Endophytic fungi associated with Mediterranean plants as a source of mycelium-bound lipases

Article abstract of DOI:10.1021/jf025786u

A screening of endophytic fungi isolated from Mediterranean plants rendered a mycelium-bound lipase from a strain of Rhizopus oryzae that catalyzed the esterification of fatty acids in isooctane. The influence of various factors (water content, temperature, and pH) on ester synthesis was investigated. Catalytic activity was inversely correlated with water content. This enzyme was active over the entire pH range studied, from pH 3 to pH 8, and activity was maximal at pH 4 and pH 7. The enzyme was thermostable, with maximal activity at 60 °C.

Full text of DOI:10.1021/jf025786u

3328 J. Agric. Food Chem. 2003, 51, 3328−3333
Endophytic Fungi Associated with Mediterranean Plants as a
Source of Mycelium-Bound Lipases
MERCEÅ TORRES,† MARTA M. DOLCET,† NURIA SALA,† AND RAMON CANELA*,‡,§
Food Technology Department, Centre UdL-IRTA, and Chemistry Department, Universitat de Lleida,
Rovira Roure, 191, 25198-Lleida, Spain
A screening of endophytic fungi isolated from Mediterranean plants rendered a mycelium-bound lipase
from a strain of Rhizopus oryzae that catalyzed the esterification of fatty acids in isooctane. The
influence of various factors (water content, temperature, and pH) on ester synthesis was investigated.
Catalytic activity was inversely correlated with water content. This enzyme was active over the entire
pH range studied, from pH 3 to pH 8, and activity was maximal at pH 4 and pH 7. The enzyme was
thermostable, with maximal activity at 60 °C.
KEYWORDS: Endophytic fungi; mycelium-bound lipase; resting cells; esterification; lipase activity;
Rhizopus oryzae
INTRODUCTION
In this study, we screened endophytic fungi displaying
lipaselike activity. We identified a mycelium-bound lipase
activity in a Rhizopus isolate and studied the effects of various
factors on this activity.
Lipases (EC 3.1.1.3) are enzymes that hydrolyze the ester
bonds of water insoluble substrates at the interface between the
substrate and the water. This reaction is reversible, and lipases
may catalyze ester synthesis and transesterification in reaction
mixtures with low water contents. Lipases catalyze a wide
diversity of reactions, which has led to their widespread use in
industrial applications (1).
Lipases are produced by animals, plants, and microorganisms.
Most of the lipases used in biotechnological applications to date
were isolated from fungi (2, 3). Commercial fungal lipases are
generally extracellular enzymes, although there is evidence for
considerable mycelium-bound lipase activity (4). The use of
naturally bound lipases is potentially cost effective because the
biomass can be used directly, thereby eliminating the need for
isolation, purification, and immobilization procedures and
minimizing the loss of enzyme activity. Naturally bound lipases
also offer other advantages, such as greater stability in the
presence of organic solvents, high temperatures, and extreme
pH values (5).
Industrial demand for new sources of lipases is continuing
to stimulate the isolation and screening of new lipolytic
microorganisms. We focused our attention on endophytic fungi
as a potential new source of lipases. The specific association
of fungal endophytes with plant hosts represents a large
unexplored domain ripe for discovery (6). The term endophyte
is commonly used for fungi and bacteria that for all or part of
their life cycle invade the tissues of living plants, causing
unapparent and asymptomatic infections entirely within plant
tissues but no symptoms of disease (7).
MATERIALS AND METHODS
Chemicals. Trifluoroacetic acid (99.5%) was purchased from Fluka.
1-Dodecanol (98%) and linoleic acid (99%) were purchased from
Aldrich. 1-Propanol (99%) and potassium hydrogenphthalate (KHC8H4O4)
were obtained from Probus. Isooctane (99%) was obtained from
Panreac. Oleic acid, potassium dihydrogenphosphate (KH2PO4), and
tris(hydroxymethyl)aminomethane (C4H11NO3) were purchased from
Merck.
Plant Material. The plants used in this study are listed in Table 1.
All samples were collected in October 2000 from The Montsec Natural
Park in Catalonia (Spain).
