Environmental and Experimental Botany 56 (2006) 108–119

Plant volatiles can minimize the growth suppression of epiphytic

bacteria by the phytopathogenic fungus Botrytis cinerea
in co-culture experiments
Daniel Abanda-Nkpwatt a , Ursula Krimm b , Heather Allison Coiner a ,
Lukas Schreiber b , Wilfried Schwab a,∗
a Biomolecular Food Technology, Technical University Munich, Lise-Meitner-Str. 34, D-85354 Freising, Germany
b Institute of Cellular and Molecular Botany (IZMB), University of Bonn, Kirschallee 1, D-53115 Bonn, Germany

Received 27 September 2004; accepted 18 January 2005

Abstract

Leaf surfaces of plants are colonized by microbes, although the ecological roles of most of these epiphytes are unknown. Eleven non-
pathogenic bacteria were isolated from strawberry (Fragaria x ananassa) plants and tested for their ability to interact with plant volatiles and
the phytopathogenic fungus Botrytis cinerea. None of the bacterial epiphytes produced antimicrobial compounds. Light microscopic and SEM
analysis of F. x ananassa leaf surfaces showed that capitate glands are densely colonized by microorganisms. Benzyl alcohol, 2-phenylethanol,
R,S-linalool and nonanal were identified as major volatiles emitted by intact strawberry leaves, while R,S-linalool and nonanal were released
by the capitate glands. The isolated epiphytes cannot utilize these leaf volatiles as sole carbon source, but some of the bacteria metabolize
them, e.g. to the corresponding acids. However, the leaf volatiles have a stronger inhibitory effect on different strains of the plant pathogenic
fungus B. cinerea than on the isolated epiphytic bacteria. In co-culture experiments, B. cinerea strains suppress the proliferation of epiphytes
but low concentrations of 1–5 ppm of R,S-linalool, 2-phenylethanol and in particular nonanal significantly enhance the progeny of a number
of epiphytic bacteria, while the growth of B. cinerea strains is retarded. Thus, native volatile compounds can affect population dynamics of
epiphytes and the phytopathogenic fungus. Our findings have significant implications for pest management notably on the use of antagonistic
bacteria as biocontrol agents.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Epiphytic bacteria; Botrytis cinerea; Volatiles; Fragaria x ananassa

1. Introduction microorganisms as such from strawberry plant surfaces have

Members of all plant phyla are colonized by microbial
epiphytes, with more than 85 different species of microor-
ganisms in 37 genera recovered from the phyllosphere of rye,
olive, sugar beet, and wheat despite the hostile environment
of the leaf surface (Morris et al., 1998; Yang et al., 2001).
Leaf surfaces often are dry and strong UV radiation, limited
nutrient availability, and large temperature swings contribute
to stressful conditions. Epiphytes have been isolated only
from a small number of commercially important crops but
∗ Corresponding author. Tel.: +49 8161 548312; fax: +49 8161 548595.
E-mail address: schwab@wzw.tum.de (W. Schwab).
not been investigated. However, the most common bacterial
contaminants of strawberry explants, probably originating
from epiphytes were recently characterized as Pseudomonas
fluorescens, P. corrugata, P. tolaasii, P. paucimobilis, X.
campestris, and Enterobacter cloacae, species also found
on other plant surfaces (Tanprasert and Reed, 1998). It is
assumed that the microbes present have a significant impact
on stress resistance and on the plant’s metabolism.
The interactions between plants and above ground epi-
phytic microorganisms have not been widely studied with
regard to metabolite exchange. Pink-pigmented facultative
methylotrophs (PPFM), however, are known to produce a
variety of compounds, e.g. cytokinins, auxins, Vitamin B12 ,

