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Journal of Plant Pathology (2011), 93 (3), 561-568
Edizioni ETS Pisa, 2011
561
INHIBITION OF OLIVE KNOT DISEASE BY POLYPHENOLS EXTRACTED FROM OLIVE MILL WASTE WATER S. Krid1,2, M. Bouaziz3, M.A. Triki2, A. Gargouri1 and A. Rhouma2 1
Laboratoire de Génétique Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax BP 1177, 3038 Sfax, Tunisia 2 Unité de Recherche Protection des Plantes Cultivées et Environnement, Institut de l’olivier, BP 1087, 3038 Sfax, Tunisia 3 Laboratoire des Bioprocédés, Centre de Biotechnologie de Sfax, BP 1177, 3038 Sfax, Tunisia
SUMMARY
Olive mill waste water (OMW) and phenolic extracts, used at different concentrations in vitro displayed a high level of antibacterial activity against Pseudomonas savastanoi pv. savastanoi, the causal agent of olive knot disease. In in planta experiments, phenolic compounds used at three concentrations (1000, 500 and 100 mg l-1) completely inhibited the formation of knots on twigs inoculated with pathogenic strains IVIA 1628 and Aw9. GC/MS analysis revealed the presence of hydroxytyrosol in high concentration, in addition to tyrosol, catechol, caffeic acid and p-coumaric acid, suggesting their antibacterial effect. Copper treatment was less effective than phenolic compounds since the inhibition percentages of knot formation were 6% and 12% for IVIA1628 and Aw9, respectively. These results show that phenolic compounds extracted from OMW and rich in hydroxytyrosol can be excellent substitutes of copper compounds for controlling olive knot disease. Key words: Pseudomonas savastanoi pv. savastanoi, hydroxytyrosol, inhibition, olive knot disease, olive mill waste water.
INTRODUCTION
Olive mill waste water (OMW) generated by the olive oil extraction process is becoming a serious environmental problem, especially for Mediterranean countries where most of the world olive oil production takes place (Khatib et al., 2009). OMW is known to have antifungal, antibacterial and phytotoxic properties (Paredes et al., 1987; Rodriguez et al., 1988; Özdemir, 2009) due to the high content of phenolics (Aziz et al., 1998) and other compounds (González et al., 1990; Capasso et al., 1992). Phenolic extracts from OMW were reported to exert antioxidant, hypoglycemic (Vassiliki et al.,
2005; Hamden et al., 2009), hypocholesterolemic (Feki et al., 2007) activities and to be active against Alternaria solani, Fusarium sambucinum and Verticillium dahliae (Yangui et al., 2009, 2010). In addition, phenolic compounds efficiently controlled some bacterial diseases such as crown gall caused by Agrobacterium tumefaciens (Yangui et al., 2008), black rot of crucifers caused by Xanthomonas campestris (Cifardini and Zullo, 2003), bacterial canker caused by Clavibacter michiganensis subsp. michiganensis and bacterial speck caused by Pseudomonas syringae pv tomato (Özdemir et al., 2009). Olive knot, caused by P. savastanoi pv savastanoi (Pss), is as a serious diseases of olive (Olea europaea L.) whose characteristic symptoms are parenchymatous galls (called knots) occurring mainly on twigs and branches, occasionally also on the leaves and fruits (Surico, 1986). Knots develop as a reaction of the plant under the influence of several bacterial virulence factors, including indole-3-acetic acid (IAA) (RodríguezMoreno et al., 2008; Matas et al., 2009), cytokinins (Surico et al., 1985) and the biosynthesis of a functional type III secretion system (TTSS) encoded by the hrp/hrc gene clusters (Sisto et al., 2004). Pss invades the host through the vascular system (Rodríguez-Moreno et al., 2009) and causes severe damage including a reduction in olive yield and quality (Schroth et al., 1968, 1973). Control of olive knot is difficult. Actually, only products based on copper compounds are available to this aim (Lavermicocca et al., 2002; Kacem et al., 2009). However, their toxicity and the possibility of resistance acquisition to copper by the pathogen incite the development of alternative control methods (Kacem et al., 2009). In this study, we analysed in vitro the effect of OMW and polyphenols present in OMW on the growth of Pss. The effect of polyphenols on olive knot symptom expression was verified in planta. To our knowledge, this is the first report on the use of polyphenols to control olive knot.