Isolation of Endophytic Fungi. Healthy plant tissues were harvested
for the isolation of endophytic fungi. The tissues were washed in
running tap water and processed within 24 h of collection. From each
washed plant, we cut six segments, each 2-3 cm long. All segments
were surface-sterilized by sequential washes in 0.53% sodium hy-
pochlorite (2 min) and 70% ethanol (2 min) and were then rinsed with
sterile distilled water and allowed to surface dry under aseptic conditions
(8, 9). The surface-sterilized segments were placed on 2% malt extract
agar in Petri dishes. All plates were incubated at room temperature for
a maximum of 2 weeks. Fungi growing out from the plant tissues were
transferred to mycological agar, composed of 10 g of glucose, 10 g of
papaic digest of soybean meal, and 15 g of agar per liter of distilled
water. The plates were incubated for 5-7 days at 30 °C.
Preliminary Screening of Lipolytic Activity of Fungi. Preparation
of Medium. The medium used for preliminary screening consisted of
5.0 g of mycological peptone, 3.0 g of yeast extract, and 10.0 g of
agar per liter of distilled water. The medium was autoclaved at 121 °C
for 15 min. After the medium was cooled to 60 °C, filter-sterilized
tributyrin was added to the medium to a final concentration of 1.0%
(v/v) and the mixture was emulsified by blending for 1-3 min. The
blended medium was dispensed into Petri dishes immediately after
homogenization and chilled rapidly (10).
* To whom correspondence should be addressed. Tel: +34 973702843.
Fax: +34 973238264. E-mail: canela@quimica.udl.es.
† Food Technology Department.
‡ Centre UdL-IRTA.
§ Chemistry Department.
10.1021/jf025786u CCC: $25.00 © 2003 American Chemical Society
Published on Web 04/16/2003

Endophytic Fungi Associated with Mediterranean Plants
J. Agric. Food Chem., Vol. 51, No. 11, 2003 3329
Table 1. Number of Strains Isolated from Each Plant Speciesa
species
N
code
species
Erinacea anthyllis
N
code
Lavandula latifolia
Rosmarinus officinalis
Salvia lavandulifolia
Salvia verbenaca
Satureja montana
Sideritis scordioides
Teucrium polium
Thymus vulgaris
6
5
2
1
5
3
6
1
2
5
5
1
2
6
1A−1F
2A−2E
3A, 3B
4
5A−5E
6A, 6C
7A−7F
8
9A, 9B
10A−10E
11A−11E
12
13A, 13B
14A−14F
3
3
2
4
6
2
3
2
2
3
5
3
6
15A−15C
16A−16C
17A, 17B
18A−18D
19A−19F
20A, 20B
21A−21C
22A, 22B
23A, 23B
24A−24C
25A−25E
26A−26C
27A−27F
Ononis tridentata
Psoralea bituminosa
Juniperus oxycedrus
Juniperus phoenicea
Arctostaphylos uva-ursi
Buxus sempervirens
Daphne gnidium
Gypsophila struthium
Limonium hibericum
Olea europaea
Thymus zygis
F. vulgare
Seseli tortuosum
Artemisia herba-alba
Jasonia glutinosa
Santolina chamaecyparissus
Plantago albicans
Quercus ilex subspecies ballota
a The identification code used is shown. N ) number of strains isolated. Code: identification code for isolated strains.
Inoculation, Incubation, and Measurement of Clearing Area. The
inoculum to be tested was cultured on mycological agar plates for 5-7
days at 30 °C. A disk of solid medium containing inoculum was excised
from this plate with a sterile plunger and placed in the middle of an
agar plate containing the substrate tributyrin. Plates were incubated at
25 °C for 5 days. The diameter (cm) of the cleared zone around the
disk was used to estimate the relative lipolytic activities of the fungi
tested.
stirrer turning at a speed of 500 rpm. Samples (1 mL) were withdrawn
after 5 min and filtered. We monitored ester formation and residual
free fatty acid levels by reverse phase high-performance liquid
chromatography (RP-HPLC), as described below. Experiments were
carried out in triplicate.
Effect of Temperature on Lipase ActiVity. To determine the effect
of temperature, three vials containing 5 mg of lyophilized mycelium
suspended in 3 mL of isooctane were prepared for each reaction time
(5 min, 20 min, and 2 h) and each temperature (30, 40, 50, 60, 70, and
80 °C). Each vial was preincubated at the appropriate temperature for
30 min, with a magnetic stirrer turning at 500 rpm, before the start of
the experiment. We then added 1 mL of a solution of 44.5 mM linoleic
acid and 89.0 mM 1-dodecanol in isooctane, warmed to the same
temperature, to each vial. The reaction mixtures were then stirred, with
a magnetic stirrer turning at a speed of 500 rpm, and 1 mL aliquots
were withdrawn at various reaction times and filtered. The amount of
dodecyl linoleate was quantified by gas chromatography, as described
below.
