Induction of phenolic compounds in two cultivars of cucumber by treatment of healthy and powdery mildew-infected plants with extracts of Reynoutria sachalinensis

Article abstract of DOI:10.1023/A:1005578510954

Accumulation of phenolic compounds (p-coumaric, caffeic, and ferulic acids and p-coumaric acid methyl ester) was followed in susceptible (Mustang) and tolerant (Flamingo) cucumber (Cucumis sativus) cultivars. The objective was to determine whether these compounds played a role in resistance against powdery mildew following a prophylactic treatment with Milsana (leaf extracts from the giant knot weed Reynoutria sachalinensis, polygonaceae). This treatment significantly reduced the incidence of powdery mildew in both cultivars. Phenolic compounds were extracted from leaves. In the hydrolyzed fraction containing phenolic aglycones, levels of p-coumaric, caffeic, and ferulic acids and of p-coumaric acid methyl ester increased in all treatments (with leaf extracts of R. Sachalinensis, powdery mildew, or both) except the control, one or two days after treatment. In the fraction containing free phenolics, from the tested compounds, only ferulic acid showed an increase in cv. Flamingo (tolerant), and was particularly evident following treatments. On the other hand, the amounts of hydroxycinnamic acids increased rapidly in the two cultivars following Milsana treatment, suggesting their role in disease reduction. All compounds showed antifungal activity when tested against common pathogens of cucumber (Botrytis cinerea, Pythium ultimum, and P. aphanidermatum), but in general methyl esters were more fungitoxic than their corresponding free acids. This study suggests that cucumber is able to release antifungal compounds that are instrumental in repressing powdery mildew infection. This response is seemingly independent from the level of genetic resistance associated with each cultivar.

Full text of DOI:10.1023/A:1005578510954

Journal of Chemical Ecology, Vol. 26, No. 7, 2000
INDUCTION OF PHENOLIC COMPOUNDS IN
TWO CULTIVARS OF CUCUMBER BY TREATMENT
OF HEALTHY AND POWDERY MILDEW-INFECTED
PLANTS WITH EXTRACTS OF Reynoutria sachalinensis
1,
1
1
´
FOUAD DAAYF, * MARC ONGENA, RENALD BOULANGER,
2
1
´
ISMAIL EL HADRAMI, and RICHARD R. BELANGER
1 Laboratoire de Biocontro
Departement de phytologie, Universite
Quebec, Canada, G1K 7P4
2 Laboratoire de Physiologie et de Biotechnologie Ve
Faculte des sciences Semlalia, Universite Caddi Ayyad
Marrakech, Morocco
ˆ
le, Centre de Recherche en Horticulture
Laval
´
´
´
´ge´tales
´
´
(Received March 15, 1999; accepted February 24, 2000)
Abstract—Accumulation of phenolic compounds (p-coumaric, caffeic, and
ferulic acids and p-coumaric acid methyl ester) was followed in susceptible
(Mustang) and tolerant (Flamingo) cucumber (Cucumis sativus) cultivars.
The objective was to determine whether these compounds played a role in
resistance against powdery mildew following a prophylactic treatment with
Milsana (leaf extracts from the giant knot weed Reynoutria sachalinensis,
polygonaceae). This treatment significantly reduced the incidence of powdery
mildew in both cultivars. Phenolic compounds were extracted from leaves. In
the hydrolyzed fraction containing phenolic aglycones, levels of p-coumaric,
caffeic, and ferulic acids and of p-coumaric acid methyl ester increased in
all treatments (with leaf extracts of R. Sachalinensis, powdery mildew, or
both) except the control, one or two days after treatment. In the fraction
containing free phenolics, from the tested compounds, only ferulic acid
showed an increase in cv. Flamingo (tolerant), and was particularly evident
following treatments. On the other hand, the amounts of hydroxycinnamic
acids increased rapidly in the two cultivars following Milsana treatment,
suggesting their role in disease reduction. All compounds showed antifungal
activity when tetsed against common pathogens of cucumber (Botrytis cinerea,
Pythium ultimum, and P. aphanidermatum), but in general methyl esters were
more fungitoxic than their corresponding free acids. This study suggests that
*To whom correspondence should be addressed at: Agriculture Canada, 440, University Avenue, PO
Box 1210, Charlottetown, PEI, C1A 7M8, Canada. e-mail: daayff@em.agr.ca
1579
0098-0331 00 0700-1579$18.00 0  2000 Plenum Publishing Corporation
/
/
/

