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The effectiveness of plant essential oils on the


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Clavibacter...

Article in Crop Protection · February 2003


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

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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


consumed in a relatively short time after harvest. The
Essential oils are naturally occurring terpenic mix- exploitation of natural substances such as the essential
tures, whose insecticidal action against specific pests and oils, safer to consumers and the environment, for the
fungicidal action against some important plant patho- control of postharvest diseases and their effective usage
gens have been recently reviewed (Isman, 2000). against both wild type and strains resistant to pesticides,
Chemical control remains the main measure to reduce is urgently needed. Furthermore, the demand for
the incidence of post harvest diseases in various fruits reduction in the use of pesticides in agriculture increases
and vegetables. Antimicrobial chemicals belonging to interest in the possibility of the application of essential
the groups of benzimidazoles, aromatic hydrocarbons oils to control plant pathogens. Our interest is focused
and sterol biosynthesis inhibitors are often used, as post on the effectiveness of essential oils against serious plant
harvest treatments. A serious problem against the pathogens (Daferera et al., 2000). The specific objectives
effective use of these chemicals is the development of in the present work were to determine the effectiveness
resistance by the fungi (Brent and Hollomon, 1998). The of the essential oils from oregano (Origanum vulgare),
application of higher concentrations of chemicals in an thyme (Thymus capitatus), dictamnus (Origanum dic-
attempt to overcome this problem increases the risk of tamnus), marjoram (Origanum majorana), lavender
high level toxic residues in the products, which is (Lavandula angustifolia), rosemary (Rosmarinus officina-
lis), sage (Salvia fruticosa), and pennyroyal (Mentha
*Corresponding author. Tel. +30-152-942-41; fax+30-152-942-65. pulegium) on the growth of Botrytis cinerea, Fusarium
E-mail address: mopol@aua.gr (M.G. Polissiou). 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 2.2. Isolation of the essential oils
oils’ activity with their chemical composition.
Grey mould disease caused by B. cinerea is one of the The classic method of hydrodistillation using the
most serious diseases of a wide range of crops of Clevenger apparatus for 4 h was used for the isolation of
worldwide importance. Post harvest losses due to the essential oils from the dried leaves or/and flowers of
development of grey mould during the storage and aromatic plants. The isolated essential oils in pure form
distribution of harvesting fruits and vegetables are very were stored at 181C until their analysis by gas
high. Fusarium solani var. coeruleum and F. sulphureum chromatography-mass spectrometry (GC-MS) or their
cause the potato tuber dry rot, which is an important usage in bioassays.
disease of stored potatoes. Clavibacter michiganensis
subsp. michiganensis is the causal agent of bacterial 2.3. Analysis conditions
canker of tomatoes. It is a soilborne pathogen and has a
particularly devastating effect on tomatoes under green The analysis of the essential oils was performed using
house conditions. a Hewlett Packard 5890 II GC, equipped with a HP-
A relatively limited number of reports were found 5MS (Crosslinked 5% PH ME Siloxane) capillary
in the literature dealing with the effect of pure column (30 m, 0.25 mm i.d., 0.25 mm film thickness)
monoterpenes, plant extracts or/and essential oils and a mass spectrometer 5972 as detector. The carrier
against B. cinerea (Caccioni and Guizzardi, 1994; Arras gas was helium, by a rate of 1 ml/min. Column
et al., 1995; Wilson et al., 1996; Thanassoulopoulos and temperature was initially kept for 3 min at 401C, then
Laidou, 1997; Reddy et al., 1998), F. solani var. gradually increased to 1801C at a 31C/min rate and
coeruleum and F. sulphureum (Gorris et al., 1994; finally increased to 2701C at 301C/min and held for
Vaughn and Spencer, 1994; Oosterhaven et al., 1996). 5 min. For GC-MS detection an electron ionization
To our knowledge, except for oregano and thyme oils, system was used with ionization energy of 70 eV.
which only have been tested against B. cinerea growth, Injector and detector (MS transfer line) temperatures
no data are in the literature for the application of these were set at 2201C and 2901C, respectively. Diluted
oils on the growth of B. cinerea, F. solani var. coeruleum, samples of 0.5 ml were injected manually and splitless.
and C. michiganensis subsp. michiganensis. Studies
on the effectiveness of the components of essential 2.4. Test microorganisms
oils or plants extracts against phytopathogenic
bacteria have been found in the literature for Erwinia Botrytis cinerea was provided by the Laboratory of
amylovora (Scortichini and Rossi, 1989, 1991; Mosch Phytopathology of the Agricultural University of
et al., 1990, 1996) and Xanthomonas campestris (Maiti Athens (AUA), Fusarium sp. (Fusarium solani var.
et al., 1985). coeruleum) was isolated from potato bulbs which
Furthermore, the chosen aromatic plants are wide- presented the dry rot symptom and Clavibacter
spread in Greek flora, they are rich in essential oil michiganensis subsp. michiganensis was provided by
content and recently a few of them are now system- the collection of Benaki Phytopathological Institute
atically cultivated in Greece. The chemical composition (Kifissia, Athens).
of essential oils can be standardized, so that results from
their application could be repeatable. 2.5. Measurement of antimicrobial activity

