Antifungal activity of pomegranate peel extract against fusarium wilt of tomato
Domenico Rongai, Patrizio Pulcini, Barbara Pesce & Filomena Milano
European Journal of Plant Pathology Published in cooperation with the European Foundation for Plant Pathology ISSN 0929-1873 Eur J Plant Pathol DOI 10.1007/s10658-016-0994-7
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Author's personal copy Eur J Plant Pathol DOI 10.1007/s10658-016-0994-7
Antifungal activity of pomegranate peel extract against fusarium wilt of tomato Domenico Rongai & Patrizio Pulcini & Barbara Pesce & Filomena Milano
Accepted: 22 June 2016 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2016
Abstract Pomegranate (Punica granatum) is an important source of bioactive compounds and has been used in folk medicine for many centuries. This paper describes the in vitro antifungal activity of pomegranate peel aqueous extract (pae) on the development of Fusarium wilt of tomato caused by Fusarium oxysporum, f. sp. lycopersici. HPLC-DAD-ESI/MS analysis was performed to identify punicalagins and ellagic acid, which are the main antifungal compounds. In vivo tests established the efficacy of pae treatments in controlling Fusarium wilt by evaluating improvements in growth variables of tomato plants. At high concentrations, pae showed allelopathic activity in tomato plants. The germination and the radicle growth of tomato seeds were significantly affected by pae. Increasing the extract concentration led to a progressive decrease in germination and in the length of the radicle. The reduction of the Fusarium population in soil and the increase in number of healthy plants obtained as a result of pae treatments indicate that this plant extract could have an important role in biologically-based management strategies for the control of Fusarium wilt in tomato crops.
Keywords Fusarium . Plant extract . Antifungal activity . Natural fungicide D. Rongai (*) : P. Pulcini : B. Pesce : F. Milano Consiglio per la Ricerca in Agricoltura e l’analisi dell’Economia Agraria, Centro di ricerca per la patologia vegetale, via C.G. Bertero, 22 -, 00156 Rome, Italy e-mail:
[email protected]
Introduction Tomato (Solanum lycopersicum) is an important crop in Italy and the fungus Fusarium oxysporum, f. sp. lycopersici is a major cause of economic loss in tomato production. The fungus is a soil-borne pathogen that causes vascular wilt in tomato by infecting plants through the roots. It is controlled by using resistant tomato cultivars, crop rotations, and fumigants or fungicides. In an attempt to reduce reliance on synthetic fungicides, alternative methods to control F. oxysporum have been tested using naturally-occurring compounds derived from plant sources (Bowers and Locke 2000; Tegegne et al. 2007; Hassanein et al. 2010; Parvu et al. 2011). For example, Singha et al. (2011) reported that betel (Piper betle) extract reduced Fusarium wilt of tomato in vivo. A 1 % (w/w) extract of P. betle was more efficient in reducing the Fusarium population in soil when compared with carbendazim, a commercial fungicide. Bioactive compounds extracted from the creosote bush (Larrea tridentate) and pomegranate (Punica granatum) were efficacious against the soil pathogens Pythium sp. and F. oxysporum (Osorio et al. 2010). Tehranifar et al. (2011) reported that the phenolic content of pomegranate peel extract was 2.8 fold higher compared to leaf extract and the high concentrations probably explained the antifungal activity of the extract. In a previous study, we described the antifungal activity of botanic extracts on the development of F. oxysporum (Rongai et al. 2012). Of extracts of 500
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plant species tested, approximately 84 % showed no inhibition, 13 % showed low inhibition, and only 3 % (including P. granatum) demonstrated a high antifungal activity. The aim of this research was to compare the antifungal activity of P. granatum peel extracts on the mycelial growth of F. oxysporum and identify the main antifungal compounds (punicalagins and ellagic acid) through a simplified HPLC-MS-MS analytical method (Fischer et al. 2011). In addition, in vivo assays were used to assess the efficacy of treatments of pomegranate peel extracts in reducing vascular wilt in tomato.
membrane filters. The solvents were evaporated using a rotary evaporator (Büchi R-210 Rotavapor, Flawil, Switzerland). The final extracts were maintained at −80 °C for 24 h, and freeze-dried for 48 h. The peel aqueous extract (pae) obtained was purified by solid phase extraction (SPE) using a BPreppy Vacuum Manifold^ (12-Port Model, Sigma-Aldrich, Milan, Italy) and SPE tubes (LC-Florisil, 2 g/12 mL). The purified pomegranate peel aqueous extract (ppae) obtained was stored in a freezer at −20 °C until required. Using this technique, 0.1 g of concentrated extract was obtained from 1 g of pomegranate powder extract.