0098-8472/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.envexpbot.2005.01.010
 D. Abanda-Nkpwatt et al. / Environmental and Experimental Botany 56 (2006) 108–119
and osmoprotectants that affect plant metabolism and
consume plant metabolic wastes, most notably methanol
(Zabetakis, 1997; Joshi and Holland, 1999; Trotsenko et al.,
2001). The composition and quantity of nutrients, including
carbohydrates, organic acids, and amino acids that support
the growth of epiphytic microorganisms, are affected by the
plant species, leaf age, and the presence of tissue damage
(Fiala et al., 1990; Hirano and Upper, 2000). Usually the car-
bon compounds or the nitrogen compounds are the limiting
factors for bacterial and yeast populations on leaves (Wilson
and Lindow, 1994; Ji and Wilson, 2002). The application
of salicylic acid can alter the population size of salicylate-
utilizing bacterial strains on leaves (Wilson and Lindow,
1995), suggesting that microbial populations of plants can be
manipulated by changing the nutrient status of the plant sur-
face. Thus, nutrient competition is a likely biological control
mechanism for pathogens with a saprophytic growth phase
or that require a nutrient source to infect plants (Brodie and
Blakeman, 1976). However, to our knowledge, the effects
of plant volatiles as potential nutrient sources or inhibitory
compounds towards epiphytic microorganisms have not been
thoroughly analyzed.
The antimicrobial properties of essential oils, odorous and
volatile products of plant secondary metabolism are known
for many centuries. A large number of essential oils and their
constituents have been investigated for their antimicrobial
properties against some bacteria and fungi in more than 500
reports. The majority of these reports deal with the suscepti-
bility of human and food-borne as well as plant pathogenic
bacteria and fungi towards different essential oils and their
constituents (Croft et al., 1993; Kalemba and Kunicka, 2003).
However, to the best of our knowledge, the effects of leaf
volatiles on non-pathogenic epiphytes isolated from the same
leaf species have not been tested.
Plant volatiles constitute a heterogeneous group of sub-
stances descending from the terpenoid, phenylpropanoid or
fatty acid pathway. These lipophilic, low molecular weight
compounds are emitted by flowers, fruits, stems, roots and
leaves and are commonly found as constituents of plant resins
and oils. Hundreds of volatile compounds have been iden-
tified as plant components, including substances, such as
alcohols, aldehydes, ketones, esters, lactones, ethers, amines
and carboxylic acids. A variety of specialized plant tissues as
well as single cells secrete volatile compounds (Gershenzon
et al., 1992; Hashidoko and Urashima, 1995). Extra-cellular
secretion occurs mainly through different types of glandular
trichomes, while internal secretion occurs through oil cells,
cavities and ducts. Found in numerous plant families, glan-
dular trichomes vary widely in shape and structure (Wagner,
1991). Two structural types of glandular trichomes are com-
mon. Peltate (subsessile) glandular trichomes, with a short,
unicellular stalk and a large, multicellular head, accumulate
secretory products in a cavity that forms between the cuti-
cle and underlying cells, whereas capitate (clavate) glandular
trichomes, with a one- or two-celled head atop a single- or
multicellular stalk, excrete secretion products outside of the
109
gland (Gershenzon et al., 1992). The exudates consist of a
diverse group of alleochemicals and comprise monoterpenes
(Rajaonarivony et al., 1992; Yamaura et al., 1992), sesquiter-
penes (Hashidoko et al., 1992), phenylpropanoids (Gang et
al., 2001) or other more unusual natural products, such as
␻5-fatty acids (Yerger et al., 1992). Glandular trichomes that
secrete terpenes and other essential oils have been found to
confer insect resistance (Carter et al., 1989) but although ter-
penes and phenylpropanoids are known as antibacterial and
antifungal agents (Moleyar and Narasimham, 1992), their
effects on epiphytes have not been well studied.
The antagonistic properties of various epiphytes on phy-
topathogenic fungi, such as Botrytis cinerea, have been inten-
sively investigated (Filonow et al., 1996; Guinebretiere et al.,
2000; Berto et al., 2001). B. cinerea causes economic losses
on a wide range of cultivated plants, stored fruits and veg-
etables. The pathogen is the major cause for the grey mould
disease of grapevine and of the decay of strawberry fruit (Fra-
garia x ananassa). It is one of the most devastating pathogens
in several crops worldwide (Paul et al., 1998). Although
the effects of some leaf volatiles on pathogenic fungi have
been described, their impact on epiphytes remains unclear
(Hamilton-Kemp et al., 1992; Filonow et al., 1996; Manohar
et al., 2001).
Owing to our experience with strawberry fruit flavor,
and to the recent isolation and characterization of straw-
berry leaf epiphytes, the strawberry leaves are likely to pro-
vide a good model for assessing the role volatiles play in
leaf surface microbial ecology. The goal of this research
was to examine possible ecological functions of volatiles
emitted by uninjured F. x ananassa leaves. We assumed
that volatiles may serve as carbon source for epiphytes or
inhibit phytopathogenic microorganisms, such as the fungus
B. cinerea. However, the data suggest that volatiles might
affect the population dynamics of epiphytic bacteria and
the phytopathogenic fungus. This novel hypothesis has been
investigated in detail using co-cultivation experiments. The
obtained results have a significant impact on the use of bac-
terial biocontrol agents, since the co-application of volatiles
and antagonistic bacteria could enhance the efficacy of the
control agents.
2. Materials and methods
2.1. Strains
Epiphytic bacteria were provided by Prof. Lukas
Schreiber, Institute of Cellular and Molecular Botany
(IZMB), University Bonn, Kirschallee 1, 53115 Bonn, Ger-
many and can be obtained there. Epiphytic bacteria were iso-
lated and identified by morphological and biochemical meth-
ods as well as by their 16S rDNA (Table 1). The strains were
maintained at −23 ◦ C in Dworkin solution (2 g (NH4 )2 SO4 ,
4 g KH2 PO4 , 6 g Na2 HPO4 , 0.2 g MgSO4 in 1 l of distilled
water) supplemented with 25% glycerol. A Botrytis cinerea
110 D. Abanda-Nkpwatt et al. / Environmental and Experimental Botany 56 (2006) 108–119
Table 1
Bacterial strains used in the study. Epiphytic bacteria were isolated from F. x
ananassa cv. Elsanta leaves and identified by morphological and biochemical
methods as well as by their 16S rDNA
Isolate Source
I2 Pseudomonas sp. NZ099 A, LLS
I3 Pseudomonas lurida A, LLS
I4 Pseudomonas rhizosphaerae A, LLS
I6 Stenotrophomonas maltophilia A, LUS
I7 Bacillus cereus Delaporte A, LUS
I8 Pseudomonas orientalis B, LUS
I21 Arthrobacter agilis A, LUS
I24 Pseudomonas parafulva A, LLS
I25 Bacillus mycoides A, LLS
B. weihenstephanensis
I29 Bacillus sp. No. 49 B, LUS
A, field grown plants; B, plant from the greenhouse; LLS leaf lower side;
LUS leaf upper side.
strain (B. c. 1: Bayer AG # 23), isolated from Fragaria x
ananassa leaves, was obtained from Bayer AG (Monheim,
Germany) and two B. cinerea strains (B. c. 2: 92/RB 11
and B. c. 3: 93/HRB 2b) were provided by Bayerische Lan-
desanstalt für Wein- und Gartenbau (Veitshöchheim, Ger-
many). B. cinerea strains were maintained on malt extract
agar (MEA) (30 g malt extract, 3 g soya peptone and 15 g
agar in 1 l of distilled water, pH to 6.5, sterilized at 121 ◦ C
for 10 min at 4 ◦ C).
2.2. Plant material and reagents
Strawberry leaves were harvested from field-grown F. x
ananassa var. Elsanta grown in the Botanical garden of the
University of Würzburg, Germany. Plants were obtained from
Plant Research International, Wageningen, The Netherlands
and grown outdoors in garden plots of approximately 4 m2
or in the greenhouse. Plants were irrigated and fertilized as
under typical commercial conditions. Healthy leaves were
sampled. Nylon mesh was obtained from Small Parts Inc.
(Miami, FL). All reagents were purchased from Research
Organics Merck (Darmstadt, Germany), Fluka (Deisenhofen,
Germany) or Sigma (Deisenhofen, Germany) and were of
analytical grade or the highest commercial grade available.
2.3. Scanning electron microscopy and light microscopy
Small pieces of leaves were fixed for 12 h in a mixture
of 3% (v/v) glutaraldehyde and 2% (v/v) paraformalde-
hyde buffered with 100 mM sodium phosphate to pH 7.2.
After washing with acetone, the leaf segments were fixed
for 3 h in 1% (w/v) aqueous osmium tetroxide. Tissue was
then dehydrated in an alcohol series, followed by criti-
cal point drying and sputter coating with palladium. Spec-
imens were viewed with a Zeiss (Jena, Germany) Digi-
tal Scanning Microscope 962 connected to a Mitsubishi
(Cypress, CA) Video Copy Processor. Epifluorescence light
microscopy was performed with a Zeiss (Jena, Germany)
Axioplan connected to a Seescan (Göttingen, Germany)
color 100 video camera after staining the samples with acri-
dine orange (0.1 mmolar) or 4,6-diamidino-2-phenylindol-
dihydrochloride (DAPI) (1 ␮molar).
NCBI numbers
AY131214 2.4. Isolation of glandular trichomes and analysis of
AY131215
AY131223
volatiles
AY131216
AY131217 Excised leaves were washed rapidly, first with 70%
AY131218 ethanol to rinse out glandular trichome exudates and dust and
AY131225 then with running tap water to remove any ethanol remain-
AY131219
AY131220
ing on the leaflets. Washed leaves (10–15 g) were placed
in a 500 ml flask, containing 200 ml of the separation solu-
AY131222 tion and about 10 g each of clean quartz and glass beads.
The separation solution consisted of 0.4 M mannitol and
50 mM sodium l-ascorbate adjusted to pH 6.8. The flask
was shaken on an orbital shaker (Bühler KL2, Tübingen,
Germany) at 200 rpm for 30 min at room temperature.
Following abrasion, clusters of glandular trichomes were
obtained by filtering the contents of the flask sequentially
through 250, 105, and 20 ␮m nylon mesh. The isolation of
pure glandular trichomes was confirmed by light microscopy.
Isolated glandular trichomes were washed several times with
the separation solution and transferred immediately to a 50 ml
flask to which 30 ml diethyl ether was added. The volatiles
were extracted by shaking the solution with an orbital shaker
at 200 rpm for 10 min. The diethyl ether solution was filtered,
carefully concentrated to 0.1 ml and directly analyzed by gas
chromatography–mass spectrometry (GC–MS).
2.5. Isolation of strawberry leaf volatiles by rinsing
Strawberry leaves (5–10 g) were dipped into 100 ml of
diethyl ether for 30 to 120 s to dissolve the volatiles on the leaf
surface. The leaves were removed and the diethyl ether was
filtered through a 0.45 ␮m Millipore filter and concentrated
to 0.1 ml using a 25 cm-Vigreux column (Roth, Karlsruhe)
(45 ◦ C). The samples were analyzed by GC–MS.
2.6. Growth conditions and biotransformation of
volatile compounds
The bacteria were cultivated in 300 ml flasks filled
with a mixture consisting of 75 ml Dworkin solution (2 g
(NH4 )2 SO4 , 4 g KH2 PO4 , 6 g Na2 HPO4 , 0.2 g MgSO4 in 1 l
of distilled water) and 375 ␮l mineral solution (0.3 g each of
CuSO4 ·7H2 O, ZnSO4 ·7H2 O, CoSO4 ·7H2 O, FeSO4 ·7H2 O,
MnSO4 ·4H2 O in 100 ml of distilled water). After autoclav-
ing (15 min at 121 ◦ C), 1 ml of 50% filter-sterilized glucose
solution was added to the cooled media. Each flask was inoc-
ulated with 100 ␮l of a bacterial culture (2 × 106 cells/ml)
and incubated on a rotary shaker (30 ◦ C, 120 rpm). For the
biotransformation experiments the Dworkin broth was sup-
plemented with 50 ␮l substrate (R,S-linalool, nonanal, benzyl
alcohol, or 2-phenylethanol), inoculated and incubated as
described above. After 3 days, the cultures were extracted
 D. Abanda-Nkpwatt et al. / Environmental and Experimental Botany 56 (2006) 108–119 111

