See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/223179155

The effectiveness of plant essential oils on the

growth of Botrytis cinerea, Fusarium sp. and
Clavibacter...

Article in Crop Protection · February 2003

DOI: 10.1016/S0261-2194(02)00095-9

CITATIONS READS

442 600

3 authors, including:

Dimitra Daferera Basil N Ziogas

Agricultural University of Athens Agricultural University of Athens
63 PUBLICATIONS 5,276 CITATIONS 48 PUBLICATIONS 1,630 CITATIONS

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Monitoring of environment in a hospital View project

Flora of Sivas View project

All content following this page was uploaded by Dimitra Daferera on 09 December 2016.

The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document
and are linked to publications on ResearchGate, letting you access and read them immediately.
 Crop Protection 22 (2003) 39–44

The effectiveness of plant essential oils on the growth

of Botrytis cinerea, Fusarium sp. and Clavibacter michiganensis
subsp. michiganensis
Dimitra J. Dafereraa, Basil N. Ziogasb, Moschos G. Polissioua,*
a
Department of Sciences, Laboratory of General Chemistry, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece
b
Laboratory of Phytopathology, Agricultural University of Athens, Iera Odos 75, 118 55, Athens, Greece
Accepted 27 May 2002

Abstract

Oregano, thyme, dictamnus, marjoram, lavender, rosemary, sage and pennyroyal essential oils were tested for their effectiveness
against Botrytis cinerea, Fusarium sp. (Fusarium solani var. coeruleum), and Clavibacter michiganensis subsp. michiganensis on
artificial growth media. The chemical composition of the oils was determined by gas chromatography-mass spectrometry (GC-MS).
The growth of Botrytis cinerea, Fusarium sp. and Clavibacter michiganensis subsp. michiganensis was completely inhibited by
oregano, thyme, dictamnus and marjoram essential oils at relatively low concentrations (85–300 mg/ml). Thymol was the main
component of oregano oil, while thyme, dictamnus, and marjoram oils were rich in carvacrol. Lavender, rosemary, sage, and
pennyroyal essential oils presented less inhibitory activity. The growth of the tested microorganisms was affected at concentrations
up to 1000 mg/ml. Lavender oil was characterized by the high content of linalool and linalyl acetate, while eucalyptol was the main
component of sage and rosemary oils. Pennyroyal oil was found rich of cis-menthone and pulegone. r 2002 Elsevier Science Ltd.
All rights reserved.

Keywords: Essential oils; Botrytis cinerea; Fusarium solani var. coeruleum; Clavibacter michiganensis subsp. michiganensis

1. Introduction particularly serious because fruits and vegetables are

Essential oils are naturally occurring terpenic mix-
tures, whose insecticidal action against specific pests and
fungicidal action against some important plant patho-
gens have been recently reviewed (Isman, 2000).
Chemical control remains the main measure to reduce
the incidence of post harvest diseases in various fruits
and vegetables. Antimicrobial chemicals belonging to
the groups of benzimidazoles, aromatic hydrocarbons
and sterol biosynthesis inhibitors are often used, as post
harvest treatments. A serious problem against the
effective use of these chemicals is the development of
resistance by the fungi (Brent and Hollomon, 1998). The
application of higher concentrations of chemicals in an
attempt to overcome this problem increases the risk of
high level toxic residues in the products, which is
*Corresponding author. Tel. +30-152-942-41; fax+30-152-942-65.
E-mail address: mopol@aua.gr (M.G. Polissiou).
consumed in a relatively short time after harvest. The
exploitation of natural substances such as the essential
oils, safer to consumers and the environment, for the
control of postharvest diseases and their effective usage
against both wild type and strains resistant to pesticides,
is urgently needed. Furthermore, the demand for
reduction in the use of pesticides in agriculture increases
interest in the possibility of the application of essential
oils to control plant pathogens. Our interest is focused
on the effectiveness of essential oils against serious plant
pathogens (Daferera et al., 2000). The specific objectives
in the present work were to determine the effectiveness
of the essential oils from oregano (Origanum vulgare),
thyme (Thymus capitatus), dictamnus (Origanum dic-
tamnus), marjoram (Origanum majorana), lavender
(Lavandula angustifolia), rosemary (Rosmarinus officina-
lis), sage (Salvia fruticosa), and pennyroyal (Mentha
pulegium) on the growth of Botrytis cinerea, Fusarium
sp. (Fusarium solani var. coeruleum), and Clavibacter