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DAAYF ET AL.
cucumber is able to release antifungal compounds that are instrumental in
repressing powdery mildew infection. This response is seemingly independent
from the level of genetic resistance associated with each cultivar.
Key Words—Cucumis sativus, phenolic, phytoalexin, Milsana, induced
resistance, aglycone.
INTRODUCTION
Plants challenged by pathogens may exhibit several biochemical defense re-
sponses (Goodman et al., 1986) such as enzyme synthesis (Stahmann and
Demorest, 1973), wall deposition of lignin and suberin (Hammerschmidt and
Kuc, 1982, Hammerschmidt et al., 1985, Borg-Olivier and Monties, 1993, Stein
et al., 1993), and accumulation of secondary metabolites (Benner, 1993; Bennett
and Wallsgrove, 1994). In cucumber, enzyme synthesis and lignification occur as
components of systemic acquired resistance (Siegrist et al., 1994). Until recently,
secondary metabolites acting as phytoalexins were generally considered to be
nonexistent in cucurbits. In 1995, we demonstrated that Milsana (Compo, Ger-
many), a commercial formulation of leaf extracts from Reynoutria sachalinen-
sis F. Schmidt stimulated the production of phenolic compounds in glycosylated
form in cucumber (Daayf et al., 1995, 1996). Their accumulation was associated
with resistance to powdery mildew, caused by Sphaerotheca fuliginea, which
indicated that they acted as phytoalexins when released in their active deglyco-
sylated form (Daayf et al., 1997b). Of the several compounds that were observed,
we characterized one as p-coumaric acid methyl ester (p-CAME) (Daayf et al.,
1997a), while most of the others appeared to be hydroxycinnamic derivatives
(Daayf et al., 1997b).
In an attempt to understand better the biological role of these antifungal
compounds in cucurbits, the objectives of this work were threefold: (1) to com-
pare the accumulation of p-CAME and of specific hydroxycinnamic acids in sus-
ceptible (cv. Mustang) and tolerant (cv. Flamingo) cultivars of cucumber infected
or not with S. fuliginea, (2) to assess the effect of Milsana on the variation of
the accumulation of these compounds, and (3) to evaluate the fungitoxicity of
these hydroxycinnamic acids and of some close derivatives on growth of three
fungal pathogens of cucumber. We also report the identities of some previously
unknown compounds.
METHODS AND MATERIALS
Plant Material and Mode of Infection. Seeds of cucumber (Cucumis sativus
L.) cultivars Mustang and Flamingo were sown in LC-1 Horticubes, fertilized
daily with a nutrient solution of N-P-K (7-11-27), and 3-week-old plants trans-
ferred to individual pots containing peat and vermiculite. Thirty-two plants were

