Research Article Received: 19 September 2013
Revised: 3 January 2014
Accepted article published: 15 January 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6576
Effect of citronella essential oil on the inhibition of postharvest Alternaria alternata in cherry tomato Qianru Chen,a,b,c Shixiang Xu,a Tao Wu,a Jun Guo,a,b Sha Sha,a Xiaodong Zhenga,b,c* and Ting Yua* Abstract BACKGROUND: Essential oils such as citronella oil exhibit antifungal activity and are potential alternative inhibitors to chemical synthetic fungicides for controlling postharvest diseases. In this study the antifungal activity of citronella oil against Alternaria alternata was investigated. RESULTS: In vitro, citronella oil showed strong inhibition activity against A. alternata. The minimum inhibitory concentration in potato dextrose agar and potato dextrose broth medium was determined as 1 and 0.8 𝛍L mL−1 respectively. In vivo the disease incidence of Lycopersicon esculentum (cherry tomato) treated with citronella oil was significantly (P < 0.05) reduced compared with the control after 5 days of storage at 25 ∘ C and 95% relative humidity. The disease incidence at oil concentrations of 0.2–1.5 𝛍L mL−1 was 88–48%. The most effective dosage of the oil was 1.5 𝛍L mL−1 , with 52% reduction, and the oil had no negative effect on fruit quality. Scanning electron microscopy observation revealed considerably abnormal mycelial morphology. CONCLUSION: Citronella oil can significantly inhibit A. alternata in vitro and in vivo and has potential as a promising natural product for controlling black rot in cherry tomato. © 2014 Society of Chemical Industry Keywords: Alternaria alternata; citronella oil; antifungal activity; cherry tomato
INTRODUCTION Fresh fruits and vegetables are readily affected by pathogenic fungi such as Alternaria, Botrytis, Fusarium, Geotrichum, Penicillium and Sclerotinia,1 especially under high-moisture and high-temperature conditions, which reduce their shelf life and cause considerable economic losses around the world. In China, cherry tomato (Lycopersicon esculentum var. cerasiforme) is perishable and susceptible to postharvest diseases owing to the warm and humid climate. Alternaria alternata is a major saprophytic pathogen causing postharvest black rot in cherry tomato and is responsible for the postharvest loss of cherry tomato.2 Chemical synthetic fungicides are mostly used as an effective postharvest disease control strategy. However, pathogen resistance to various types of fungicides can be developed after frequent usage.3 In addition, synthetic fungicides are not environmentally friendly reagents and are potential hazards to human health owing to their long degradation time and teratogenic and carcinogenic properties.4 Thus natural antifungal products should be developed to meet health demands. Essential oil, the aromatic volatile oily liquid of plant secondary metabolism, represents part of the defense system of plants against infection by microorganisms.5 The antifungal activity of an essential oil depends on the type and amount of phenolic compounds it contains.6 Several essential oils with biologically active components have been applied as effective disease control agents, since these components have a broad spectrum against J Sci Food Agric (2014)
plant pathogens or Insects.7 Furthermore, essential oils extracted from plants are considered to be non-phytotoxic agents, suggesting that the majority of them are safe for consumers, and also have a low risk of pathogens developing resistance to them.1,8 – 10 Previous studies have proved the activities of certain essential oils against fungal pathogens to control postharvest diseases in vitro and in vivo.4,10 – 13 The antimicrobial properties of the oils were attributed to the presence of mono- and sesquiterpenes, monoand sesquiterpene hydrocarbons and phenolic compounds such as thymol, carvarol, citral, trans-2-decenal and p-cymene.1 Cymbopogon nardus (L.) Rendle (citronella grass or Ceylon citronella), an aromatic grass belonging to the Poaceae family, originated from Sri Lanka and southern India and is now grown in many areas of the world.14 The citronella oil extracted from C. nardus (L.) Rendle is mainly composed of monoterpenes (up to 80%), including citronellal, citronellol and geraniol,
∗
Correspondence to: Xiaodong Zheng, Department of Food Science and Nutrition, Zhejiang University, Hangzhou, 310058, China, and Ting Yu, E-mail: [email protected]; [email protected]
a Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, China b Zhejiang Key Laboratory for Agro-Food Processing, Hangzhou 310058, China c Fuli Institute of Food Science, Zhejiang University, Hangzhou 310058, China
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www.soci.org and some sesquiterpenes.15 Citronella oil has been used in mosquito repellent,16 perfume, soap and cosmetics.14 Studies have demonstrated inhibitory effects of the oil on ectoparasites and pathogenic microorganisms, including bacteria and fungi.17,18 The antimicrobial properties of citronella oil may be due to its ingredients such as geraniol and citronellol.19 Thus citronella oil may have potential activity to inhibit A. alternata (the main spoilage fungus on fruits and vegetables, especially cherry tomato). Owing to the lack of information on the anti-A. alternata activity of citronella oil, it is necessary to systematically evaluate the antifungal efficacy of the oil against A. alternata.
MATERIALS AND METHODS Fungal strain Alternaria alternata obtained from the Institute of Microbiology, Chinese Academy of Science was cultured on a potato dextrose agar (PDA) slant at 25 ∘ C for 10–12 days. Essential oil Pure citronella oil extracted from C. nardus (L.) Rendle (from Jiang Xi province, China) by water distillation was purchased from Yue Yu Essential Oil Corporation (Guang Dong province, China). The oil was stored at 4 ∘ C before use. In vitro antifungal activity assay The antifungal activity of the essential oil against A. alternata was determined by the method of Singh et al.20 with some modifications. The oil was dissolved and diluted tenfold in 5 mL L−1 Tween 20 and mixed aseptically with PDA medium at 40–45 ∘ C to obtain oil concentrations of 0 (control), 0.2, 0.4, 0.6, 1 and 1.2 μL mL−1 . The mixed media were transferred onto 90 mm Petri plates (15 mL per plate). A mycelium disk cut from a 10-day-old culture of A. alternata grown on PDA was placed on the center of each Petri plate and then incubated at 25 ∘ C. The mean diameter of mycelial growth of the fungus was determined each day for 7 days. All treatments consisted of five replicates and the whole experiment was performed three times. Mean growth values were converted into percentage mycelial inhibition using the formula ) ] [( percentage inhibition = dc − dt ∕dc × 100% where dc (mm) and dt (mm) represent the mean colony diameters of the control and treated groups respectively. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of the oil that inhibited mycelial growth completely. In vivo antifungal activity assay on cherry tomato fruit The antifungal assay of the essential oil against A. alternata in vivo on cherry tomato was conducted by the modified method of Feng and Zheng10 (fruit pathology test). Mature and healthy fresh cherry tomatoes from Zhejiang Academy of Agricultural Sciences were dipped in 1 mL L−1 sodium hypochlorite for 1 min, washed in running water and dried at room temperature. Each fruit was cut at the equatorial region with a 4 mm diameter and 2 mm deep hole using a sterile puncher to generate the wound. The fruits were then divided randomly into six groups and placed in containers (21 cm × 15 cm white plastic boxes). A 10 μL aliquot of spore suspension (104 spores mL−1 ) was inoculated into each wound. After 30 min, 20 μL aliquots of the oil with concentrations of 0 (control), 0.2, 0.5, 0.8, 1, 1.5 and 2 μL mL−1 were pipetted into