Thermostability of Mycelium-Bound Lipase. A set of vials, each
containing 5 mg of lyophilized mycelium suspended in 3 mL of
isooctane, was incubated at 60 or 70 °C for 2.5 h. The vials were cooled
to 30 °C, and 1 mL of a solution of 44.5 mM linoleic acid and 89.0 M
1-dodecanol in isooctane, warmed to 30 °C, was added to each vial.
The reaction mixtures were then incubated at 30 °C, with a magnetic
stirrer turning at a speed of 500 rpm. Samples (1 mL) were withdrawn
at various reactions times and filtered. The amount of dodecyl linoleate
was quantified by gas chromatography, as described below. Experiments
were carried out by triplicate.
Effect of pH on Lipase ActiVity. The effect of pH on the activity of
mycelium-bound lipase was measured at several pH values. A potassium
hydrogenphthalate (KHC8H4O4) buffer was used to control pH in the
3-5.5 range. A potassium dihydrogenphosphate (KH2PO4) buffer was
used to control pH in the 6-7.5 range. Finally, a tris(hydroxymethyl)-
aminomethane (C4H11NO3) buffer was used to control pH at 8. All the
buffers were made up from stock solutions of 0.05 M salts using either
0.1 M NaOH or 0.1 M HCl to reach the required pH. The mycelium
was first incubated in buffered solutions for 3 h at 26 °C. It was then
filtered, washed three times with distilled water and three times with
acetone, lyophilized for 2 h, and ground to a powder. To assay enzyme
activity, we prepared three vials for each reaction time (5 and 20 min),
each containing 4 mL of 11.1 mM linoleic acid and 22.2 mM 1-propanol
in isooctane. The reaction mixtures were stirred with a magnetic stirrer
turning at 500 rpm. Aliquots (1 mL) were withdrawn at different
reaction times and filtered. The amount of propyl linoleate was
quantified by gas chromatography, as described below.
Chromatographic Analyses. HPLC Analysis. HPLC analyses were
carried out with a Waters series 600 pumping system, a Waters 710
autosampler, and a Waters 2690 UV detector at 210 nm (Waters
Cromatografia SA, Spain). Twenty microliters of each sample was
injected into a Simmetry C18 5 µm (150 mm × 3.9 mm) (Waters
Cromatografia SA) reverse-phase column at 40 °C. The eluent was
95% ethanol/5% 0.1% trifluoroacetic acid in Milli-Q water (95:5 v/v).
Secondary Screening. We assessed the catalytic activity of the
selected fungi by monitoring propyl oleate synthesis.
Culture of Microorganisms. Microorganisms were cultured in a
synthetic liquid medium consisting of 2 g of asparagine, 2 g of glucose,
1 g of K2HPO4, 0.5 g of MgSO4, 5 mg of thiamine hydrochloride,
1.45 mg of Fe(NO3)3‚9H2O, 0.88 mg of ZnSO4‚7H2O, and 0.235 mg
of MnSO4‚H2O per liter of distilled water (11). The pH of the medium
was adjusted to 5.5-6.0. The medium was sterilized (121 °C for 15
min) in 250 mL aliquots, to which we added 2% (v/v) refined sunflower
oil in aseptic conditions. The medium was inoculated with 2.5 mL of
a spore suspension ((1-4) × 106 spores mL-1) for sporulating isolates
or with four disks cut out from mycological agar for nonsporulating
cultures. It was then incubated at 28 °C for 5 days on an orbital shaker
(200 rpm).
Preparation of Resting Cells and ActiVity Assay. Mycelium was
harvested from the culture medium with a Buchner funnel and was
washed with distilled water and then with acetone. The mycelium was
dried under vacuum for 18 h and ground to a powder (12).
To study the mycelium-bound lipase activity, 50 mg of lyophilized
mycelium was suspended in 10 mL of a solution of 89 mM oleic acid
and 666 mM 1-propanol in isooctane. This suspension was incubated
for 3 h at 25 °C, with shaking at 200 rpm, on an orbital shaker. Samples
(1 mL) were withdrawn and filtered. The amount of propyl oleate was
quantified by gas chromatography, as described below. Experiments
were carried out in triplicate.