CUCUMBER PHENOLICS
1581
placed in a growth chamber and divided equally into four groups separated by
plastic curtains. Plants from two groups were inoculated artificially by shaking
off conidia from powdery mildew-infected leaves. Upon appearance of the first
signs of powdery mildew, Milsana (M+) and water (M ) were applied each to
eight infected (B+) and eight healthy (B ) plants, which resulted in four different
treatments: B M , B M+, B+M , and B+M+. The Milsana treatment comprised
an application of leaf extracts from Reynoutria sachalinensis with the recom-
mended dosage of concentrated extracts diluted in water (2%).
Estimation of Disease Severity. The severity of powdery mildew was esti-
mated one, two, and seven days after treatment by calculating the percent
infected leaf area for all leaves on each plant and the mean for each treatment
standard deviation (P < 0.05). In addition, means were compared by anal-
ysis of variance (ANOVA), at P 0.05, with the software Genstat 5 (IACR,
Rothamsted, UK).
Extraction and Fractionation of Phenolic Compounds. Leaves from four
plant replicates were used for extraction from each of the four tested treatments
(B M , B M+, B+M , and B+M+) one, two, and seven days after treatment. The
method of extraction was adopted as described by Daayf et al. (1997b), allow-
ing for the determination of free and glycosidic-linked phenolics. Chlorophylls,
carotenoids, waxes, and lipids were contained in fraction I. Free phenolics were
contained in fraction II, and aglycones obtained from glycosidically bound phe-
nolics were in fraction III.
HPLC Apparatus and Determination of Compounds. Compounds were sep-
arated by HPLC. The chromatograph was equipped with a photodiode array
detector, an autosampler (Waters model 717), and fitted with a C18 reverse-phase
column (Waters model RCM (8× 100 mm). Results were analyzed with the pro-
gram Millenium Software 2.1. The column was eluted with a gradient of 0–100%
acetonitrile as follows: [time (minutes) acetonitrile (percent) flow (milliliters
/
/
per minute)] [0 0 1, 10 0 0.9, 20 15 0.9, 40 15 0.9, 50 25 0.9, 60 25 0.65,
/ / / /
/
/
/
/
/
/
/
/
70 40 0.5, 80 50 0.5, 85 70 0.5, 90 80 0.9, 95 80 1, 100 0 1]. Two column
/
/
/
/
/
/
/
/
/
/
/ /
cartridges were used to insure optimum separation of molecules. p-Coumaric
(p-CA), m-coumaric (m-CA), o-coumaric (o-CA), ferulic (FA), sinapic (SA),
and caffeic (CA) acids were identified based on their retention times, their
absorbance spectra (220–400 nm), and cochromatography with commercial stan-
dards (Sigma Chemicals, St-Louis, Missouri). Standard and extracted com-
pounds had the following retention times (minutes): p-CA: 51, m-CA: 63, o-
CA: 73, CA: 38, FA: 59, SA: 58, p-CAME: 92, m-CAME: 89, and o-CAME:
94. Concentration of each compound was estimated on the basis of five-point
calibration curves established with corresponding standards and averaged from
three separate HPLC runs. Concentration means were compared by analysis of
variance with the software Genstat 5 (IACR, Rothamsted, UK).
Esterification. Methyl esters of p-CA (p-CAME), m-CA (m-CAME), and

