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Tropical Plant Pathology

Print version ISSN 1982-5676

Trop. plant pathol. vol.35 no.3 Brasília May/June 2010

http://dx.doi.org/10.1590/S1982-56762010000300001 

RESEARCH ARTICLE ARTIGO

 

Antifungal activity of eugenol against Botrytis cinerea

 

 

Chunmei WangI, II, III; Jie ZhangIV; Hao ChenI, II, III; Yongjian FanI, II, III; Zhiqi ShiI, II, III

IFood Safety Research and Service Institute, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, P.R. China
IIKey Laboratory of Food Safety and Quality of Jiangsu Province (State Key Laboratory Breeding Base), Nanjing 210014, P.R. China
IIIKey Laboratory of Food Safety and Management, Ministry of Agriculture, Nanjing 210014, P.R. China
IVGuabu High School in Jiangsu Province, Nanjing 211511, P.R. China

 

 


ABSTRACT

The antifungal properties of eugenol were tested against ten plant pathogenic fungal species and detailed studies were conducted regarding in vitro activity of eugenol on Botrytis cinerea. The EC50 value of eugenol on mycelial radial growth of B. cinerea was 38.6 μg/mL; however, eugenol had no bioactivity against conidia germination. B. cinerea hyphae treated with eugenol showed strong propidium iodide fluorescence in the cytosol. Eugenol increased the concentration of potassium ion and cellular materials in the medium. Furthermore, light and scanning electron microscopy observations on hyphae exposed to eugenol revealed considerable morphological alterations in hyphae, such as cytoplasmic coagulation, vacuolation, and hyphal shriveling. Eugenol induced the generation of H2O2 and increased free Ca2+ in the cytoplasm. These results strongly support the idea that the antifungal activity of eugenol is due to membrane binding and permeability alteration, leading to destabilization and disruption of the plasma membrane.

Keywords: natural compound; fungal diseases; mode of action; plasma membrane.


 

 

INTRODUCTION

Botrytis cinerea causes gray mold in a variety of fruits, vegetables, and field crops. The pathogen infects leaves, stems, flowers and fruits, and severe damage can occur due to gray mold epidemics (Soulie et al., 2003; Milena & Evelyn, 2005). The control of B. cinerea is still based upon multiple applications of fungicides during the flowering and fruiting periods. Currently, there is a worldwide trend to explore new alternatives to synthetic fungicides in order to minimize the risks associated with the development of populations insensitive to these chemical compounds (Elad, 1991; Yourman & Jeffers, 1999) and also to comply with food safety standards (Liu et al., 2007). Frequent applications of site-specific fungicides can result in the emergence of resistant strains of B. cinerea (Elad, 1991). Furthermore, the use of some synthetic chemicals to control fungal diseases is restricted due to their high toxicity, long degradation periods, and environmental pollution.

The use of natural compounds as plant extracts may be an alternative to fungicides to control plant pathogens (Tsair-Bor & Shang-Tzen, 2008). Eugenol (4-allyl-2-methoxyphenol) is a naturally occurring phenolic compound which is used as a food flavor and fragrance agent. It is a major component of clove oil and is also present in the essential oils or extracts of many other plants, including cinnamon, basil, and nutmeg (Ghosh et al., 2005). Eugenol has been reported to inhibit the growth of Escherichia coli, Listeria monocytogenes and Lactobacillus sakei (Blaszyk & Holley, 1998; Gill & Holley, 2004) and also exhibits antifungal activity against wood decay fungi (Tsair-Bor & Shang-Tzen, 2008). It has also been demonstrated that the compound induced morphological alterations in Candida albicans and Saccharomyces cerevisiae (Dalleau et al., 2007). However, there is little work on the effect of eugenol on plant pathogens and its mode of action is poorly understood.