0261-2194/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 2 6 1 - 2 1 9 4 ( 0 2 ) 0 0 0 9 5 - 9
40 D.J. Daferera et al. / Crop Protection 22 (2003) 39–44
michiganensis subsp. michiganensis and to correlate the
oils’ activity with their chemical composition.
Grey mould disease caused by B. cinerea is one of the
most serious diseases of a wide range of crops of
worldwide importance. Post harvest losses due to
development of grey mould during the storage and
distribution of harvesting fruits and vegetables are very
high. Fusarium solani var. coeruleum and F. sulphureum
cause the potato tuber dry rot, which is an important
disease of stored potatoes. Clavibacter michiganensis
subsp. michiganensis is the causal agent of bacterial
canker of tomatoes. It is a soilborne pathogen and has a
particularly devastating effect on tomatoes under green
house conditions.
A relatively limited number of reports were found
in the literature dealing with the effect of pure
monoterpenes, plant extracts or/and essential oils
against B. cinerea (Caccioni and Guizzardi, 1994; Arras
et al., 1995; Wilson et al., 1996; Thanassoulopoulos and
Laidou, 1997; Reddy et al., 1998), F. solani var.
coeruleum and F. sulphureum (Gorris et al., 1994;
Vaughn and Spencer, 1994; Oosterhaven et al., 1996).
To our knowledge, except for oregano and thyme oils,
which only have been tested against B. cinerea growth,
no data are in the literature for the application of these
oils on the growth of B. cinerea, F. solani var. coeruleum,
and C. michiganensis subsp. michiganensis. Studies
on the effectiveness of the components of essential
oils or plants extracts against phytopathogenic
bacteria have been found in the literature for Erwinia
amylovora (Scortichini and Rossi, 1989, 1991; Mosch
et al., 1990, 1996) and Xanthomonas campestris (Maiti
et al., 1985).
Furthermore, the chosen aromatic plants are wide-
spread in Greek flora, they are rich in essential oil
content and recently a few of them are now system-
atically cultivated in Greece. The chemical composition
of essential oils can be standardized, so that results from
their application could be repeatable.
2. Materials and methods
2.1. Plant material
The aromatic plants were collected from different
areas of Greece: oregano from Fokida (South-Central
Greece), thyme from Naxos island (Central Aegean sea),
dictamnus from Crete island (South Greece), marjoram
from Attiki (Central Greece), rosemary from the area of
the Agricultural University of Athens, sage from Epirus
(North-West Greece) and pennyroyal from Rodopi
(North Greece). Lavender was purchased from
the Athens market. All the samples were air dried
and stored at room temperature in darkness until
distillation.
2.2. Isolation of the essential oils
The classic method of hydrodistillation using the
Clevenger apparatus for 4 h was used for the isolation of
the essential oils from the dried leaves or/and flowers of
aromatic plants. The isolated essential oils in pure form
were stored at 181C until their analysis by gas
chromatography-mass spectrometry (GC-MS) or their
usage in bioassays.
2.3. Analysis conditions
The analysis of the essential oils was performed using
a Hewlett Packard 5890 II 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 5972 as detector. The carrier
gas was helium, by a rate of 1 ml/min. Column
temperature was initially kept for 3 min at 401C, then
gradually increased to 1801C at a 31C/min rate and
finally increased to 2701C at 301C/min and held for
5 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 2201C and 2901C, respectively. Diluted
samples of 0.5 ml were injected manually and splitless.
2.4. Test microorganisms
Botrytis cinerea was provided by the Laboratory of
Phytopathology of the Agricultural University of
Athens (AUA), Fusarium sp. (Fusarium solani var.
coeruleum) was isolated from potato bulbs which
presented the dry rot symptom and Clavibacter
michiganensis subsp. michiganensis was provided by
the collection of Benaki Phytopathological Institute
(Kifissia, Athens).
2.5. Measurement of antimicrobial activity
Stocks of pure essential oils of the aromatic plants
were prepared in order to be used in all bioassays.
Inhibition of the mycelial growth of B. cinerea and
Fusarium sp. was determined by daily measuring the
radial growth on potato dextrose agar (PDA) plates
containing the respective essential oil at a range of
concentrations, for 4 and 7 days, respectively (Ziogas
and Girgis, 1993). The experiments were conducted in
90 mm petri dishes, inoculated with 5 mm PDA plugs
from cultures on the same medium on which conidia had
been allowed to germinate. Three replicates were used
per treatment, and the incubation was at 251C in the
dark. The antibacterial activity was determined by
counting colonies on nutrient agar (NA) plates contain-
ing the respective essential oil at a range of concentra-
tions (Mosch et al., 1990). The solidified medium was
 D.J. Daferera et al. / Crop Protection 22 (2003) 39–44 41