Factors Affecting Mycelium-Bound Lipase Activity. Growth
Media and Culture Conditions. Fungi were cultured as described for
secondary screening, except that glucose was omitted from the culture
medium (12).
Effect of Water Content on Lipase ActiVity. Lyophilized mycelia were
dried under vacuum over P2O5 at room temperature for 2 h (13).
Reactions were carried out immediately after drying to prevent the
mycelium from taking up water. We added 5 mg of lyophilized
mycelium to one vial and 5 mg of lyophilized and dried mycelium to
another vial, each vial containing 4 mL of a solution of 11.1 mM linoleic
acid and 22.2 mM 1-propanol in isooctane. The reaction mixtures were
incubated at 26 °C for 5 min, with a magnetic stirrer turning at a speed
of 500 rpm. Samples (1 mL) were withdrawn and filtered, and the
resting cells that accumulated on the filter were allowed to dry. We
suspended 5 mg of dried resting cells for each sample in several vials
with 3 mL of isooctane and either 0, 5, 7, 10, 15, or 20 µL of water
and incubated them for 15 h at 26 °C. We then added 1 mL of a solution
of 44.5 mM linoleic acid and 89.0 mM 1-dodecanol in isooctane to
each vial and incubated the reaction mixtures at 26 °C, with a magnetic

3330 J. Agric. Food Chem., Vol. 51, No. 11, 2003
Torres et al.
Secondary Screening. We assessed mycelium-bound lipase
activity by monitoring the esterification of an alcohol with a
carboxylic acid in an organic solvent (14). We assessed the
ability of all 76 strains selected in the first screening to act as
biocatalysts for the synthesis of propyl oleate. Figure 1 shows
the diameter of the halo formed in tributirin assays plotted
against lipase activity as evaluated on the basis of propyl oleate
concentration. Ten of the tested strains (8, 9A, 10A, 10E, 16C,
18A, 23A, 24A, 24C, and 27F) gave significant ester yields.
However, comparison of the results of preliminary and second-
ary screening showed an absence of general correlation between
hydrolytic and synthetic activities, as reported in other studies
(3, 18). Thus, the lipase from strain 16C, which displayed low
levels of hydrolytic activity, was very active in the synthesis of
propyl oleate. In contrast, lipases from strains 10E, 18A, 23A,
24A, and 24C, which were highly active against tributyrin,
displayed only moderate levels of activity in propyl oleate
synthesis. Nevertheless, strain 10A clearly gave the highest
hydrolytic and synthetic activities of any of the tested strains.
The differences between levels of hydrolytic and synthetic
activity may be accentuated by the fact that the tributirin assay
measures both extracellular and mycelium-bound hydrolase
activity on a hydrophilic medium whereas propyl oleate
synthesis indicates the presence of mycelium-bound lipase
activity on a hydrophobic medium.
Table 2. Diameter of the Cleared Zone (Halo) after 5 Days of
Incubation at 25 °C for Each Isolated Straina
strain
H
strain
0.9 6C
6A
H
strain
H
strain
H
strain
23B
H
1A
1B
1C
1D
1E
1F
2A
2B
2C
2D
2E
3A
3B
4
1.1 11C
11D
1.6 17B
1.6 18A
1.5 18B
18C
2.1 18D
1.8 19A
1.2 19B
1.1 19C
1.3 19D
1.2 19E
1.2 19F
1.2 20A
20B
1.6 21A
1.5 21B
1.2 21C
1.3 22A
1.2 22B
23A
1
2.1
1.9
1
1.9 24A
2 24B
1.6 24C
1.9 25A
2.5 25B
25C
1.2 6B
1.2 7A
1.2 7B
1.3 7C
1.2 7D
7E
1.1 7F
1.2
1.3 9A
9B
1.2 10A
10B
1.4 11E
1.6 12
1.2 13A
1.4 13B
1.3 14A
14B
1.2 14C
1.4 14D
1
1.8
1.1
1.1
1.6 25D
25E
1.3
0.9
1.1
1.3
1.9
0.9
8
1.8 26A
1.7 26B
1.8 26C
1.7 27A
1.8 27B
27C
1.6 27D
1.9 27E
1.6 27F
1.8
14E
1.1 14F
8.6 15A
15B
5A
5B
5C
5D
5E
1.2 10C
1.2 10D
1.1 10E
2
15C
1.8 16A
1.8 16B
1.6 16C
1.5 17A
1
11A
1.1
1.2 11B
a The identification code used is shown. H ) halo diameter (cm).
