As. J. Food Ag-Ind.

2009, Special Issue, S229-S233

Asian Journal of
Food and Agro-Industry
ISSN 1906-3040
Available online at www.ajofai.info

Antifungal activity of essential oils derived from some medicinal

plants against grey mould (botrytis cinerea)
Sirirat Siripornvisal*, Wimolpun Rungprom and Sanit Sawatdikarn

Phranakhon Si Ayutthaya Rajabhat University, Rojana Road,

Phranakhon Si Ayutthaya, 13000 Thailand.

*Author to whom correspondence should be addressed, email: [email protected]

This article was originally presented at the International Symposium “GoOrganic2009”, Bangkok, Thailand,
August 2009.

Abstract

Preventing fungal decay in organic fruits after harvest is an increasingly challenge and novel
preservation approaches which comply with organic food standards need to be developed.
Preliminary experiments have shown that the essential oils of various plants possess some
antifungal activities. In this study, the essential oils derived from four medicinal plants were
evaluated and compared for their antifungal efficacies against Botrytis cinerea, an aggressive
postharvest pathogen on a wide range of fruit. According to a modified in vitro micro-
atmospheric test, vapors of clove oil, cinnamon oil and lemongrass oil exhibited strong
inhibitory effects on B. cinerea, with a MIQ (minimal inhibitory quantity) equal to 15 µL.
However the headspace vapors of galingale oil exhibited weaker inhibitory effect (MIQ = 25
µL). The results of the nature of this inhibition of these oils indicated that clove oil, cinnamon
oil and lemongrass oil all exhibited fungicidal effect on the pathogen, while galingale oil
exhibited some fungistatic properties. These results have shown that the essential oils derived
from clove, cinnamon and lemongrass might be used as alternative options for the control of
gray mould on postharvest organic fruits.

Keywords : antifungal activity; biological control; Botrytis cinerea; essential oil

Introduction

Gray mould decay caused by the fungus Botrytis cinerea is a serious worldwide problem in
flower, fruit and vegetable production (Masih and Paul, 2002). Gray mould can cause severe
As. J. Food Ag-Ind. 2009, Special Issue, S229-S233 230

postharvest losses, particularly in organic produces due to the prohibition of synthetic

fungicides. Therefore, the developments of alternative non-synthetic chemical strategies, which
better comply with organic food standards, are needed. Among the various strategies proposed,
biological control using natural products such as plant extracts and essential oils show most
promise.
Many plant essential oils and their volatile constituents have been reported to possess potent
antifungal activities. The advantage of plant essential oils is their bioactivity in the vapor
phase, which makes them attractive as possible fumigants for the postharvest control of decay
fungi in fruit and grains (Paster et al., 1995; Hammer et al., 1999; Tripathi et al., 2008). For
example, peppermint and sweet basil oils have reported to be effective fumigants to the control
of decay fungi in stored peach fruits (Ziedan and Farrag, 2008). Tripathi et al. (2008) recently
reported the effective control of the gray mould fungi using essential oils derived from Ocimum
sanctum, Prunus persica and Zingiber officinale. In this study, the essential oils derived from
four medicinal plants (clove, cinnamon, lemongrass and galingal) were evaluated for efficacy
against B. cinerea under a modified atmosphere environment in vitro.

Materials and Methods

Pathogen
B. cinerea, was isolated from decayed strawberry fruits and maintained on potato dextrose agar
(PDA) at 4 °C. Fresh cultures of the fungus were prepared by subculture of mycelia onto new
PDA plate and the incubated at 23 °C for 3-5 days.

Extraction of essential oils

Essential oils of selected plants including clove, cinnamon, lemongrass and galingale, were
obtained by hydro-distillation in a Clevenger-type apparatus as described by Tripathi et al.,
(2008). The extracted crude essential oils were stored in sealed glass bottles or flasks, and
protected from the light by wrapping in aluminum foil and stored at 4 °C.

Antifungal bioassay
The volatile component of the essential oils from clove, cinnamon, lemongrass and galingale
were tested for antifungal activity against B. cinerea by using modified micro-atmospheric
method (in vitro) previously described by Singh et al. (1999) with some modification.
PDA plates were prepared using 5 cm Petri dishes containing 5 mL of PDA. A 4 mm
(diameter) agar disc of B. cinerea was cut from the periphery of the active growth culture (3 - 5
days old) and the mycelial surface was placed upside down on the centre of the dish. The Petri
dish was then inverted and a small paper disc (6 mm diameter, Whatman No.1) was placed
inside on the lid of each Petri dish. An aliquot amount (5, 10, 15, 20 and 25 µl) of each
essential oil was applied to the paper disc. Incubation of the fungus and test was conducted in a
growth chamber at 23 °C with 12/12 dark-light regime. Each test was replicated for three
times. The antifungal activity was determined after 3 day incubation by means of the
percentage of inhibited redial growth as following equation:
∆d − ∆d
% inhibition = o × 100
∆d o
Where ∆do and ∆d are the average diameter of the fugal colonies in the control and treatment
sets, respectively

Determination of the nature of the inhibition of essential oil

To determine the nature of the inhibition of each essential oil, the mycelial discs which were
totally suppressed by the essential oil were transferred to a PDA plate which was not
As. J. Food Ag-Ind. 2009, Special Issue, S229-S233 231

supplemented with the essential oil. The treatment was fungistatic if the growth of the fungus
began again and was considered fungicidal if the fungus did not re-grow.

