Mycologia, 97(3), 2005, pp. 576–579.
2005 by The Mycological Society of America, Lawrence, KS 66044-8897
Altered growth and polyamine catabolism following exposure of the chocolate
spot pathogen Botrytis fabae to the essential oil of Ocimum basilicum
Senga K. Oxenham1 a variety of biological properties (Maruzzella and
Katja P. Svoboda Robbins 1961), including antimicrobial activity. The
Scottish Agricultural College, Ayr Campus, majority of research on the antimicrobial effects of
Auchincruive Estate, Ayr KA6 5HW, UK essential oils relates to human pathogens, dermato-
phytes and spoilage micro-organisms, with relatively
Dale R. Walters2
little work on plant pathogens. Nevertheless essential
Crop & Soil Research Group, Scottish Agricultural oils have been shown to possess in vitro activity
College, West Mains Road, Edinburgh EH9 3JG, UK
against plant-pathogenic bacteria (Maruzzella et al
1963) and fungi (Letessier et al 2001, Maruzzella et
al 1963, Shimoni et al 1993, Yegen et al 1992), and
Abstract: Biomass of the fungal pathogen Botrytis fa-
although less work has been performed in vivo essen-
bae in liquid culture amended with two chemotypes
tial oils have been shown to reduce fungal infections
of the essential oil of basil, Ocimum basilicum, was
on various plants (Asthana et al 1989, Awuah 1994,
reduced signiﬁcantly at concentrations of 50 ppm or
Letessier et al 2001). However their mode of action
less. The methyl chavicol chemotype oil increased the
remains unknown despite data demonstrating anti-
activity of the polyamine biosynthetic enzyme S-aden-
microbial effects of essential oils and their constitu-
osylmethionine decarboxylase (AdoMetDC), but
ents. In recent work examining the effects of the es-
polyamine concentrations were not signiﬁcantly al-
sential oil of Ocimum basilicum on fungal pathogens
tered. In contrast, the linalol chemotype oil de-
of broad bean (Oxenham 2003), the whole oils of
creased AdoMetDC activity in B. fabae, although
two chemotypes were shown to possess powerful an-
again polyamine concentrations were not altered sig-
tifungal activity both in vitro and in vivo. Essential
niﬁcantly. However activities of the polyamine cata-
oils and their individual constituents have been ex-
bolic enzymes diamine oxidase (DAO) and poly-
amined for anticancer properties. Thus geraniol, a
amine oxidase (PAO) were increased signiﬁcantly in
monoterpene found in the essential oil of various
B. fabae grown in the presence of the essential oil of
plants, was shown to inhibit proliferation of human
the two chemotypes. It is suggested that the elevated
colon cancer cells (Carnesecchi et al 2001). More-
activities of DAO and PAO may be responsible, in
part, for the antifungal effects of the basil oil, possibly over, these workers found that geraniol decreased the
via the generation of hydrogen peroxide and the sub- intracellular pool of the diamine putrescine and ac-
sequent triggering of programmed cell death. tivated polyamine catabolism in such cells. Based on
Key words: basil, essential oil, linalol, methyl these observations, they suggested that polyamine
chavicol, polyamines metabolism may be a target in the antiproliferative
properties of geraniol (Carnesecchi et al 2001).
Against this background, studies were undertaken to
examine the effects of two chemotypes of the essen-
INTRODUCTION tial oil of O. basilicum on polyamine metabolism in
Essential oils are complex mixtures of naturally oc- the plant pathogenic fungus Botrytis fabae.