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individual wounds. The containers were sealed with plastic wrap to maintain a high relative humidity (RH) of 90–95% during storage. The disease incidence of the fruits was measured after 5 days of storage at 25 ∘ C. Twenty fruits were used in a treatment and each treatment was repeated three times in an experiment. The quality of oil-treated/untreated fruits was evaluated for mass loss, fruit firmness, total soluble solids (TSS) and titratable acidity (TA) after fungal infection.21 All quality indices were measured before and after 15 days of storage (13 ∘ C, 85% RH). Mass loss was determined by monitoring the weight of the fruits and calculated as a percentage. Fruit firmness was measured at one point of the equatorial region using a texture analyzer (GY-3, Top Instrument Co., Ltd, Hang Zhou, Zhe Jiang provinve, China). TSS was evaluated by measuring the refractive index of cherry tomato juice with a hand-held refractometer (WYT-32, Top Instrument Co., Ltd). TA was obtained by titration with 0.05 mol L−1 NaOH to pH 8.1 and reported as g citric acid kg−1 fresh fruit weight. Effect of essential oil on spore germination assay The spore germination of A. alternata was tested by the method of Singh et al.20 The essential oil was dissolved and diluted tenfold in 5 mL L−1 Tween 20 and mixed aseptically with 2 mL of potato dextrose broth (PDB) medium in test tubes to obtain oil concentrations of 0 (control) 0.1, 0.3, 0.5, 1 and 1.5 μL mL−1 . A 100 μL aliquot of spore suspension of A. alternata (106 spores mL−1 ) was added to each test tube and the test tubes were placed on a rotary shaker at 0.58 × g for 12 h at 25 ∘ C. Approximately 150 spores of pathogen were counted in each replicate to determine the germination rate of the pathogen. The spore germination assay was estimated under a microscope. Each treatment consisted of three replicates and the experiments were performed three times. Effect of essential oil on wet and dry mycelium weight The effect of the essential oil on the wet and dry mycelium weight of the pathogen was measured by the method of Dikbas et al.22 The oil was dissolved and diluted tenfold in 5 mL L−1 Tween 20 and transferred into Erlenmeyer flasks with 50 mL of PDB medium to obtain oil concentrations of 0 (control), 0.1, 0.3, 0.5, 0.7 and 0.9 μL mL−1 . Then 100 μL of spore suspension of the pathogen (106 spores mL−1 ) was inoculated into each flask and the flasks were incubated on a rotary shaker at 0.58 × g and 25 ∘ C. The mycelia were harvested on days 5 and 10 by centrifugation at 3260 × g for 15 min and precipitates were washed twice by dipping them in distilled sterile water and filtering for 30 min. The wet mycelia were weighed. The dry mycelia were weighed after drying the wet mycelia at 60 ∘ C for 12 h. Each treatment consisted of three replicates and the experiments were performed three times. Scanning electron microscopy (SEM) examination of effect of essential oil on hyphal morphology SEM (S-3000N, Hitachi Limited, Tokyo, Japan) analysis was performed using the method of Lu et al.23 with some modifications. Ten-day-old fungal materials of A. alternata cultured in PDB medium only (control) and PDB medium with the most effective concentration of the oil were observed by SEM. Glutaraldehyde (250 mL L−1 , Ted Pella, Inc., Redding, CA, USA) at a concentration of 25 mL L−1 in 0.1 mol L−1 phosphate-buffered saline (PBS) (pH 7.2) was added to the fungal samples and fixed overnight at 4 ∘ C. The fixed samples were then treated with following steps. Each sample was rinsed with 0.1 mol L−1 PBS (pH 7.2) three times for 15 min, fixed again with 5 mL L−1 osmium tetroxide solution (40 mL L−1
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Inhibition of A. alternata in cherry tomato by citronella oil
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aqueous, Ted Pella, Inc.) for 1–2 h and washed again with PBS (pH 7.2). The fixed samples were subjected to a series of graded acetone dehydration processes (500, 700, 800, 900 and 950 mL L−1 , 15 min for each concentration) and then dehydrated twice with pure ethanol for 20 min. After treatment with a mixture of ethanol and isoamyl acetate for 30 min, samples were treated with pure isoamyl acetate for 1–2 h. After dehydration, samples were dried in critical point dryer (HCP-2, Hitachi Limited) at critical point according to the manufacturer’s instructions and mounted on stubs using a carbon adhesive. Finally, samples were coated with a gold/palladium layer by sputter coating with an ion sputter (E-1010, Hitachi Limited) and scanned and photographed by SEM at 15 kV. Identification of oil components by gas chromatography/mass spectrometry (GC/MS) The chemical composition of the essential oil was analyzed by GC/MS (5975I, Agilent Co., Santa Clara, CA, USA). Constituents were separated on a 30 m × 250 μm × 0.25 μm capillary column (HP-5MS, Agilent Co.). High-purity helium was used as carrier gas at a constant flow rate of 1 mL min−1 . The pressure at the column inlet was 9.37 psi. The GC column temperature was initially held 40 ∘ C for 1 min and then increased at a rate of 10 ∘ C min−1 to 280 ∘ C and held for 6 min. The temperature of the auxiliary heater was maintained at 280 ∘ C. Eluted components were detected using an electron ionization (EI) mass spectrometer (EI mode, 70 eV). Data acquisition was in full-scan mode in the range m/z 45–500. The temperatures of the ion source and quadrupole were set at 230 and 150 ∘ C respectively. The relative contents of the oil were calculated as percentages following the peak area normalization method. The oil compounds were identified by comparing retention indices obtained according to a standard solution of n-alkanes (C7–C26) with known compounds in a reference of authentic standards24 and by comparing their mass spectra with the NIST/NIH/EPA spectral database and also by manual interpretation. Statistical analysis Data are presented as mean ± standard deviation (n = 3). All data were subjected to single-factor analysis of variance (ANOVA) using SPSS Statistics 17.0 (SPPS Inc., Chicago, IL, USA). Significant differences in mean values were assessed by Duncan’s multiple range test at the level P < 0.05.
RESULTS In vitro antifungal activity assay The antifungal activity of the oil on A. alternata mycelial growth during 7 days of incubation is shown in Fig. 1. The results indicated that the diameter of the fungal mycelia increased with increasing incubation time. Also, the mycelial growth of all treatments was considerably inhibited compared with the control in a dose-dependent manner. The beginning of visible mycelial growth was delayed 2 days at 0.6 μL mL−1 and 3 days at 0.8 μL mL−1 , while mycelial growth was completely inhibited at 1 μL mL−1 during the entire incubation time. The MIC of the oil was 1 μL mL−1 after 7 days of incubation. The inhibition rate of oil treatment calculated after 7 days of cultivation was 26–100% with oil concentrations of 0.2–1 μL mL−1 . Antifungal activity of essential oil in vivo on cherry tomato The antifungal activity of the essential oil in vivo on cherry tomato is shown in Fig. 2. The disease incidence of all treatments except J Sci Food Agric (2014)
Figure 1. Effect of citronella oil on inhibition of mycelial growth of Alternaria alternata in vitro. The diameter of the fungal mycelial growth of the control and different concentrations of the oil was measured from day 2 to day 7 during the cultivation time. Values are mean ± standard deviation (n = 3).
Figure 2. Effect of citronella oil on inhibition of Alternaria alternata in vivo (cherry tomato). The disease incidence of the control and treatments with different concentrations of the oil was measured after 5 days of storage at 25 ∘ C. Values are mean ± standard deviation (n = 3). Values with different letters are significantly different (P < 0.05).
0.2 μL mL−1 was significantly (P < 0.05) reduced compared with the control. The oil concentration of 1.5 μL mL−1 exhibited the greatest antifungal activity, reducing the disease incidence to 48% compared with the control. The disease incidence at oil concentrations of 0.2–1.5 μL mL−1 was 88–48%. The disease incidence with the treatment at 2 μL mL−1 was significantly higher than that at 1.5 μL mL−1 . Table 1 shows the effect of citronella oil on fruit quality. After 15 days of storage, increased mass loss and decreased fruit firmness, TSS and TA were observed, while the oil had no significant effect on these four indicators after the storage period. Effect of essential oil on A. alternata spore germination The effect of the oil on spore germination of the fungus is shown in Fig. 3. The results indicated that oil treatment significantly (P < 0.05) inhibited fungal spore germination compared with the control in a dose-dependent manner after 12 h of incubation at 25 ∘ C. Spore germination was incompletely inhibited at oil concentrations of 0.1–1.5 μL mL−1 in PDB medium, with the spore germination rate decrease ranging from 54 to 13%.