surface-inoculated with 0.1 ml suspension of C. michi- 3. Results and discussion

ganensis subsp. michiganensis from tubes which were
prepared by the method of the serial dilutions. Three
replicates were used per treatment, and the incubation
was at 261C in the dark. The bacterial growth recorded
after 3 days. The respective essential oil was incorpo-
rated in each sterilized medium cooled until 501C. Oil
solubility was enhanced by using ethanol as emulsifier.
Ethanol concentrations never exceeded 0.5% (v/v) in the
case of fungi or 0.1% (v/v) in the case of bacterium and
an equal amount was added to the control. The effective
concentration causing a 50% reduction in the linear
growth of the fungi on PDA or in the colony forming
ability of the bacterium on NA (ED50 value) was
calculated by Probit analysis (StatSoft 1995) using the
values of dose-response experiments.
3.1. Essential oils composition
The chemical composition of the essential oils was
determined by the GC-MS analysis. The identification
of the unknown compounds was based on their relative
retention time and their mass spectra in comparison
with those observed by standards. Some compounds
were tentatively identified, by using the NBS75K library
data of the GC-MS system and literature data (Adams,
2001). The results are presented in Table 1. The
compounds are listed according to their elution order
which is in agreement with their Kovats Index (KI) on
non-polar phases reported in the literature (Adams,
2001). In Table 1 are also reported the oil yields from the

Table 1
Chemical composition for the essential oils from oregano (O), thyme (T), dictamnus (D), marjoram (M), lavender (L), rosemary (R), and sage (S)

Component KIa Composition (%)

O T D M L R S

a-Pineneb 939 0.5 0.8 0.5 0.7 — 12.7 6.3

Camphenec 954 — 0.1 — tr 0.5 4.2 1.7
b-Pineneb 979 0.3 0.1 — 0.4 — 1.1 2.4
b-Myrceneb 991 1.0 1.1 0.5 1.3 1.6 1.6 2.6
a-Terpineneb 1017 1.4 0.8 1.1 5.4 — 0.1 —
p-Cymeneb 1025 13.0 5.4 20.3 5.8 0.5 0.5 1.3
b-Phellandrenec 1030 — tr — 1.8 — — —
Eucalyptolb 1031 — — — — 2.4 31.5 59.5
a-Ocimenec — — — — — 1.1 0.1 —
g-Terpineneb 1060 7.5 2.6 3.9 9.3 0.1 0.7 —
trans-Linalool oxide (furanoid)c 1073 — — — — 3.0 — 0.2
cis-Linalool oxide (furanoid)c 1087 — — — — 2.1 — 0.2
Terpinolenec 1089 0.1 0.2 0.1 1.5 0.1 0.5 —
Linaloolb 1097 — 0.4 1.3 5.6 25.5 1.7 1.5
Thujoneb 1102 — — — — — — 3.1
3,7-Dimethyl-1,5,7-octatrien-3-olc — — — — — 2.1 — —
Octen-1-ol, acetatec — — — — — 1.6 — —
Camphorc 1146 — — — — 0.6 4.4 10.2
Borneolb 1169 — 0.2 0.1 0.5 3.2 14.2 —
Terpinen-4-olb 1177 0.3 0.5 0.9 9.4 1.7 1.3 0.6
Cryptonec 1186 — — — — 1.4 — —
a-Terpineolb 1189 — 0.1 — 2.4 5.0 4.3 3.9
Verbenonec 1205 — — — — 0.2 4.4 —
Nerolb 1230 — — — — 1.0 — —
Linalyl acetatec 1257 — — — 0.8 17.7 — —
Bornyl acetatec 1289 — — — — 1.0 2.9 0.1
Lavandulyl acetatec 1290 — — — — 3.9 — —
Thymolb 1290 63.7 0.2 2.6 0.8 3.2 0.8 1.2
Carvacrolb 1299 8.6 81.5 64.1 45.1 3.2 5.7 0.7
Neryl acetatec 1362 — — — 0.3 1.3 — —
Geranyl acetatec 1381 — — — 0.5 2.6 — —
b-Caryophylleneb 1419 1.0 2.7 1.0 1.0 1.0 1.7 0.4
Caryophyllene oxidec 1583 0.2 0.5 0.5 — 3.3 0.2 —
Total 97.6 97.2 96.7 92.6 90.9 94.6 95.9
Essential oil yield (ml/100 g) 5.2 4.0 1.7 4.4 1.0 0.8 1.6
a
KI on DB-5 in reference to n-alkanes tr o0.05%.
b
Identified injecting standard compound.
c
Tentatively identified.
42 D.J. Daferera et al. / Crop Protection 22 (2003) 39–44

