic homopolysaccharide composed by four differently linked galactose molecules

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and a glucouronic acid residue, that affects the plant mainly by plugging the vascular tissue, thus inducing the wilting of the twigs (Bugert and Geider, 1995). Moreover, the virulence of Ralstonia solanacearum, the causal agent of bacterial wilt of many valuable crops, largely depends on an acidic high molecular mass heteropolysaccharide that promotes the systemic colonization of tomato plants and induces wilt symptom (Denny, 1995). Whether the exopolysaccharides found in the Psa culture filtrates are responsible for twig die-back that characterizes bacterial canker of kiwifruit is still unknown. This aspect, however, deserve further studies together with the identification of the chemical structure of the phytotoxic metabolites here preliminary described and their possible production in kiwifruit naturally infected by Psa. In fact, if the exopolysaccharides are produced also in vivo, they could putatively be used as biomarkers to detect the pathogen in infected plants, as reported by Andolfi et al. (2009) for Phaemoniella chlamydospora, a fungus involved in grapevine trunk diseases, that can be detected by a flow cytometry technique recognizing the exopolysaccharides produced by such a pathogen. ACKNOWLEDGEMENTS

The NMR spectra were recorded at the CERMANU Centre at the University of Naples, “Federico II”, Italy by Dr. Pierluigi Mazzei. The authors also thanks Dr. Riccardo Spaccini (Department of Agriculture, University of Naples “Federico II”) for GC-MS analysis. The research was in part supported by a grant from Italian Ministry of the University and Research and by the Italian Ministry of Agriculture, Food and Forestry, project INTERACT: “Interventi di coordinamento ed implementazione delle azioni di ricerca, lotta e difesa al cancro batterico dell’actinidia”. Prof. A. Evidente is associated to the “Istituto di Chimica Biomolecolare del CNR, Pozzuoli, Italy” REFERENCES Andolfi A., Cimmino A., Evidente A., Iannacone M., Capparelli R., Mugnai L., Surico G., 2009. A new flow cytometry technique to identify Phaeomoniella chlamydospora exopolysaccharides and study mechanisms of esca grapevine foliar symptoms. Plant Disease 93: 680-684. Arrebola E., Cazorla F.M., Perez-Garcia A., De Vicente A., 2011. Chemical and metabolic aspects of antimetabolites toxins produced by Pseudomonas syringae pathovars. Toxins 3: 1089-1110. Bugert P., Geider K., 1995. Molecular analysis of the ams operon required for exopolysaccharide synthesis of Erwinia amylovora. Molecular Microbiology 15: 917-933. Butler M.I., Stockwell P.A., Black M.A., Day R.C., Lamont I.R., Poulter T.M., 2013. Pseudomonas syringae pv. actinidiae from recent outbreaks of kiwifruit bacterial canker belong to different clones that originated in China. PLoS ONE 8: e57464.


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Received July 17, 2013 Accepted September 8, 2013

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