Antifungal activity of lemongrass (Cympopogon citratus L.

) essential oil against key postharvest pathogens

Nikos G. Tzortzakis , Costas D. Economakis
Department of Hydroponics and Aromatic plants, Institute of Olive Tree and Subtropical Plants, National Agricultural Research Foundation (N.AG.RE.F.), Agrokipion, 73100, Chania, Greece

Abstract Lemongrass (Cympopogon citratus L.) oil (ranging between 25 and 500 ppm) was tested for antifungal activity against Colletotrichum coccodes, Botrytis cinerea, Cladosporium herbarum, Rhizopus stolonifer and Aspergillus niger in vitro. Oil-enrichment resulted in significant (P b 0.05) reduction on subsequent colony development for the examined pathogens. Fungal spore production inhibited up to 70% at 25 ppm of lemongrass oil concentration when compared with equivalent plates stored in ambient air. In the highest oil concentration (500 ppm) employed, fungal sporulation was completely retarded. Lemongrass oil reduced spore germination and germ tube length in C. coccodes, B. cinerea, C. herbarum and R. stolonifer with the effects dependent on oil concentration. However, lemongrass oil (up to 100 ppm) accelerated spore germination for A. niger. Work is currently focussing on the mechanisms underlying the impacts of essential oil volatiles on disease development with a major contribution to limiting the spread of the pathogen by lowering the spore load in the storage/transit atmospheres as well as the use of essential oil as an alternative food preservative.

Keywords: Antifungal activity; Essential oils; Fungal growth; Lemongrass Industrial relevance: The present study suggests that the use of pure lemongrass essential oil is an innovative and useful tool as alternative to the use of synthetic fungicides or other sanitation techniques in storage/packaging. Oil enrichment may reduce disease development with a major contribution to limiting the spread of the pathogen by lowering the spore load (spore production) in the storage/transit atmospheres as well as the use of essential oil as an alternative food preservative. The effectiveness (oil concentration) of the oil depends on the target pathogen. The effects of natural compounds on individual microorganisms (fungi and bacteria), both responsible for spoilage and food-borne pathogens, as well as the minimum concentration to gain effectiveness without affecting fresh produce quality and storage deserve further research.

1. Introduction The widespread use of pesticides has significant drawbacks including increased cost, handling hazards, concern about pesticide residues on food, and threat to human health and environment (Paster & Bullerman, 1988). Public awareness of these risks has increased interest in finding safer alternatives protectants to replace synthetic chemical pesticides. One such alternative is the use of natural plant protectants with pesticidal
Corresponding author. Tel.: +30 28210 83435, +30 6973531250; fax: +30 28210 93963. E-mail addresses: Nikos.Tzortzakis@ncl.ac.uk, ntzortzakis@Googlemail.com (N.G. Tzortzakis). 1466-8564/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ifset.2007.01.002

activity, as well as they tend to have low mammalian toxicity, less environmental effects and wide public acceptance (DonPedro, 1996; Hamilton-Kemp et al., 2000; Liu & Ho, 1999; Paranagama, Abeysekera, Abeywickrama, & Nugaliyadd, 2003; Paster, Menasherov, Ravid, & Juven, 1995). Essential oils are complex volatile compounds produced in different plant parts, which are known to have various functions in plants including conferring pest and disease resistance (Goubran & Holmes, 1993). The complexity in essential oils is due to terpene hydrocarbons as well as their oxygenated derivatives, such as alcohols, aldehydes, ketones, acids and esters (Wijesekara, Ratnatunga, & Durbeck, 1997). Lemongrass (Cympopogon citratus L.) is a plant in the grass family that contains 1 to 2% essential oil on a dry basis with

