Abstract Olive quick decline syndrome (OQDS) causes severe damages to the olive trees in Salento (Apulia, Italy) and poses a severe threat for the agriculture of Mediterranean countries. DNA-based typing methods have pointed out that OQDS is caused by a single outbreak strain of Xylella fastidiosa subsp. pauca referred to as CoDiRO or ST53. Since no effective control measures are currently available, the objective of this study was to evaluate in vitro antimicrobial activities of different classes of compounds against Salento-1 isolated by an OQDS affected plant and classified as ST53. A bioassay based on agar disk diffusion method revealed that 17 out of the 32 tested antibiotics did not affect bacterial growth at a dose of 5 μg disk−1. When we assayed micro-, ultra- and nano-filtered fractions of olive mill wastewaters, we found that the micro-filtered fraction resulted to be the most effective against the bacterium. Moreover, some phenolics (4-methylcathecol, cathecol, veratric acid, caffeic acid, oleuropein) were active in their pure form. Noteworthy, also some fungal extracts and fungal toxins showed inhibitory effects on bacterial growth. Some of these compounds can be further explored as potential candidate in future applications for curative/preventive treating OQDS-affected or at-risk olive plants. microbial metabolites, antibiotics, phenolics, olive mill wastewaters, fungal extracts, toxins INTRODUCTION In 2013, the xylem-limited phytopathogenic bacterium Xylella fastidiosa (Wells et al.1987) was first detected in oleander and olive plants of Salento area, Apulia (Italy) (Saponari et al.2013; Cariddi et al.2014; Loconsole et al.2014), and then recognized as the etiological agent of the ‘olive quick decline syndrome’ (OQDS) (Saponari et al.2016). According to multilocus sequence typing (MLST), all strains isolated so far, regardless from the host of provenience, share the same sequence type, ST53, which has been tentatively ascribed to X. fastidiosa subsp. pauca (Nunney et al.2014; Loconsole et al.2016; Giampetruzzi et al.2017b). The same phylogenetic lineage was previously found in Central America and very recently detected in coffee plants intercepted in France and in the Netherlands (Nunney et al.2014; Bergsma-Vlami et al.2017; Denancé et al.2017). Xylella fastidiosa is insect vector-transmitted and thus the measures adopted by EU Commission so far to counteract the spread of OQDS (Commission Implementing Decision (EU) 2015/789 of 18 May 2015) are focused on the use of insecticides to control the vectors population, which in turn reducing bacterial transmission, and on the eradication of infected plants to reduce inoculum sources. To date, these methods have been only partially successful and, recently, EFSA reported that in Apulia, the number of diseased trees is increasing despite the treatments with insecticides formulations as well as the use of good crop management practices (EFSA 2016). At present, OQDS has spread to the whole province of Lecce and to part of Brindisi and Taranto provinces (April 2017, http://webapps.sit.puglia.it/freewebapps/DatiFasceXF/index.html), underlying the urgent need of more efficient management practices. Among the possible strategies that have at least in part been explored against X. fastidiosa, there is the production of transgenic plants armed with proteins or peptides able to kill the pathogen (Agüero et al.2005; Dandekar et al.2012; Li, Hopkins and Gray 2015), applications of phage cocktails (Das et al.2015) or of selected xylem endophytes for symbiontic control of disease (Azevedo, Araújo and Lacava 2016). Moreover, since X. fastidiosa is limited to the plant xylem, particular attention has been given to small molecules that can reach the xylem sap flow and that can inhibit the growth and movement of the pathogen. To this regard, early attempts have been made with antibiotics (Hopkins 1979) as potential bactericides of X. fastidiosa to be applied directly in planta to reduce the infective capacity of insect vectors or as prophylactic treatment to prevent infection of grapes and other hosts (Kuzina, Miller and Cooksey 2006). Xylella fastidiosa subsp. fastidiosa, subsp. multiplex and subsp. pauca, as identified by Schaad et al. (2004) by DNA-DNA relatedness assays and sequencing of the 16S–23S intergenic spacer (ITS) region, also showed a different response to antibiotics. Subspecies multiplex and pauca resulted to be susceptible to penicillin and resistant to carbenicillin, whereas subsp. fastidiosa strains showed an opposite behavior. Moreover, the use of antibiotics as selective markers for laboratory activities, i.e. the isolation of the pathogen and the development of new transformation systems, was also verified by determining the susceptibility of different isolates of X. fastidiosa to these compounds (Lacava et al.2001). However, Italy has banned the use of antibiotics for plant diseases management (D.M. 10.08.1971, in G.U. 212 del 23.08.1971) and also in EU a contentious debate has been opened concerning the possible development of antibiotic resistance in human pathogens (McManus et al.2002). Because of this and given the epiphytotic trend of OQDS, other managing strategies urgently need to be explored taking also into account the costs of proactive measures that can be sustained by the growers. To this