The flow rate was 1 mL/min. Chromatographic peaks were recorded
and integrated with Millenium 32 computer software (Waters Cro-
matografia).
As shown in Figure 1, strain 16C presented considerable cell-
bound catalytic activity. However, this strain proved to be very
difficult to maintain on agar after repeated platings. We therefore
used only strain 10A for subsequent experiments. Strain 10A,
isolated from Foeniculum Vulgare (fennel), was identified in
our laboratory as Rhizopus oryzae, an identification confirmed
by the CBS (Central Bureau voor Schimmelcultures, Baarn,
Holland).
GC Analysis. Samples were analyzed with a ThermoQuest series
2000 chromatograph equipped with an FID detector, an EEP system
(Fisons, Barcelona, Spain), a split/splitless injection system, and an
autosampler. The analytical column (Supelco, Madrid, Spain) was a
30 m × 0.25 mm fused silica capillary coated with a 0.25 µm film of
poly(80% biscyanopropyl-20% cyanopropylphenyl siloxane) (SP-
2330). The GC run was programmed such that the temperature increased
from 50 to 150 °C (180 °C for dodecyl ester) at a rate of 5 °C/min,
then remained constant for 1 min at 150 °C, increasing thereafter to
220 °C (230 °C for dodecyl ester) at a rate of 10 °C/min. A 1:20 split
injection ratio was used with He as the carrier gas. Chromatographic
peaks were recorded and integrated with Trace-ThermoQuest computer
software. Before GC analysis, we added 0.5 mL of methyl palmitate
in isooctane (10 mM) as an internal standard and 1 mL of aqueous
saturated NaHCO3 solution to 0.5 mL of each sample. These mixtures
were incubated at room temperature for 24 h on an orbital shaker (200
rpm). The organic phase was separated by centrifugation at 3000 rpm.
Improvement of the Mycelium-Bound Lipase Activity of
the Selected Strain (R. oryzae). Effect of Water Content on
Lipase ActiVity. Water activity (aw) as a measure of water
content is one of the most important factors, other than the nature
of the solvent, affecting enzyme activity in organic solvents.
Once the nature of the biocatalyst and the nature of the solvent
are fixed, the water sorption isotherm depends on the amount
of added water (13). A minimum amount of water, depending
on the type of organic solvent used and the characteristics of
the immobilization support, is known to be strictly required for
the solvation of enzymes. However, the presence of excess water
favors hydrolysis over synthesis reactions (19). Water content
may therefore affect reaction rate, yield, and the stability of
enzymes (20). Thus, water content must generally be strictly
controlled for ester synthesis (21). Moreover, the water content
requirements for biocatalysis are highly dependent on the solvent
(22), with highly polar solvents tending to strip essential water
from the enzyme (23). The mycelium-bound lipase of R. oryzae
was most efficient at the esterification of linoleic acid with
1-propanol if used dried or after adding small amounts of water
(5 µL) (Figure 2). The addition of 10 or 20 µL of water led to
a clear decrease in ester formation after 5 min of reaction. The
addition of 5 µL of water to the reaction system corresponds to
the same amount of mycelium used. Considering the low
solubility of the water in isooctane, it is possible to assume that
biocatalyst acts as a sorbent of the added water without any
great influence on its activity, determined considering the initial
esterification rate. Addition of 7 µL of water does not cause
any considerable loss of activity either. However, when 10 µL
or more water was added, enzymatic activity showed a clear
decrease. Although we had not measure the water activity of
RESULTS AND DISCUSSION
Isolation of Endophytic Fungi. In this work, we isolated a
total of 94 fungi from plant samples. Table 1 shows the number
of strains isolated from each plant species and gives the
corresponding identification codes.