with 3 × 40 ml of diethyl ether and the extract concentrated
to 0.1 ml using a Vigreux column (45 ◦ C). After addition of a
standard solution consisting of 0.1% (v/v) vanillin in diethyl
ether, the samples were analyzed by GC–MS. Control exper-
iments without bacteria were carried out in a similar manner.
The broth had a pH of 6.7.
2.7. Test for antimicrobial compounds
Malt extract agar (MEA) plates were inoculated with 1 ml
of a suspension of B. cinerea mycelia (106 CFU/ml) in saline
solution (0.85% NaCl), and 100 ␮l of culture suspensions
(2 × 106 CFU/ml) or culture filtrates were pipetted into wells
(1 cm diameter) punched in the center of the Petri dish. The
culture filtrate was prepared by centrifugation (9000 × g,
25 min at 4 ◦ C), followed by sterile filtration of the super-
natant with a 0.2 ␮m filter. Autoclaved distilled water was
used in place of culture extracts as a control. The plates were
incubated at 22–25 ◦ C for 7 days.
2.8. Growth inhibition test
Bacterial and fungal strains were grown on MEA plates,
containing 1, 10, 100, 1000 ppm of R,S-linalool, nonanal,
benzyl alcohol and 2-phenylethanol. Plates were inoculated
with plugs of mycelium and 100 ␮l of a bacterial cul-
ture (2 × 106 CPU ml−1 ), which was pipetted into a well
punched in the center of the MEA, and incubated at room
temperature. Colony diameter was measured after 7 days.
Controls were cultures grown on MEA without volatile
compounds.
2.9. Co-cultivation
ware (version 1.3). A J & W DB Wax 20 M fused silica
capillary column (30 m × 0.25 mm i.d.; df = 0.25 ␮m), which
was programmed from 50 ◦ C for 3 min, then increased to
240 ◦ C at 4 ◦ C min−1 intervals, was used with helium gas at
a flow rate of 3 ml min−1 . The EI-MS-operating parameters
were: ionization voltage, 70 eV (electron impact ionization);
ion source and interface temperature, 230 and 240 ◦ C, respec-
tively. Compounds were identified by their mass spectra and
retention indices in comparison with the NIST mass spec-
tra library (Thermo Finnigan, San Jose, CA) and reference
compounds. The metabolites produced were quantified as
equivalents of vanillin.
3. Results
3.1. Light and scanning electron microscopy
Glandular trichomes are distributed along vascular bun-
dles on the adaxial F. x ananassa leaf surface, but randomly
on the abaxial surface (Fig. 1). These trichomes are capitate
glandular trichomes with a one- or two-celled head atop a
single or multi-celled stalk and extrude secretory products
(Gershenzon et al., 1992). Microorganisms were detected by
light microscopy and were concentrated on the adaxial tri-
chomes (Fig. 1B and C). In general, other parts of the leaf
surfaces were not as densely colonized by bacteria as the
trichomes.
3.2. Isolation of strawberry leaf volatiles
Table 2 summarizes the compounds isolated from Fra-
garia x ananassa leaves of the variety Elsanta. The con-