regard, N-acetylcysteine (NAC) has showed antibacterial effects against X. fastidiosa causing citrus variegated chlorosis (Muranaka, Giorgiano and Takita 2013); sets of antimicrobial peptides isolated from plant seeds, spiders, Xenopus and human neutrophils resulted to be effective in vitro against several strains of X. fastidiosa (Kuzina, Miller and Cooksey 2006; Fogaça et al.2010); and various endophytic microorganisms have been used as sources of bioactive natural products suitable to counteract this bacterium (Kuzina, Miller and Cooksey 2006; Aldrich et al.2015). Recently, olive growers in Salento reported that in orchards where olive mill wastewaters (OMWs) were spread, olive trees showed limited OQDS symptoms, even if they were very close to heavily symptomatic infected plants. It is worthwhile considering that OMWs are known to possess antimicrobial properties mainly related to the presence of phenolic compounds (Tafesh et al.2011). Phenolics are a class of secondary metabolites produced in many plant species for defensive functions against a wide range of pathogens (Boudet 2006). Research studies showed that phenolic compounds, single or in combination, were able to inhibit the growth of several bacterial species (Obied et al.2005; Tafesh et al.2011). Moreover, the growth of different X. fastidiosa strains from grape and almond (namely Temecula, Conn Creek, Dixon and Tulare) was reported to be affected in vitro by various phenolic compounds such as hydroxytyrosol, tyrosol, cathecol, 4-methyl catechol, oleuropein, verbascoside, 4-hydroxybenzoic acid, vanillic acid and p-coumaric acid (Maddox, Laur and Tian 2010). Since OQDS first report, although MLST analyses have been extensively performed and two genomes describing ‘CoDiRO’ and ‘De Donno’ isolates have been deposited (Giampetruzzi et al.2015, 2017a), the phenotypic characterization of X. fastidiosa existing in Salento has not yet been carried out. In this study, for the first time, X. fastidiosa Salento-1 (NCCPB No. 4595 LMG 29352; Bleve et al.2016), representative of the outbreak strain causing OQDS in Apulia, was tested in vitro in order to characterize its susceptibility/resistance to different antibiotics, phenols, fungal toxins and crude culture extracts. Moreover, we set up an experimental work aimed to ascertain the potential role of OMWs obtained by different filtration steps, per se or as source of candidate compounds, for the therapy of diseased olive trees. MATERIALS AND METHODS Strain and growth conditions Xylella fastidiosa Salento-1 isolate (NCCPB No. 4595 LMG 29352; Bleve et al.2016) was grown in BCYE agar medium (LaBM) for 15–20 days at 28°C. For long-term storage, glycerol stocks of X. fastidiosa cells were prepared in 1X PBS buffer (Sigma-Aldrich, Milan, Italy) with a final glycerol concentration of 15% (v/v) and stored at –80°C. Antibiotics, fungal toxins and phenolic compounds All antibiotics were obtained from Sigma (Milan, Italy) and prepared in stock solutions at a concentration of 10 mg mL−1. Nystatin and rifampicin were suspended in methanol; spiramycin, tyrothricin and chloramphenicol were suspended in ethanol; cephalexin hydrate was suspended in 1M NH4OH; nalidixic acid was suspended in CHCl3; oxytetracycline was dissolved in 1M HCl; sulfapyridine was suspended in 0.5 M NaOH; all other antibiotics were suspended in sterile water and were dissolved in methanol. Fungal toxins produced by different fungal species were a kind gift of Prof. A. Evidente, Università degli Studi di Napoli Federico II. They were selected on the basis that these secondary metabolites were reported to possess antimicrobial activity but were never assayed before against X. fastidiosa. Before the use, they were dissolved in methanol at a final concentration of 1 mM. All phenolic compounds were obtained by Sigma (Milan, Italy), with the exception of oleuropein, verbascoside and luteolin-7-O-glucoside that were purchased from Extrasynthèse (Lyon, France). All phenolics were suspended in ethanol at a final concentration of 10 mM, with the exception of cathecol and 4-methyl cathecol that were suspended in sterile water. Verbascoside and luteolin-7-O-glucoside were suspended in methanol at 10 mM final concentration. Preparation of Trichoderma spp. culture extracts. Eight isolates of Trichoderma spp. were cultured in 500 mL Erlenmeyer flasks containing 200 g of autoclaved rice kernels previously adjusted to ∼60% water content and inoculated with 10–15 small pieces (2 × 2 mm) of a 5- 7-day-old culture of the fungus on potato dextrose agar. The cultures were incubated at 25 ± 1°C with a photoperiod of 12 h for 3 weeks. The harvested culture material was dried in a forced draft oven at 45°C for 48 h, finely ground in a laboratory mill (Molino Cyclone LMLF; PBI International, Milan, Italy) to a particle size ≤ 0.2 mm and stored at 4°C until extraction. A 20 g sample of each dried culture was extracted in a blender with 100 mL of methanol–1% aqueous NaCl (55:45 v/v) for 3 min, filtered through a filter paper (Whatman No. 1), and 50 mL of the filtrate were transferred into a separatory funnel and defatted twice with 50 mL of n-hexane. The upper n-hexane layer was discarded and the methanol layer was extracted three times with 30 mL methylene chloride. The methylene chloride portions were collected, combined, evaporated to dryness and the residue