curring compounds, mostly monoterpenes and ses-
quiterpenes. However, although in some plant spe- MATERIALS AND METHODS
cies one main constituent of the oil may predomi-
nate, in many plant species no single compound pre- Basil (Ocimum basilicum) was grown in a glasshouse as de-
dominates and rather there is a balance of various scribed previously (Oxenham 2003) and was harvested and
components (Svoboda and Hampson 1999). It has dried at 35 C distillation. Essential oils were extracted by
been known for some time that essential oils possess hydrodistillation and analysed by gas chromatography and
gas chromatography-mass spectrometry as described by Svo-
Accepted for publication 7 February 2005. boda and Hampson (1999). Essential oils of two chemoty-
1 Current address: Faculty of Life Sciences, University of Dundee, pes were used: methyl chavicol chemotype containing meth-
Dundee, UK. yl chavicol and linalol (76.1% and 18.6% of the whole oil,
2 Corresponding author. E-mail: email@example.com respectively) and linalol chemotype (linalol, eugenol, eu-
OXENHAM ET AL: ANTIFUNGAL ACTIVITY OF ESSENTIAL OIL OF BASIL 577
calyptol and caryophyllene) (53%, 12.4%, 7.7% and 5% of 1). The linalol chemotype oil also reduced fungal
the whole oil, respectively). biomass signiﬁcantly in liquid culture at 30 ppm, and
In an initial experiment a growth curve for Botrytis fabae at 50 ppm growth was reduced by 82% (FIG. 2).
was constructed to determine the timing of its exponential In studies of the effects of xenobiotics on fungal
growth phase. This information then was used in all sub-
growth and metabolism, it is necessary to choose a
sequent experiments conducted with liquid culture. Conical
ﬂasks (250 mL) containing 100 mL of liquid medium (Last
concentration of the xenobiotic that reduces growth
and Hamley 1956) were inoculated with 7 mm diam plugs sufﬁciently for any effects on metabolism to be de-
of B. fabae taken from stock plates of the fungus growing tectable while providing sufﬁcient fungal material for
on potato-dextrose agar. Flasks were placed in an orbital metabolic study. Because 2 ppm methyl chavicol-type
incubator set at 90 rpm and 19 C. For determination of the oil and 30 ppm linalol-type oil fulﬁlled these criteria,
growth curve, three ﬂasks were harvested every 12 h for these concentrations were chosen for studies of poly-
108 h. Fungal material was harvested with a ﬁne mesh sieve, amine metabolism. Growth of B. fabae in liquid me-
centrifuged and weighed. Based on the growth curve ob- dium amended with 2 ppm of the methyl chavicol-
tained ﬂasks were harvested after 3 d in all subsequent ex- type oil did not signiﬁcantly affect ODC activity but
periments. To examine the effect of the two chemotypes of led to signiﬁcant increases in AdoMetDC activity and
basil oil on growth of B. fabae, the oils were added to ﬂasks
activities of the two catabolic enzymes DAO and PAO
containing liquid medium to obtain concentrations of 10–
50 ppm. Essential oils are considered to be sterile (Zaika
(TABLE I). The methyl chavicol-type oil had no sig-
1988), and so ﬂasks containing oils were not autoclaved but niﬁcant effect on the incorporation of labeled orni-
used immediately in experiments. thine into polyamines or concentrations of free poly-
The activities of the polyamine biosynthetic enzymes, or- amines in B. fabae (data not shown). The linalol
nithine decarboxylase and S-adenosylmethionine decarbox- chemotype oil also led to signiﬁcant increases in
ylase (AdoMetDC), the catabolic enzymes diamine oxidase DAO and PAO activities in the fungus, and although
(DAO) and polyamine oxidase (PAO), intracellular concen- ODC activity was not altered AdoMetDC activity was
trations of free polyamines and incorporation of radiola- reduced signiﬁcantly (TABLE II). As observed with
beled ornithine into polyamines were determined as de- the methyl chavicol-type oil, B. fabae exposed to the
scribed by Oxenham (2003) and Walters et al (1995). linalol-type oil showed no signiﬁcant effects on the
All values presented are the means of four replicates. All
ﬂux of label into polyamines or concentrations of
experiments were repeated with similar results and statisti-
cal signiﬁcance was assessed with Student’s t-test. free polyamines (data not shown).