MATERIALS AND METHODS Corresponding author: S. Krid Fax: +216.74.241442 E-mail:
[email protected]
Origin of OMW. OMW, stored in the dark for 6 months at 25°C, came from a three-phase continuous
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extraction oil mill located in the region of Sfax (southern Tunisia). The physical and chemical characteristics of OMW were: pH 4.9, electrical conductivity 9 mS cm-1, chemical oxygen demand (COD) 89 g l-1, total organic carbon (TOC) 25 g l-1, total polyphenols 5.5 g l-1, K 5.7 mg l-1, Fe 3 mg l-1, P 6 mg l-1 C 15.2 mg l-1and Na 220 mg l-1. Bacterial strains. Two strains of Pss (Aw9 and IVIA 1628) were used in this study. IVIA 1628 was supplied by the Centro de Proteccion Vegetal y Biotecnología, IVIA, Valencia (Spain), whereas Aw9 was isolated from olives of cv. Chemlali grown in the region of Ouedna (south-eastern Tunisia). Bacterial strains were routinely streaked on King’s medium B [KB (20 g peptone; 1.5 g K2HPO4, 3H2O; 1.15 g MgSO4, 7H2O; 15 ml glycerol; 20 g agar; 1 l distilled water)] and incubated for 48 h at 26°C. Purified colonies were stored in KB supplemented with 20% glycerol at -20°C. Bacterial strains were identified by sequencing the 16S rRNA gene (rrs) and the sequences were compared to those from GenBank with the BLAST program. Pathogenicity of the strains was assessed by inoculation of a bacterial suspension (108 CFU ml-1) into the stems of 1-year-old cv. Chemlali trees. Three olive trees were used in each inoculation experiment. Symptom development was followed for up to 2 months post inoculation and was rated by visual observation of knot formation. Effect of OMW on multiplication of Pss cells. Bacterial growth was tested under different concentrations of OMW, viz 0.5, 1, 3, 5 and 10% (v/v) added to KB medium at 45°C before pouring on Petri dishes. After solidification, plates were inoculated by streaking a colony from a young pure culture of Pss (48 h) and incubated at 26°C for 2 days. Controls consisted of inoculated KB plates without OMW addition. Antibacterial activity of total polyphenols extracted from OMW. Polyphenols extraction and analysis. OMW was centrifuged at 8,000 rpm for 20 min. The supernatant was extracted with 1/2 vol. ethyl acetate. The organic layer was collected and reduced to 10 ml by rotary evaporation (40°C). The extract was then stored immediately at -20°C to avoid auto-oxidation and subsequent polymerisation of the phenolic compounds. The concentration of total phenolics was measured according to Ryan et al. (1999). Briefly, an aliquot (1 ml) of appropriately diluted extracts or standard solutions of tannic acid (0, 1.2, 2.4, 3.6, 4.8 and 6 mg l-1) was added to a 50 ml volumetric flask containing 35 ml of double-distilled water (ddH2O). A reagent blank consisted of pure ddH2O. A volume of 2.5 ml of Folin Ciocalteu’s phenol reagent was added to the mixture and shaken. After 3 min, 5 ml of 6% Na2CO3 solution was added with mixing. The solution was then immediately
Journal of Plant Pathology (2011), 93 (3), 561-568
diluted to volume (50 ml) with ddH2O and mixed thoroughly. After incubation for 90 min at room temperature, the absorbance versus the ddH2O blank was read at 725 nm. A total phenolic content was expressed as mg tannic acid equivalent per litre of OMW. The sample was analyzed in three replications. GC-MS analysis was performed with a HP model Hewlett-Packard 6980/HP5973MSD, equipped with a capillary HP5MS column (30 m length, 0.32 mm internal diameter, 0.25 mm film thickness). The carrier gas H2 was used at 1 ml min-1 flow rate. The oven temperature program was as follows: 2 min at 100°C, from 100 to 280°C at 5°C min-1 and 5 min at 280°C. For the silylation procedure, a mixture of 40 µl pyridine and 200 µl BSTFA [bis(trimethylsilyl) trifluoroacetamide (Promega, France)] was added to 200 µl of ethyl acetate extract and vortexed in screw cap glass tubes, then placed in a water bath at 80°C for 45 min. One µl of the silylated mixture was directly analyzed by GC-MS. The constituents of the extract were identified by matching their mass spectra with Wiley and NIST library data and pure standards. Antimicrobial activity of phenolic and their effect on bacterial viability. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined by the NCCLS (2000) broth dilution method. Phenolic compounds were twofold serially diluted in liquid KB medium. Duplicate tubes of each dilution (10, 5, 2.5, 1.25 and 0.625 mg l-1) were inoculated with about 106 CFU ml-1 of the bacterial culture in the exponential phase of growth. The tubes were incubated at 26°C for 18 h. MIC was retained as the highest dilution (least concentration) of phenolic extract showing no detectable growth. MIC determination by the broth dilution method was converted to determine the minimum bactericidal concentrations (MBC) by sub-culturing all tubes that showed no visible growth on KB medium plates. MBC was retained as the lowest concentration of phenolic extract killing more than 99.9% of bacteria. The effect of phenolic extract on bacterial growth of Pss was also investigated using the agar-well diffusion method (Tagg and Given, 1971). Different concentrations of total polyphenols ranging from 20 mg l-1 to 1000 mg l-1 were prepared. The cell suspension of the target bacterial strain to be tested, was prepared in sterile distilled water (1 ml) from a culture grown on KB plates for 48 h and adjusted to OD600 = 0.5 (corresponding to about 108 CFU ml-1), then mixed with 3 ml of KB (0.6% agar) at 45°C and overlain to LB medium (10 g tryptone, 5 g yeast extract, 5 g NaCl, 20 g agar). After cooling, wells of 8 mm in diameter were punched in the agar with a sterile steel borer. A volume of 40 µl of phenolic compounds and controls (ethyl acetate and CuSO4 at 1%) tested, was filled separately in the wells. The inoculated plates were incubated for 24 h at 26°C. Each experiment was repeated at least three times and the di-