MEA plates were prepared, containing 0, 1, 5 or 10 ppm

nonanal R,S-linalool, benzyl alcohol or 2-phenylethanol. A Table 2
Volatile compounds isolated from leaf surfaces and glandular trichomes of
small plug of mycelium (6 mm diameter) from a 7-day- Fragaria x ananassa cv. Elsanta
old culture grown on MEA plates was placed approxi-
Compound Leaf surfaces Glandular trichomes
mately 4.5 cm apart in the agar from a well, containing
100 ␮l of a bacteria suspension, adjusted to 106 CPU ml−1 . Nonanal ++ +
Decanal + −
The controls were plates with fungi and 100 ␮l distilled 1-Penten-3-ol + −
water, and bacteria with no fungus, at all tested volatile (Z)-2-Buten-1-ol + −
concentrations. All plates were incubated at room temper- (Z)-3-Hexenol +++ −
ature and colony growth was measured daily along the R,S-linalool +++ +
axis bisecting the two colonies. Bacterial colony radius was Nonanol + −
␣-Terpineol + −
measured from the edge of the agar well to the edge of Citronellol ++ −
the colony, while fungal growth was measured as colony Myrtenol + −
radius from the center of the initial plug to the edge of the Geraniol + −
colony. Benzyl alcohol +++ −
2-Phenylethanol ++ −
(Z)-3-Hexenyl acetate + −
2.10. Capillary gas chromatography–mass spectrometry Acetic acid + −
(GC–MS) analysis Nonanoic acid + −
Benzoic acid + −
GC-analyses were performed with a Fisons MD 800 Decanoic acid + −
Quadrupol mass spectrometer coupled to a Fisons GC 8000 Concentration (mg/kg fresh weight): (+) 0.01-0.1; (++) 0.1-1.0; (+++) >1.0.
with split injector (1:20) equipped with Fisons MassLab soft- (−) not identified.
112 D. Abanda-Nkpwatt et al. / Environmental and Experimental Botany 56 (2006) 108–119

Fig. 1. Fluorescence light microscopy of capitate glands stained with acridine orange (A) and DAPI (B and C) located on the adaxial side of F. x ananassa
leaves. Scanning electron micrograph of a F. x ananassa leaf surface showing the abaxial (D and E) and the adaxial (F and G) side. On the abaxial surface,
glandular trichomes are randomly distributed, whereas on the adaxial surface trichomes are found only on top of the vascular bundles. Capitate glands (C), leaf
hairs (H) and microbial cells (M) are labeled by arrows.

centration ranges of volatiles are indicated. Major volatiles
were nonanal, (Z)-3-hexenol, R,S-linalool, citronellol, benzyl
alcohol, and 2-phenylethanol. Their concentrations ranged
from 0.1 to 10 mg per kg fresh weight. The concentra-
tions of the other volatiles, including decanal, 1-penten-3-ol,
(Z)-2-buten-1-ol, nonanol, ␣-terpineol, myrtenol, geraniol,
(Z)-3-hexenyl acetate, acetic acid, nonanoic acid, benzoic
acid, and decanoic acid never exceeded 0.1 mg kg−1 fresh
weight.
3.3. Isolation of glandular trichomes and identification
of volatiles
Only R,S-linalool and nonanal were unambiguously
present in the extracts obtained from the isolated glandu-
lar trichomes (Table 2). In contrast, benzyl alcohol and 2-
phenylethanol were detected in the flow through of the 20 ␮m
mesh cloth filtration used for the separation of the trichomes.
Thus, the glandular trichomes contain at least R,S-linalool
and nonanal, but benzyl alcohol and 2-phenylethanol migrate
from the leaf cuticle.
3.4. Biotransformation of R,S-linalool, nonanal, benzyl
alcohol and 2-phenylethanol by bacteria isolated from
F. x ananassa leaves
Epiphytic bacteria used in this study are listed in Table 1.
The bacteria were isolated from leaves of strawberry plants
cultivated in the field or in the greenhouse and identified
by morphological and biochemical methods as well as by
their 16S rDNA (unpublished). Since none of these bacte-
rial strains could use R,S-linalool, nonanal, benzyl alcohol
or 2-phenylethanol as a sole carbon source (data not shown)
biotransformation experiments were conducted. The extracts
obtained after incubation of R,S-linalool with the bacteria
were similar in composition and total recovered amount of
metabolites to the control. Thus, we concluded that R,S-
linalool was not biotransformed by the bacteria examined
(data not shown). Nonanal is oxidized to nonanoic acid in
the control and in the microbial test flasks (Fig. 2A). I2,
I3, I4, I24 and I29 could degrade nonanal and/or nonanoic
acid. 2-Heptyl-2-undecenal was a major biotransformation
product, which can be formed by an aldol condensation of
 D. Abanda-Nkpwatt et al. / Environmental and Experimental Botany 56 (2006) 108–119 113