Table 1 Percentage composition (> 1%) of the lemongrass essential oil Components Limonene Citronellal n.i. Borneol n.i. Neral Geranial Neryl acetate Z-caryophyllene Identified components (%) n.i.: not identified. Retention time (min) 15.789 20.804 21.248 21.248 21.902 24.055 25.120 28.204 30.011 Lemongrass oil (%) 4.39 1.32 2.16 2.16 1.66 31.85 40.79 2.95 2.71 96

widely variation of the chemical composition as a function of genetic diversity, habitat and agronomic treatment of the culture (Carlson, Machado, Spricigo, Periera, & Bolzan, 2001). Lemongrass essential oil is characterized by a high content of citral (composed of neral and geranial isomers (c. 69%)), which is used as a raw material for the production of ionone, vitamin A and betacarotene (Paviani, Pergher, & Dariva, 2006). Several studies reported antimicrobial activities (even for human pathogenic fungi) by lemongrass oil (Appendini & Hotchkiss, 2002; Daferera, Ziogas, & Polissiou, 2003; Hammer, Carson, & Riley, 1999; Plotto, Roberts, & Roberts, 2003; Saikia, Khanuja, Kahol, Gurta, & Kumar, 2001; Serrano, Martinez-Romero, Castillo, Guillen, & Valero, 2005). Indeed, the lemongrass oil exhibited a broad spectrum of fungitoxicity by inhibiting completely growth of 35, 45, and 47 fungal species at 500, 1000, and 1500 ppm, respectively, and its fungitoxic potency remained unaltered for 210 days of storage, after which it started to decline, with considerable interests in the application of lemongrass oil for the preservation of stored food crops (Mishra & Dubey, 1994). Moreover, the essential oil of C. citratus was superior to synthetic fungicides like Agrosan GN, Dithane M-43 and copper oxychloride (Mishra & Dubey, 1994; Adegoke & Odesola, 1996). Lemongrass as well as oregano and bay oil inhibited all microorganisms examined at 2% (v/v) (Adegoke & Odesola, 1996; Hammer et al., 1999). Moreover, lemongrass oil was nonphytotoxic in nature, since it did not exhibit any adverse effects on germination and seedling growth of wheat and rice (Mishra & Dubey 1994). Interestingly, lemongrass oil showed higher activity than pure isolate (citral) as reported by Saikia et al. (2001). The aim of this study was to determine the efficacy of lemongrass oil against postharvest pathogens with emphasis for the possible future use of the essential oil as alternative antimould compounds. 2. Material and methods 2.1. Plants and oils constituents Essential oil derived from lemongrass (C. citratus L.) was obtained from the Natural Product Division (Neal's Yard Remedies, Manchester, UK). Essential oil extracted by hydro-

distillation and its quality and stability was certified by suppliers. The analysis of the essential oil was performed using a Hewlett Packard 6890 GC, equipped with a HP-5MS (crosslinked 5% PH ME Siloxane) capillary column (30 m, 0.25 mm i.d., 0.25 mm film thickness) and a mass spectrometer 5973 as detector (Tzortzakis, unpublished data). The carrier gas was helium, by a rate of 1 ml/min. Column temperature was initially kept for 3 min at 50 C, then gradually increased to 300 C at a 4 C/min rate and then held to 300 C for 20 C/min. For GC MS detection an electron ionization system was used with ionization energy of 70 eV. Injector and detector (MS transfer line) temperatures were set at 230 C and 310 C, respectively. Diluted samples of 0.1 ml were injected manually and splitless. The relative percentage-concentration of compounds was obtained by integrating the peak area of the chromatograms (see Table 1). 2.2. Inocula Colletotrichum coccodes, Cladosporium herbarum and Aspergillus niger isolated from tomato fruit (Lycopersicon esculentum L.) were supplied by DSMZ (Deutsche Sammlung von Mikroorganismen und Zellekulturen GmbH, Mascheroder Weg, Braunschweig, Germany). Botrytis cinerea and Rhizopus stolonifer isolated from tomato were supplied by CABI (Cabi Bioscience UK Centre, Bakeham Lane, Egham, England). Isolates were aseptically sub-cultured on standard triple-vented Petri dishes containing 20 ml of Potato Dextrose Agar (PDA, Oxoid Ltd, Hampshire, UK). Plates were incubated in the dark at 25 C for 1 week and cultures were stored at 4 C for longterm use. 2.3. Impact of lemongrass oil on pathogen development in vitro Antifungal activity on fungal colony development was obtained by dilution method (25 ppm, 50 ppm, 100 ppm and 500 ppm) of essential oil (C. citratus) in the appropriate culture media-PDA. The oils were dissolved in 5% Tween 20 and added to the 20 ml of PDA before solidified into Petri dish. One disc (0.5 cm diameter) of mycelial plug, taken from the edge of fourto-six-day-old fungal cultures, was placed into the Petri dish. Petri dishes were placed in containers with filter paper moistened with water maintaining high relative humidity (RH 9095%) during the inoculation period. The containers were then transferred to storage at 13 C in a cold room and incubated for six days for C. coccodes, C. herbarum and A. niger, four days for B. cinerea and three days for R. stolonifer. Controls consisted with 5% Tween 20 mixed with PDA and were handled similarly with the exception of the volatile treatment. The efficacy of treatments was evaluated by measuring fungal colony development (in cm2). 2.4. Impact of lemongrass oil on fungal spore production and spore germination Spores from six- to ten-day-old colonies (until spores formed) of C. coccodes, B. cinerea, C. herbarum, R. stolonifer and A. niger