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Table 1. Effect of citronella oil on postharvest quality characteristics of cherry tomato before and after 15 days of storage (85% RH) at 13 ∘ C Treatment (μL mL−1 ) Before storage After storage
– 0 0.8 1.5 2
Mass loss (%)
Firmness (105 Pa)
0 1.42 ± 0.12a 1.40 ± 0.08a 1.22 ± 0.14a 1.53 ± 0.18a
4.22 ± 0.02 4.83 ± 0.21a 4.91 ± 0.02a 4.86 ± 0.11a 4.73 ± 0.07a
Total soluble solids (TSS) (∘ Brix)
Titratable acidity (TA) (g kg−1 )
7.06 ± 0.35 6.57 ± 0.96a 6.02 ± 0.28a 6.73 ± 0.42a 6.30 ± 0.75a
9.01 ± 0.72 8.82 ± 1.30a 10.30 ± 1.27a 9.22 ± 0.76a 9.42 ± 0.69a
Values are mean ± standard deviation (n = 3). Values in the same column with different letters are significantly different (P < 0.05).
inhibited. The 0.8 μL mL−1 concentration of citronella oil was thus most effective and considered as the MIC for A. alternata in PDB medium.
Figure 3. Effect of citronella oil on inhibition of spore germination of Alternaria alternata. The fungal germination rate of the control and treatments with different concentrations of the oil was measured after 12 h of cultivation. Values are mean ± standard deviation (n = 3). Values with different letters are significantly different (P < 0.05).
Effect of essential oil on A. alternata wet and dry mycelium weight The effect of citronella oil on A. alternata wet and dry mycelium weight in PDB medium was measured on days 5 and 10 after inoculation and the results are shown in Table 2. On day 5 the biomass of the fungal mycelium for all treatments except 0.2 μL mL−1 was significantly (P < 0.05) reduced compared with the control, and no mycelium was produced at oil concentrations of 0.6 and 0.8 μL mL−1 . On day 10 the fungal mycelium biomass for the treatments at 0.4 and 0.6 μL mL−1 increased substantially in comparison with that on day 5. No growth of mycelium was observed on either day 5 or day 10 for the treatment at 0.8 μL mL−1 , i.e. mycelium production was completely
SEM examination of hyphal morphology SEM examination of hyphal morphology was conducted to determine the influence of the essential oil on the microstructural surface of A. alternata (Fig. 4). The A. alternata mycelium grown in PDB medium as the control showed regular and normal morphology with rich conidia (Fig. 4A). Furthermore, the control clearly exhibited healthy development of conidia and intact, plump hyphae with a smooth external surface (Fig. 4C). In contrast, the mycelial morphology changed severely after treatment with the most effective essential oil concentration determined in PDB medium. The hyphae folded and flattened with a rough surface and deformed with absence of conidia, and the treated hyphae also showed abnormal mycelial morphology with shrivelled and wrinkled flattened empty hyphae (Figs 4B and 4D). Chemical composition of essential oil To determine the active ingredients in citronella oil, its chemical composition was analyzed by GC/MS and the results are listed in Table 3. A complex mixture of 23 compounds accounting for 96.68% of the total oil were identified, consisting of monoterpenes (75.38%) and sesquiterpenes (21.30%). The major individual compounds were citronellal (26.23%), geraniol (19.75%) and citronellol (12.96%), followed by elemol (5.07%), 2,6-dimethyl-2,6-octadiene (4.31%), 𝛿-cadinene (4.21%), geranyl acetate (3.58%), D-limonene (2.96%), 𝛽-elemene (2.80%), 𝛼-cadinol (2.75%) and other components in lower amount.
Table 2. Effect of citronella oil on biomass of Alternaria alternata in PDB medium at days 5 and 10 during incubation Mycelium weight Concentration of essential oil (μL mL−1 )
0 0.2 0.4 0.6 0.8
Day 5
Day 10
Wet (g)
Dry (mg)
Wet (g)
Dry (mg)
14.92 ± 1.27a 18.84 ± 0.85a 2.95 ± 0.76b 0.00 ± 0.00c 0.00 ± 0.00c
568.73 ± 45.51a 580.27 ± 19.09a 81.60 ± 17.41b 0.00 ± 0.00c 0.00 ± 0.00c
14.63 ± 1.02a 15.12 ± 0.18a 12.89 ± 0.65b 0.34 ± 0.06c 0.00 ± 0.00d
560.13 ± 22.63a 535.00 ± 24.69a 430.97 ± 32.59b 8.63 ± 2.23c 0.00 ± 0.00d
Values are mean ± standard deviation (n = 3). Values in the same column with different letters are significantly different (P < 0.05).
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Inhibition of A. alternata in cherry tomato by citronella oil
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Figure 4. Scanning electron micrographs of Alternaria alternata mycelium grown in PDB medium without (control) or with (treated) 0.8 μL mL−1 citronella oil during 7 days of incubation at 25 ∘ C: A, control with rich conidiophore and regular hyphae of A. alternata (magnification × 500, bar = 100 μm); B, treated variant with absence of conidia and unhealthy hyphal morphology (magnification × 500, bar = 100 μm); C, control of intact conidia and plump hyphae with smooth external surface (magnification × 5000, bar = 10 μm); D, treated shrivelled and wrinkled hyphae with rough surface (magnification × 5000, bar = 10 μm). Table 3. The composition of citronella oil obtained by water distillation from Cymbopogon nardus (L.) Rendle
Number
Retention time (min)
Peak area (Ab*s)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
7.688 8.782 9.542 9.634 9.721 10.711 11.112 11.325 11.851 12.419 12.552 12.824 13.058 14.151 14.243 14.440 14.632
3293948 788870 1558038 29139061 637172 14404889 21942928 749497 883112 4789168 1607440 3974360 3107166 659667 1099347 1371575
18 19 20 21 22 23
14.719 14.907 15.037 16.043 16.139 16.293
4678365 325434 5630677 773216 1530192 3052473
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Compound name
Composition (%)
D-Limonene
Linalool 5-Methyl-2-(1-methylethenyl)cyclohexanol Citronellal Isopulegol Citronellol Geraniol Citral 3,7-Dimethyl-6-octenoic acid 2,6-Dimethyl-2,6-octadiene Eugenol Geranyl acetate 𝛽-Elemene 𝛾-Muurolene Germacrene D 𝛼-Muurolene 1,2,4a,5,6,8a-Hexahydro-4,7-dimethyl1-(1-methylethyl)naphthalene 𝛿-Cadinene 𝛼-Cadinene Elemol Machilol 𝜏-Cadinol 𝛼-Cadinol
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2.96 0.71 1.40 26.23 0.57 12.96 19.75 0.67 0.79 4.31 1.45 3.58 2.80 0.59 0.99 1.23 1.29 4.21 0.29 5.07 0.70 1.38 2.75
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DISCUSSION Previous studies have focused on the inhibitory effect of essential oils against fungal growth in vitro, and some have been tested in vivo to control postharvest diseases.11,25 In this study, citronella oil was systematically investigated in vitro and in vivo on cherry tomato to demonstrate the antifungal inhibition activity against A. alternata. Citronella oil showed pronounced antifungal efficacy against A. alternata mycelial growth in vitro. The MIC in PDA medium was determined as 1 μL mL−1 , which was similar to the result reported by Tian et al.13 (2 μL mL−1 Anethum graveolens L. essential oil against A. alternata), revealing that citronella oil had a strong inhibition activity in controlling A. alternata. In addition, citronella oil effectively suppressed the biomass of A. alternata (Table 2). A lower concentration (0.4 μL mL−1 ) of the oil could suppress mycelium production strongly in the early period of incubation (day 5), though its inhibition activity weakened gradually with prolonged incubation (day 10). These results indicated that the active compounds of the oil at lower concentration delayed the growth of A. alternata effectively. The results of in vivo investigations showed that citronella oil concentrations of 0.5–2 μL mL−1 had a significant inhibition effect against black rot in cherry tomato. An increased percentage of disease incidence at 2 μL mL−1 was found in comparison with 1.5 μL mL−1 . Thus the most effective concentration of citronella oil against A. alternata in vivo was 1.5 μL mL−1 . Tian et al.13 reported that 120 μL mL−1 Cicuta virosa L. var. latisecta Celak essential oil inhibited about 80% disease incidence of Aspergillus flavus, Aspergillus oryzae, Aspergillus niger and A. alternata on cherry tomato, while Xing et al.26 found that 30 μL mL−1 clove oil completely inhibited the disease incidence of A. flavus and Penicillium citrinum on orange fruit. The present