The objective of this work was to assess the antifungal activity of eugenol against ten plant pathogenic fungi, more specifically how it affects B. cinerea in vitro, and to understand its mechanism of action by closely examining the interaction of this compound with fungal cell membrane. The results strongly support the idea that the antifungal activity of eugenol is due to the disruption of the membrane, leading to cell death.

 

MATERIALS AND METHODS

Fusarium moniliforme, Sclerotinia sclerotiorum, Cercospora beticola, Mycogone perniciosa, Phytophthora capsici, Fusarium graminearum, Macrophoma kawatsukai, Thanatephorus cucumeris, Alternaria alternata, and B. cinerea were originally isolated from diseased plants and isolates were maintained on potato dextrose agar (PDA) in the dark at 25ºC. A 6-mm diameter agar plug containing actively growing fungus was obtained from the edge of the colony of each isolate and placed on potato dextrose agar medium (PDA) in plates containing different concentrations of eugenol (0, 25, 50, 100, 150, and 200 μg/mL). Eugenol (99.0 %) used in this study was purchased from Sigma-Aldrich (Shanghai, Trading Co. Ltd. China) and it was prepared as a stock solution at 40 mg/mL in 70 % ethanol, and stored in the dark at 4ºC. Plates in three replicates were used for each treatment, and all plates were placed in an incubation chamber at 25ºC for 3 to 10 days. When mycelial growth on the control plate reached more than 2/3 of the total diameter of the plate, mycelial radial growth was measured and activity was expressed as EC50 (the concentration inhibiting growth by 50%). The EC50 value was calculated according to the relationship of eugenol concentrations and inhibition rate of mycelial growth (Taylor et al., 2002). First, the inhibition rate was transformed to probability value (Y), concentrations of the compound were transformed to logarithm (X), then linear regression equation (Y=a+bx) was fit and the coefficient (r) was estimated. The logarithm value of X was calculated according to the regression when Y=5. This logarithm value of X was the EC50 value.

To examine the effect of eugenol on mycelial growth, 50 mL of potato dextrose broth (PDB) was inoculated with three 6 mm-diameter mycelial agar discs from 6 to 7 day-old cultures of B. cinerea. Eugenol was added to PDB from stock solution to reach final concentrations of 0, 25, 50, 100, 150 and 200 μg/mL. The flasks were kept at 25ºC under gentle shaking and after 96 h mycelial growth was checked by measuring the dry weight according to the method of Vicedo et al. (2006).

The effect of pH on eugenol efficacy (100μg/mL) was tested against B. cinerea under the same conditions described above except for culture medium pH values from 5 to 8 (with one pH unit interval) that were adjusted with 1 M NaOH or HCl. All experiments were repeated three times. All the treatments were applied in three experimental units. Means and standard errors were calculated for all treatments. B. cinerea was grown on PDA and conidia suspensions were prepared from the sporulating edges of 2-week-old cultures. Conidia were gently removed with a bacteriological loop suspended in sterile distilled water containing 0.01% Tween-20, and filtered through four layers of sterile cheesecloth to remove remaining mycelia. The concentration was determined with a hemocytometer and adjusted to 106 conidia mL-1 (Richard et al., 2002).

Spore germination assay: 50 μL of conidia suspension were transferred to a concave slide and a drop of 50 μL of the stock solution of eugenol was added to yield a final concentration of 4×104μg eugenol/mL. Total volumes were increased to 100 μL per slide using sterile distilled water. After 18 h, the percentage of germinated conidia was determined from at least 100 conidia per well in four replicate wells by microscopic examination. The effect of eugenol was compared to pyrimethanil (5μg/mL), a standard fungicide for controlling B. cinerea (Vicedo et al., 2006).

Conidia and mycelia plasma membrane integrity assay

Conidia and mycelia from B.cinerea were incubated in the presence of eugenol at the concentration of 100 μg/mL. After 15, 30, 60, 120, and 240 min of incubation, conidia and mycelia were harvested by centrifugation at 10,000 ×g for 15min, washed in PB buffer (50 mM, pH 7.0), and stained with 10 μg/mL propidium iodide for 15 min. Conidia and mycelia were observed under a fluorescence microscope (Olympus BX-60) (Liu et al., 2007).