aromatic plants expressed in ml/100 g of dry wt. The Table 2

chemical composition and the oil yield of the pennyroyal Toxicity of the tested essential oils to Botrytis cinerea, Fusarium sp.,
essential oil are mentioned in the text (not presented in and Clavibacter michiganensis subsp. michiganensis
Table 1), because the detected oil components were Essential oils ED50a ED50b
found only in this plant material.
B. cinerea Fusarium. sp. C. michiganensis
Oregano, thyme and dictamnus essential oils were subsp. michiganensis
characterized by the presence of p-cymene, g-terpinene,
Oregano 50 50 67
thymol and carvacrol. Thymol was found as the main
Thyme 83 71 39
compound in oregano oil, while carvacrol was found in Dictamnus 67 76 88
thyme and dictamnus oils. The analysis of the marjoram Marjoram 143 120 119
oil gave a large number of constituents which partici- Lavender 223 520 252
pated in the mixture in percentages more than 1.5%. Sage p1000 X1000 211
Rosemary 606 668 —
Among them were detected a-terpinene, p-cymene,
Pennyroyal 216 400 92
b-phellandrene, g-terpinene, linalool, terpinen-4-ol,
a
a-terpineol, and carvacrol. Carvacrol was also the major The concentration of the essential oil (mg/ml) causing a 50%
component in marjoram oil. In lavender essential oil reduction in the linear growth of the fungi on potato dextrose agar.
b
The concentration of the essential oil (mg/ml) causing a 50%
more than 60 compounds have been recorded from reduction in the colony forming ability of the bacterium on nutrient
which 41 have been identified. In Table 1 are presented agar.
only the compounds which participated in the mixture in
percentages higher than 0.1%. Lavender oil was
characterized by the relatively high content of linalool 100
and linalyl acetate. Bicyclic terpenes give a different
profile in the rosemary and the sage essential oil 80
composition. Eucalyptol was the main compound in
(% of control)

60
radial growth

rosemary oil, followed by borneol and a-pinene. In sage

oil, the predominant constituent was also eucalyptol in 40
percentage almost two times higher compared with that
of rosemary. Camphor and a-pinene were also present in 20
high amounts in sage oil. In the case of pennyroyal oil
cis-menthone and pulegone were the main compounds. 0
Trans-menthone (1.2%), cis-menthone (15.0%), pule- 0 100 200 300 400 500
gone (76.5%) and piperitone (0.6%) consisted the essential oil (µg/mL)
93.3% of the total oil. The oil yield from pennyroyal oregano dictamnus
was only 0.3 ml/100 g of dry wt. thyme marjoram
pennyroyal
3.2. Toxicity of essential oils on B. cinerea, Fusarium sp.,
Fig. 1. Effect of oregano, thyme, dictamnus, marjoram and penny-
and C. michiganensis subsp. michiganensis royal essential oils on radial growth of Botrytis cinerea.