Materials and methods
Total phenolic and flavonoid content, acidity and pH analysis
Plants material and fungal isolates Fruit of P. granatum, var. Dente di Cavallo, were collected from CRA-FRUT (Fruit Tree Research Center, via di Fioranello, Rome, Italy) orchards (latitude 41°47′ 19″ N; longitude 12°34′03″ E), located in Rome, Italy. An isolate of F. oxysporum f. sp. lycopersici from tomato plants held in the CRA-PAV (Plant Pathology Research Center, via C. G. Bertero, Rome, Italy) collection (Isolate No. ER1372) was used in these studies. The fungus was maintained on potato dextrose agar (PDA, Oxoid CM 0139, Basingstoke, England) and stored at 4 °C. When inoculum was required, the isolate was subcultured on potato dextrose agar (PDA) and grown in the dark at 25 ± 2 °C for 7–10 days. Preparation of powder extracts Extracts were prepared from 50 g of pomegranate peel, cut into small pieces and added to 500 mL of solvent: water (bidistilled water from a Milli-Q-System, Millipore, Bedford, UK), ethanol, methanol, propanol (analytical grade RPE, Carlo Erba Reagents, Milan, Italy). The solvents were used separately and mixed together (WPEM): water 25 %, propanol 25 %, ethanol 25 % and methanol 25 %. The mixture (solvent and pomegranate pieces) was agitated overnight on a magnetic stirrer at 40 °C, before sonicating at 80 % amplitude for 3 min (3 s on and 7 s off) (Ney 300 Ultrasonic Bath, New York, USA) and centrifuging at 5000 rpm for 10 min at room temperature (Allegra 21 R, Beckman Coulter, Milan, Italy). The supernatant was filtered through 0.22 μm polytetrafluoroethylene (PTFE)
Total phenolic content (TPC) of pae was determined using Folin-Ciocalteu reagent, according to Slinkard and Singleton (1977). The measurement was taken using a spectrophotometer (Varian Cary 100 Conc UVVisible, Santa Clara, CA, USA) at λ = 760 nm against a blank. Gallic acid was used as the standard phenolic compound for the calibration curve (0 and 500 mg L−1, R2 = 0.99). The results were expressed as gallic acid equivalent (GAE) in g of dry weight of lyophilized plant extracts. Total flavonoid content (TFC) was estimated using the AlCl3 method (Nongalleima et al. 2013). TFC was expressed as the rutin equivalent (mg L−1) of the extract and a calibration curve (0 and 500 mg L−1, R2 = 1.00) was generated. Acidity was determined by titration with a 0.01 N alkaline sodium hydroxide solution using phenolphthalein (1 %) as the indicator, and was expressed in g L−1 citric acid. The pH value of each extract was determined with a Hamilton pH electrode sensor (Hanna Instruments, Italy). All measurements were repeated once within a period of 10 days. HPLC-DAD-ESI/MS analysis of pomegranate peel aqueous extract (pae) and purified pomegranate peel aqueous extract (ppae) The pae and ppae analyses were performed using a Micromass 4 μ triple quadrupole system (Waters Corp. Milford MA, USA), equipped with a Waters Alliance 2695 HPLC separation module, 2695 Alliance autosampler, Waters 2487 dual λ absorbance detector (UV-DAD) and a Waters 4 μ mass spectrometry detector (MSD) with an electro spray ionisation (ESI) interface, connected in series.