was dissolved in 1 mL of methanol and stored at –20°C until used in the agar disk diffusion assays. OMW samples OMWs were obtained by local olive mills that used a three-phase system. The OMWs fractions were obtained as described by D’Antuono et al. (2014). Briefly, acetic acid was added to pH 5 to OMW samples in order to avoid phenolic compounds oxidation and then they were sieved through a sieve with 425 μm porosity to remove large particles and colloids and then they were processed with a laboratory-scale system (Permeare s.r.l., Milano, Italy). Microfiltration process (MF) was performed using the Pilot Plant N022/N256C with continuous recirculation of the sample and by using a ceramic membrane, PERMAPORE EOV 1046, with a cut-off of about ∼100 000 Da (membrane porosity 0.1 μm). Then, the Pilot Plant N021/N256C unit was used to carry out ultrafiltration step (UF), using a polyethersulfone membrane, PERMAPORE DGU 1812 BS EM with a cut-off of 5000 Da and the nanofiltration (NF) process, using a polyamide membrane, PERMAPORE AEN 1812 BS with cut-off of 200 Da. MF, UF and NF samples were filter sterilized by passing through 0.2-μm cellulose acetate membrane syringe filters (GVS). HPLC-DAD analysis Analytical-scale HPLC analyses of MF, UF and NF OMWs fractions were performed using an Agilent Technologies series 1100 liquid chromatography (Waldbronn, Germany) supplied of a binary gradient pump G1312A, a G1315A photodiode array detector and a G1316A column thermostat set at 45°C. For spectra analysis and data processing, ChemStation for LC3D (Rev. A. 10.02) software was used. For polyphenols separation, an analytical (5 μm) column Phenomenex (Torrance, CA) Luna C18, 4.6 × 250 mm was used. The solvent system of (A) methanol and (B) acetic acid/water (5:95, v/v) was used for polyphenols elution, following the here reported linear gradient elution profile: 0–21 min, 15%–40% A; 21–30 min, 40% A (isocratic); 30–45 min, 40%–63% A; 45–47 min, 63% A (isocratic); and 47–51 min, 63%–100% A (Lattanzio 1982). The flow rate was 1 mL/min and the injection volume was 20 μL. The polyphenols identification was performed comparing the spectra and retention time of the pure standards. Agar dilution assay MF, UF and NF fraction samples were added in different quantities (1, 2.5, 5, 10% v/v) to BCYE agar medium. Xylella fastidiosa Salento-1 was grown on BCYE agar plates, suspended in 1X PBS buffer and adjusted, prior plating, to a final concentration of 106 CFU mL−1 corresponding to a culture optical density of ≈ 0.8 at 600 nm. Ten microliters of decimal dilutions from 105 to 101 CFU mL−1 of X. fastidiosa inoculum were applied to the BCYE agar plates with or without OMWs. The agar plates were incubated at 28°C for 20–30 days. Each assay was performed in triplicate, and each experiment was repeated at least two times. Agar disk diffusion method Xylella fastidiosa Salento-1 suspended in 1X PBS buffer and adjusted to a final concentration of 106 CFU mL−1 was streaked onto solid BCYE medium. Antibiotics, fungal Trichoderma spp. crude extracts, fungal toxins and phenolics were applied to sterile filter disks (Difco) to achieve a dose of 10–20 μL disk−1 in the case of each crude fungal extract, 3.6–36 μg disk−1 in the case of each fungal toxin, 1–10 mM disk−1 of each phenolic compound and 5–100 μg disk−1 for each antibiotic (with the exception of cephalothin sodium salt that was added at a concentration of 1–5 μg disk−1). After each solvent was evaporated, the filter disks were placed onto the plates previously inoculated with X. fastidiosa. In each test, plates were incubated at 28°C for 20–30 days and then observed for an inhibition halo around the paper disk. The inhibition zone was measured in mm. In order to verify the bactericidal/bacteriostatic activity of phenolics compounds, fungal extracts and toxins, cell material around the disk within the inhibition halos was sampled by an inoculation loop and streaked in a fresh BCYE agar plate to test the ability of X. fastidiosa cells to grow or not in the absence of the inhibiting compound. The plates were incubated as above. Each assay was performed in triplicate and each experiment was repeated at least two times. In the case of antibiotics, we arbitrarily considered Salento-1 to be resistant when, in the medium containing 5 μg antibiotic, there was no inhibition halo; to be susceptible when in the presence of 5 and 100 μg the inhibition halos were ≥2 and ≥5 mm, respectively; to have an intermediate behavior when there was an inhibition halo ≤ 2 mm for 5 μg and ≥5 mm for 100 μg. RESULTS Antibiotic resistance/susceptibility The Gram-negative bacterium X. fastidiosa Salento-1 was resistant to anti-fungal compound nystatin (Table 1). Among cephalosporins, the strain showed high level of susceptibility to cephaloridine, cephalothin sodium salt and ceftriaxone, an intermediate sensitivity to cephaloglycin and it resulted to be resistant to cephalexin hydrate. Resistance was observed against cycloserine D, the fluoroquinolone of first generation nalidixic acid and the aminocoumarin novobiocin sodium salt. The bacterial strain resulted to be slightly susceptible to gentamycin, to streptomycin sulfate salt and to bramycin, while it showed resistance to neomycin trisulfate salt hydrate, kanamycin, amikacin and kasugamycin. Salento-1 isolate was susceptible to