Preliminary Screening. We screened all of the microbial
strains isolated for hydrolytic activity in plates containing solid
medium supplemented with tributyrin as a substrate. In this test,
activity is detected as the formation of a clear halo around the
colony (14). The use of tributyrin as a substrate for the detection
of microbial lipolysis has been called into question by some
authors (15) because, in addition to most lipases, some esterases
also hydrolyze this substrate. However, tributyrin is a good
substrate for a rapid screening test because it disperses in water
more easily than true lipase substrates such as triolein (16). A
total of 76 microorganisms, corresponding to 80% of the
investigated strains, generated a clear halo (Table 2). Halos of
this type are observed only if the fatty acids released are at
least partially soluble in water. The observed clarification of
the medium results from a decrease in the size of the hydrolyzed
emulsified particles, which in turn causes a decrease in light
diffusion (17).

Endophytic Fungi Associated with Mediterranean Plants
J. Agric. Food Chem., Vol. 51, No. 11, 2003 3331
Figure 1. Semilogarithmic plot of hydrolytic activity vs synthetic activity of the isolated strains. Hydrolytic activity was measured as the diameter of the
cleared zone after 5 days of incubation at 25 °C. Synthetic activity was measured as the concentration of 1-propyl oleate obtained following the incubation
of the lyophilized mycelium in a solution of oleic acid and 1-propanol in isooctane for 3 h at 26 °C.
Figure 3. Effect of temperature on the esterification of linoleic acid with
1-dodecanol catalyzed by R. oryzae resting cells. Reactions were carried
out in isooctane for 5 min, 20 min, and 2 h.
Figure 2. Effect of water content on the esterification of linoleic acid with
1-propanol catalyzed by R. oryzae resting cells. Reactions were carried
out in isooctane at 26 °C for 5 min.
in 20 min, and 100% conversion after 2 h. The enzyme
transformed 100% of the linoleic acid after 2 h of reaction,
whether the reaction was performed at 50 or 70 °C. Above 80
°C, this membrane-bound lipase retained about 80% activity,
giving yields similar to those obtained at 30 °C. Yields of
dodecyl linoleate greater than 100% were obtained in some
cases, indicating the presence of residual linoleic acid originating
from the sunflower oil component of the growth medium (12).
the system, it is well-known that an increase of the contents of
water causes an increase of the aw of the system (13). These
results are similar to the ones found by Koller et al. (24). These
authors, using extracellular enzymes isolated from R. oryzae
and Rhizopus niVeus and adsorbed onto a microporous polypro-
pylene support, showed that lipases displayed optimal activity
at very low aw levels. However, the amount of water required
for high levels of catalytic activity differs greatly between
lipases. Some, such as the enzymes from Rhizomucor miehei,
Candida antarctica, and Pseudomonas glumae, retain high levels
of activity even after drying over a molecular sieve. Other
lipases, such as those from Candida rugosa and Candida
deformans, seem to require higher levels of water for high levels
of activity (21, 22).
This mycelium-bound lipase was stable at 60 and 70 °C
(Figure 4). Increases in preincubation time (30 min to 2.5 h)
and preincubation temperature (30 to 60 or 70 °C) slightly
increased synthetic activity. Ester yields of 87.6, 96.5, and 93%,
respectively, were obtained after 2 h of reaction at 30 °C
depending on the preincubation temperature used.
Effect of Temperature on Lipase ActiVity. We carried out
dodecyl linoleate synthesis reactions to study the effect of
temperature on the mycelium-bound lipase activity of R. oryzae
(Figure 3). The optimal temperature for esterification was 60
°C, with over 20% transformation in 5 min, 60% transformation
In a similar study, Razak et al. (25) found that the optimal
temperature for activity of the membrane-bound lipase obtained
from a R. oryzae strain was 37 °C. At 50 °C, it retained more
than 90% of its initial activity after 30 min, but at 70 °C, only
10% of the initial activity was observed. Our mycelium-bound

3332 J. Agric. Food Chem., Vol. 51, No. 11, 2003
Torres et al.
catalyzed by the R. oryzae mycelium was optimal at pH 3.5-4
and pH 6-7. Resting cells of R. oryzae displayed marked heat
stability because activity began to decrease markedly only at
temperatures above 80 °C.
ACKNOWLEDGMENT
We thank Mr. Joan Pedrol for botanical advice.
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Endophytic Fungi Associated with Mediterranean Plants
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Received for review July 3, 2002. Revised manuscript received March
12, 2003. Accepted March 25, 2003. This research was supported by
the Spanish Government (INIA, IRTA01-075).
JF025786U