Release of cellular material

Three 6 mm-diameter mycelial plugs of B.cinerea were taken from the edge of 3-day-old colonies and placed in flasks with 50 mL of PDB. The flasks were incubated at 25°C with gentle shaking (140 rpm), and mycelia was harvested by centrifugation at 10,000 ×g for 10 min, washed three times and suspended in 20 mL PBS buffer (0.05 mol/L, pH 7.0). After 1 h incubation with different eugenol concentrations, samples were centrifuged at 10,000 ×g at 4°C for 10 min. The UV (260nm) absorbing materials in each supernatant were measured (Christopher & Isao, 2000).

Determination of extracellular K+ contents of the hypha: Mycelia were incubated for 2 days in 50mL PDB, at 25°C with gentle shaking (140 rpm) and washed three times with Hepes buffer (2.5 mM, pH 7.0) and then suspended in 60 mL of the same buffer. The hyphae were then incubated with 100μg/mL eugenol under room temperature, for predetermined times. Samples (10 mL) were taken at 0, 3, 5, 10 min after treatment and immediately chilled on ice. The samples were centrifuged at 10,000 ×g at 4°C for 10 min, and the supernatant was removed and stored for the determination of extracellular K+. The K+ concentration in the samples was determined by flame photometry (A Analyst 300 Atomic Absorption Spectrometer) (Olivia et al., 1998).

A mycelial agar disc from a three-day-old culture was placed in the center of plates containing PDA before adding 100 μg/mL eugenol and incubated at 25ºC for 3 days under dark. Thin layers (1mm) of agar blocks ( 2 to 3 cm2) containing mycelia were cut off from the growing edges of the colonies for examination by light microscopy. The blocks were placed in water on a microscope slide and covered with coverslip. The microscope slides were examined with a light microscope (Nikon YS100, Japan) to observe recognizable cytological changes that were photographed with a digital camera (Nikon COOLPIX 4500, Japan). The mycelia incubated in the absence of eugenol were used as control (Cristesu et al., 2002 )

For scanning electron microscopy (SEM) analysis, mycelial discs (6 mm in diameter) were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 12 h at 4ºC. The samples were washed twice, each time for 10 min, in the same buffer, and then dehydrated in a graded ethanol series (30, 50, 70, 80, 90, and three times at 100%) for 15 min in each series. The samples were critical point dried in a drying apparatus (EMITECH TS-7 K850, UK). The fixed material was mounted on aluminum stubs using double-sided carbon tape and coated with gold in a sputter coater system (EMITECH K575X, UK) for 420 s at 10 mA. Finally, the samples were examined with the scanning electron microscope at an accelerating voltage of 10 kV as described (Agizzio et al., 2006).

Detection of H2O2 in hyphae

The generation of H2O2 after addition of eugenol was measured by using a previously described method with few modifications (Rosa et al., 2004). The pathogen was treated with eugenol and incubated as described for the measurements of cellular material release. Mycelia (0.5 g) were ground into a fine powder with a mortar and pestle using liquid nitrogen and then resuspended with 4 mL of PBS buffer (0.05 mol/L, pH 7.0). The suspension was centrifuged (10,000 ×g, 4ºC, 10 min) and H2O2 concentration was measured in the supernatant. An aliquot of the supernatant (100 μL) was added to 100 μL of assay reagent (500 μM ferrous ammonium sulfate, 50 mM H2SO4, 200 μM xylenol orange, and 200 mM sorbitol). After 30 min of incubation under room temperature, the peroxide-mediated oxidation of Fe2+ to Fe3+ was determined by measuring the A570 of the Fe3+-xylenol orange complex.