Stocks of pure essential oils of the aromatic plants


2. Materials and methods were prepared in order to be used in all bioassays.
Inhibition of the mycelial growth of B. cinerea and
2.1. Plant material Fusarium sp. was determined by daily measuring the
radial growth on potato dextrose agar (PDA) plates
The aromatic plants were collected from different containing the respective essential oil at a range of
areas of Greece: oregano from Fokida (South-Central concentrations, for 4 and 7 days, respectively (Ziogas
Greece), thyme from Naxos island (Central Aegean sea), and Girgis, 1993). The experiments were conducted in
dictamnus from Crete island (South Greece), marjoram 90 mm petri dishes, inoculated with 5 mm PDA plugs
from Attiki (Central Greece), rosemary from the area of from cultures on the same medium on which conidia had
the Agricultural University of Athens, sage from Epirus been allowed to germinate. Three replicates were used
(North-West Greece) and pennyroyal from Rodopi per treatment, and the incubation was at 251C in the
(North Greece). Lavender was purchased from dark. The antibacterial activity was determined by
the Athens market. All the samples were air dried counting colonies on nutrient agar (NA) plates contain-
and stored at room temperature in darkness until ing the respective essential oil at a range of concentra-
distillation. 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 3.1. Essential oils composition
replicates were used per treatment, and the incubation
was at 261C in the dark. The bacterial growth recorded The chemical composition of the essential oils was
after 3 days. The respective essential oil was incorpo- determined by the GC-MS analysis. The identification
rated in each sterilized medium cooled until 501C. Oil of the unknown compounds was based on their relative
solubility was enhanced by using ethanol as emulsifier. retention time and their mass spectra in comparison
Ethanol concentrations never exceeded 0.5% (v/v) in the with those observed by standards. Some compounds
case of fungi or 0.1% (v/v) in the case of bacterium and were tentatively identified, by using the NBS75K library
an equal amount was added to the control. The effective data of the GC-MS system and literature data (Adams,
concentration causing a 50% reduction in the linear 2001). The results are presented in Table 1. The
growth of the fungi on PDA or in the colony forming compounds are listed according to their elution order
ability of the bacterium on NA (ED50 value) was which is in agreement with their Kovats Index (KI) on
calculated by Probit analysis (StatSoft 1995) using the non-polar phases reported in the literature (Adams,
values of dose-response experiments. 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 43

100 with, apparently, different mechanisms of antimicrobial


activity.
80 Further research is needed in order to obtain
number of colonies
(% of control)

information regarding the practical effectiveness of


60 essential oils to protect the plants or the plant products
40 without phytotoxic effects, but our study gives support
for the application of certain essential oils to control
20 plant pathogens such as B. cinerea and Fusarium sp. or
to eliminate the C. michiganensis subsp. michiganensis
0 under specific application conditions.
0 50 100 150
essential oil (µg/mL)
oregano thyme dictamnus Acknowledgements
Fig. 3. Effect of oregano, thyme and dictamnus essential oils on
colony forming ability of Clavibacter michiganensis subsp. michiga- This research was supported financially by the
nensis. Greek Ministry of Development, General Secretariat
of Research and Technology.

and by pennyroyal oil at 400 mg/ml. The radial


growth of Fusarium sp. was inhibited 83% and 72% at References
1000 mg/ml by lavender and rosemary essential oils,
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