Fig. 2. Composition of the extract after transformation of nonanal (A), benzyl alcohol (B), and 2-phenylethanol (C) by epiphytic bacteria. (A) Nonanal (in
green) was transformed to nonanoic acid (
), 2-heptyl-2-undecenal (in yellow), nonanol (in red) and others (). (B) Benzyl alcohol (
) was metabolized to
benzoic acid (in green) benzaldehyde (in yellow), and others (). (C) 2-Phenylethanol (
) was transformed to 1-phenyl-1,2-ethandiol (in green), phenylacetic
acid (in yellow), and others (). Vertical bars represent ± S.E. of the mean (n = 2). (For interpretation of the references to colour in this figure legend, the reader
is referred to the web version of the article.)

two molecules of nonanal, but was not detected in control
experiments (without bacteria) or in biotransformation exper-
iments where nonanoic acid was used instead of nonanal. E-
2-nonenol, methyl nonanoate, E-2-nonenal, ethyl nonanoate
and nonanamide were identified as minor metabolites. More
than 98% of the benzyl alcohol was recovered in the control
sample and in the extract obtained after the incubation with
strain I21 (Fig. 2B). All of the other epiphytic bacteria could
metabolize benzyl alcohol to some degree as evidenced by
the formation of benzoic acid in the extracts. Benzaldehyde,
methyl benzoate and benzyl acetate were present as minor
metabolites. Of the eleven strains, I3 was the most successful
in degrading benzyl alcohol with less than 10% of the initial
amount of benzyl alcohol remaining. A similar metabolic
pattern was observed for 2-phenylethanol (Fig. 2C). More
than 99% of the alcohol was recovered from the control
sample and the extracts obtained from strain I21 and I25.
Significant amounts of 1-phenyl-1,2-ethandiol and pheny-
lacetic acid were formed by the other epiphytic bacteria.
Phenylacetaldehyde, methyl phenylacetate, and 2-hydroxy-
1-phenylethanone were identified as minor metabolites. Sum-
marizing, it can be stated that nonanal, or rather its oxidation
product nonanoic acid, was effectively metabolized by half
of the studied bacteria. Benzyl alcohol and 2-phenylethanol
were mainly oxidized by the bacteria to their correspond-
ing acids, while R,S-linalool was not degraded by these
microbes. Strains I2, I3, I4, I24 and I29 metabolized more
of the volatiles than did I21 and I25.
3.5. Inhibition of B. cinerea by epiphytic bacteria
Several of the bacteria caused a zone of inhibition, but
culture filtrates obtained by sterile filtration did not affected
fungal growth. Thus, the observed inhibitory effect proba-
bly is not caused by antifungal compounds excreted by the
bacteria but by competition for nutrients.
3.6. Effects of F. x ananassa leaf volatiles on epiphytic
bacteria and B. cinerea
R,S-linalool at 1 or 10 ppm, depending on the strain
inhibited the growth of B. cinerea, but concentrations up
114 D. Abanda-Nkpwatt et al. / Environmental and Experimental Botany 56 (2006) 108–119

Table 3
Effect of plant volatiles on the growth of Botrytis cincera strains (B. c. 1: Bayer AG # 23, B. c. 2: Bayerische Landesanstalt 92/RB 11, B. c. 3: Bayerische
Landesanstalt 93/HRB 2b) and epiphytic bacteria I3, I4, I7, I8, I24, I26, I29
Plant volatiles Microorganisms Colony diameters at volatile concentration of

0 ppm 1 ppm 10 ppm 100 ppm 1000 ppm

R,S-linalool B. c. 1 7.3 ± 0.1 2.2 ± 0.1 0.8 ± 0.1 0 0
B. c. 2 6.8 ± 0.1 6.8 ± 0.3 3.0 ± 0.3 0 0
B. c. 3 7.5 ± 0.4 6.3 ± 0.4 3.3 ± 0.5 0 0
I3 8.5 ± 0.1 8.5 ± 0.1 8.5 ± 0.1 3.2 ± 0.1 1.1 ± 0.1
I4 6.5 ± 0.5 6.7 ± 0.3 6.0 ± 0.1 1.5 ± 0.1 0
I7 3.4 ± 0.4 3.0 ± 0.1 2.6 ± 0.1 0 0
I8 6.1 ± 0.5 6.8 ± 1.1 3.6 ± 0.1 0.8 ± 0.2 0
I24 6.1 ± 1.9 6.6 ± 0.3 2.4 ± 0.8 1.1 ± 0.5 0.7 ± 0.1
I25 4.5 ± 0.1 2.8 ± 0.4 0.9 ± 0.4 0 0
I29 3.0 ± 1.0 2.3 ± 0.4 1.8 ± 0.2 1.1 ± 0.1 0.7 ± 0.2

Nonanal B. c. 1 7.3 ± 0.1 4.5 ± 0.1 0 0 0

B. c. 2 6.8 ± 0.1 6.2 ± 0.4 0 0 0
B. c. 3 7.5 ± 0.5 8.2 ± 0.4 0 0 0
I3 8.1 ± 0.1 8.1 ± 0.1 5.0 ± 0.1 4.7 ± 0.1 0
I4 8.2 ± 0.1 8.2 ± 0.1 6.3 ± 0.6 0 0
I7 2.6 ± 0.1 2.5 ± 0.6 0 0 0
I8 8.3 ± 0.1 8.3 ± 0.1 5.7 ± 0.6 2.8 ± 0.5 0
I24 8.4 ± 0.1 8.4 ± 0.1 8.5 ± 0.1 4.6 ± 0.5 0
I25 5.0 ± 0.1 0 0 0 0
I29 8.5 ± 0.1 8.5 ± 0.1 8.5 ± 0.1 2.7 ± 0.3 0

Benzyl alcohol B. c. 1 7.3 ± 0.1 7.0 ± 0.2 6.0 ± 0.1 0 0

B. c. 2 6.8 ± 0.1 6.9 ± 0.1 6.0 ± 0.1 0 0
B. c. 3 7.5 ± 0.3 8.0 ± 0.5 6.8 ± 0.2 0 0
I3 8.1 ± 0.1 8.1 ± 0.1 8.1 ± 0.1 0 0
I4 8.2 ± 0.1 8.2 ± 0.1 8.2 ± 0.1 0 0
I7 2.7 ± 1.3 4.7 ± 1.2 3.2 ± 0.5 0 0
I8 8.3 ± 0.1 8.3 ± 0.1 8.3 ± 0.1 0 0
I24 8.4 ± 0.1 8.4 ± 0.1 0 0 0
I25 5.0 ± 0.3 0 0 0 0
I29 8.5 ± 0.1 8.5 ± 0.1 8.5 ± 0.1 0 0