Fig. 1. Impacts of lemongrass (Cympopogon citratus L.) essential oil-enrichment on colony growth (cm2) of Colletotrichum coccodes, Botrytis cinerea, Cladosporium herbarum, Rhizopus stolonifer and Aspergillus niger raised on PDA. Plates were incubated in controlled environment champers maintained at 13 C and 95% RH. Values represent means ( SE) of measurements made on six independent plates per treatment.

previously exposed to lemongrass oil-enrichment (25 ppm, 50 ppm, 100 ppm and 500 ppm) were collected by adding 5 ml of sterile water containing 0.1% (v/v) Tween 80 (for better spore separation) to each Petri dish and rubbing the surface with a sterile L-shaped spreader (3 times). The suspension was collected and then centrifuged at room temperature at 2000 g (Sorvall RC-5B Plus, Dupont, Wilmington, USA) for 5 min. The supernatant was discarded and re-centrifuged until 1 ml of highly concentrated spore solution remained. A haemocytometer slide was used to count spore production. Fungistatic or fungicidal effects were examined on spore viability following oil treatments. Spores from six- to ten-day-old colonies of C. coccodes, B. cinerea, C. herbarum, R. stolonifer and A. niger previously exposed to lemongrass oil-enrichment (25 ppm, 50 ppm, 100 ppm and 500 ppm) were collected as

described above. Spore suspension was inoculated on fresh PDA medium (23 mm thick). Plates were exposed to ambient air at 13 C for 24 h and for each of six replicates, 100 spores were examined and the extent of spore germination assessed by looking for the presence of germ tubes. Results were expressed in terms of the percentage of spores germinated. Moreover, germ tube length (m) was also evaluated. All experiments were repeated twice. 2.5. Statistical analysis Data were first tested for normality, and then subjected to analysis of variance (ANOVA). Significant differences between mean values were determined using Duncan's Multiple Range test (P = 0.05) following one-way ANOVA. Statistical analyses

Table 2 Impacts of lemongrass (Cympopogon citratus L.) essential oil-enrichment on spore production (number of spores 106) by Colletotrichum coccodes, Botrytis cinerea, Cladosporium herbarum, Rhizopus stolonifer and Aspergillus niger raised on PDA Treatment Control 25 ppm 50 ppm 100 ppm 500 ppm C. coccodes 12.10 5.13b 2.55c 1.53cd 0.00d
a

B. cinerea 14.28 4.30b 5.23b 5.75b 0.00c

a

C. herbarum 79.35 47.70b 45.38b 39.88b 0.00c

a

R. stolonifer 14.13 9.23b 8.63b 5.00c 0.00d

a

A. niger 169.50a 99.35b 62.93b 63.13b 0.00c

Plates were incubated in controlled environment champers maintained at 13 C and 95% RH. Values represent means of measurements made on six independent plates per treatment. In each column, values followed by the same letter do not differ significantly at P = 0.05 according to Duncan's Range Test.