The antimicrobial activity of the tested essential oils is

shown in Table 2 and Figs. 1–3.
In both fungal pathogens, oregano, thyme, and 100
dictamnus oils had strong activities followed by
marjoram oil. Lavender and pennyroyal oils exhibited 80
(% of control)

a moderate action while sage and rosemary oils

radial growth

60
presented weak activity to both pathogens. A dose
dependent inhibition of B. cinerea and Fusarium sp. 40
mycelial growth was caused by oregano, thyme,
dictamnus and marjoram essential oils (Figs. 1 and 2). 20
Mycelial growth of B. cinerea was totally inhibited by
these oils at 200, 150, 200 and 300 mg/ml, respectively, 0
and of Fusarium sp. at 150, 200, 250 and 300 mg/ml, 0 100 200 300 400
respectively. Lavender, rosemary, sage and pennyroyal essential oil (µg/mL)
essential oils were also fungitoxic on mycelial growth of oregano dictamnus
B. cinerea and Fusarium sp. but at higher concentrations thyme marjoram
(Table 2). The radial growth of B. cinerea was totally Fig. 2. Effect of oregano, thyme, dictamnus and marjoram essential
inhibited by lavender and rosemary oils at 1000 mg/ml, oils on radial growth of Fusarium sp.
 D.J. Daferera et al. / Crop Protection 22 (2003) 39–44
100
80
number of colonies
(% of control)
60
40
20
0 under specific application conditions.
0 50 100
essential oil (µg/mL)
oregano thyme
Fig. 3. Effect of oregano, thyme and dictamnus essential oils on
colony forming ability of Clavibacter michiganensis subsp. michiga-
nensis.
and by pennyroyal oil at 400 mg/ml. The radial
growth of Fusarium sp. was inhibited 83% and 72% at
1000 mg/ml by lavender and rosemary essential oils,
respectively.
The bacterial pathogen of tomatoes, C. michiganensis
subsp. michiganensis, is more sensitive to the biological
activity of the essential oils (Table 2, Fig. 3) compared
with the fungal pathogens tested in the present work.
Colony forming ability of the bacterium was completely
inhibited by oregano, thyme and dictamnus oils at 100,
85 and 100 mg/ml, respectively. Marjoram and pennyr-
oyal oils exhibited moderate activity with an 80%
growth inhibition observed at 200 mg/ml. Lavender oil
caused complete inhibition of bacterial growth at
600 mg/ml, while sage oil caused 89% inhibition of
bacterial growth at the concentration of 1000 mg/ml.
Although, the antimicrobial activity of an essential oil
is attributed mainly to its major compounds, the
synergistic or antagonistic effect of one compound in
minor percentage in the mixture has to be considered.
Each of the essential oil components has its own
contribution on biological activity of the oil. For
example, rosemary oil has half the amount of eucalyptol
(the main compound) compared with the sage oil but it
is proved in the present work to be more effective
against the fungal inhibition. The analysis of their
chemical composition showed significant quantitative
differences in the oil components (Table 1). Apparently,
the presence of borneol in rosemary oil (14.2%) and its
absence in sage oil is the reason for the difference in
fungitoxicity, previously mentioned.
The essential oils as antimicrobial agents present two
main characters: the first is their natural origin which
means more safety to the people and the enviroment and
the second is that they have be considered at low risk for
resistance development by pathogenic microorganisms.
It is believed that it is difficult for the pathogens to
develop resistance to such a mixture of oil components
43
with, apparently, different mechanisms of antimicrobial
activity.
Further research is needed in order to obtain
information regarding the practical effectiveness of
essential oils to protect the plants or the plant products