Cytosolic Ca2+ measurements

In order to determine if the different biological effects elicited by eugenol in mycelia are related to changes in internal Ca2+ concentration, the concentrations of free cytosolic Ca2+ at different incubation times were measured. Cytosolic Ca2+ measurement in mycelia was carried out by using the fluorescent Ca2+ indicator Fluo3-AM (Giudice et al., 2006). The final concentration of Fluo3-AM was adjusted to 5 μM and prepared from a 5 mM Me2SO stock solution (DMSO). The buffer used was 10 mM Hepes, pH 7.4 and samples were observed with a Confocal Laser Scanning Microscope (LEICA TCS SP2), and images were processed using Leica Confocal Software Lite Version.

Statistical analysis

All experiments were performed two times with three replicates of each treatment. Data were subjected to analysis of variance (ANOVA) and Duncan's multiple range test was used for multiple comparisons of treatment means.

 

RESULTS

Effect of eugenol on mycelial growth

The EC50 values of eugenol on mycelial radial growth were examined to determine its fungicidal spectrum (Table 1). The mycelial growths of B. cinerea and S. sclerotiorum were the most sensitive to eugenol and the EC50 values were 38.6 and 39.9 μg/mL, respectively. The EC50 values for F. graminearum and P. capsici were above 100 μg/mL, therefore these species were not highly sensitive to eugenol. The other species were moderately sensitive to eugenol with EC50 values ranging from 46.7 to 96.9 μg/mL. Mycelial growth of B. cinerea was inhibited by eugenol in a concentration-dependent manner (Figure 1).

 

 

The medium pH did not change the effects of eugenol on mycelial growth (Figure 2). Mycelial growth of B. cinerea in the absence of eugenol was not significantly affected by pH values (data not shown). The germination rate of conidia in untreated control was 98.6%. Conidia germination was not affected by eugenol at the concentrations tested. Pyrimethanil was used as a positive control and there was no germination of B. cinerea conidia in this treatment.

 

 

Assay of plasma membrane integrity of spores and mycelia

The ability of eugenol to permeabilize conidia plasma membrane and mycelia of B. cinerea was examined. When observed with a fluorescence microscope, mycelia of B. cinerea showed strong PI fluorescence in the presence of eugenol (100μg/mL) as compared to the control treatment (Figure 3). Conidia of B. cinerea showed no PI fluorescence in the presence of eugenol (data not shown).

 

 

Release of cellular material (OD260 and K+)

There was massive leakage of K+ when mycelia of B. cinerea were exposed to eugenol (Figure 4). The level of K+ reached a maximum after 3 min of treatment with 100 μg/mL eugenol, and remained constant over time.

 

 

Effect of eugenol on hyphal morphology

Important morphological damage was detected in the hyphae exposed to eugenol compared to the hyphae in the controls. Hyphae of B. cinerea grown in the absence of eugenol showed typical features of the genus. After exposure to eugenol, hyphae appeared degraded and large vesicles were visible (Figure 5). The SEM micrographs showed important morphological damage due to eugenol (Figure 6). Shriveled hyphae were commonly observed in eugenol treated mycelia compared with the normal mycelia.

Detection of H2O2 in hyphae

The oxidative burst belongs to the fastest active defense responses. It is defined as a rapid and transient production of large amounts of reactive oxygen species (ROS) including hydrogen peroxide (H2O2). The accumulation of H2O2 was induced by eugenol and the level of H2O2 increased up to 1,000% after 4h of treatment (Figure 7).

 

 

Cytosolic Ca2+ measurements

The concentration of Ca2+ increased at the beginning of the treatment with eugenol and reached the highest level after 30 min, suggesting that either directly or indirectly cytosolic Ca2+ is related to the biological effects elicited by eugenol. However, it was reduced from 30 to 120 min of treatment (Figure 8).