usage of citronella oil was relatively low compared with the results of Tian et al.13 and Xing et al.26 It was found that citronella oil treatment did not impair fruit quality during 15 days of storage, similarly to a previous report.27 These results indicate that citronella oil might also have potential for commercialization to control black rot in cherry tomato. Moreover, a low concentration of citronella oil for inhibition of fungi reduces costs effectively. However, it is widely accepted that application in foods requires higher concentrations of essential oil than in laboratory media.22,28,29 This could be explained by altered reaction temperature changing the site of action of the oil in foods, leading to decreased oil invasion of the interior of cells.22 The SEM images of the microstructure of A. alternata revealed a degenerative alteration in hyphal morphology, such as absence of conidia, flattened and shriveled hyphae, swelling and rough hyphal walls. These results resemble those of a previous study in which the microstructure of different fungal hyphae treated with essential oils was analyzed.4 The mode of action of essential oils and their components has not been fully investigated, but one possible mode may be attributable to the hydrophobicity of essential oils, which could partition the lipids of fungal cell membranes and damage the integrity of cell walls and cell membranes, causing an imbalance in intracellular osmotic pressure and the leakage of vital intracellular ingredients such as ATP, ions, nucleic acids and amino acids.8,14,30 One study showed that active components may cause structural and metabolic changes in fungi, such as cytoplasm membrane burst, cytoplasm granulation, uncoupling of oxidative phosphorylation, cytoplasm hyperacidity, disruption of the electron transport chain, loss of pool metabolites and inhibition of H+ -ATPase and transport channels, breakdown of RNA, DNA, protein, lipid and polysaccharide synthesis and, eventually, initiation of autolytic processes.8
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In addition, the main compounds of citronella oil were similar to those found in previous studies, with variations in percentage composition. The main compounds reported in citronella oil from Benin were citronellal (41.3%), geraniol (23.4%) and citronellol (9.2%), from Congo were citronellal (37.5%), geraniol (29.4%) and citronellol (7.5%) and from India were citronellal (29.7%) and geraniol (24.2%).31,32 The variation in percentage composition of different samples of the same essential oil may be due to differences in habitat, genetic diversity and agronomic treatment of the original aroma plants33 and seasonal, climatic and geographical conditions, harvest time and distillation techniques.34 The antifungal activities of citronella oil may depend on the presence of the main components (citronellal, geraniol and citronellol) and other minor components. It was reported that geraniol, citronellol and citral exhibited the highest antifungal activities among terpenoids,19 and some reports revealed that the inhibitory activity of the essential oil was mainly ascribable to the presence of those abundant components.35,36 Mahmoud37 found that geraniol and citronellol contained in citronella oil at 500 mg L−1 inhibited A. flavus growth effectively. Additionally, minor components such as eugenol, linalool and isopulegol could be involved in some antimicrobial synergism with other active components.38 Generally, the essential oil presents greater antimicrobial activities than its individual components and/or mixtures of its major components.39
CONCLUSION Our results indicate that citronella oil possesses strong antifungal activity against A. alternata in vitro and in vivo. The MIC in PDA and PDB medium was 1 and 0.8 μL mL−1 respectively. In vivo the most effective dosage of the oil was 1.5 μL mL−1 , with 52% reduction, and the oil had no negative effect on fruit quality. SEM observation revealed considerably abnormal mycelial morphology. Citronella oil could be a promising natural product for use as an anti-A. alternata agent to control black rot in cherry tomato. However, further studies are required to investigate the mode of action of the essential oil.
ACKNOWLEDGEMENTS This research was partially supported by the National Public Benefit (Agricultural) Research Foundation of China (200903044), the Program for Key Innovative Research Team of Zhejiang Province (2009R50036) and the National Natural Science Foundation of China (31260402). The authors have declared no conflict of interest.
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Inhibition of A. alternata in cherry tomato by citronella oil 6 Tripathi A, Sharma N and Sharma V, In vitro efficacy of Hyptis suaveolens L. (Poit.) essential oil on growth and morphogenesis of Fusarium oxysporum f.sp. gladioli (Massey) Snyder & Hansen. World J Microbiol Biotechnol 25:503–512 (2009). 7 Isman MB, Plant essential oils for pest and disease management. Crop Protect 19:603–608 (2010). 8 Burt S, Essential oils: their antibacterial properties and potential applications in food – a review. Int J Food Microbiol 94:223–253 (2004). 9 Cardilea V, Russo A, Formisano C, Rigano D, Senatore F, Arnold NA, et al., Essential oils of Salvia bracteata and Salvia rubifolia from Lebanon: chemical composition, antimicrobial activity and inhibitory effect on human melanoma cells. J Ethnopharmacol 126:265–272 (2009). 10 Feng W and Zheng XD, Essential oils to control Alternaria alternata in vitro and in vivo. Food Control 18:1126–1130 (2007). 11 Dan Y, Liu HY, Gao WW and Chen SL, Activities of essential oils from Asarum heterotropoides var. mandshuricum against five phytopathogens. Crop Protect 29:295–299 (2010). 12 Pérez-Alfonso CO, Martínez-Romero D, Zapata PJ, Serrano M, Valero D and Castillo S, The effects of essential oils carvacrol and thymol on growth of Penicillium digitatum and P. italicum involved in lemon decay. Int J Food Microbiol 158:101–106 (2012). 13 Tian J, Ban X, Zeng H, Huang B, He J and Wang Y, In vitro and in vivo activity of essential oil from dill (Anethum graveolens L.) against fungal spoilage of cherry tomatoes. Food Control 22:1992–1999 (2011). 14 Chan LK, Dewi PR and Boey PL, Effect of plant growth regulators on regeneration of plantlets from bud cultures of Cymbopogon nardus L. and the detection of essential oils from the in vitro plantlets. J Plant Biol 48:142–146 (2005). 15 Beneti SC, Rosset E, Corazza ML, Frizzo CD, Luccio MD and Oliveira JV, Fractionation of citronella (Cymbopogon winterianus) essential oil and concentrated orange oil phase by batch vacuum distillation. J Food Eng 102:348–354 (2011). 16 Revay EE, Junnila A, Xue RD, Kline DL, Bernier UR, Kravchenko VD, et al., Evaluation of commercial products for personal protection against mosquitoes. Acta Trop 125:226–230 (2013). 17 Billerbeck VG, Roques CG, Bessière JM, Fonvieille JL and Dargent R, Effects of Cymbopogon nardus (L.) W. Watson essential oil on the growth and morphogenesis of Aspergillus niger. Can J Microbiol 47:9–17 (2011). 18 Clemente MA, de Oliveira Monteiro CM, Scoralik MG, Gomes FT, de Azevedo Prata MC and Daemon E, Acaricidal activity of the essential oils from Eucalyptus citriodora and Cymbopogon nardus on larvae of Amblyomma cajennense (Acari: Ixodidae) and Anocentor nitens (Acari: Ixodidae). Parasitol Res 107:987–992 (2010). 19 Viollon C and Chaumont J, Antifungal properties of essential oils and their main components upon Cryptococcus neoformans. Mycopathologia 128:151–153 (1994). 20 Singh P, Sivastava B, Kumar A, Dubey NK and Gupta R, Assessment of Pelargonium graveolens oil as plant-based antimicrobial and aflatoxin suppressor in food preservation. J Sci Food Agric 88:2421–2425 (2008). 21 Maqbool M, Ali A, Alderson PG, Mohamed MTM, Siddiqui Y and Zahid N, Postharvest application of gum arabic and essential oils for controlling anthracnose and quality of banana and papaya during cold storage. Postharv Biol Technol 62:71–76 (2011).