2-Phenylethanol B. c. 1 7.3 ± 0.1 3.5 ± 0.3 0 0 0

B. c. 2 6.8 ± 0.1 6.8 ± 0.2 3.9 ± 0.3 0 0
B. c. 3 7.5 ± 0.6 8.2 ± 0.6 5.3 ± 0.1 0 0
I3 8.1 ± 0.1 8.1 ± 0.1 8.1 ± 0.1 0 0
I4 6.8 ± 0.1 7.0 ± 0.1 6.4 ± 0.1 0 0
I7 3.4 ± 1.8 5.2 ± 1.3 2.7 ± 0.3 0 0
I8 8.2 ± 0.1 8.2 ± 0.1 4.5 ± 0.7 0 0
I24 8.3 ± 0.1 8.3 ± 0.1 8.3 ± 0.1 0 0
I25 1.6 ± 0.1 2.5 ± 0.1 2.2 ± 0.1 0 0
I29 8.4 ± 0.1 8.4 ± 0.1 5.4 ± 0.1 0 0
MEA plates, containing 0, 1, 10, 100, and 1000 ppm of the respective plant volatile were inoculated with plugs of mycelium and 100 ␮l of a bacterial culture
(2 × 106 cells/ml). Colony diameters were determined after 7 days of incubation at room temperature.

to 10 ppm showed no significant influence on the growth
of most of the epiphytic bacteria (Table 3). R,S-linalool
at 1000 ppm inhibited completely the growth of almost all
of the bacterial strains tested. Nonanal, benzyl alcohol and
2-phenylethanol gave similar results (Table 3). Benzyl alco-
hol and 2-phenylethanol were very effective inhibitors
at 100 ppm. B. cinerea strains were totally inhibited at
100 ppm R,S-linalool, 10 ppm nonanal, 100 ppm benzyl alco-
hol, and 10 or 100 ppm 2-phenylethanol, depending on the
strain.
3.7. Inhibition of B. cinerea strains by epiphytic
bacteria in the presence of plant volatiles
B. cinerea strain B. c. 1 (Bayer AG # 23) overgrew an
I3 bacterial colony on MEA plates within 4 days, but in the
presence of 1 ppm nonanal I3 overgrew the fungal mycelium
(Fig. 3). Increasing concentrations of 2-phenylethanol also
permitted the growth of the bacterial colony (Fig. 4), but
inhibited the growth of the fungal mycelium (B. c. 3: 93/HRB
2b). Similar results were obtained with I8 (data not shown).
 D. Abanda-Nkpwatt et al. / Environmental and Experimental Botany 56 (2006) 108–119 115

Fig. 3. Effect of nonanal on the growth of I3 and B. cinerea (B. c. 1: Bayer

AG # 23). The radius of mycelium and of bacterial colony was measured
daily. Without nonanal B. cinerea (-
-) overgrew the bacterial colony (--),
while at 1 ppm nonanal I3 (-䊉-) overgrew the fungal mycelium (--) within
6 days. Vertical bars represent ± S.E. of the mean (n = 2).