1582
DAAYF ET AL.
TABLE 1. POWDERY MILDEW INFECTION OF CUCUMBER CULTIVARS MUSTANG
a
(SUSCEPTIBLE) AND FLAMINGO (TOLERANT) IN RESPONSE TO TREATMENT WITH MILSANA
Percent infection
M B+ M+B
Days after
treatment
M B
M+B+
Flamingo
1
2
7
0
0
2
0 (a)
0 (a)
1 (ab)
5
5
1 (b)
2 (b)
0
0
2
0 (a)
0 (a)
1 (a)
5
5
9
1 (b)
1 (b)
3 (b)
30 3 (c)
Mustang
1
2
7
0
1
2
0 (a)
2 (a)
2 (a)
5
7
3 (b)
2 (b)
0
0 (a)
5
6
2 (b)
2 (ab)
0.5 2 (a)
2.5 3 (ab)
43 2 (d)
11 2 (b)
+
a
Control consisted of water application (B M ), B inoculation with powdery mildew pathogen,
M+, treatment with Milsana extract. Each value represents the mean infection rate of eight plants
(%) SD. Data were submitted to analysis of variance. Values followed by the same letter do not
differ significantly at P ≤ 0.05 (LSD: 4.4).
o-CA (o-CAME) were synthesized as follows: in a condenser-equipped flask
containing 20 ml of methanol, we added 1 g of p-CA, m-CA, or o-CA (Sigma)
and 10 ml (0.12 mol) of BF3–MeOH complex (Aldrich). The mixture was heated
60 min at 60 C. After cooling to room temperature, the methanol was removed
under vacuum. The residue was diluted with water and extracted with ethyl
acetate (3 × 20 ml). The organic fractions were combined, washed with water,
dried over anhydrous magnesium sulfate, filtered, and evaporated under vacuum.
The residue was purified by recrystallization or flash chromatography. Analysis
of the synthesized molecules gave the following spectral data: EI-MS (70 eV),
m z (relative intensity): p-CAME 178 M+(65), 179 M+1(8), 147 M+-CH3O(100),
/
119 M+-C2H3O2(28), 91 M+-C4H7O2); m-CAME 178 M+(19), 179 M++1(2),
146 M+-CH3OH(100), 118 M+-C2H4O2(99), 91 M+-C4H7O2(42); o-CAME 178
M+(78), 179 M++1(10), 146 M+-CH3OH(100), 119 M+-C2H3O2(35), 91 M+-
C4H7O2(40).
The identity of these molecules was confirmed on the basis of data stored
in the GC-MS compounds bank.
Effects of Compounds on Fungal Growth. Authentic samples of p-CA, m-
CA, o-CA, CA, FA, SA, p-CAME, m-CAME, and o-CAME were tested for their
fungitoxicity against Pythium ultimum, P. aphanidermatum, and Botrytis cinerea
as root and foliar pathogens. Briefly, for each replicate, a mycelial disk (3 mm)
was placed in the center of a Petri dish and three aliquots of each compound
were placed 4 cm away from the fungal disk. Five concentrations (0, 1, 3, 5, or
10 mg ml) were tested. Growth of mycelium was then observed daily or hourly,
/
depending on the speed of fungal growth. Reported results were obtained for

FIG. 1. Concentrations of p-coumaric acid over time in its aglycone form (FIII) from cucumber leaves obtained from healthy
ꢀ
ꢀ
ꢀ
ꢁ
ꢁ
ꢀ
ꢁ ꢁ
control (B
M ), healthy Milsana-treated (B M ), powdery mildew-infected (B M ), and Milsana-treated (B M )
plants of two cultivars: Mustang (susceptible) and Flamingo (tolerant). Each value is the mean ꢂ SD of three separate HPLC
runs. FM: fresh weight. LSD: least significant difference for days ꢃ treatments based on ANOVA at P = 0.05. F-II- and
F-III- represent results for free phenolics and for aglycone phenolics obtained after hydrolysis, respectively.

FIG. 2. Concentrations of caffeic acid over time in its free (FII) and aglycone (FIII) forms, from cucumber leaves obtained from
ꢀ
ꢀ
ꢀ
ꢁ
ꢁ ꢀ
healthy control (B
M ), healthy Milsana-treated (B M ), powdery mildew-infected (B M ), and Milsana-treated
ꢁ
ꢁ
(B M ) plants of two cultivars: Mustang (susceptible) and Flamingo (tolerant). Each value is the mean ꢂ SD of three separate
HPLC runs. FM: fresh weight. LSD: least significant difference for days ꢃ treatments based on ANOVA at P = 0.05. F-II- and
F-III- represent results for free phenolics and for aglycone phenolics obtained after hydrolysis, respectively.