were performed using SPSS (SPSS Inc., Chicago, USA) and graph was produced using Prism v.2.0 (Graph Pad Inc., San Diego, USA). 3. Results and discussion Culture PDA media with lemongrass oil-enrichment resulted in significant (P b 0.05) reduction on subsequent colony development of C. herbarum (up to 18%) at 100 ppm as well as B. cinerea (up to 33%) and R. stolonifer (up to 16%) at 25 ppm (Fig. 1). Moreover, the highest oil concentration employed (500 ppm) revealed complete (100%) inhibition on fungal colony development for all the pathogens per se. This concentration was fungicidal for C. coccodes, C. herbarum, R. stolonifer and A. niger after 10 days inoculation. However, in case of B. cinerea at 500 ppm, fungal colony development initiated after 8 days of inoculation and fungal colony growth inhibited up to 60% following 10 days inoculation (data not presented). Baratta et al. (1998) reported 91% inhibition of the growth of A. niger in liquid culture media, when treated with 1000 ppm lemongrass oil. Lemongrass oil decreased Fusarium verticillioides growth in PDA by 90 and 100% at 500 and 1000 ppm, respectively (Mishra & Dubey, 1994) being in accordance with the present study. However, Adegoke & Odesola

Fig. 2. Illustration (400 magnification) of (A) control and (B) lemongrass (Cympopogon citratus L.) essential oil-enrichment (100 ppm) on germ tube length of Botrytis cinerea, raised on PDA and measured after 24 h incubation in controlled environment champers maintained at 13 C and 95% RH.

Table 3 Impacts of lemongrass (Cympopogon citratus L.) essential oil-enrichment on spore germination (%) and germ tube length (in m) in parenthesis of Colletotrichum coccodes, Botrytis cinerea, Cladosporium herbarum, Rhizopus stolonifer and Aspergillus niger raised on PDA and measured after 24 h incubation in controlled environment champers maintained at 13 C and 95% RH Treatment C. coccodes B. cinerea Control 25 ppm 50 ppm 100 ppm 500 ppm 97 (146 ) 95a (165a) 72b (63b) 73b (61b) 0c (0c)
a a

C. herbarum R. stolonifer A. niger 60a 42b 21c 19c 0d (31a) (31a) (34a) (42a) (0b) 100a 100a 100a 98b 0c (222a) 9c (15b) (165c) 52b (20ab) (215ab) 50b (26a) (179bc) 71a (24a) (0d) 0c (0c)

100 (211 ) 99a (195a) 94b (164a) 93b (161a) 0c (0 b)

Values represent means of measurements made on six independent plates per treatment. In each column, values followed by the same letter do not differ significantly at P = 0.05 according to Duncan's Range Test.

(1996) reported contrasting results, that F. verticillioides growth was not affected when lemongrass oil was added in culture medium. In vitro studies of oregano, thyme, lemongrass, and cilantro vapours (5001000 ppm) showed complete growth inhibition of B. cinerea and Alternaria arborescens. Geotrichum candidum was more sensitive to lemongrass oil vapours than to thyme or oregano oils (Plotto et al., 2003). Lemongrass oil was only effective at 1000 ppm, whereas no inhibition observed for Rhizopus fungi (Plotto et al., 2003). Indeed, antimicrobial activity and preservative of lemongrass oil are believed to be associated with phytochemical components of the lemongrass powder, like alkaloids, tannins and cardiac glycosides (Adegoke & Odesola, 1996). The impact of lemongrass oil-enrichment on fungal sporulation in PDA revealed spore production to be significantly (P b 0.05) inhibited when compared with equivalent plates stored in ambient air, with spore production depressed by 70% for B. cinerea, 58% for C. coccodes, 41% for A. niger, 40% for C. herbarum, and 35% for R. stolonifer at 25 ppm (Table 2). Moreover, spore production was completely inhibited at the highest oil concentration (500 ppm) examined for all of the pathogens. Previous studies reported that the sporulation of Aspergillus flavous was completely inhibited by C. citrates (2800 ppm) when used as fumigant whereas aflatoxin production inhibited at 100 ppm of C. citrates treatments (Paranagama et al., 2003). Table 3 shows the impact of lemongrass oil on spore germination and germ tube length. ANOVA revealed spore germination to be significantly (P b 0.05) reduced by lemongrass oil in C. coccodes, B. cinerea, C. herbarum and R. stolonifer with the impacts of oil dependent on different oil concentrations. The greater inhibition on spore germination was observed in