As little as 1 ppm nonanal significantly reduced the prolif-

eration of B. cinerea strain B. c. 2 (92/RB 11) in favor of
the epiphytic bacteria I3, I6, I8, and I24 (Fig. 5A). The
effect of R,S-linalool on the dual incubation system was
less pronounced but statistically significant at 5 ppm R,S-
linalool (Fig. 5B). Although growth of B. cinerea strain B. c. 3
(93/HRB 2b) was inhibited only at 5 ppm nonanal (Fig. 6A),
this strain was susceptible to 1 ppm R,S-linalool (Fig. 6B).
However, B. cinerea strain B. c. 3 (93/HRB 2b) showed no
statistically significant effect in dual incubation experiments
with I3, I6, I8 and I24 in the presence of benzyl alcohol and
2-phenylethanol (data not shown). Comparison of the results
obtained by the single culture experiments (Table 3) with the
results of the in vitro dual culture studies (Figs. 3–6) demon-
strated that in the absence of volatiles the B. cinerea strains
inhibit the growth of the bacteria. In the co-culture experi-
ments, the mycelia reached almost the same sizes as in the
single culture studies (Table 3) but the colony radii of the
bacteria were reduced. However, with increasing compound
concentrations the volatiles inhibited fungal growth and sup-
ported the proliferation of the bacteria, but their effectiveness
varied depending on the strains. In general, particularly in the
case of nonanal, reduced proliferation of B. cinerea strains
caused by the plant volatiles led to the enhanced growth of
epiphytic bacteria.
Fig. 4. The effect of 2-phenylethanol on the colony size of I3 and mycelium
size of the phytopathogenic fungus B. cinerea (B. c. 3: Bayerische Lan-
desanstalt 93/HRB 2b). The radius of the mycelium and the colony was
measured after 5 days when they came into contact. Vertical bars rep-
resent ± S.E. of the mean (n = 4). When exposed to 2-phenylethanol, the
growth of B. cinerea was reduced, whereas the colony diameter of I3 was
increased.
Fig. 5. The effect of nonanal (A) and R,S-linalool (B) on the colony sizes
of I3 (in yellow), I6 (in green), I8 (in red) and I24 (in blue) as well as
the mycelium size of the phytopathogenic fungus B. cinerea (
) (B. c. 2:
Bayerische Landesanstalt 92/RB 11). The radius of the mycelium and the
colony was measured after 7 days. Vertical bars represent ± S.E. of the mean
(n = 4). The growth of B. cinerea 92/RB 11 decreases (a) and the bacterial
colony increases (b). Bars marked with (a) or (b) imply that values are
statistically different according to the t-test (P < 0.001). (For interpretation
of the references to colour in this figure legend, the reader is referred to the
web version of the article.)
4. Discussion
4.1. Colonization sites and nutrient sources for
epiphytic bacteria
R,S-linalool, nonanal, benzyl alcohol and 2-phenylethanol
were the major native volatiles released by strawberry leaves
confirming the data of Pyysalo et al., 1979. R,S-linalool and
nonanal are emitted from capitate glands, which are located
on vascular bundles on the adaxial surface and randomly
distributed on the abaxial leaf surfaces. The significance
of the different arrangement of the capitate glands on the
ad- and abaxial leaf surface remains unclear. Glandular
trichomes have been examined in several species and can
produce flavonoids, monoterpenes, sesquiterpenes and
phenylpropenes (Gershenzon et al., 1992; Bosabalidis and
Skoula, 1998; Gang et al., 2001). Since large bacterial
aggregates can form when nutrients are available, localized
leakage glands and vascular bundles constitute good sites for
colonization (Lindow and Brandl, 2003; Monier and Lindow,
2004). The vascular bundles transport high concentrations
of plant nutrients, and therefore, it is conceivable that
they also deliver appreciable amounts of nutrients, such as
carbohydrate and amino acids to the leaf surface supporting
the growth of epiphytic bacteria. For microbial colonization
116 D. Abanda-Nkpwatt et al. / Environmental and Experimental Botany 56 (2006) 108–119
Fig. 6. The effect of nonanal (A) and R,S-linalool (B) on the colony sizes
of I3 (in yellow), I6 (in green), I8 (in red) and I24 (in blue) as well as
the mycelium size of the phytopathogenic fungus B. cinerea (
) (B. c. 3:
Bayerische Landesanstalt 93/HRB 2b). The radius of the mycelium and the
colony was measured after 7 days. Vertical bars represent ± S.E. of the mean
(n = 8). The growth of B. cinerea 93/HRB 2b decreases (a) and the bacterial
colony increases (b). Bars marked with (a) or (b) imply that values are
statistically different according to the t-test (P < 0.001). (For interpretation
of the references to colour in this figure legend, the reader is referred to the
web version of the article.)
to occur, a carbon source for energy generation and growth,
a nitrogen source, and certain essential inorganic molecules
must be present on the leaves. Since plants usually are
colonized by bacteria, nutrients released from the plant to
its intact surface must be sufficient to support microbial
populations (Mercier and Lindow, 2000). Molecules leached
from plant leaves include a variety of organic and inorganic
substances, such as sugars, organic acids, amino acids,
methanol and various salts (Fiala et al., 1990). Our data
indicate that several epiphytic bacteria can also utilize plant
volatiles, such as nonanal that are emitted from glandular
trichomes as an energy or carbon source. This result can
explain the observation that the capitate glands are more
densely colonized than other parts of the leaf surfaces.
4.2. Metabolism of volatiles by epiphytic bacteria
Although R,S-linalool, nonanal, benzyl alcohol and 2-
phenylethanol cannot serve as sole carbon source for the iso-
lated epiphytes, they can be metabolized, with the exception
of R,S-linalool, by at least some of the bacteria. Biotransfor-
mation experiments have shown that R,S-linalool is degraded
especially by fungi and yeasts (Schrader and Berger, 2001;
Demyttenaere et al., 2001), while benzyl alcohol and 2-
phenylethanol are transformed also by bacteria (Horiuchi
et al., 1993; Celik et al., 2004; Gandolfi et al., 2001). The
metabolism of nonanal is not well investigated (Abanda-
Nkpwatt and Schwab, 2004). The major metabolites from
the transformation experiments (nonanoic acid and benzoic
acid) were also found in all of the strawberry leaf extracts.
Thus, microbial transformation of the leaf volatiles certainly
could occur on the leaf surface, although we cannot currently
rule out the formation of benzoic acid and nonanoic acid by
the leaf tissue. The capitate glands are intensively colonized
by microorganisms supporting the hypothesis that the bacte-
ria can use the compounds synthesized by the glands.
4.3. Antifungal and antibacterial effect of plant volatiles
and their metabolites
R,S-linalool, nonanal, benzyl alcohol and 2-phenylethanol
are known antifungal and antibacterial agents (Corre et
al., 1990; Trombetta et al., 2002). Benzyl alcohol and 2-
phenylethanol were the most effective substances and totally
inhibited growth of all strains at 100 ppm. B. cinerea was the
most susceptible microorganism tested. The concentrations
of the compounds tested exceeds those released from straw-
berry leaves, which normally fall between 0.15 mg kg−1 for
R,S-linalool and 0.45 mg kg−1 for benzyl alcohol (Pyysalo
et al., 1979). However, volatiles are not evenly distributed
on the leaf surface. R,S-linalool and nonanal are emitted by
capitate glands, and their local concentrations in the vicin-
ity of the glandular trichomes are higher than the averages
calculated for an entire leaf (Iijima et al., 2004). Also, an
additive or even synergistic effect of the volatiles cannot be
excluded. On the other hand, benzoic acid, the major metabo-
lite of benzyl alcohol, is intensively used as a preservative
and has a stronger effect on yeast and fungi than on bacteria
