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 activity, as well as they tend to have low mammalian toxicity,

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
less environmental effects and wide public acceptance (Don-
Pedro, 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 distillation and its quality and stability was certified by suppliers.
Percentage composition (> 1%) of the lemongrass essential oil The analysis of the essential oil was performed using a Hewlett
Components Retention time (min) Lemongrass oil (%) Packard 6890 GC, equipped with a HP-5MS (crosslinked 5%
Limonene 15.789 4.39 PH ME Siloxane) capillary column (30 m, 0.25 mm i.d.,
Citronellal 20.804 1.32 0.25 mm film thickness) and a mass spectrometer 5973 as
n.i. 21.248 2.16 detector (Tzortzakis, unpublished data). The carrier gas was
Borneol 21.248 2.16
helium, by a rate of 1 ml/min. Column temperature was initially
n.i. 21.902 1.66
Neral 24.055 31.85 kept for 3 min at 50 °C, then gradually increased to 300 °C at a
Geranial 25.120 40.79 4 °C/min rate and then held to 300 °C for 20 °C/min. For GC–
Neryl acetate 28.204 2.95 MS detection an electron ionization system was used with
Z-caryophyllene 30.011 2.71 ionization energy of 70 eV. Injector and detector (MS transfer
Identified components (%) 96
line) temperatures were set at 230 °C and 310 °C, respectively.
n.i.: not identified. 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
widely variation of the chemical composition as a function of (see Table 1).
genetic diversity, habitat and agronomic treatment of the culture
(Carlson, Machado, Spricigo, Periera, & Bolzan, 2001). Lemon- 2.2. Inocula
grass essential oil is characterized by a high content of citral
(composed of neral and geranial isomers (c. 69%)), which is used Colletotrichum coccodes, Cladosporium herbarum and
as a raw material for the production of ionone, vitamin A and beta- Aspergillus niger isolated from tomato fruit (Lycopersicon
carotene (Paviani, Pergher, & Dariva, 2006). Several studies esculentum L.) were supplied by DSMZ (Deutsche Sammlung
reported antimicrobial activities (even for human pathogenic von Mikroorganismen und Zellekulturen GmbH, Mascheroder
fungi) by lemongrass oil (Appendini & Hotchkiss, 2002; Weg, Braunschweig, Germany). Botrytis cinerea and Rhizopus
Daferera, Ziogas, & Polissiou, 2003; Hammer, Carson, & Riley, stolonifer isolated from tomato were supplied by CABI (Cabi
1999; Plotto, Roberts, & Roberts, 2003; Saikia, Khanuja, Kahol, Bioscience UK Centre, Bakeham Lane, Egham, England).
Gurta, & Kumar, 2001; Serrano, Martinez-Romero, Castillo, Isolates were aseptically sub-cultured on standard triple-vented
Guillen, & Valero, 2005). Indeed, the lemongrass oil exhibited a Petri dishes containing 20 ml of Potato Dextrose Agar (PDA,
broad spectrum of fungitoxicity by inhibiting completely growth Oxoid Ltd, Hampshire, UK). Plates were incubated in the dark
of 35, 45, and 47 fungal species at 500, 1000, and 1500 ppm, at 25 °C for 1 week and cultures were stored at 4 °C for long-
respectively, and its fungitoxic potency remained unaltered for term use.
210 days of storage, after which it started to decline, with
considerable interests in the application of lemongrass oil for the 2.3. Impact of lemongrass oil on pathogen development in vitro
preservation of stored food crops (Mishra & Dubey, 1994).
Moreover, the essential oil of C. citratus was superior to synthetic Antifungal activity on fungal colony development was
fungicides like Agrosan GN, Dithane M-43 and copper obtained by dilution method (25 ppm, 50 ppm, 100 ppm and
oxychloride (Mishra & Dubey, 1994; Adegoke & Odesola, 500 ppm) of essential oil (C. citratus) in the appropriate culture
1996). Lemongrass as well as oregano and bay oil inhibited all media-PDA. The oils were dissolved in 5% Tween 20 and added
microorganisms examined at ≤2% (v/v) (Adegoke & Odesola, to the 20 ml of PDA before solidified into Petri dish. One disc
1996; Hammer et al., 1999). Moreover, lemongrass oil was (0.5 cm diameter) of mycelial plug, taken from the edge of four-
nonphytotoxic in nature, since it did not exhibit any adverse to-six-day-old fungal cultures, was placed into the Petri dish.
effects on germination and seedling growth of wheat and rice Petri dishes were placed in containers with filter paper moistened
(Mishra & Dubey 1994). Interestingly, lemongrass oil showed with water maintaining high relative humidity (RH ∼ 90–95%)
higher activity than pure isolate (citral) as reported by Saikia et al. during the inoculation period. The containers were then
(2001). transferred to storage at 13 °C in a cold room and incubated
The aim of this study was to determine the efficacy of for six days for C. coccodes, C. herbarum and A. niger, four
lemongrass oil against postharvest pathogens with emphasis for days for B. cinerea and three days for R. stolonifer. Controls
the possible future use of the essential oil as alternative antimould consisted with 5% Tween 20 mixed with PDA and were handled
compounds. similarly with the exception of the volatile treatment. The
efficacy of treatments was evaluated by measuring fungal colony
2. Material and methods development (in cm2).