all tested tetracyclines (oxytetracycline, tetracycline hydrochloride, hyclate). Among penicillins, a susceptibile behavior was observed for penicillin G sodium salt and carbenicillin, ampicillin and polymyxin B sulfate salt, while resistance was detected against penicillin V potassium salt. Although the isolate Salento-1 resulted to be susceptible to rifampicin, it was resistant, in the tested experimental conditions, to several compounds active against Gram-positive bacteria such as vancomycin hydrochloride, spiramycin, sulfapyridine, tyrothricin, teicoplanin, lincomycin and chloramphenicol. No bacteriostatic effects were ever observed. Table 1. Susceptibility/resistance of X. fastidiosa Salento-1 to antibiotics. Antibiotics Classificationa Inhibition zone (mm) 5 μg disk−1 Inhibition zone (mm) 100 μg disk−1 Resistance Nystatin Polyene antifungal drug 0 0 R Ceftriaxone Cephalosporins – third generation 10 ± 0.1 12 ± 0.3 S Cephalexin hydrate Cephalosporins – first generation 0 12 ± 0.4 R Cephaloglycin Cephalosporins 1.5 ± 0.1 5.6 ± 0.3 I Cephaloridine Cephalosporin C-first generation 3.4 ± 0.3 10 ± 0.3 S Cephalotin sodium salt Cephalosporins – first generation 4 ± 0.2b 12 ± 0.2c S Cycloserine (D) Cycloserine 0 0 R Nalidixic acid Fluoroquinolones—first generation 0 0 R Amikacin Aminoglycosides 0 0 R Gentamycin Aminoglycosides 2 ± 0.1 12 ± 0.4 S Kanamycin Aminoglycosides 0 0 R Kasugamycin Aminoglycosides 0 12 ± 0.5 R Neomycin trisulfate salt hydrate Aminoglycosides 0 5.5 ± 0.3 R Streptomycin sulfate salt Aminoglycosides 1 ± 0.1 11 ± 0.4 I Tobramycin Aminoglycosides 1 ± 0.2 12 ± 0.3 I Novobiocin sodium salt Aminocoumarin 0 11 ± 0.2 R Doxycycline hyclate Tetracyclines 5.7 ± 0.3 11 ± 0.2 S Oxytetracycline Tetracyclines 3.7 ± 0.2 7.5 ± 0.4 S Tetracycline hydrochloride Tetracyclines 9 ± 0.4 12 ± 0.5 S Ampicillin Penicillins—Aminopenicillins 7 ± 0.4 11 ± 0.5 S Carbenicillin Antipseudomonal penicillins 9 ± 0.6 11 ± 0.3 S Penicillin G sodium salt Penicillin 4 ± 0.3 11 ± 0.5 S Penicillin V potassium salt Penicillins 0 10 ± 0.4 R Polymyxin B sulfate salt Polymyxins 2.5 ± 0.3 11 ± 0.6 S Rifampicin Rifamycin 3.5 ± 0.2 11 ± 0.5 S Vancomycin hydrochloride from Streptomyces orientalis Vancomycin 0 10 ± 0.4 R Spiramycin Macrolide 0 10 ± 0.3 R Sulphapyridine Sulfonamide antibiotic 0 10 ± 0.4 R Tyrothricin from Bacillus aneurinolyticus (B. brevis) Polypeptide antibiotic mixture 0 10 ± 0.6 R Teicoplanin Glycopeptides 0 0 R Lincomycin Lincomycin derivatives 0 0 R Chloramphenicol Chloramphenicol 0 0 R Antibiotics Classificationa Inhibition zone (mm) 5 μg disk−1 Inhibition zone (mm) 100 μg disk−1 Resistance Nystatin Polyene antifungal drug 0 0 R Ceftriaxone Cephalosporins – third generation 10 ± 0.1 12 ± 0.3 S Cephalexin hydrate Cephalosporins – first generation 0 12 ± 0.4 R Cephaloglycin Cephalosporins 1.5 ± 0.1 5.6 ± 0.3 I Cephaloridine Cephalosporin C-first generation 3.4 ± 0.3 10 ± 0.3 S Cephalotin sodium salt Cephalosporins – first generation 4 ± 0.2b 12 ± 0.2c S Cycloserine (D) Cycloserine 0 0 R Nalidixic acid Fluoroquinolones—first generation 0 0 R Amikacin Aminoglycosides 0 0 R Gentamycin Aminoglycosides 2 ± 0.1 12 ± 0.4 S Kanamycin Aminoglycosides 0 0 R Kasugamycin Aminoglycosides 0 12 ± 0.5 R Neomycin trisulfate salt hydrate Aminoglycosides 0 5.5 ± 0.3 R Streptomycin sulfate salt Aminoglycosides 1 ± 0.1 11 ± 0.4 I Tobramycin Aminoglycosides 1 ± 0.2 12 ± 0.3 I Novobiocin sodium salt Aminocoumarin 0 11 ± 0.2 R Doxycycline hyclate Tetracyclines 5.7 ± 0.3 11 ± 0.2 S Oxytetracycline Tetracyclines 3.7 ± 0.2 7.5 ± 0.4 S Tetracycline hydrochloride Tetracyclines 9 ± 0.4 12 ± 0.5 S Ampicillin Penicillins—Aminopenicillins 7 ± 0.4 11 ± 0.5 S Carbenicillin Antipseudomonal penicillins 9 ± 0.6 11 ± 0.3 S Penicillin G sodium salt Penicillin 4 ± 0.3 11 ± 0.5 S Penicillin V potassium salt Penicillins 0 10 ± 0.4 R Polymyxin B sulfate salt Polymyxins 2.5 ± 0.3 11 ± 0.6 S Rifampicin Rifamycin 3.5 ± 0.2 11 ± 0.5 S Vancomycin hydrochloride from Streptomyces orientalis Vancomycin 0 10 ± 0.4 R Spiramycin Macrolide 0 10 ± 0.3 R Sulphapyridine Sulfonamide antibiotic 0 10 ± 0.4 R Tyrothricin from Bacillus aneurinolyticus (B. brevis) Polypeptide antibiotic mixture 0 10 ± 0.6 R Teicoplanin Glycopeptides 0 0 R Lincomycin Lincomycin derivatives 0 0 R Chloramphenicol Chloramphenicol 0 0 R ahttps://www.drugbank.ca/drugs b1 μg disk−1. c5 μg disk−1. All data represent mean ± SD of three independent experiments. R: resistant; S: susceptible; I: intermediate sensitivity. View Large Susceptibility in vitro against fungal toxins and Trichoderma spp. culture extracts The fungal toxins reported in Table 2 were assayed for their ability to inhibit in vitro X. fastidiosa growth. All these metabolites were used at quantity of 3.6 or 36 μg disk−1. Among these compounds, ophiobolin A and gliotoxin showed a bacteriostatic effect against X. fastidiosa when used at 3.6 μg disk−1. The same results were observed when ophiobolin A and gliotoxin were used at 36 μg disk−1 (not shown). No inhibition halos were observed for the other compounds reported in Table 2 at the two tested quantities. Table 2. Susceptibility of X. fastidiosa Salento-1 to fungal toxins. Compound Source MW Inhibition (mm) (3.6 μg disk−1) Reference Chenopodolan A Phoma chenopodicola 268 0 Cimmino et al. (2013a) Chenopodolan C Phoma chenopodicola 234 0 Cimmino et