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ameter of the inhibition zone was measured with a calliper. The viability of Pss cells exposed to the phenolic extract was examined according to Lavermicocca et al. (2002). Strain IVIA 1628 and Aw9 were grown overnight with shaking in KB broth. One milliliter of the suspension adjusted to OD600=0.5 (108 CFU ml-1) was inoculated in each 15 ml flask containing 9 ml of fresh medium with 0, 1, 5 and 10 mg l-1 of phenolic extract or copper sulphate [final concentration, 0.05% (wt/vol)]. The number of viable bacterial cells was determined at different intervals on KB medium agar plates. Each essay was performed in duplicate in three separate experiments. Phenolic extract suppressive effect on olive knot. In vivo assays were performed to determine the effect of polyphenols extracted from OMW on the development of knots. A 10 µl aliquot of bacterial suspension containing 108 CFU ml-1 of strain IVIA 1628 or Aw9 was slit inoculated into stems of 2-year-old cv. Chemlali olive. The slits (ca. 2-3 mm wide and deep) were protected by parafilm. Three days after inoculation, the parafilm was removed and 10 µl of phenolic extract at concentration of 1000, 500 and 100 ppm or copper sulphate (0.5%) was added to the wounds and covered again with parafilm. Phenolic extract was added also to uninoculated wounds as negative control. The experiment was repeated twice. For each treatment, three inoculations were performed on the stem of three plants. Inoculated and treated plants were kept in a greenhouse at 25°C with a 16 h photoperiod. After 2 months, knots (when present) were excised and their weights compared. The percentage of inhibition was
Krid et al.
calculated by counting sites that did not develop knots with respect to the total of inoculated not treated sites. Data analysis. Data were subjected to analysis of variance using the SPSS software (version 11). Mean values among treatments were compared by Duncan’s multiple range test at the 5% (P=0.05) level of significance.
RESULTS
In vitro effect of olive mill waste water and phenolic extracts. The incorporation of 10% OMW into the culture medium resulted in a strong antibacterial activity against Pss, i.e. a complete inhibition of cell multiplication (Fig. 1). However, the doses of 0.5, 1, 3 and 5% were less efficient in preventing cell multiplication and a slow growth of the bacteria was observed (not shown).
Fig. 1. Effect of OMW on P. savastanoi pv. savastanoi IVIA 1628. A Bacterial growth in the control (no OMW). B Growth inhibition at the dosage of 10%.
Table 1. Diameter of inhibition zones of P. savastanoi pv savastanoi treated with different concentrations of phenolic extract. Diameter of inhibition zones (mm) Treatment mg tannic acid eq (20 mgl-1)
IVIA 1628
AW9
1.16±0.15
1.12±0.10
-1
6.55±0.16
6.53±0.11
-1
mg tannic acid eq (60 mgl )
7.65±0.26
7.42±0.08
mg tannic acid eq (80 mgl-1)
8.85±0.07
7.73±0.08
mg tannic acid eq (100 mgl-1
mg tannic acid eq (40 mgl )
12.5±0.08
10.31±0.15
-1
24.47±0.10
22.18±0.07
-1
mg tannic acid eq (500 mgl )
30.3±0.15
27.12±0.18
mg tannic acid eq (700 mgl-1)
33.3±0.18
32.34±0.1
mg tannic acid eq (1000 mgl-)))))1mg/l)
35.5±0.27
35.18±0.07
Ethyl acetate
0
0
Copper sulfate (1%)
5.08±0.25
5.11±0.07
mg tannic acid eq (300 mgl )
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Journal of Plant Pathology (2011), 93 (3), 561-568
Fig. 2. Growth inhibition of P.savastanoi pv. savastanoi IVIA 1628 by 1000 mg l-1 phenolic extract (a) and 1% CuSO4 (b).
The results with OMW were confirmed by the effect of the extracted polyphenols that caused an inhibition zone of 35.5±0.27 mm and 35.18±0.07 mm at an extract concentration of 1000 mg l-1 for strain IVIA 1628 and Aw9, respectively (Table 1, Fig. 2). Phenolic extracts exhibited antibacterial activity and showed the same MIC and MBC (MIC=MBC=5 mg l-1) on both IVIA 1628 and Aw9 strains. The GC-MS analysis of the ethyl acetate extract carried out for identifying the phenolic compounds responsible for these activities disclosed the presence of 3,4-dihydroxyphenylethanol (hydroxytyrosol), 4-hydroxyphenylethanol (tyrosol), p-coumaric acid, catechol and caffeic acid. Hydroxytyrosol and tyrosol were the major compounds (Fig. 3). The main phenolic compounds identified and the obtained mass fragments (Table 2) agreed with those described previously (Bouaziz et al., 2005). Bactericidal effect of total polyphenols. The addition of 1 mg l-1 (