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-
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 (2–3 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 (μm) 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 C. coccodes B. cinerea C. herbarum R. stolonifer A. niger
a a a a
Control 12.10 14.28 79.35 14.13 169.50a
25 ppm 5.13b 4.30b 47.70b 9.23b 99.35b
50 ppm 2.55c 5.23b 45.38b 8.63b 62.93b
100 ppm 1.53cd 5.75b 39.88b 5.00c 63.13b
500 ppm 0.00d 0.00c 0.00c 0.00d 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). 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
3. Results and discussion controlled environment champers maintained at 13 °C and 95% RH.

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 (1996) reported contrasting results, that F. verticillioides growth
25 ppm (Fig. 1). Moreover, the highest oil concentration was not affected when lemongrass oil was added in culture
employed (500 ppm) revealed complete (100%) inhibition on medium. In vitro studies of oregano, thyme, lemongrass, and
fungal colony development for all the pathogens per se. This cilantro vapours (500–1000 ppm) showed complete growth
concentration was fungicidal for C. coccodes, C. herbarum, inhibition of B. cinerea and Alternaria arborescens. Geotrichum
R. stolonifer and A. niger after 10 days inoculation. However, in candidum was more sensitive to lemongrass oil vapours than to
case of B. cinerea at 500 ppm, fungal colony development thyme or oregano oils (Plotto et al., 2003). Lemongrass oil was
initiated after 8 days of inoculation and fungal colony growth only effective at 1000 ppm, whereas no inhibition observed for
inhibited up to 60% following 10 days inoculation (data not Rhizopus fungi (Plotto et al., 2003). Indeed, antimicrobial activity
presented). Baratta et al. (1998) reported 91% inhibition of the and preservative of lemongrass oil are believed to be associated
growth of A. niger in liquid culture media, when treated with with phytochemical components of the lemongrass powder, like
1000 ppm lemongrass oil. Lemongrass oil decreased Fusarium alkaloids, tannins and cardiac glycosides (Adegoke & Odesola,
verticillioides growth in PDA by 90 and 100% at 500 and 1996).
1000 ppm, respectively (Mishra & Dubey, 1994) being in The impact of lemongrass oil-enrichment on fungal sporu-
accordance with the present study. However, Adegoke & Odesola lation 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%
Table 3
for B. cinerea, 58% for C. coccodes, 41% for A. niger, 40% for
Impacts of lemongrass (Cympopogon citratus L.) essential oil-enrichment on C. herbarum, and 35% for R. stolonifer at 25 ppm (Table 2).
spore germination (%) and germ tube length (in μm) in parenthesis of Moreover, spore production was completely inhibited at the
Colletotrichum coccodes, Botrytis cinerea, Cladosporium herbarum, Rhizopus highest oil concentration (500 ppm) examined for all of the
stolonifer and Aspergillus niger raised on PDA and measured after 24 h pathogens. Previous studies reported that the sporulation of
incubation in controlled environment champers maintained at 13 °C and 95%
RH
Aspergillus flavous was completely inhibited by C. citrates
(2800 ppm) when used as fumigant whereas aflatoxin
Treatment C. coccodes B. cinerea C. herbarum R. stolonifer A. niger
production inhibited at 100 ppm of C. citrates treatments
a a a a
Control 97 (146 ) 100 (211 ) 60a (31a) 100a (222a) 9c (15b) (Paranagama et al., 2003).
25 ppm 95a (165a) 99a (195a) 42b (31a) 100a (165c) 52b (20ab)
Table 3 shows the impact of lemongrass oil on spore
50 ppm 72b (63b) 94b (164a) 21c (34a) 100a (215ab) 50b (26a)
100 ppm 73b (61b) 93b (161a) 19c (42a) 98b (179bc) 71a (24a) germination and germ tube length. ANOVA revealed spore
500 ppm 0c (0c) 0c (0 b) 0d (0b) 0c (0d) 0c (0c) germination to be significantly (P b 0.05) reduced by lemongrass
Values represent means of measurements made on six independent plates per oil in C. coccodes, B. cinerea, C. herbarum and R. stolonifer
treatment. In each column, values followed by the same letter do not differ with the impacts of oil dependent on different oil concentrations.
significantly at P = 0.05 according to Duncan's Range Test. 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-carcinogen-
ic 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.
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