Mycologia
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DOI: 10.3852/mycologia.97.3.576
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Mycologia, 97(3), 2005, pp. 576-579.
© 2005 by The Mycological Society of America

Altered growth and polyamine catabolism following exposure of the chocolate spot pathogen Botrytis fabae to the essential oil of Ocimum basilicum


Senga K. Oxenham 1
Katja P. Svoboda

     Scottish Agricultural College, Ayr Campus, Auchincruive Estate, Ayr KA6 5HW, UK

Dale R. Walters 2

     Crop & Soil Research Group, Scottish Agricultural College, West Mains Road, Edinburgh EH9 3JG, UK


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

Biomass of the fungal pathogen Botrytis fabae in liquid culture amended with two chemotypes of the essential oil of basil, Ocimum basilicum, was reduced significantly at concentrations of 50 ppm or less. The methyl chavicol chemotype oil increased the activity of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC), but polyamine concentrations were not significantly altered. In contrast, the linalol chemotype oil decreased AdoMetDC activity in B. fabae, although again polyamine concentrations were not altered significantly. However activities of the polyamine catabolic enzymes diamine oxidase (DAO) and polyamine oxidase (PAO) were increased significantly in B. fabae grown in the presence of the essential oil of the two chemotypes. It is suggested that the elevated activities of DAO and PAO may be responsible, in part, for the antifungal effects of the basil oil, possibly via the generation of hydrogen peroxide and the subsequent triggering of programmed cell death.

Key words: basil, essential oil, linalol, methyl chavicol, polyamines


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Essential oils are complex mixtures of naturally occurring compounds, mostly monoterpenes and sesquiterpenes. However, although in some plant species one main constituent of the oil may predominate, in many plant species no single compound predominates and rather there is a balance of various components (Svoboda and Hampson 1999Go). It has been known for some time that essential oils possess a variety of biological properties (Maruzzella and Robbins 1961Go), including antimicrobial activity. The majority of research on the antimicrobial effects of essential oils relates to human pathogens, dermatophytes and spoilage micro-organisms, with relatively little work on plant pathogens. Nevertheless essential oils have been shown to possess in vitro activity against plant-pathogenic bacteria (Maruzzella et al 1963Go) and fungi (Letessier et al 2001Go, Maruzzella et al 1963Go, Shimoni et al 1993Go, Yegen et al 1992Go), and although less work has been performed in vivo essential oils have been shown to reduce fungal infections on various plants (Asthana et al 1989Go, Awuah 1994Go, Letessier et al 2001Go). However their mode of action remains unknown despite data demonstrating antimicrobial effects of essential oils and their constituents. In recent work examining the effects of the essential oil of Ocimum basilicum on fungal pathogens of broad bean (Oxenham 2003Go), the whole oils of two chemotypes were shown to possess powerful antifungal activity both in vitro and in vivo. Essential oils and their individual constituents have been examined for anticancer properties. Thus geraniol, a monoterpene found in the essential oil of various plants, was shown to inhibit proliferation of human colon cancer cells (Carnesecchi et al 2001Go). Moreover, these workers found that geraniol decreased the intracellular pool of the diamine putrescine and activated polyamine catabolism in such cells. Based on these observations, they suggested that polyamine metabolism may be a target in the antiproliferative properties of geraniol (Carnesecchi et al 2001Go). Against this background, studies were undertaken to examine the effects of two chemotypes of the essential oil of O. basilicum on polyamine metabolism in the plant pathogenic fungus Botrytis fabae.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Basil (Ocimum basilicum) was grown in a glasshouse as described previously (Oxenham 2003Go) and was harvested and dried at 35 C distillation. Essential oils were extracted by hydrodistillation and analysed by gas chromatography and gas chromatography-mass spectrometry as described by Svoboda and Hampson (1999)Go. Essential oils of two chemotypes were used: methyl chavicol chemotype containing methyl chavicol and linalol (76.1% and 18.6% of the whole oil, respectively) and linalol chemotype (linalol, eugenol, eucalyptol and caryophyllene) (53%, 12.4%, 7.7% and 5% of the whole oil, respectively).

In an initial experiment a growth curve for Botrytis fabae was constructed to determine the timing of its exponential growth phase. This information then was used in all subsequent experiments conducted with liquid culture. Conical flasks (250 mL) containing 100 mL of liquid medium (Last and Hamley 1956Go) were inoculated with 7 mm diam plugs of B. fabae taken from stock plates of the fungus growing on potato-dextrose agar. Flasks were placed in an orbital incubator set at 90 rpm and 19 C. For determination of the growth curve, three flasks were harvested every 12 h for 108 h. Fungal material was harvested with a fine mesh sieve, centrifuged and weighed. Based on the growth curve obtained flasks were harvested after 3 d in all subsequent experiments. To examine the effect of the two chemotypes of basil oil on growth of B. fabae, the oils were added to flasks containing liquid medium to obtain concentrations of 10–50 ppm. Essential oils are considered to be sterile (Zaika 1988Go), and so flasks containing oils were not autoclaved but used immediately in experiments.