1586
DAAYF ET AL.
each fungus when the control culture (0 mg) reached confluence on the Petri
dish. This experiment was replicated three times and means were compared by
analysis of variance with the software Genstat 5 (IACR, Rothamsted, UK).
RESULTS AND DISCUSSION
Pathogenicity Test. Application of Milsana resulted in a reduction of pow-
dery mildew incidence in the tested cultivars, relative to control, seven days after
the treatment (Table 1). Untreated plants of cv. Flamingo (tolerant) showed a
lower rate of infection than those of cv. Mustang (susceptible), but these dif-
ferences subsided in treated plants (B+M+). These results confirm the prophy-
lactic properties of Milsana as previously reported (Daayf et al., 1995, Dik and
VanDerStraay, 1995). When cucumber leaves were analyzed for their pheno-
lic content, high variations in the concentrations of p-CAME and of some hy-
droxycinnamic acids were shown in cucumber leaves. These concentrations were
affected by infection with S. fuliginea, by the Milsana treatment, and by the cul-
tivar tested.
Changes in Phenolic Compounds. In the fraction containing free phenolics
(fraction II), p-CA was present in traces only. In the hydrolyzed fraction (fraction
III), p-CA aglycone concentration increased the first day and mostly the second
day after treatment in both cultivars (Figure 1). These increases were higher in
B+M+, with higher accumulation in the susceptible cv. Mustang. On day 7 after
treatment, these concentrations dropped significantly.
Concentration of caffeic acid (CA) in fraction II showed no difference
among treatments in cv. Flamingo, while an increase was recorded the second
day in cv. Mustang in all treatments but the control (Figure 2). In fraction III,
the increase in CA levels induced by powdery mildew infection and or Milsana
/
application occurred on day 2 after treatment in the two cultivars (Figure 2) with
a higher level in cv. Mustang. As with p-CA, the highest level was found in the
treatment B+M+.
FA and SA showed very similar and retention times and absorbance spectra;
as a result they were eluted as two distinct peaks only when present in very low
quantities. However, they could be distinguished on the basis of their absorbance
spectra. They each have maximum absorbance at 325.1 nm, but only FA has
a shoulder at 295 nm. The concentration of FA estimated in fraction III was
shown to increase on day 2 in all treatments except the control (Figure 3). This
increase was particularly important in the tolerant cv. Flamingo (Figure 3), which
may explain in part the difference in powdery mildew resistance between the
two cultivars. Hydroxycinnamic acids are known to have antimicrobial activ-
ity (Baranowski and Nagel, 1982), and some of them, including FA and SA,
are components of the lignification process, which has been shown to occur as a

FIG. 3. Concentrations of ferulic acid over time in its aglycone form (FIII) from cucumber leaves obtained from healthy
ꢀ
ꢀ
ꢀ
ꢁ
ꢁ
ꢀ
ꢁ ꢁ
control (B M ), healthy Milsana-treated (B M ), powdery mildew-infected (B M ), and Milsana-treated (B M )
plants of two cultivars: Mustang (susceptible) and Flamingo (tolerant). Each value is the mean ꢂ SD of three separate
HPLC runs. FM: fresh weight. LSD: least significant difference for days ꢃ treatments based on ANOVA at P = 0.05.
F-II- and F-III- represent results for free phenolics and for aglycone phenolics obtained after hydrolysis, respectively.

FIG. 4. Concentrations of p-coumaric acid methyl ester (p-CAME) over time in its free (FII) and aglycone (FIII) forms,
ꢀ
ꢀ
ꢀ
ꢁ
ꢁ ꢀ
obtained from healthy control (B
M ), healthy Milsana-treated (B M ), powdery mildew-infected (B M ), and
ꢁ
ꢁ
Milsana-treated (B M ) cucumber leaves of two cultivars: Mustang (susceptible) and Flamingo (tolerant). Each value is
the mean ꢂ SD of three separate HPLC runs. FM: fresh weight. LSD: least significant difference for days ꢃ treatments
based on ANOVA at P = 0.05. F-II- and F-III- represent results for free phenolics and for aglycone phenolics obtained
after hydrolysis, respectively.