al. (2013a) Chenopodolin Phoma chenopodicola 446 0 Cimmino et al. (2013b) Phyllostictin A Phyllosticta cirsii 311 0 Zonno et al. (2008) Ophiobolin A Drechslera gigantea 400 10 ± 0.3a Evidente et al. (2006) AC toxins Ascochyta caulina mix 0 Vurro et al. (2012) Gliotoxin Various fungal species 326 10 ± 0.2a Compound Source MW Inhibition (mm) (3.6 μg disk−1) Reference Chenopodolan A Phoma chenopodicola 268 0 Cimmino et al. (2013a) Chenopodolan C Phoma chenopodicola 234 0 Cimmino et al. (2013a) Chenopodolin Phoma chenopodicola 446 0 Cimmino et al. (2013b) Phyllostictin A Phyllosticta cirsii 311 0 Zonno et al. (2008) Ophiobolin A Drechslera gigantea 400 10 ± 0.3a Evidente et al. (2006) AC toxins Ascochyta caulina mix 0 Vurro et al. (2012) Gliotoxin Various fungal species 326 10 ± 0.2a aType of action: bacteriostatic. View Large The inhibitory effects of crude culture extracts of Trichoderma spp. strains to X. fastidiosa Salento-1 are shown in Table 3. Six out of seven tested extracts inhibited the growth of X. fastidiosa at the dose of 10 μL disk−1. However, while the effect of ITEM 908, ITEM 908–5, ITEM 4484, ITEM 1317 and CFA 53 was bactericidal, CFA 54 resulted to be bacteriostatic. The extract CFA 33 was ineffective. The most active extract was from the isolate T. citrinoviride ITEM 4484, which displayed the largest inhibition zone associated to bactericidal effect. Table 3. Susceptibility of X. fastidiosa Salento-1 to Trichoderma spp. crude culture extracts. Extract Source Inhibition (mm) (10 μL disk−1) Reference ITEM 908 WT r Trichoderma atrobrunneuma 4 ± 0.3b Marzano, Gallo and Altomare (2013) ITEM 908 5-r Trichoderma spp. 5 ± 0.3b Marzano, Gallo and Altomare (2013) ITEM 4484 Trichoderma citrinoviride 10 ± 0.4b Evidente et al. (2008, 2009); Ganassi et al. (2016) ITEM 1317 Trichoderma citrinoviride 5 ± 0.2b CFA 33 Trichoderma spp. 0.5 ± 0.1 CFA 53 Trichoderma spp. 8 ± 0.3b CFA 54 Trichoderma spp. 3.5 ± 0.2c Extract Source Inhibition (mm) (10 μL disk−1) Reference ITEM 908 WT r Trichoderma atrobrunneuma 4 ± 0.3b Marzano, Gallo and Altomare (2013) ITEM 908 5-r Trichoderma spp. 5 ± 0.3b Marzano, Gallo and Altomare (2013) ITEM 4484 Trichoderma citrinoviride 10 ± 0.4b Evidente et al. (2008, 2009); Ganassi et al. (2016) ITEM 1317 Trichoderma citrinoviride 5 ± 0.2b CFA 33 Trichoderma spp. 0.5 ± 0.1 CFA 53 Trichoderma spp. 8 ± 0.3b CFA 54 Trichoderma spp. 3.5 ± 0.2c aTrichoderma harzianum species complex, formerly reported as T. harzianum ITEM 908. bType of action: bactericidal. cType of action: bacteriostatic. View Large Susceptibility to OMWs-derived fractions The main identified phenolics in OMWs fractions are reported in Table 4. The most abundant compounds were oleuropein, followed by verbascoside, hydroxytyrosol and tyrosol. Table 4. Main phenolic compounds associated micro-filtrated (MF), ultra-Filtrated (UF) and nano-filtrated (NF) OMWs fractions. Phenolic compound Concentrations (mg/L) MF UF NF Hydroxytyrosol 187.1 ± 7.8 172.3 ± 2.8 152.5 ± 3.3 Tyrosol 121.2 ± 1.7 64.0 ± 1.1 49.7 ± 1.3 Caffeic acid 3.0 ± 0.5 2.5 ± 0.4 2.1 ± 0.4 Vanillic acid 5.9 ± 0.4 7.4 ± 0.6 3.3 ± 0.6 p-Coumaric acid 1.3 ± 0.1 1.1 ± 0.2 1.2 ± 0.3 Verbascoside 1457.0 ± 37.0 429.7 ± 7.1 158.4 ± 4.2 Isoverbascoside 87.3 ± 1.7 23.7 ± 3.2 8.6 ± 0.8 Oleuropein 2482.5 ± 23.9 1635.0 ± 91.3 1093.7 ± 22.3 Total 4345.0 ± 48 2335.7 ± 100 1469.5 ± 20.7 Phenolic compound Concentrations (mg/L) MF UF NF Hydroxytyrosol 187.1 ± 7.8 172.3 ± 2.8 152.5 ± 3.3 Tyrosol 121.2 ± 1.7 64.0 ± 1.1 49.7 ± 1.3 Caffeic acid 3.0 ± 0.5 2.5 ± 0.4 2.1 ± 0.4 Vanillic acid 5.9 ± 0.4 7.4 ± 0.6 3.3 ± 0.6 p-Coumaric acid 1.3 ± 0.1 1.1 ± 0.2 1.2 ± 0.3 Verbascoside 1457.0 ± 37.0 429.7 ± 7.1 158.4 ± 4.2 Isoverbascoside 87.3 ± 1.7 23.7 ± 3.2 8.6 ± 0.8 Oleuropein 2482.5 ± 23.9 1635.0 ± 91.3 1093.7 ± 22.3 Total 4345.0 ± 48 2335.7 ± 100 1469.5 ± 20.7 View Large To evaluate whether OMWs were able to inhibit the growth of X. fastidiosa Salento-1, a phenotype analysis was performed plating serial dilutions of the bacterium on plates supplemented with increasing quantities (1, 2.5, 5, 10% v/v) of OMWs-derived MF, UF and NF fractions. As positive control, the bacterial strain was plated on BCYE medium not containing any OMWs supplement (Fig. 1). The presence of OMWs in the growth medium clearly affected the X. fastidiosa growth even at the minimum quantities added to the medium (10 μL mL−1). However, reduction of death phenotype was observed only when UF and NF fractions were added at the minimum quantity (10 μL mL−1), even if, also in this case, the bacterium had a reduced growth with respect to the control (Fig. 1). Figure 1. View largeDownload slide Growth of X. fastidiosa subsp. pauca was inhibited by OMWs purified derived fractions. Xylella fastidiosa Salento-1 isolate was grown on BCYE medium (lane C) and on the same medium supplemented with 1, 2.5, 5, 10% (v/v) of OMWs MF fraction (lanes 1–4), UF fraction (lanes 5–8) and NF fraction (lanes 9–12). Cells were grown in BCYE medium to A600 ∼0.8 corresponding to 106 CFU/mL and then 10 μL of 105, 104, 103, 102 and 101 diluted strain was spotted onto the different media described above. Cells were incubated at 28°C for 20 days. Figure 1. View largeDownload slide Growth of X. fastidiosa subsp. pauca was inhibited by OMWs purified derived fractions. Xylella fastidiosa Salento-1 isolate was grown on BCYE medium (lane C) and