Results and Discussion

Of the four essential oils tested, clove oil, cinnamon oil and lemongrass oil exhibited strong
inhibitory effects on B. cinerea, since they completely suppressed the mycelial growth of the
fungus at 15 µL (MIQ = 15 µL). However the headspace vapors of galingale oil exhibited
weaker inhibitory effect (MIQ = 25 µL) (Table 1).
The lemongrass oil and clove oil appeared to possess the strongest antifungal activity against B.
cinerea since it allow only minimal growth of the fungi at 10 µL and a few growth at 5 µL
dose (Table 1). Lee et al. (2007) tested lemongrass oil (Cymbopogon citrates) as a fumigant
against B. cinerea, but in contrast to this study their results were negative. This may be because
Lee et al. (2007) only applied 1 µL of oil for each treatment, which less than the MIQ level we
found in this study.
Although cinnamon oil exhibited lower antifungal activity, it maybe a better potential
candidate as fumigant as it is more widely available. Moreover, it essential oils may be used in
mixtures to improve their efficacy. Sukatta et al. (2008) previously showed that mixing of
clove oil and cinnamon oils at appropriate ratios result in an improvement of the efficacy
against the postharvest decay fungi of grapes Aspergillus niger, Alternaria alternata,
Colletotrichum gloeosporioides, Lasiodiplodia theobromae, Phomopsis viticola and Rhizopus
stolonifer.
The results of the nature of this inhibition of the oils tested indicated that clove oil, cinnamon
oil and lemongrass oil exhibited fungicidal effect on the pathogen, while galingale oil exhibited
fungistatic properties.

Table 1: Effect of essential oils on the mycelial growth of B. cinerea.

Treatment Dose (µL/disc) mycelial growth (mm)
clove oil 5 15.4 ± 3.71
10 N/M
15 I
20 I
25 I
cinnamon oil 5 30.8 ± 5.54
10 20.2 ± 3.20
15 I
20 I
25 I
galingale oil 5 29.0 ± 2.55
10 24.8 ± 2.39
15 24.0 ± 4.53
20 26.4 ± 3.85
25 I
lemongrass oil 5 12.0 ± 5.10
10 N/M
15 I
20 I
25 I
Control No treatment 31.4 ± 2.41
Note: N/M = Minimal growth was observed but can not measure.
I = Total inhibition
As. J. Food Ag-Ind. 2009, Special Issue, S229-S233 232

It should be noted that the diameter of the fungus growth on the treated plates were nearly the
same diameters as those of the untreated controls (Fig. 1). However the treated colony
appeared flatter and was considerably thinner in appearance. This observation suggests that the
current assessment of essential oils fumigant efficacy against fungi by measurement of growth
diameter alone is not suitable. We proposed that alternative method, such as the measurement
of mycelial dried weight would be a better measure of efficacy.

A B C

Figure 1: Radial growth of B. cinerea after 3 days incubation with different dosage of clove
oil. (A) untreated control, (B) 5 µL treatment : the fungal colony is flatter and thinner, (C) 10
µL treatment : little growth was observed but the colony sized is unmeasurable, (D) 5 µL
treatment : totally inhibited

We also investigated the effect of clove, lemongrass and cinnamon oils by direct investigation
under light microscope. The results showed that the oils caused cutotoxicity to the fungus
resulting in abnormal development of hyphae of the target fungus (Fig. 2).

A B

Figure 2. Abnormal growth of B. cinerea treated with lemongrass oil (A) compared with the
untreated (B).

Conclusions

In conclusion this preliminary study showed that the essential oils derived from clove,
cinnamon and lemongrass maybe used as alternative for the control of gray mould on
postharvest organic fruits especially as a natural fumigant in closed container or packaging.
As. J. Food Ag-Ind. 2009, Special Issue, S229-S233 233

Acknowledgements

The authors acknowledge the Office of the Higher Education Commission of Thailand for
financial support (under the program Strategic Scholarships for Frontier Research Network)

References

Hammer, K.A., Carson, C.F. and Riley, T.V. (1999). Antimicrobial activity of essential oils
and other plant extracts. Journal of Applied Microbiology, Vol. 86, pp. 985-990.

Lee, S.O., Choi, G.J., Jang, K.S. Lim, H.K., Cho, K.Y., Kim, J.C. (2007). Antifungal Activity
of five plant essential oils as fumigant against postharvest and soilborne plant pathogenic fungi.
Plant Pathology Journal, Vol. 23(2), pp. 97-102.

Masih, E.I. and Paul, B. (2002). Secretion of β-1,3-glucanases by the yeast Pichia
membranifaciens and its possible role in the biocontrol of Botrytis cinerea causing grey mold
disease of the grapevine. Current Microbiology, Vol. 44, pp. 391–395.

Paster, N., Menasherov, M., Ravid, U. and Juven, B. (1995). Antifungal activity of oregano and
thyme essential oils applied as fumigants against attacking stored grain. Journal Food
Protection, Vol. 58(1), pp. 81-85.

Singh, G., Sumitra, M., de Lampasona, M.P. and Catalan, C. (2006). Chemical constituents,
antifungal and antioxidative potential of Foeniculum vulgare volatile oil and its acetone
extract. Food Control, Vol. 17, pp. 745–752.

Sukatta,U., Haruthaithanasan, V., Chantarapanont, W., Dilokkunanant, U. and Suppakul, P.

(2008). Antifungal activity of clove and cinnamon oil and their synergistic against postharvest
decay fungi of grape in vitro. Kasetsart Journal (Natural Science), Vol. 42, pp. 169–174.

Tripathi, P., Dubey, N.K. and Shukla, A.K. (2008). Use of some essential oils as post-harvest
botanical fungicides in the management of grey mould of grapes caused by Botrytis cinerea.
World Journal of Microbiology and Biotechnology, Vol. 24, pp. 39–46.

Ziedan, E.H.E. and Farrag, E.S.H. (2008). Fumigation of peach fruits with essential oils to
control postharvest decay. Research Journal of Agriculture and Biological Sciences, Vol.
4(5), pp. 512-519.