The activities of the polyamine biosynthetic enzymes, ornithine decarboxylase and S-adenosylmethionine decarboxylase (AdoMetDC), the catabolic enzymes diamine oxidase (DAO) and polyamine oxidase (PAO), intracellular concentrations of free polyamines and incorporation of radiolabeled ornithine into polyamines were determined as described by Oxenham (2003)Go and Walters et al (1995)Go.

All values presented are the means of four replicates. All experiments were repeated with similar results and statistical significance was assessed with Student’s t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
The methyl chavicol chemotype oil reduced biomass of B. fabae significantly at just 2 ppm and inhibited production of fungal biomass by 84% at 20 ppm (FIG. 1Go). The linalol chemotype oil also reduced fungal biomass significantly in liquid culture at 30 ppm, and at 50 ppm growth was reduced by 82% (FIG. 2Go).



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  		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.

 


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  		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.

 
In studies of the effects of xenobiotics on fungal growth and metabolism, it is necessary to choose a concentration of the xenobiotic that reduces growth sufficiently for any effects on metabolism to be detectable while providing sufficient fungal material for metabolic study. Because 2 ppm methyl chavicol-type oil and 30 ppm linalol-type oil fulfilled these criteria, these concentrations were chosen for studies of polyamine metabolism. Growth of B. fabae in liquid medium amended with 2 ppm of the methyl chavicol-type oil did not significantly affect ODC activity but led to significant increases in AdoMetDC activity and activities of the two catabolic enzymes DAO and PAO (TABLE IGo). The methyl chavicol-type oil had no significant effect on the incorporation of labeled ornithine into polyamines or concentrations of free polyamines in B. fabae (data not shown). The linalol chemotype oil also led to significant increases in DAO and PAO activities in the fungus, and although ODC activity was not altered AdoMetDC activity was reduced significantly (TABLE IIGo). As observed with the methyl chavicol-type oil, B. fabae exposed to the linalol-type oil showed no significant effects on the flux of label into polyamines or concentrations of free polyamines (data not shown).


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  		TABLE I. Effects of 2 ppm methyl chavicol type basil oil on activities of enzymes of polyamine biosynthesis and catabolism

 

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  		TABLE II. Effects of 30 ppm linalol type basil oil on activities of enzymes of polyamine biosynthesis and catabolism

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Growth of B. fabae in liquid medium was reduced significantly by concentrations of methyl chavicol chemotype oil and linalol chemotype oil of 30 ppm or less. The methyl chavicol chemotype oil exerted a more powerful effect than the linalol-type oil, reducing fungal biomass significantly at just 2 ppm. These results are in line with other studies demonstrating antifungal activities of essential oils against phyto-pathogenic fungi. For example, volatile components of the essential oil of basil have been shown to possess in vitro activity against a number of plant pathogenic fungi (Reuveni et al 1984Go), while the essential oil of hyssop as well as many of its individual constituents reduced in vitro growth of Pyrenophora avenae and Pyricularia oryzae (Letessier et al 2001Go).

The significant increases in DAO and PAO activities in B. fabae grown in the presence of the methyl chavicol and the linalol chemotype oils suggest that polyamine biosynthesis increases in these tissues, requiring polyamine catabolism to prevent accumulation of polyamines. Indeed AdoMetDC activity increased significantly in fungus treated with methyl chavicol-type oil and the flux of labeled ornithine into polyamines increased, albeit not significantly. However, with the linalol chemotype oil, polyamine biosynthesis either was unchanged or decreased. Whether the increased DAO and PAO activities were a cellular response to increasing polyamine levels or the result of a direct effect of the oils on the enzymes is not known. Nevertheless enhanced activities of DAO and PAO will lead to the production of hydrogen peroxide (H2O2, Walters 2003Go) and H2O2 has been implicated in programmed cell death in human melanoma cells (Chen et al 2001Go), plant cells (Levine et al 1994Go) and plants expressing resistance to pathogens (Angelini et al 1993Go; Cowley and Walters 2002aGo, bGo). Moreover, a number of workers have shown that some essential oil constituents can induce apoptosis in human cells (Hata et al 2003Go, Kitamura et al 2003Go). Based on the above it is possible to hypothesize that the increased activities of DAO and PAO result in the formation of H2O2, triggering programmed cell death in B. fabae. In this way the increased polyamine catabolic activity could be responsible, at least in part, for the antifungal activity of the basil oils. In view of the interest in plant essential oils as novel agents for plant disease control, and as potential leads for the development of new fungicides, further studies to test this hypothesis are warranted.


   ACKNOWLEDGMENTS
 
SKO is grateful for the award of a WJ Thomson postgraduate scholarship. SAC receives grant-in-aid from the Scottish Executive Environment and Rural Affairs Department.