C. herbarum (81%) and the least in R. stolonifer (2%). However, lemongrass oil (up to 100 ppm) accelerated spore germination for A. niger. Indeed, the greatest oil concentration (500 ppm) inhibited spore germination due to failure of spore production. Lemongrass oil-enriched PDA reduced germ tube length for C. coccodes, whereas no major differences were observed for B. cinerea, C. herbarum and R. stolonifer (see Table 3 and Fig. 2 for B. cinerea). Increased spore germination accelerated by increased lemongrass oil concentration resulted in increased germ tube length for A. niger. However, oil treatments revealed no differences on fungal spore viability among the treatments when five essential oils (lemongrass, cinnamon, rosemary, lavender and basil) were tested for antifungal activity against green mould (P. digitatum) implying that effects were fungistatic (Tzortzakis, unpublished data) being in accordance with the previous studies (Alzoreky & Nakahara, 2003; Mishra & Dubey, 1994). Moreover, it was reported that lemongrass oil has not demonstrated fungicidal activity against P. digitatum and P. italicum when applied as a fumigant (Goubran & Holmes, 1997) contrasting the present results when examined in different pathogens probably due to variation of the treatment and/or pathogen per se. This study indicated that essential oils may possess antifungal activity and can be exploited as an ideal treatment for future plant disease management programs eliminating fungal spread. Recently, there has been a considerable interest in extracts and essential oils from aromatic plants with antimicrobial activities for controlling pathogens and/or toxin producing microorganisms in foods (Reddy, Angers, Gosselin, & Arul, 1998; Soliman & Badeaa, 2002; Valero & Salmeron, 2003). Essential oils are natural products extracted from vegetal materials, which because of their antibacterial, antifungal, antioxidant and anti-carcinogenic properties can be used as natural additives in many foods (Teissedre & Waterhouse, 2000). In general, the levels of essential oils and their compounds necessary to inhibit microbial growth are higher in foods than in culture media. This is due to interactions between phenolic compounds and the food matrix (Nuchas & Tassou, 2000) and should be considered for commercial applications. Treatment with basil oil controlled crown rot and anthracnose prolonging storage of bananas (Anthony, Abeywickrama, & Wijeratnam, 2003) as well as cinnamon and eucalyptus oil-enrichment reduced fruit decay and improved fruit quality of tomato and strawberries (Tzortzakis, in press). However, phytotoxicity on the fresh commodity is also to be considered while lemongrass emulsions were more damaging to the tomato tissue than thyme or oregano essential oils (Plotto et al., 2003). Suppression on spore production by oil treatment could make a major contribution to limiting the spread of the pathogen by lowering the spore load in the storage atmosphere and on surfaces. The mechanism underlying the action of essential oil-enrichment on the switch between vegetative and reproductive phases of fungal development remains to be understood. The impacts of oils on sporulation may reflect effects of the volatiles emitted by oils on surface mycelial development (and thus the platform to support spore production) and/or the perception/ transduction of signals involved in the switch from vegetative to reproductive development.

Acknowledgement We thank Dr. Paul Donohoe and colleagues, Environmental Mass Spectrometry Unit, Newcastle University, UK, for their respective technical inputs on oil analysis. References
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