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The HPLC separation was carried out on a RP C18 analytical column (Phenomenex Gemini C18 150 × 46 mm 5 μm, Bologna, Italy), operated at 30 °C. The mobile phase consisted of 2 % (v/v) acetic acid in water (eluent A) and 2 % (v/v) acetic acid in methanol (eluent B). The flow rate was constant at 0.9 mL min−1 split for the MSD in a ratio of 7:3. The UV-DAD performed simultaneous detection at 280 nm and 320 nm. The Waters 4 μ triple quadrupole MSD data acquisition and processing were performed using MassLynx 4.1 software (Waters Corp. Milford, MA). The ESI interface operated in the Negative (ES-) using the MRM (Multiple Reaction Monitoring) method, monitoring two m/z mass transitions, the first for identification and the second for confirmation (Fischer et al. 2011) of the molecules investigated. Analytical standards of ellagic acid and punicalagins (SigmaAldrich, Milan, Italy) were used. In vitro tests Antifungal activity of pae and ppae was tested in 50 mm Petri dishes. A total of 5, 10, 15 or 20 mg of the powdered extract was added to 10 mL−1 of PDA as the media was about to gel (approx. 50 C°) such that the concentration corresponded to 0.5, 1, 1.5 and 2 % (w/v), respectively. PDAwith sterile water added was the control. In addition, a plate containing a standard fungicide (Marisan 50 PB, Dichloran 60 %, SIAPA, Milan, Italy) was used at the recommended concentration as a positive control to determine the effectiveness of the extracts by comparison to an industry standard. A 5 mm diameter plug of inoculum was taken from the actively growing margin of a colony of F. oxysporum and placed face down in the center of each Petri plate. There were four replicates of each treatment and the test was repeated twice. Plates were incubated at 25 °C, radial growth was measured each day, starting 3 days after inoculation until the plates were overgrown. Inhibition by each extract was calculated: % inhibition = colony diameter (mm) of control - colony diameter (mm) of sample / colony diameter (mm) of control × 100. In vivo experiments – control of fusarium wilt by pomegranate peel extracts To prepare the inoculum sterile distilled water was added to an 8-day-old culture of F. oxysporum, dislodging spores gently with a sterile glass rod. The
suspension was subsequently filtered through four layers of cheesecloth to remove the mycelia. The concentration of conidia was determined using a hemocytometer and adjusted to (1 × 106 spore mL−1). The conidia suspension (15 mL) was mixed with 150 g of soil (profit substrat - Gramoflor GmbH & Co. KG, Vechta, Germany). After a 7-day incubation period, the soil was treated by incorporating the pae at a concentration of 0.5 % (w/w). After an additional seven days of incubation, soil from each treatment was placed in four 10-cm-diameter standard plastic pots, and ten tomato seeds (var. Corbarino ISCI 05) were planted in the soil in each pot. Pots were placed randomly on a greenhouse bench (25 ± 2 °C, 70– 80 % relative humidity and 12 h photoperiod) and were watered from below as needed. Each treatment consisted of 40 seeds (10 seeds × 4 replicates) and included: a) non infested control (no F. oxysporum); b) F. oxysporum infested control; c) F. oxysporum infested soil treated with pae; and d) F. oxysporum infested soil treated with Marisan 50 PB fungicide at a concentration of 0.15 % (w/ v). Disease severity and growth variables of the tomato plants (shoot length, root length, fresh weight, dry weight) were recorded at four weeks after two to three true leaves had emerged. Disease severity was recorded on a 0–5 scale where: 0 = no symptoms; 1 = slight yellowing of one or two leaves; 2 = more extensive yellowing of basal and median leaves, with some leaves wilted; 3 = severe yellowing of leaves, 50 % of leaves wilted and growth inhibited; 4 = widespread symptoms, all leaves yellow, rot on roots, vascular discoloration in the stem, and severe stunting; 5 = dead plants. The percentage disease severity of each treatment was calculated by the formula: % disease severity = sum of all disease ratings × 100 / total number of ratings × maximum disease grade. Disease incidence was determined according to Song et al. (2004). Population density of F. oxysporum was determined before and after the four weeks of soil treatment. One g of soil from each pot was placed in 10 mL of sterile water and was serially diluted. One mL from the 4th dilution was pipetted onto Petri plates containing a selective substrate for F. oxysporum (Komada 1975). Plates were incubated at 25 ± 2 °C in the dark for six days, when colonies of F. oxysporum were counted. The average population density for the three replications per treatment at each assay date was calculated. The experiments were performed once in 2013 and repeated in 2014.