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www.soci.org 22 Dikbas N, Kotan R, Dadasoglu F and Sahin F, Control of Aspergillus flavus with essential oil and methanol extract of Satureja hortensis. Int J Food Microbiol 124:179–182 (2008). 23 Lu M, Han ZQ, Xu Y and Yao L, Effects of essential oils from Chinese indigenous aromatic plants on mycelial growth and morphogenesis of three phytopathogens. Flav Fragr J 28:84–92 (2013). 24 Adams RP, Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry. Allured Publishing, New York, NY, pp. 423–426 (2007). 25 Al-Reza SM, Rahman A, Ahmed Y and Kang SC, Inhibition of plant pathogens in vitro and in vivo with essential oil and organic extracts of Cestrum nocturnum L. Pestic Biochem Physiol 96:86–92 (2010). 26 Xing YG, Xu QL, Li XH, Che ZM and Yun J, Antifungal activities of clove oil against Rhizopus nigricans, Aspergillus flavus and Penicillium citrinum in vitro and in wounded fruit test. J Food Saf 32:84–93 (2012). 27 Tzortzakis NG, Maintaining postharvest quality of fresh produce with volatile compounds. Innovat Food Sci Emerg Technol 8:111–116 (2007). 28 Farbood MI, MacNeil JH and Ostovar K, Effect of rosemary spice extractive on growth of micro-organisms in meats. J Milk Food Technol 39:675–679 (1976). 29 Yu C, Zhou T, Sheng K, Zeng LZ, Ye CZ, Yu T, et al., Effect of pyrimethanil on Cryptococcus laurentii, Rhodosporidium paludigenum, and Rhodotorula glutinis biocontrol of Penicillium expansum infection in pear fruit. Int J Food Microbiol 164:155–160 (2013). 30 Gao C, Tian C, Lu Y, Xu J, Luo J and Guo X, Essential oil composition and antimicrobial activity of Sphallerocarpus gracilis seeds against selected food-related bacteria. Food Control 22:517–522 (2011). 31 Abena AA, Gbenou JD, Yayi E, Moudachirou M, Ongoka RP, Ouamba JM, et al., Comparative chemical and analgesic properties of essential oils of Cymbopogon nardus (L) Rendle of Benin and Congo. Afr J Tradit Compl Altern Med 4:267–272 (2007). 32 Mahalwal VS and Ali M, Volatile constituents of Cymbopogon nardus (Linn.) Rendle. Flav Fragr J 18:73–76 (2003). 33 Carlson LHC, Machado RAF, Spricigo CB, Pereira LK and Bolzan A, Extraction of lemongrass essential oil with dense carbon dioxide. J Supercrit Fluids 21:33–39 (2001). 34 Panizzi L, Flamini G, Cioni PL and Morelli I, Composition and antimicrobial properties of essential oils of four Mediterranean Lamiaceae. J Ethnopharmacol 39:167–170 (1993). 35 Rasooli I, Fakoor MH, Yadegarinia D, Gachkar L, Allameh A and Rezaei MB, Antimycotoxigenic characteristics of Rosmarinus officinalis and Trachyspermum copticum L. essential oils. Int J Food Microbiol 122:135–139 (2008). 36 Zuzarte M, Gonçalves MJ, Cavaleiro C, Cruz MT, Benzarti A, Marongiu B, et al., Antifungal and anti-inflammatory potential of Lavandula stoechas and Thymus herba-barona essential oils. Ind Crops Prod 44:97–103 (2013). 37 Mahmoud ALE, Antifungal action and antiaflatoxigenic properties of some essential oil constituents. Lett Appl Microbiol 19:110–113 (1994). 38 Marino M, Bersani C and Comi G, Impedance measurements to study the antimicrobial activity of essential oils from Lamiaceae and Compositae. Int J Food Microbiol 67:187–195 (2011). 39 Gill AO, Delaquis P, Russo P and Holley RA, Evaluation of antilisterial action of cilantro oil on vacuum packed ham. Int J Food Microbiol 73:83–92 (2002).
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Revised: 3 January 2014
Accepted article published: 15 January 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6576
Effect of citronella essential oil on the inhibition of postharvest Alternaria alternata in cherry tomato Qianru Chen,a,b,c Shixiang Xu,a Tao Wu,a Jun Guo,a,b Sha Sha,a Xiaodong Zhenga,b,c* and Ting Yua* Abstract BACKGROUND: Essential oils such as citronella oil exhibit antifungal activity and are potential alternative inhibitors to chemical synthetic fungicides for controlling postharvest diseases. In this study the antifungal activity of citronella oil against Alternaria alternata was investigated. RESULTS: In vitro, citronella oil showed strong inhibition activity against A. alternata. The minimum inhibitory concentration in potato dextrose agar and potato dextrose broth medium was determined as 1 and 0.8 𝛍L mL−1 respectively. In vivo the disease incidence of Lycopersicon esculentum (cherry tomato) treated with citronella oil was significantly (P < 0.05) reduced compared with the control after 5 days of storage at 25 ∘ C and 95% relative humidity. The disease incidence at oil concentrations of 0.2–1.5 𝛍L mL−1 was 88–48%. The most effective dosage of the oil was 1.5 𝛍L mL−1 , with 52% reduction, and the oil had no negative effect on fruit quality. Scanning electron microscopy observation revealed considerably abnormal mycelial morphology. CONCLUSION: Citronella oil can significantly inhibit A. alternata in vitro and in vivo and has potential as a promising natural product for controlling black rot in cherry tomato. © 2014 Society of Chemical Industry Keywords: Alternaria alternata; citronella oil; antifungal activity; cherry tomato
INTRODUCTION Fresh fruits and vegetables are readily affected by pathogenic fungi such as Alternaria, Botrytis, Fusarium, Geotrichum, Penicillium and Sclerotinia,1 especially under high-moisture and high-temperature conditions, which reduce their shelf life and cause considerable economic losses around the world. In China, cherry tomato (Lycopersicon esculentum var. cerasiforme) is perishable and susceptible to postharvest diseases owing to the warm and humid climate. Alternaria alternata is a major saprophytic pathogen causing postharvest black rot in cherry tomato and is responsible for the postharvest loss of cherry tomato.2 Chemical synthetic fungicides are mostly used as an effective postharvest disease control strategy. However, pathogen resistance to various types of fungicides can be developed after frequent usage.3 In addition, synthetic fungicides are not environmentally friendly reagents and are potential hazards to human health owing to their long degradation time and teratogenic and carcinogenic properties.4 Thus natural antifungal products should be developed to meet health demands. Essential oil, the aromatic volatile oily liquid of plant secondary metabolism, represents part of the defense system of plants against infection by microorganisms.5 The antifungal activity of an essential oil depends on the type and amount of phenolic compounds it contains.6 Several essential oils with biologically active components have been applied as effective disease control agents, since these components have a broad spectrum against J Sci Food Agric (2014)
plant pathogens or Insects.7 Furthermore, essential oils extracted from plants are considered to be non-phytotoxic agents, suggesting that the majority of them are safe for consumers, and also have a low risk of pathogens developing resistance to them.1,8 – 10 Previous studies have proved the activities of certain essential oils against fungal pathogens to control postharvest diseases in vitro and in vivo.4,10 – 13 The antimicrobial properties of the oils were attributed to the presence of mono- and sesquiterpenes, monoand sesquiterpene hydrocarbons and phenolic compounds such as thymol, carvarol, citral, trans-2-decenal and p-cymene.1 Cymbopogon nardus (L.) Rendle (citronella grass or Ceylon citronella), an aromatic grass belonging to the Poaceae family, originated from Sri Lanka and southern India and is now grown in many areas of the world.14 The citronella oil extracted from C. nardus (L.) Rendle is mainly composed of monoterpenes (up to 80%), including citronellal, citronellol and geraniol,
∗
Correspondence to: Xiaodong Zheng, Department of Food Science and Nutrition, Zhejiang University, Hangzhou, 310058, China, and Ting Yu, E-mail: [email protected]; [email protected]
a Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, China b Zhejiang Key Laboratory for Agro-Food Processing, Hangzhou 310058, China c Fuli Institute of Food Science, Zhejiang University, Hangzhou 310058, China
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www.soci.org and some sesquiterpenes.15 Citronella oil has been used in mosquito repellent,16 perfume, soap and cosmetics.14 Studies have demonstrated inhibitory effects of the oil on ectoparasites and pathogenic microorganisms, including bacteria and fungi.17,18 The antimicrobial properties of citronella oil may be due to its ingredients such as geraniol and citronellol.19 Thus citronella oil may have potential activity to inhibit A. alternata (the main spoilage fungus on fruits and vegetables, especially cherry tomato). Owing to the lack of information on the anti-A. alternata activity of citronella oil, it is necessary to systematically evaluate the antifungal efficacy of the oil against A. alternata.