without phytotoxic effects, but our study gives support
for the application of certain essential oils to control
plant pathogens such as B. cinerea and Fusarium sp. or
to eliminate the C. michiganensis subsp. michiganensis
150
dictamnus Acknowledgements
This research was supported financially by the
Greek Ministry of Development, General Secretariat
of Research and Technology.
References
Adams, P.R., 2001. Identification of Essential Oils components by Gas
Chromatography/Quadrupole Mass Spectroscopy. Allured Pub-
lishing Corporation, IL, USA.
Arras, G., Agabbio, M., Piga, A., D’hallewin, G., 1995. Fungicide
effect of volatile compounds of Thymus capitatus essential oil. Acta
Hortic. 379, 593–600.
Brent, K.J., Hollomon, D.W., 1998. Fungicide resistance: the
assessment of risk. FRAC, Global Crop Protection Federation,
Brussels, Monograph No. 2, pp. 1–48.
Caccioni, D., Guizzardi, M., 1994. Inhibition of germination and
growth of fruit and vegetable postharvest pathogenic fungi by
essential oil components. J. Essent. Oil Res. 6, 173–179.
Daferera, J.D., Ziogas, N.B., Polissiou, G.M., 2000. GC-MS analysis
of essential oils from some Greek aromatic plants and their
fungitoxicity on Penicillium digitatum. J. Agric. Food Chem. 48,
2576–2581.
Gorris, L.G.M., Oosterhaven, K., Hartmans, K.J., De Witte, Y.,
Smid, E.J., 1994. Control of fungal storage diseases of potato by
use of plant-essential oil components. Pests Dis. 3D-20, 307–312.
Isman, B.M., 2000. Plant essential oils for pest and disease manage-
ment. Crop Prot. 19, 603–608.
Maiti, D., Kole, R.C., Sen, C., 1985. Antimicrobial efficacy of some
essential oils. J. Plant Dis. Prot. 92 (1), 64–68.
Mosch, J., Klingauf, F., Zeller, W., 1990. On the effect of plant
extracts against fireblight (Erwinia amylovora). Acta Hortic. 273,
355–361.
Mosch, J., Zeller, W., Rieck, M., Ullrich, W., 1996. Further studies on
plant extracts with a resistance induction effect against Erwinia
amylovora. Acta Hortic. 411, 361–366.
Oosterhaven, K., Chambel Leitao, A., Gorris, L.G.M., Smid, E.J.,
1996. Comparative study on the action of S-(+)-carvone, in situ,
on the potato storage fungi Fusarium solani var. coeruleum and F.
sulphureum. J. Appl. Bacteriol. 80, 535–539.
Reddy, M.V.Bh., Angers, P., Gosselin, A., Arul, J., 1998. Character-
ization and use of essential oil from Thymus vulgaris against
Botrytis cinerea and Rhizopus stolonifer in strawberry fruits.
Phytochemistry 47 (8), 1515–1520.
Scortichini, M., Rossi, M.P., 1989. In vitro activity of some essential
oils toward Erwinia amylovora (burrill) Winslow et al. Acta
Phytopathol. Entomol. Hung. 24 (3–4), 423–431.
Scortichini, M., Rossi, M.P., 1991. Preliminary in vitro evaluation of
the antimicrobial activity of terpenes and terpenoids towards
44 D.J. Daferera et al. / Crop Protection 22 (2003) 39–44
Erwinia amylovora (burrill) Winslow et al. J. Appl. Bacteriol. 71,
109–112.
Thanassoulopoulos, C.C., Laidou, Y., 1997. On the biological control
of Botrytis cinerea on kiwifruit CV ‘‘Hayward’’ during storage.
Acta Hortic. 444 (2), 757–764.
Vaughn, F.S., Spencer, F.G., 1994. Antifungal activity of natural
compounds against thiabendazole-resistant Fusarium sambucinum
strains. J. Agric. Food Chem. 42, 200–203.
View publication stats
Wilson, L.C., Solar, M.J., Ghaouth, EI.A., Wisniewski, M.,
1996. Rapid evaluation of plant extracts and essential oils for
antifungal activity against Botrytis cinerea. Plant Dis. 81 (2),
204–210.
Ziogas, B.N., Girgis, S.M., 1993. Cross-resistance relationships
between benzimidazole fungicides and diethofencarb in Botrytis
cinerea and their genetical basis in Ustilago maydis. Pestic. Sci. 39,
199–205.