1590
DAAYF ET AL.
cucumber defense reaction in response to microbial attack (Hammerschmidt and
Kuc, 1985; Stein, 1993). However, in our study, the increase of FA in the tolerant
cultivar was associated only with treatment with Milsana (Figure 3).
Concentrations of p-CAME, previously described as a phytoalexin-like
compound in cv. Mustang (Daayf et al., 1997a, 1997b), also were evaluated
along with those of the isomers m-CAME and o-CAME. The latter two were not
detected in the tested cultivars in either fraction. One and two days after treat-
ment, no significant variation could be recorded in concentration of p-CAME
in fraction II (Figure 4). On the other hand, analysis of these fractions seven
days after treatment showed a marked increase in this compound within infected
leaves of the two cultivars (Figure 4). The highest levels were reached when
plants had both treatments (inoculated and treated with Milsana) (B+M+). In
fraction III, an increase in p-CAME level occurred one day after treatment in
cv. Mustang B+M and B+M+ and in cv. Flamingo B+M+. Accumulation of
p-CAME in response to infection and treatment with Milsana was more marked
the second day after treatment and was higher in cv. Mustang (Figure 4).
Antifungal Activity of Phenolic Compounds. When tested for their antifun-
gal activity, p-CA, m-CA, and o-CA were more fungitoxic on B. cinerea, a foliar
and stem pathogen, than on P. ultimum and P. aphanidermatum, which are root
rot agents. FA and SA had the opposite effect (Figure 5), and CA was found to
be the least fungitoxic of the tested compounds. Methyl esters generally showed
higher inhibitory activities than their corresponding acids (Figure 5), which con-
firm data previously reported for other microorganisms (Baranowski and Nagel,
1982, 1984). In addition, alkyl esters inhibit fungal (Macko et al., 1972) as well
as bacterial growth (Baranowski and Nagel, 1982, 1983) and also have antioxi-
dant activities in food products (Baranowski and Nagel, 1984). p-CAME was
previously reported in the rhizome of Costus speciosus as a constitutive princi-
ple with antifungal activity against Cladosporium cucumerinum, Colletotricum,
Curvularia sp., and Penicillium sp. (Bandara et al., 1988). However, methyl
esters are rarely cited as components of plant tissues (Bandara et al., 1988, Daayf
et al., 1997b). This pattern of accumulation is in line with the results obtained
previously with cv. Mustang (Daayf et al., 1997b).
The accumulation of compounds with antifungal activity suggests the non-
FIG. 5. (Opposite) Percent in vitro inhibition of radial growth of three fungal pathogens
of cucumber by different phenolic compounds. A, B, C, and D represent results at four
concentrations (1, 3, 5, and 10 mg ml). Percent corresponds to reduction of fungal radial
/
growth as compared to the control (medium with no phenolics added). p-CA, m-CA,
and o-CA: p-, m-, and o-coumaric acids; CA, FA, and SA: caffeic, ferulic, and sinapic
acids; and p-CAME, m-CAME, and o-CAME: p-, m-, and o-coumaric acid methyl esters,
respectively. LSD: least significant difference based on ANOVA at P 0.05.

CUCUMBER PHENOLICS
1591

1592
DAAYF ET AL.
specific role that cucumber antifungal compounds may play in the defense pro-
cess against pathogens. Indeed, in addition to their possible protective role
against powdery mildew (Daayf et al., 1995, 1996, 1997a,b), it appears that these
compounds can have activity against both foliar (B. cinerea) and root (P. ulti-
mum and P. aphanidermatum) pathogens. Considering that only foliar extracts
were tested here, it is nonetheless relevant to compare these results with those of
Cherif et al. (1994), who demonstrated accumulation of antifungal compounds
´
in crude extracts of cucumber roots in response to a Pythium attack.
The cultivars we tested could not be distinguished in this study on the basis
of differential accumulation of phenolics that may be related to their differences
in susceptibility to powdery mildew. Both cultivars reacted to the infection and
to the Milsana application by increasing concentrations of their antifungal com-
pounds. This suggests that tolerance of cv. Flamingo is predetermined also by
other factors, such as cuticle thickness and lignification, which would be in line
with FA production.
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