on the same medium supplemented with 1, 2.5, 5, 10% (v/v) of OMWs MF fraction (lanes 1–4), UF fraction (lanes 5–8) and NF fraction (lanes 9–12). Cells were grown in BCYE medium to A600 ∼0.8 corresponding to 106 CFU/mL and then 10 μL of 105, 104, 103, 102 and 101 diluted strain was spotted onto the different media described above. Cells were incubated at 28°C for 20 days. Single phenolic compounds activities against Xylella In order to elucidate the specific role of more representative phenolic species reported to be associated with OMWs, susceptibility test assays were performed by the agar disk diffusion method. In addition to previous studies (Ribeiro et al.2008; Maddox, Laur and Tian 2010), other phenolic compounds were tested for the first time in this study against X. fastidiosa (Table 5). Isolate Salento-1 resulted to be not susceptible to several compounds at the two different tested concentrations (1 and 10 mM). The most active compounds resulted in the two simple phenols cathecol and its methylated form 4-methyl cathecol. Also caffeic acid and veratric acid showed to possess some inhibitory activity. Among polyphenols, oleuropein (at 10 mM concentration) revealed the strongest anti-Xylella activity, followed by luteolin-7-O-glucoside (flavonoid) and verbascoside. For all of these phenolic compounds, it was demonstrated that their inhibition activity against X. fastidiosa is due to a bacteriostatic effect. In fact, the bacterium was able to recover its ability to grow when it was transferred from the inhibition halo region in the plate containing each of the tested phenolic compounds in a fresh BCYE medium not containing any of them (Table 5). Table 5. Susceptibility in vitro of X. fastidiosa Salento-1 to phenolic compounds. Phenolic compound Familya Inhibition zone (mm) Reference Concentration on disk 1 mM 10 mM Monophenol Gallic acid PA 0 0 Di Gioia et al. (2001, 2002) 2,6-Dihydroxybenzoic acid PA 0 0 Di Gioia et al. (2001, 2002) 4-Hydroxybenzoic acid PA 0 0 Di Gioia et al. (2001, 2002) Syringic acid PA 0 0 Di Gioia et al. (2001, 2002) Vanillic acid PA 0 0 Di Gioia et al. (2001, 2002) Veratric acid PA 1.7 ± 0.1a 4.7 ± 0.2a Di Gioia et al. (2001, 2002) Cathecol SP 2.5 ± 0.5a 11 ± 0.6a Casa et al. (2003) 4-Methyl cathecol SP 3.6 ± 0.5a 12 ± 0.3a Casa et al. (2003) Tyrosol SP 0 0 Casa et al. (2003) Caffeic acid CA 1.2 ± 0.2a 5.7 ± 0.4a Di Gioia et al. (2001, 2002) p-Coumaric acid CA 0 0 Bianco et al. (2003); Casa et al. (2003) Ferulic acid CA 0 0 Tafesh et al. (2011) t-Cinnamic acid CA 0 0 Di Gioia et al. (2001, 2002) 4-Hydroxyphenylacetic acid PAA 0 0 Di Gioia et al. (2001, 2002) Polyphenol Luteolin-7-O-glucoside F 1 ± 0.2a 2.7 ± 0.2a Servili et al. (1999); Tafesh et al. (2011) Quercetin F 0 0 Leouifoudi, Harnafi and Zyad (2015) Rutin hydrate F 0 0 Servili et al. (1999); Mulinacci et al. (2001) Oleuropein Ester of elenolic acid glucoside with hydroxytyrosol 0 3.8 ± 0.2a Servili et al. (1999); Garcia-Castello et al. (2010) Verbascoside Ester of caffeic acid with hydroxytyrosol 1.8 ± 0.1a 1.8 ± 0.1a De Marco et al. (2007); Tafesh et al. (2011) Phenolic compound Familya Inhibition zone (mm) Reference Concentration on disk 1 mM 10 mM Monophenol Gallic acid PA 0 0 Di Gioia et al. (2001, 2002) 2,6-Dihydroxybenzoic acid PA 0 0 Di Gioia et al. (2001, 2002) 4-Hydroxybenzoic acid PA 0 0 Di Gioia et al. (2001, 2002) Syringic acid PA 0 0 Di Gioia et al. (2001, 2002) Vanillic acid PA 0 0 Di Gioia et al. (2001, 2002) Veratric acid PA 1.7 ± 0.1a 4.7 ± 0.2a Di Gioia et al. (2001, 2002) Cathecol SP 2.5 ± 0.5a 11 ± 0.6a Casa et al. (2003) 4-Methyl cathecol SP 3.6 ± 0.5a 12 ± 0.3a Casa et al. (2003) Tyrosol SP 0 0 Casa et al. (2003) Caffeic acid CA 1.2 ± 0.2a 5.7 ± 0.4a Di Gioia et al. (2001, 2002) p-Coumaric acid CA 0 0 Bianco et al. (2003); Casa et al. (2003) Ferulic acid CA 0 0 Tafesh et al. (2011) t-Cinnamic acid CA 0 0 Di Gioia et al. (2001, 2002) 4-Hydroxyphenylacetic acid PAA 0 0 Di Gioia et al. (2001, 2002) Polyphenol Luteolin-7-O-glucoside F 1 ± 0.2a 2.7 ± 0.2a Servili et al. (1999); Tafesh et al. (2011) Quercetin F 0 0 Leouifoudi, Harnafi and Zyad (2015) Rutin hydrate F 0 0 Servili et al. (1999); Mulinacci et al. (2001) Oleuropein Ester of elenolic acid glucoside with hydroxytyrosol 0 3.8 ± 0.2a Servili et al. (1999); Garcia-Castello et al. (2010) Verbascoside Ester of caffeic acid with hydroxytyrosol 1.8 ± 0.1a 1.8 ± 0.1a De Marco et al. (2007); Tafesh et al. (2011) aType of action: bacteriostatic. All data represent mean ± SD of three independent experiments. PA: phenolic acid; SP: simple phenolic; CA: cinnamic acid; PAA: phenilacetic acid; F: flavonoid (Obied et al.2005; Vermerris and Nicholson 2006). View Large DISCUSSION From 2013, several studies have been carried out in order to produce molecular and genetic characterization of X. fastidiosa from olive plants of Salento area, Apulia (Italy) (Saponari et al.2013, 2016; Cariddi et al.2014; Loconsole et al.2014; Giampetruzzi et al.2015, 2017a; Bleve et al.2016). Most interestingly, whole genome comparisons between strain CoDiRO and strains isolated from Costa Rica from coffee and oleander suggest that they all belong to the same clonal complex (Marcelletti and Scortichini 2016). Building evidence suggests that a single pathogen introduction of X. fastidiosa subsp. pauca, referred to in the literature as ST53 or CoDiRO, is