MATERIALS AND METHODS Fungal strain Alternaria alternata obtained from the Institute of Microbiology, Chinese Academy of Science was cultured on a potato dextrose agar (PDA) slant at 25 ∘ C for 10–12 days. Essential oil Pure citronella oil extracted from C. nardus (L.) Rendle (from Jiang Xi province, China) by water distillation was purchased from Yue Yu Essential Oil Corporation (Guang Dong province, China). The oil was stored at 4 ∘ C before use. In vitro antifungal activity assay The antifungal activity of the essential oil against A. alternata was determined by the method of Singh et al.20 with some modifications. The oil was dissolved and diluted tenfold in 5 mL L−1 Tween 20 and mixed aseptically with PDA medium at 40–45 ∘ C to obtain oil concentrations of 0 (control), 0.2, 0.4, 0.6, 1 and 1.2 μL mL−1 . The mixed media were transferred onto 90 mm Petri plates (15 mL per plate). A mycelium disk cut from a 10-day-old culture of A. alternata grown on PDA was placed on the center of each Petri plate and then incubated at 25 ∘ C. The mean diameter of mycelial growth of the fungus was determined each day for 7 days. All treatments consisted of five replicates and the whole experiment was performed three times. Mean growth values were converted into percentage mycelial inhibition using the formula ) ] [( percentage inhibition = dc − dt ∕dc × 100% where dc (mm) and dt (mm) represent the mean colony diameters of the control and treated groups respectively. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of the oil that inhibited mycelial growth completely. In vivo antifungal activity assay on cherry tomato fruit The antifungal assay of the essential oil against A. alternata in vivo on cherry tomato was conducted by the modified method of Feng and Zheng10 (fruit pathology test). Mature and healthy fresh cherry tomatoes from Zhejiang Academy of Agricultural Sciences were dipped in 1 mL L−1 sodium hypochlorite for 1 min, washed in running water and dried at room temperature. Each fruit was cut at the equatorial region with a 4 mm diameter and 2 mm deep hole using a sterile puncher to generate the wound. The fruits were then divided randomly into six groups and placed in containers (21 cm × 15 cm white plastic boxes). A 10 μL aliquot of spore suspension (104 spores mL−1 ) was inoculated into each wound. After 30 min, 20 μL aliquots of the oil with concentrations of 0 (control), 0.2, 0.5, 0.8, 1, 1.5 and 2 μL mL−1 were pipetted into
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individual wounds. The containers were sealed with plastic wrap to maintain a high relative humidity (RH) of 90–95% during storage. The disease incidence of the fruits was measured after 5 days of storage at 25 ∘ C. Twenty fruits were used in a treatment and each treatment was repeated three times in an experiment. The quality of oil-treated/untreated fruits was evaluated for mass loss, fruit firmness, total soluble solids (TSS) and titratable acidity (TA) after fungal infection.21 All quality indices were measured before and after 15 days of storage (13 ∘ C, 85% RH). Mass loss was determined by monitoring the weight of the fruits and calculated as a percentage. Fruit firmness was measured at one point of the equatorial region using a texture analyzer (GY-3, Top Instrument Co., Ltd, Hang Zhou, Zhe Jiang provinve, China). TSS was evaluated by measuring the refractive index of cherry tomato juice with a hand-held refractometer (WYT-32, Top Instrument Co., Ltd). TA was obtained by titration with 0.05 mol L−1 NaOH to pH 8.1 and reported as g citric acid kg−1 fresh fruit weight. Effect of essential oil on spore germination assay The spore germination of A. alternata was tested by the method of Singh et al.20 The essential oil was dissolved and diluted tenfold in 5 mL L−1 Tween 20 and mixed aseptically with 2 mL of potato dextrose broth (PDB) medium in test tubes to obtain oil concentrations of 0 (control) 0.1, 0.3, 0.5, 1 and 1.5 μL mL−1 . A 100 μL aliquot of spore suspension of A. alternata (106 spores mL−1 ) was added to each test tube and the test tubes were placed on a rotary shaker at 0.58 × g for 12 h at 25 ∘ C. Approximately 150 spores of pathogen were counted in each replicate to determine the germination rate of the pathogen. The spore germination assay was estimated under a microscope. Each treatment consisted of three replicates and the experiments were performed three times. Effect of essential oil on wet and dry mycelium weight The effect of the essential oil on the wet and dry mycelium weight of the pathogen was measured by the method of Dikbas et al.22 The oil was dissolved and diluted tenfold in 5 mL L−1 Tween 20 and transferred into Erlenmeyer flasks with 50 mL of PDB medium to obtain oil concentrations of 0 (control), 0.1, 0.3, 0.5, 0.7 and 0.9 μL mL−1 . Then 100 μL of spore suspension of the pathogen (106 spores mL−1 ) was inoculated into each flask and the flasks were incubated on a rotary shaker at 0.58 × g and 25 ∘ C. The mycelia were harvested on days 5 and 10 by centrifugation at 3260 × g for 15 min and precipitates were washed twice by dipping them in distilled sterile water and filtering for 30 min. The wet mycelia were weighed. The dry mycelia were weighed after drying the wet mycelia at 60 ∘ C for 12 h. Each treatment consisted of three replicates and the experiments were performed three times. Scanning electron microscopy (SEM) examination of effect of essential oil on hyphal morphology SEM (S-3000N, Hitachi Limited, Tokyo, Japan) analysis was performed using the method of Lu et al.23 with some modifications. Ten-day-old fungal materials of A. alternata cultured in PDB medium only (control) and PDB medium with the most effective concentration of the oil were observed by SEM. Glutaraldehyde (250 mL L−1 , Ted Pella, Inc., Redding, CA, USA) at a concentration of 25 mL L−1 in 0.1 mol L−1 phosphate-buffered saline (PBS) (pH 7.2) was added to the fungal samples and fixed overnight at 4 ∘ C. The fixed samples were then treated with following steps. Each sample was rinsed with 0.1 mol L−1 PBS (pH 7.2) three times for 15 min, fixed again with 5 mL L−1 osmium tetroxide solution (40 mL L−1
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Inhibition of A. alternata in cherry tomato by citronella oil
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aqueous, Ted Pella, Inc.) for 1–2 h and washed again with PBS (pH 7.2). The fixed samples were subjected to a series of graded acetone dehydration processes (500, 700, 800, 900 and 950 mL L−1 , 15 min for each concentration) and then dehydrated twice with pure ethanol for 20 min. After treatment with a mixture of ethanol and isoamyl acetate for 30 min, samples were treated with pure isoamyl acetate for 1–2 h. After dehydration, samples were dried in critical point dryer (HCP-2, Hitachi Limited) at critical point according to the manufacturer’s instructions and mounted on stubs using a carbon adhesive. Finally, samples were coated with a gold/palladium layer by sputter coating with an ion sputter (E-1010, Hitachi Limited) and scanned and photographed by SEM at 15 kV. Identification of oil components by gas chromatography/mass spectrometry (GC/MS) The chemical composition of the essential oil was analyzed by GC/MS (5975I, Agilent Co., Santa Clara, CA, USA). Constituents were separated on a 30 m × 250 μm × 0.25 μm capillary column (HP-5MS, Agilent Co.). High-purity helium was used as carrier gas at a constant flow rate of 1 mL min−1 . The pressure at the column inlet was 9.37 psi. The GC column temperature was initially held 40 ∘ C for 1 min and then increased at a rate of 10 ∘ C min−1 to 280 ∘ C and held for 6 min. The temperature of the auxiliary heater was maintained at 280 ∘ C. Eluted components were detected using an electron ionization (EI) mass spectrometer (EI mode, 70 eV). Data acquisition was in full-scan mode in the range m/z 45–500. The temperatures of the ion source and quadrupole were set at 230 and 150 ∘ C respectively. The relative contents of the oil were calculated as percentages following the peak area normalization method. The oil compounds were identified by comparing retention indices obtained according to a standard solution of n-alkanes (C7–C26) with known compounds in a reference of authentic standards24 and by comparing their mass spectra with the NIST/NIH/EPA spectral database and also by manual interpretation. Statistical analysis Data are presented as mean ± standard deviation (n = 3). All data were subjected to single-factor analysis of variance (ANOVA) using SPSS Statistics 17.0 (SPPS Inc., Chicago, IL, USA). Significant differences in mean values were assessed by Duncan’s multiple range test at the level P < 0.05.
