Altered growth and polyamine cat

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					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: d.walters@ed.sac.ac.uk                     respectively) and linalol chemotype (linalol, eugenol, eu-

                                                                     576
                          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).


                         RESULTS                                                          DISCUSSION

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.
578                                                       MYCOLOGIA




  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
ODC                                                              ODC
 (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]
AdoMetDC                                                         AdoMetDC
 (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)
DAO                                                              DAO
 (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
PAO                                                              PAO
 (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

et al 1994) and plants expressing resistance to path-                   mildew fungus, Blumeria graminis f. sp. hordei. J Phy-
ogens (Angelini et al 1993; Cowley and Walters                          topathol 150:1–7.
2002a, b). Moreover, a number of workers have                      Hata T, Sagaguchi I, Mori M, Ikeda N, Kato Y, Minamino
shown that some essential oil constituents can induce                   M, Watabe K. 2003. Induction of apoptosis by Citrus
                                                                        paradisi essential oil in human leukemic (HL-60) cells.
apoptosis in human cells (Hata et al 2003, Kitamura
                                                                        In Vivo 17:553–559.
et al 2003). Based on the above it is possible to hy-              Kitamura C, Ogawa Y, Morotomi T, Terashita M. 2003. Dif-
pothesize that the increased activities of DAO and                      ferential induction of apoptosis by capping agents dur-
PAO result in the formation of H2O2, triggering pro-                    ing pulp wound healing. J Endodontics 29:41–43.
grammed cell death in B. fabae. In this way the in-                Last FT, Hamley RE. 1956. A local lesion technique for mea-
creased polyamine catabolic activity could be respon-                   suring the infectivity of conidia of Botrytis fabae. Ann
sible, at least in part, for the antifungal activity of the             App Biol 44:410–418.
basil oils. In view of the interest in plant essential oils        Letessier MP, Svoboda KP, Walters DR. 2001. Antifungal ac-
as novel agents for plant disease control, and as po-                   tivity of the essential oil of hyssop (Hyssopus officinalis).
tential leads for the development of new fungicides,                    J Phytopathol 149:673–678.
                                                                   Levine A, Tenhaken R, Dixon R, Lamb C. 1994. H2O2 from
further studies to test this hypothesis are warranted.
                                                                        the oxidative burst orchestrates the plant hypersensi-
                                                                        tive disease resistance response. Cell 79:583–593.
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