   FOOTNOTES
 
Accepted for publication February 7, 2005.

1 Current address: Faculty of Life Sciences, University of Dundee, Dundee, UK. Back

2 Corresponding author. E-mail: d.walters{at}ed.sac.ac.uk


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Angelini R, Bragaloni M, Federico R, Infantino A, Porta-Puglia A. 1993. Involvement of polyamines, diamine oxidase and peroxidase in resistance of chick-pea to Ascochyta rabei. J Plant Physiol 142:704–709.

Asthana A, Dixit N, Tripathi S, Dixit N. 1989. Efficacy of Ocimum oil against fungi attacking chilli seeds during storage. Trop Sci 29:15–20.

Awuah RT. 1994. In vivo use of extracts from Ocimum gratissimum and Cymbopogon citratus against Phytophthora palmivora causing blackpod disease of cocoa. Ann App Biol 124:173–178.[CrossRef]

Carnesecchi S, Schneider Y, Ceraline J, Duranton B, Gosse F, Seiler N, Raul F. 2001. Geraniol, a component of plant essential oils, inhibits growth and polyamine biosynthesis in human colon cancer cells. J Pharmacol Exp Ther 298:197–200.[Abstract/Free Full Text]

Chen Y, Kramer DL, Diegelman P, Vujcic S, Porter CW. 2001. Apoptotic signaling in polyamine analogue treated SK-MEL-28 human melanoma cells. Cancer Res 61:6437–6444.[Abstract/Free Full Text]

Cowley T, Walters DR. 2002a. Polyamine metabolism in barley reacting hypersensitively to the powdery mildew fungus Blumeria graminis f.sp. hordei. Plant, Cell & Environ 25:461–468.[CrossRef]

———, ———. 2002b. Polyamine metabolism in an incompatible interaction between barley and the powdery mildew fungus, Blumeria graminis f. sp. hordei.J Phytopathol 150:1–7.[CrossRef]

Hata T, Sagaguchi I, Mori M, Ikeda N, Kato Y, Minamino M, Watabe K. 2003. Induction of apoptosis by Citrus paradisi essential oil in human leukemic (HL-60) cells. In Vivo 17:553–559.[Medline]

Kitamura C, Ogawa Y, Morotomi T, Terashita M. 2003. Differential induction of apoptosis by capping agents during pulp wound healing. J Endodontics 29:41–43.[CrossRef][Medline]

Last FT, Hamley RE. 1956. A local lesion technique for measuring the infectivity of conidia of Botrytis fabae. Ann App Biol 44:410–418.[CrossRef]

Letessier MP, Svoboda KP, Walters DR. 2001. Antifungal activity of the essential oil of hyssop (Hyssopus officinalis). J Phytopathol 149:673–678.[CrossRef]

Levine A, Tenhaken R, Dixon R, Lamb C. 1994. H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79:583–593.[CrossRef][Medline]

Maruzzella JC, Robbins AL. 1961. Effects of essential oils on seed germination. Naturwissenschaften 48:383.

———, Kleinberg CC, Urso CJ. 1963. Effects of vapors of aromatic chemicals on phytopathogenic bacteria. Plant Dis Reptr 47:1067–1070.

Oxenham S. 2003. Studies on the chemotaxonomy of an Ocimum basilicum germplasm collection and the antifungal activity of essentials oils of basil [Doctoral thesis]. University of Glasgow, UK.

Reuveni R, Fleischer A, Putievsky E. 1984. Fungistatic activity of essential oils from Ocimum basilicum chemotypes. Phytopath Z 110:20–22.

Shimoni M, Putievsky E, Ravid U, Reuveni R. 1993. Antifungal activity of volatile fractions of essential oils from four aromatic wild plants in Israel. J Chem Ecol 19:1129–1133.[CrossRef]

Svoboda KP, Hampson J. 1999. Bioactivity of essential oil of selected temperate aromatic plants: antibacterial, anti-oxidant, anti-inflammatory and other related pharmacological activities. In: Speciality chemicals for the 21st century, ADEME/IENICA Seminar, 16–17 Sep 1999:43–49. ADEME, Paris.

Walters DR, Keenan JP, Cowley T, McPherson A, Havis ND. 1995. Inhibition of polyamine biosynthesis in Phytophthora infestans and Pythium ultimum. Plant Pathol 44:80–85.[CrossRef]

———. 2003. Resistance to plant pathogens: possible roles for free polyamines and polyamine catabolism. New Phytol 159:109–115.[CrossRef]

Yegen O, Bergen B, Heitefuss R. 1992. Untersuchungen zur fungitoxischen Wirkungden Extriakte sechs ausgewahlten Pflanzen aus der Turkei auf Phytopathogene Pilze. Z. Pflkr. U. Pflsch 99:349–359.

Zaika LL. 1988. Spices and herbs: their antimicrobial activity and its determination. J Food Safety 9:97–118.





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