RESULTS In vitro antifungal activity assay The antifungal activity of the oil on A. alternata mycelial growth during 7 days of incubation is shown in Fig. 1. The results indicated that the diameter of the fungal mycelia increased with increasing incubation time. Also, the mycelial growth of all treatments was considerably inhibited compared with the control in a dose-dependent manner. The beginning of visible mycelial growth was delayed 2 days at 0.6 μL mL−1 and 3 days at 0.8 μL mL−1 , while mycelial growth was completely inhibited at 1 μL mL−1 during the entire incubation time. The MIC of the oil was 1 μL mL−1 after 7 days of incubation. The inhibition rate of oil treatment calculated after 7 days of cultivation was 26–100% with oil concentrations of 0.2–1 μL mL−1 . Antifungal activity of essential oil in vivo on cherry tomato The antifungal activity of the essential oil in vivo on cherry tomato is shown in Fig. 2. The disease incidence of all treatments except J Sci Food Agric (2014)
Figure 1. Effect of citronella oil on inhibition of mycelial growth of Alternaria alternata in vitro. The diameter of the fungal mycelial growth of the control and different concentrations of the oil was measured from day 2 to day 7 during the cultivation time. Values are mean ± standard deviation (n = 3).
Figure 2. Effect of citronella oil on inhibition of Alternaria alternata in vivo (cherry tomato). The disease incidence of the control and treatments with different concentrations of the oil was measured after 5 days of storage at 25 ∘ C. Values are mean ± standard deviation (n = 3). Values with different letters are significantly different (P < 0.05).
0.2 μL mL−1 was significantly (P < 0.05) reduced compared with the control. The oil concentration of 1.5 μL mL−1 exhibited the greatest antifungal activity, reducing the disease incidence to 48% compared with the control. The disease incidence at oil concentrations of 0.2–1.5 μL mL−1 was 88–48%. The disease incidence with the treatment at 2 μL mL−1 was significantly higher than that at 1.5 μL mL−1 . Table 1 shows the effect of citronella oil on fruit quality. After 15 days of storage, increased mass loss and decreased fruit firmness, TSS and TA were observed, while the oil had no significant effect on these four indicators after the storage period. Effect of essential oil on A. alternata spore germination The effect of the oil on spore germination of the fungus is shown in Fig. 3. The results indicated that oil treatment significantly (P < 0.05) inhibited fungal spore germination compared with the control in a dose-dependent manner after 12 h of incubation at 25 ∘ C. Spore germination was incompletely inhibited at oil concentrations of 0.1–1.5 μL mL−1 in PDB medium, with the spore germination rate decrease ranging from 54 to 13%.
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Table 1. Effect of citronella oil on postharvest quality characteristics of cherry tomato before and after 15 days of storage (85% RH) at 13 ∘ C Treatment (μL mL−1 ) Before storage After storage
– 0 0.8 1.5 2
Mass loss (%)
Firmness (105 Pa)
0 1.42 ± 0.12a 1.40 ± 0.08a 1.22 ± 0.14a 1.53 ± 0.18a
4.22 ± 0.02 4.83 ± 0.21a 4.91 ± 0.02a 4.86 ± 0.11a 4.73 ± 0.07a
Total soluble solids (TSS) (∘ Brix)
Titratable acidity (TA) (g kg−1 )
7.06 ± 0.35 6.57 ± 0.96a 6.02 ± 0.28a 6.73 ± 0.42a 6.30 ± 0.75a
9.01 ± 0.72 8.82 ± 1.30a 10.30 ± 1.27a 9.22 ± 0.76a 9.42 ± 0.69a
Values are mean ± standard deviation (n = 3). Values in the same column with different letters are significantly different (P < 0.05).
inhibited. The 0.8 μL mL−1 concentration of citronella oil was thus most effective and considered as the MIC for A. alternata in PDB medium.
Figure 3. Effect of citronella oil on inhibition of spore germination of Alternaria alternata. The fungal germination rate of the control and treatments with different concentrations of the oil was measured after 12 h of cultivation. Values are mean ± standard deviation (n = 3). Values with different letters are significantly different (P < 0.05).
Effect of essential oil on A. alternata wet and dry mycelium weight The effect of citronella oil on A. alternata wet and dry mycelium weight in PDB medium was measured on days 5 and 10 after inoculation and the results are shown in Table 2. On day 5 the biomass of the fungal mycelium for all treatments except 0.2 μL mL−1 was significantly (P < 0.05) reduced compared with the control, and no mycelium was produced at oil concentrations of 0.6 and 0.8 μL mL−1 . On day 10 the fungal mycelium biomass for the treatments at 0.4 and 0.6 μL mL−1 increased substantially in comparison with that on day 5. No growth of mycelium was observed on either day 5 or day 10 for the treatment at 0.8 μL mL−1 , i.e. mycelium production was completely
SEM examination of hyphal morphology SEM examination of hyphal morphology was conducted to determine the influence of the essential oil on the microstructural surface of A. alternata (Fig. 4). The A. alternata mycelium grown in PDB medium as the control showed regular and normal morphology with rich conidia (Fig. 4A). Furthermore, the control clearly exhibited healthy development of conidia and intact, plump hyphae with a smooth external surface (Fig. 4C). In contrast, the mycelial morphology changed severely after treatment with the most effective essential oil concentration determined in PDB medium. The hyphae folded and flattened with a rough surface and deformed with absence of conidia, and the treated hyphae also showed abnormal mycelial morphology with shrivelled and wrinkled flattened empty hyphae (Figs 4B and 4D). Chemical composition of essential oil To determine the active ingredients in citronella oil, its chemical composition was analyzed by GC/MS and the results are listed in Table 3. A complex mixture of 23 compounds accounting for 96.68% of the total oil were identified, consisting of monoterpenes (75.38%) and sesquiterpenes (21.30%). The major individual compounds were citronellal (26.23%), geraniol (19.75%) and citronellol (12.96%), followed by elemol (5.07%), 2,6-dimethyl-2,6-octadiene (4.31%), 𝛿-cadinene (4.21%), geranyl acetate (3.58%), D-limonene (2.96%), 𝛽-elemene (2.80%), 𝛼-cadinol (2.75%) and other components in lower amount.
Table 2. Effect of citronella oil on biomass of Alternaria alternata in PDB medium at days 5 and 10 during incubation Mycelium weight Concentration of essential oil (μL mL−1 )
0 0.2 0.4 0.6 0.8
Day 5
Day 10
Wet (g)
Dry (mg)
Wet (g)
Dry (mg)
14.92 ± 1.27a 18.84 ± 0.85a 2.95 ± 0.76b 0.00 ± 0.00c 0.00 ± 0.00c
568.73 ± 45.51a 580.27 ± 19.09a 81.60 ± 17.41b 0.00 ± 0.00c 0.00 ± 0.00c
14.63 ± 1.02a 15.12 ± 0.18a 12.89 ± 0.65b 0.34 ± 0.06c 0.00 ± 0.00d
560.13 ± 22.63a 535.00 ± 24.69a 430.97 ± 32.59b 8.63 ± 2.23c 0.00 ± 0.00d
Values are mean ± standard deviation (n = 3). Values in the same column with different letters are significantly different (P < 0.05).