The methyl chavicol chemotype oil reduced biomass Growth of B. fabae in liquid medium was reduced
of B. fabae signiﬁcantly at just 2 ppm and inhibited signiﬁcantly by concentrations of methyl chavicol
production of fungal biomass by 84% at 20 ppm (FIG. chemotype oil and linalol chemotype oil of 30 ppm
FIG. 1. Effects of various concentrations of methyl chavicol-type basil oil on mycelial growth of Botrytis fabae in liquid
culture. Values are the means of four replicates. Signiﬁcant differences from controls are shown as * P 0.5; ** P 0.01;
*** P 0.001.
FIG. 2. Effects of various concentrations of linalol-type basil oil on mycelial growth of Botrytis fabae in liquid culture.
Values are the means of four replicates. Signiﬁcant differences from controls are shown as * P 0.5; *** P 0.001.
or less. The methyl chavicol chemotype oil exerted a polyamine biosynthesis increases in these tissues, re-
more powerful effect than the linalol-type oil, reduc- quiring polyamine catabolism to prevent accumula-
ing fungal biomass signiﬁcantly at just 2 ppm. These tion of polyamines. Indeed AdoMetDC activity in-
results are in line with other studies demonstrating creased signiﬁcantly in fungus treated with methyl
antifungal activities of essential oils against phyto- chavicol-type oil and the ﬂux of labeled ornithine
pathogenic fungi. For example, volatile components into polyamines increased, albeit not signiﬁcantly.
of the essential oil of basil have been shown to pos- However, with the linalol chemotype oil, polyamine
sess in vitro activity against a number of plant path- biosynthesis either was unchanged or decreased.
ogenic fungi (Reuveni et al 1984), while the essential Whether the increased DAO and PAO activities were
oil of hyssop as well as many of its individual constit- a cellular response to increasing polyamine levels or
uents reduced in vitro growth of Pyrenophora avenae the result of a direct effect of the oils on the enzymes
and Pyricularia oryzae (Letessier et al 2001). is not known. Nevertheless enhanced activities of
The signiﬁcant increases in DAO and PAO activi- DAO and PAO will lead to the production of hydro-
ties in B. fabae grown in the presence of the methyl gen peroxide (H2O2, Walters 2003) and H2O2 has
chavicol and the linalol chemotype oils suggest that been implicated in programmed cell death in human
melanoma cells (Chen et al 2001), plant cells (Levine
TABLE I. Effects of 2 ppm methyl chavicol type basil oil on
activities of enzymes of polyamine biosynthesis and catabo- TABLE II. Effects of 30 ppm linalol type basil oil on activ-
lism ities of enzymes of polyamine biosynthesis and catabolism
Methyl chavicol Methyl chavicol
Enzyme Control type oil Enzyme Control type oil
(pmol CO2 [mg protein] 1 47 5.0 58 4.7 (pmol CO2 [mg protein] 1 64 29.9 61 15.2
h 1) h 1]
(pmol CO2 [mg protein] 1 1
5 0.6 9 0.6** (pmol CO2 [mg protein] 21 4.0 8 3.1*
h 1) h 1)
(pmol product 170 15.7 392 14.0*** (pmol product 201 8.0 358 21***
[mg protein] 1 h 1 [mg protein] 1 h 1
(pmol product 57 1.9 121 6.7*** (pmol product 83 3.4 107 3.4***
[mg protein] 1 h 1 [mg protein] 1 h 1
Values are the means of four replicates. Signiﬁcant differ- Values are the means of four replicates. Signiﬁcant differ-
ences from controls are shown as ** P 0.01; *** P ences from controls are shown as * P 0.5; ** P 0.01;
0.001. *** P 0.001.
OXENHAM ET AL: ANTIFUNGAL ACTIVITY OF ESSENTIAL OIL OF BASIL 579
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