the causal agent of OQDS in Apulia (EFSA PLH Panel 2016; Loconsole et al.2016; Baù et al.2017; Giampetruzzi et al.2017b). Nevertheless, no phenotypic and physiological description of this strain has been produced yet. In this study, for the first time, a preliminary in vitro susceptibility screening of X. fastidiosa subsp. pauca ST53, here represented by the isolate Salento-1, affecting olive plant in Salento area against different classes of antibacterial compounds is presented. The agar disk diffusion method was used to assess susceptibility/resistance of Salento-1 to a large number of antibiotics. This approach is routinely adopted in clinical microbiology laboratories for the testing of both common rapidly growing and some fastidious pathogenic bacterial species (Jenkins and Schuetz 2012). The data here reported can be useful for the physiological characterization of this isolate, though MIC and MBC breakpoints determinations are needed. In order to be able to precisely estimate these indices and to evaluate possible synergistic effects among different compounds, we are currently setting up the conditions necessary to grow X. fastidiosa in liquid culture. Two different amount of antibiotics on disk were used: the first one (1–5 μg) chosen according to the concentration range generally used in laboratory media for selecting bacteria in cell transformation strategies and the second one (100 μg) as the highest quantities suggested by the CLSI (2015) guidelines for disk diffusion quality control for fastidious organisms. This test was customized in order to be able to screen various antibiotics of different sources other than those commercially available. By these analyses, a specific profile was determined for Salento-1 (Table 1) suitable for future studies directed to the determination of specific resistance mechanisms and possibly to elucidate if these traits are intrinsic features, a result of mutations or due to acquirement of exogenous resistance genes (Hogan and Kolter 2002; Normark and Normark 2002). The use of antibiotic susceptibility testing as one of the specific physiological traits for strain characterization was demonstrated by the study of Lacava et al. (2001). Data deriving from previous genetic analyses suggested that X. fastidiosa isolates from Italy and Costa Rica belong to an atypical pauca (Giampetruzzi et al.2017b). Indeed in our study we found some phenotypic traits that support this hypothesis. According to Schaad et al. (2004), subspecies fastidiosa can be distinguished from subspecies pauca and multiplex in relation to a differential sensitivity to penicillin and carbenicillin: subspecies pauca and multiplex are highly sensitive to penicillin and exhibit low susceptibility to carbenicillin; subspecies fastidiosa shows low and medium sensitivity to penicillin and carbenicillin, respectively. On the basis of our preliminary results, Salento-1 seems to be highly sensitive to both carbenicillin and penicillin and it shows resistance to kanamycin, similar to strains from different hosts (Chang and Schaad 1982; de PMA Ribeiro, de TF Dellias and Tsai et al.2005). As far as fungal toxin assayed, ophiobolin A was the first member of the group of ophiobolins, secondary metabolites belonging to the family of sesterterpenoid compounds produced by phytopathogenic fungi, mainly of the genus Bipolaris, to be isolated and characterized in the mid-1960s (Nozoe 1965). Currently, more than 25 biogenic ophiobolins have been identified (Au, Chick and Leung 2000), including marine-derived fungal ophiobolins (Arai Niikawa and Kobayashi 2013). It is reported to possess a number of biological activities, including changes in cell membrane permeability, stimulation of β-cyanin leakage, protein and nucleic acid synthesis (Au, Chick and Leung 2000). Although the direct practical use of ophiobolin A would probably be not feasible due to its toxicological properties, new ophiobolins isolated by other fungal strains, or new derivatives obtained starting from the natural compounds, having a lower undesired toxicity or stronger bactericidal properties, or even nanotechnologically improved ophiobolins could deserve further attention. Gliotoxin is a potent metabolite belonging to the epipolythiodioxopiperazine (ETP), a class of toxins produced by several fungi, e.g. Trichoderma, Gladiocladium, Aspergillus and Penicillium species. It is known since long time (Brian and Hemming 1945), as well as its antifungal and antibacterial properties. Its total synthesis has been established long time ago too (Fukuyama and Kishi 1976). For the first time, in this study, indications about the effectiveness in vitro of ophiobolin A and gliotoxin against the bacterium X. fastidiosa have been reported, even if further studies need to be developed. Members of the genus Trichoderma are among the most widely utilized biocontrol agents of plant diseases (Woo et al.2014). Trichoderma spp. owe their success in competing with other microorganisms in a variety of environments to their metabolic versatility and to the ability to produce an array of bioactive metabolites that act as chemical weapons within ecological interactions with other organisms, particularly microorganisms (Reino et al.2008). Trichoderma are also able to establish intimate, anatomic connections with plants, so that they have been defined ‘opportunistic avirulent plant symbionts’ (Harman et al.2004). The results herein shown demonstrate for the first time that Trichoderma metabolites, still unidentified to date, are highly active against X. fastidiosa, and a possible application of Trichoderma strains or their bioactive compounds against X. fastidiosa can be conceived and envisaged. In vitro antibacterial activity of OMW-derived fractions was for the first time demonstrated against X. fastidiosa also at the minimum tested concentration of 1% (v/v). The most active fraction in terms of inhibitory activity against X. fastidiosa resulted in the MF probably due to its highest total phenolic content (Table 4). When some phenolics were administered to X. fastidiosa Salento-1, they significantly affected its in vitro growth. These data confirmed the antibacterial activities of plant phenolics and plant extracts against a wide range of Gram-positive and Gram-negative bacteria (Taguri, Tanaka and Kouno 2006). In particular, single phenolic compounds, representative of phenolic species present in OMWs (simple phenolics, phenolic acids, cinnamic acids, phenilacetic acids, phenylpropanoids, secoiridoids and flavonoids), were tested for their antibacterial activities. Among phenolic acids, only veratric acid (3,4-dimethoxybenzoic acid) characterized by two methoxy (–OCH3) substitutions, was able to inhibit growth of X. fastidiosa with a bacteriostatic effect (Table 5). Moreover, the simple phenols cathecol and 4-methyl cathecol showed the highest bacteriostatic activity against X. fastidiosa Salento-1. Caffeic acid was the only cinnamic acid showing antibacterial activity against the pathogen. The phenolic species that resulted to be effective in inhibiting X. fastidiosa growth belong to ortho-dihydroxylated simple phenols (cathecol and 4-methyl cathecol); meta- and para-dimethoxylated phenolic acids (veratric acid); and meta- and para-dihydroxylated cinnamic acids (caffeic acid). Hydroxytyrosol was one of the most represented compounds in OMWs, but it was not considered in this study because of its high cost that would render very difficult any practical application. Some of the results on efficacy of specific phenolic compounds against X. fastidiosa observed in this study, in particular data obtained using cathecol and caffeic acid, are in accordance with previous results reported by Maddox, Laur and Tian (2010) for Temecula and Conn Creek (grape), Dixon and Tulare (almond) strains. Indeed, also on X. fastidiosa Salento-1, cathecol revealed a strong inhibitory activity although this was observed at a concentration higher than that reported by Maddox, Laur and Tian (2010). Different results were observed for other phenolic compounds in terms of ability to counteract bacterial growth. In our experimental conditions, X. fastidiosa Salento-1 growth was not inhibited by ferulic, p-coumaric and gallic acids that were previously reported to be able to affect the growth of Temecula, Dixon, Conn Creek and Tulare strains (Maddox, Laur and Tian 2010). These observed differences probably depend on the different experimental conditions used, although it cannot be excluded that they could depend on strain-specific bacterial responses. The results obtained using flavonoids (luteolin-7-O-glucoside), secoiridoids (oleuropein) and phenylpropanoid (verbascoside) also showed some inhibitory activity against X. fastidiosa Salento-1. The potential use of phenolic compounds as bactericides against this pathogen, in particular flavonoids, coumarins, terpenoids, phenolic acids and alkaloids, present in several plant species, was proposed for CVC-affected trees (Ribeiro et al.2008). It is also important to consider that the inhibitory activity of a complex phenolic mixture against X. fastidiosa could result enhanced thanks to a synergistic effect. The relationship between phenolic structure and antibacterial activity and the mechanism for X. fastidiosa inhibition are still unknown. However, we are planning to test the efficacy of these anti-Xylella compounds (OMWs, single phenolics and microbial toxins) by administering them by fertigation or by leaf spraying to artificially infected plants and measuring the plant uptake and the possible efficacy of the treatment on X. fastidiosa counts and on symptoms reversion. Another interesting perspective is to immobilize some of these compounds in nanoparticles able to deliver them and/or to allow a controlled slow release inside the plant, in order to set up a new concept of long-term prevention and/or treatment of plants. Results obtained in this study represent the first description of some of the chemotypic features of the outbreak strain of X. fastidiosa subsp. pauca phylotype ST53, causing OQDS in Apulia. Acknowledgements The authors are very grateful to Federazione Coldiretti Lecce for helpful collaboration. We wish also to thank Regione Toscana for supporting in part this study. FUNDING This study was in part supported by ‘Tecnologie abilitanti per produzioni agroalimentari sicure e sostenibili-TAPASS’ project Nr. 516 [Regione Puglia FSC 2007–2013 ‘Cluster Tecnologici Regionali 2014’]. Conflict of Interest. None declared. REFERENCES Agüero CB, Uratsu SL, Greve C et al. 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Published: Mar 1, 2018
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