 

 

DISCUSSION

Eugenol has antimicrobial activity against a variety of food-borne (Rhayour et al., 2003), wood decay fungi, and human pathogens (Vázquez et al., 2001; Gayoso et al., 2005; Ghosh et al., 2005). However, little attention has been paid to its antifungal activity against plant pathogens. It is interesting to point out that eugenol only inhibited the growth of B. cinerea mycelia. Conidia germination is the growth stage most sensitive to inhibition by many compounds, but eugenol was not effective in preventing germination of conidia of B. cinerea. This suggests that eugenol interferes with processes taking place after the stages of germination. Many fungicides such as carbendazim and N-phenylarbmates have little or no effect on spore germination but strongly inhibit mycelial growth (Sherald et al., 1973; Suzuki et al., 1984). Nevertheless, the reason why eugenol was not effective in reducing conidia germination of B. cinerea needs to be further investigated.

Recent investigations about antimicrobial action of eugenol showed disruption of fungal and bacterial membranes (Gill & Holley, 2006). All these reports suggest that this antimicrobial mechanism is due to membrane damage. Eugenol, known to be a lipophilic compound, can enter between the fatty acid chains that make up the membrane lipid bilayers, thus altering the fluidity and permeability of cell membranes (Braga et al., 2007). These findings were supported by the intensive staining of eugenol-treated hyphae of B. cinerea, and release of OD260nm absorbing material, although no substantial changes took place in mycelial morphology. B. cinerea mycelia treated with eugenol resulted in the leakage of ultraviolet-absorbing materials compared with the controls and the release was concentration dependent. Changes in the membrane permeability occurred simultaneously with cell death, in contrast to ultraviolet-absorbing materials that are released after cell death. However, no staining of conidia was observed. From this experiment, we concluded that Eugenol had no effect on membrane permeability of B. cinerea conidia. This might explain the specific composition of the cell wall of conidia.

Few studies of the effects of eugenol on the morphology and ultrastructure of yeast and bacteria have been carried out (Rhayour et al., 2003). In this study, the hyphae of B. cinerea grown on PDA with eugenol showed morphological changes, including cytoplasmic coagulation and vesiculation. Shriveled hyphae were commonly observed in eugenol-treated mycelia, compared with the normal mycelia.

Although the order of events leading to cell death provoked by eugenol is not exactly known, membrane permeabilization should be an early effect. Indeed there is a relationship among hyphae viability, H2O2 production, eugenol concentration and treated times as shown in the present study, which would indicate that H2O2 production and subsequent cell death may be a consequence of membrane changes. It was also demonstrated that eugenol induced an increase in internal Ca2+ concentration, and this Ca2+ was probably liberated from internal stores (vacuoles, endoplasmic reticulum, etc.). It has been previously shown that the increase in free cytosolic Ca2+ concentration induced by the presence of eugenol is related to H2O2 production (Marcela et al., 2006). Perhaps eugenol induces either hyperpolarization of the inner mitochondrial membrane or mitochondrial swelling or both. Usually, cells in which mitochondria are destabilized and finally broken down suffer a decrease in the coupling efficiency of the electron-transport chain and therefore can generate ROS intermediates which can lead to oxidative stress (Martindale & Holbrook, 2002). Finally, the present study suggests that eugenol could directly inhibit the growth of B. cinerea in vitro. Eugenol can be used in the control of B. cinerea and other phytopathogenic fungi and can also be considered as a potential alternative for synthetic fungicides. A further investigation is under progress in our laboratory to find the exact action site of eugenol.

 

ACKNOWLEDGEMENTS

This work was supported by Hi-Tech Research of Jiangsu (Nos. BG2006325) and Innovation Fund for Agricultural Sciences and Technology of Jiangsu (Nos. CX(08)125). Jie Zhang and Chunmei Wang contributed equally to this work.

 

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Received 28 April 2009
Accepted 28 May 2010

 

 

Author for correspondence: Zhiqi Shi, e-mail: nytrpz@yahoo.cn
TPP 9057
Section Editor: Eduardo S.G. Mizubuti

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