(Lattanzio et al., 1994). Thus, the transformation of benzyl
alcohol into benzoic acid should increase a plant’s resistance
towards phytopathogenic fungi, such as B. cinerea.
4.4. Interactions among non-pathogenic and pathogenic
epiphytes
Interactions among the non-pathogenic and pathogenic
microorganisms, such as antibiosis and nutrition competition
may lead to natural biological control of foliar pathogens
(Andrews, 1992). The competition of epiphytic bacterial
populations for nutritional resources is known (Wilson and
Lindow, 1994) and antagonism via the production of antimi-
crobial compounds appears to be an uncommon mechanism
of interaction of bacteria on leaf surfaces (Lindow and Brandl,
2003). We found that plant volatiles affect the population
dynamics of non-pathogenic bacteria and a pathogenic fun-
gus. Our data indicate that leaf volatiles have a stronger
inhibitory effect on B. cinerea strains than on the epiphytic
bacteria (Table 3). When the bacteria and the fungus were
grown on the same plate the fungus suppressed the prolif-
eration of the bacteria as their colonies grew larger in the
absence of the fungus. But this effect was minimized when
bacterial strains I3, I6, I8 and I24 and B. cinerea strains pro-
 D. Abanda-Nkpwatt et al. / Environmental and Experimental Botany 56 (2006) 108–119
liferated in the presence of already 1 ppm nonanal, a realistic
concentration of the volatile in the leaf (Figs. 5 and 6). A
similar, but less pronounced effect was observed with 2-
phenylethanol (Fig. 4), and R,S-linalool (Figs. 5B and 6B)
at 1 to 5 ppm even though the bacterial strains cannot use
these volatiles as a sole carbon source. While these results
are preliminary, they are consistent with the inhibitory effect
of the volatiles (Table 3), the ability of the epiphytic bacteria
to metabolize most of the volatiles (Fig. 2) (Schrader and
Berger, 2001; Horiuchi et al., 1993; Celik et al., 2004;
Gandolfi et al., 2001), and the bacterial concentration around
the trichomes. Effective concentrations of volatiles were
those, which also occur in the native leaves. The selective
inhibitory effect of the plant volatiles against pathogenic
fungi, the ability of the epiphytes to catabolize the plant
volatiles and consequently the depletion of nutrients by
epiphytic bacteria, can explain the observed results. Addi-
tionally, some leaf epiphytes may detoxify plant secondary
metabolites resulting in compounds that play further alleo-
chemical roles in the phylloplane (Hashidoko et al., 2002).
By emitting volatiles from glands or the epidermis the straw-
berry plants may create a micro-environment that generally
supports the growth of beneficial bacteria, while inhibiting
fungal pathogens.
4.5. Efficacy in biological control
Our data suggest that plant volatiles minimize the growth
suppression of epiphytes by B. cinerea, and thus, they can
alter the biological control efficacy of non-pathogenic epi-
phytes. For a non-pathogenic, leaf-associated bacterium,
effectiveness in the control of bacterial speck of tomato is
correlated with the similarity in the nutritional needs of the
non-pathogenic bacterium and the pathogen Pseudomonas
syringae pv. tomato (Wilson and Lindow, 1994). However, in
the case of the pathogen Xanthomonas campestris pv. vesica-
toria nutritional similarity with non-pathogenic bacteria was
not correlated with reductions in pathogen population size
on tomato leaves (Dianese et al., 2003). We believe that the
effects of plant secondary metabolites might also contribute
to the observed result. Tomato leaves are a rich source of
plant volatiles, which could serve, beside carbohydrates and
amino acids as carbon sources for a range of non-pathogenic,
leaf-associated bacteria but could also specifically inhibit
the proliferation of some strains (Andersson et al., 1980;
Lundgren et al., 1985; Buttery et al., 1987; Kim and Kil,
2001). Effective colonization and high population size of
introduced bacterial biological control agents on plant sur-
faces are important factors in the successful control of plant
diseases (Wilson and Lindow, 1995). Hence, many strategies
have been developed to enhance colonization and population
size of bacterial control agents in order to improve biolog-
ical control efficacy, including attempts to use nutritional
amendments to favor the growth and activity of those bio-
logical control agents. Our results suggest that plants already
produce volatiles for this purpose. Plant volatiles are con-
117
stantly emitted by leaf surfaces and could serve as antimi-
crobial compounds that specifically reduce the number of
pathogenic microorganisms. On the other side volatiles may
act as energy sources by which beneficial bacterial strains
can increase their population size. The relative spatial dis-
tribution of B. cinerea and the potential, naturally occurring
competitors are not known. However, during the last years
methods became available to observe single cells of a partic-
ular species on the leaf surface. Specific cells become visible
after in situ hybridization with dye-labeled, rRNA-targeted
oligonucleotide probes (Schonholzer et al., 2002). In another
approach, strains are engineered to express a green fluores-
cent protein (Gfp) constitutively. The labeled cells can be
observed by confocal laser-scanning microscopic analysis
after colonization (Newman et al., 2003). It will be impor-
tant to apply these methods to determine whether the isolated
epiphytes and B. cinerea colonize the same physical niche.
5. Conclusion
Control of plant diseases with beneficial bacteria has
become reality, and products for biocontrol of posthar-
vest diseases of fruits are commercially available. However,
knowledge of the mechanism of biocontrol by these beneficial
microorganisms is rudimentary. Various observations suggest
that competition for nutrients between beneficial organisms
and pathogens could be an important mechanism of biocon-
trol in the system. Studies have shown that many benefi-
cial species can effectively deplete the sugar and/or amino
acids occurring on leaf and fruit and inhibit propagation of
pathogens. Thus, effective colonization and high population
size of introduced biological control agent is an important fac-
tor in the successful control of plant diseases. The selective
increase in the population size, and thus, the biological con-
trol efficacy of a biocontrol agent is achieved through nutri-
tional amendment and is based on the observation that avail-
ability of nutrients is a limiting factor for growth of microbial
populations in various plant habitats. Our data demonstrate
for the first time that also plant volatiles emitted by leaf sur-
faces and glandular trichomes affect the population dynamics
of non-pathogenic bacteria and can selectively inhibit the
propagation of phytopathogens, such as B. cinerea. Volatiles,
neglected until now can increase the population size of ben-
eficial bacteria, and thus, the efficacy of biocontrol agents.
We demonstrated this effect the first time by in vitro experi-
ments. The findings have significant implications on the use
of antagonistic bacteria as biocontrol agents since volatiles
can be used to enhance colonization and population size.
Acknowledgements
Financial support for this work was provided by SFB 567.
We thank PD Dr. Rainer Wolf for performing the scanning
electron microscope measurements, Frank Heckel, Thomas
118 D. Abanda-Nkpwatt et al. / Environmental and Experimental Botany 56 (2006) 108–119
Wickert, Bianka Pink and Márti Dregus for GC–MS anal-
ysis, Bayerische Landesanstalt für Wein- und Gartenbau,
Veitshöchheim (Erna Schindler) for technical assistance and
providing B. cinerea strains, and Bayer AG for providing
additional B. cinerea strains.
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