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Inhibition of A. alternata in cherry tomato by citronella oil
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Figure 4. Scanning electron micrographs of Alternaria alternata mycelium grown in PDB medium without (control) or with (treated) 0.8 μL mL−1 citronella oil during 7 days of incubation at 25 ∘ C: A, control with rich conidiophore and regular hyphae of A. alternata (magnification × 500, bar = 100 μm); B, treated variant with absence of conidia and unhealthy hyphal morphology (magnification × 500, bar = 100 μm); C, control of intact conidia and plump hyphae with smooth external surface (magnification × 5000, bar = 10 μm); D, treated shrivelled and wrinkled hyphae with rough surface (magnification × 5000, bar = 10 μm). Table 3. The composition of citronella oil obtained by water distillation from Cymbopogon nardus (L.) Rendle
Number
Retention time (min)
Peak area (Ab*s)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
7.688 8.782 9.542 9.634 9.721 10.711 11.112 11.325 11.851 12.419 12.552 12.824 13.058 14.151 14.243 14.440 14.632
3293948 788870 1558038 29139061 637172 14404889 21942928 749497 883112 4789168 1607440 3974360 3107166 659667 1099347 1371575
18 19 20 21 22 23
14.719 14.907 15.037 16.043 16.139 16.293
4678365 325434 5630677 773216 1530192 3052473
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Compound name
Composition (%)
D-Limonene
Linalool 5-Methyl-2-(1-methylethenyl)cyclohexanol Citronellal Isopulegol Citronellol Geraniol Citral 3,7-Dimethyl-6-octenoic acid 2,6-Dimethyl-2,6-octadiene Eugenol Geranyl acetate 𝛽-Elemene 𝛾-Muurolene Germacrene D 𝛼-Muurolene 1,2,4a,5,6,8a-Hexahydro-4,7-dimethyl1-(1-methylethyl)naphthalene 𝛿-Cadinene 𝛼-Cadinene Elemol Machilol 𝜏-Cadinol 𝛼-Cadinol
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2.96 0.71 1.40 26.23 0.57 12.96 19.75 0.67 0.79 4.31 1.45 3.58 2.80 0.59 0.99 1.23 1.29 4.21 0.29 5.07 0.70 1.38 2.75
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DISCUSSION Previous studies have focused on the inhibitory effect of essential oils against fungal growth in vitro, and some have been tested in vivo to control postharvest diseases.11,25 In this study, citronella oil was systematically investigated in vitro and in vivo on cherry tomato to demonstrate the antifungal inhibition activity against A. alternata. Citronella oil showed pronounced antifungal efficacy against A. alternata mycelial growth in vitro. The MIC in PDA medium was determined as 1 μL mL−1 , which was similar to the result reported by Tian et al.13 (2 μL mL−1 Anethum graveolens L. essential oil against A. alternata), revealing that citronella oil had a strong inhibition activity in controlling A. alternata. In addition, citronella oil effectively suppressed the biomass of A. alternata (Table 2). A lower concentration (0.4 μL mL−1 ) of the oil could suppress mycelium production strongly in the early period of incubation (day 5), though its inhibition activity weakened gradually with prolonged incubation (day 10). These results indicated that the active compounds of the oil at lower concentration delayed the growth of A. alternata effectively. The results of in vivo investigations showed that citronella oil concentrations of 0.5–2 μL mL−1 had a significant inhibition effect against black rot in cherry tomato. An increased percentage of disease incidence at 2 μL mL−1 was found in comparison with 1.5 μL mL−1 . Thus the most effective concentration of citronella oil against A. alternata in vivo was 1.5 μL mL−1 . Tian et al.13 reported that 120 μL mL−1 Cicuta virosa L. var. latisecta Celak essential oil inhibited about 80% disease incidence of Aspergillus flavus, Aspergillus oryzae, Aspergillus niger and A. alternata on cherry tomato, while Xing et al.26 found that 30 μL mL−1 clove oil completely inhibited the disease incidence of A. flavus and Penicillium citrinum on orange fruit. The present usage of citronella oil was relatively low compared with the results of Tian et al.13 and Xing et al.26 It was found that citronella oil treatment did not impair fruit quality during 15 days of storage, similarly to a previous report.27 These results indicate that citronella oil might also have potential for commercialization to control black rot in cherry tomato. Moreover, a low concentration of citronella oil for inhibition of fungi reduces costs effectively. However, it is widely accepted that application in foods requires higher concentrations of essential oil than in laboratory media.22,28,29 This could be explained by altered reaction temperature changing the site of action of the oil in foods, leading to decreased oil invasion of the interior of cells.22 The SEM images of the microstructure of A. alternata revealed a degenerative alteration in hyphal morphology, such as absence of conidia, flattened and shriveled hyphae, swelling and rough hyphal walls. These results resemble those of a previous study in which the microstructure of different fungal hyphae treated with essential oils was analyzed.4 The mode of action of essential oils and their components has not been fully investigated, but one possible mode may be attributable to the hydrophobicity of essential oils, which could partition the lipids of fungal cell membranes and damage the integrity of cell walls and cell membranes, causing an imbalance in intracellular osmotic pressure and the leakage of vital intracellular ingredients such as ATP, ions, nucleic acids and amino acids.8,14,30 One study showed that active components may cause structural and metabolic changes in fungi, such as cytoplasm membrane burst, cytoplasm granulation, uncoupling of oxidative phosphorylation, cytoplasm hyperacidity, disruption of the electron transport chain, loss of pool metabolites and inhibition of H+ -ATPase and transport channels, breakdown of RNA, DNA, protein, lipid and polysaccharide synthesis and, eventually, initiation of autolytic processes.8
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In addition, the main compounds of citronella oil were similar to those found in previous studies, with variations in percentage composition. The main compounds reported in citronella oil from Benin were citronellal (41.3%), geraniol (23.4%) and citronellol (9.2%), from Congo were citronellal (37.5%), geraniol (29.4%) and citronellol (7.5%) and from India were citronellal (29.7%) and geraniol (24.2%).31,32 The variation in percentage composition of different samples of the same essential oil may be due to differences in habitat, genetic diversity and agronomic treatment of the original aroma plants33 and seasonal, climatic and geographical conditions, harvest time and distillation techniques.34 The antifungal activities of citronella oil may depend on the presence of the main components (citronellal, geraniol and citronellol) and other minor components. It was reported that geraniol, citronellol and citral exhibited the highest antifungal activities among terpenoids,19 and some reports revealed that the inhibitory activity of the essential oil was mainly ascribable to the presence of those abundant components.35,36 Mahmoud37 found that geraniol and citronellol contained in citronella oil at 500 mg L−1 inhibited A. flavus growth effectively. Additionally, minor components such as eugenol, linalool and isopulegol could be involved in some antimicrobial synergism with other active components.38 Generally, the essential oil presents greater antimicrobial activities than its individual components and/or mixtures of its major components.39
CONCLUSION Our results indicate that citronella oil possesses strong antifungal activity against A. alternata in vitro and in vivo. The MIC in PDA and PDB medium was 1 and 0.8 μL mL−1 respectively. In vivo the most effective dosage of the oil was 1.5 μL mL−1 , with 52% reduction, and the oil had no negative effect on fruit quality. SEM observation revealed considerably abnormal mycelial morphology. Citronella oil could be a promising natural product for use as an anti-A. alternata agent to control black rot in cherry tomato. However, further studies are required to investigate the mode of action of the essential oil.
ACKNOWLEDGEMENTS This research was partially supported by the National Public Benefit (Agricultural) Research Foundation of China (200903044), the Program for Key Innovative Research Team of Zhejiang Province (2009R50036) and the National Natural Science Foundation of China (31260402). The authors have declared no conflict of interest.
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