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Allelopathic activity of pomegranate peel aqueous extract (pae) Germination and root length of tomato seeds were assessed in Petri dishes (9 cm diameter) under aseptic conditions with four replications. Each dish contained three layers of filter paper (Whatman No. 1), 5 mL of extract at concentration of 0.25, 0.75, 1.5 and 2.5 % (w/ v) or 5 mL of distilled water as the control. Each Petri dish had 20 seeds. The germination data and root length were measured eight days after starting the experiment. The trial was repeated once. Statistical analysis Mycelial growth inhibition, soil population densities, shoot height, root length, fresh weight, dry weight, disease severity and disease incidence were analysed by ANOVA with means separation by Fisher’s protected LSD test at α = 0.05. Because the results from two independent experiments were consistent and there were no interactions between experiment run and treatments, data from two independent experiments were combined with experiment treated as a block term in the analysis. To correct for heterogeneity of variance, the data were arcsine-transformed prior to analysis. SigmaPlot V10 (London, UK) was used to create graphics and Sigma Stat V3.5 (London, UK) was used for ANOVA.
Results Chemical analyses of pomegranate peel aqueous extract (pae) The total phenolic and flavonoid contents of pae were 542.5 mg GAE g−1 DW and 102.8 mg RE g−1 DW, respectively. The total acidity was 1.376 meq NaOH g−1, while the pH was 4.08. The pae was also analyzed with HPLC-UV-ESI-MS to identify punicalagins and ellagic acid, the main components of P. granatum peel (Fig. 1). In vitro antifungal activity of P. granatum peel extract against F. oxysporum Five days after inoculation, the control plates had been entirely colonised by F. oxysporum while on the plates containing extracts, mycelia growth was inhibited by
62 % (propanol extract) to 78 % (water extract) (Fig. 2). There were no significant differences in mycelial growth inhibition among the solvents used for the extraction procedures. For example, at 3, 4 and 5 days after inoculation, there were no significant differences among water, ethanol, methanol and the WPEM extract, although the propanol extract had numerically slightly lower efficacy (Fig. 2). Consequently, the antifungal activity of only the pomegranate peel aqueous extract (pae) was tested further (Fig. 3). By increasing the concentration of the extract, inhibition increased significantly (F = 195.5, P < 0.001; F = 80.5, P < 0.001; F = 50.1, P < 0.001; F = 35.8, P < 0.001 at 3, 4, 5 and 6 days respectively). At a concentration of 1 %, fungal growth inhibition was 73 % on the fourth day and 64 % on the sixth day, which were statistically different compared with the control plates. In addition, on the plates with 2 % pae, fungal growth was completely inhibited at 3 and 4 days after inoculation (Fig. 3). Finally, at a concentration of 0.5 %, the fungicide was more effective than pae. The pae extract showed much lower inhibition percentages (28, 39 and 50 %) compared to the ppae extract (91, 82 and 83 %) on the third, fourth and fifth days, respectively (Fig. 4). The HPLC chromatograms of pae and ppae showed that ppae had much greater concentrations (higher peaks) of punicalagins. In vivo experiments – control of fusarium wilt by pomegranate peel extracts The ANOVA test for experiment and experiment × treatment interactions showed no significant effects, thus data were combined in the analysis. The inoculated control, had 5.65 log10 CFU g−1 F. oxysporum four weeks after treatments, which was significantly (F = 93.0, p < 0.001) higher than soil treated with fungicide (2.4 log10 CFU g−1) and pae (2.6 log10 CFU g−1) (Fig.5). There were no significant differences between the fungicide and pae. The growth variables of the tomato plants indicated that plants from the inoculated control were undersized compared to the plants from the non-inoculated control and plants from soil treated with pae (Table 1). Plants grown in soil treated with 0.5 % (w/ w) pae had longer shoot compared to diseased tomato plants from the inoculated control. The mean shoot height of plants in the inoculated control was 40.1 cm,
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Fig. 1 a HPLC- UV-DAD chromatogram of pomegranate peel aqueous extract (pae); b overlaid mass MRM chromatograms of punicalagins and ellagic acid, as identified in the pae; c overlaid
mass MRM chromatograms of punicalagin standards (10 mg L−1) and ellagic acid standard (2 mg L−1)
which was statistically (F = 10.4, P = 0.004) shorter compared with plants grown in soil treated with pae (45.7 cm). The beneficial effects of soil
treatment with pae were statistically significant for root length (F = 8.3, P = 0.008), fresh (F = 9.1, P = 0.006) and dry weight (F = 8.2, P = 0.008).
Author's personal copy Eur J Plant Pathol Methanol extract Ethanol extract Water extract Propanol extract WPEM extract Mycelial growth inhibition (%)
100 (F=32.75; P
