Abstract: The efficacy of Trilogy, a natural product from neem (Azadirachta indica Fuss.), against cucumber (Cucumis sativus L.) powdery mildew (Podosphaera xanthii) was studied in detached leaf and intact plant experiments. Trilogy significantly retarded several growth parameters of the pathogen, viz. multiple germ tube formation, number of germ tubes, haustoria and colony size on cucumber leaves. Trilogy induced hypersensitive reaction (HR), as evidence by browning of host cell associated with appressoria. Furthermore, increase in protein concentration of intercellular fluid followed treatment with Trilogy. The elicited leaves exhibited significantly high activity of enzymes: Phenylalanine Ammonia Lyase (PAL) and Tyrosine Ammonia Lyase (TAL) along with rapid and distinct accumulation of fungitoxic phenolic compounds (phytoalexin). Based on analysis of foliar tissues, at least five separate phenolic compounds were identified as intrinsic components of cucumber plants. The pathogen P. xanthii alone induced the production of other seven identifiable phenolic compounds within the leaves of the cucumber plants. These compounds, displayed a significant increase in concentration as a result of elicitation with Trilogy when the plant was stressed by the pathogen especially cucumerin A and B. The combined amount of these antifungal compounds in treated plants was nearly five times the level found in control plants. Phytoalexin production was triggered by the combination of an eliciting/inoculation treatment. These results provide direct evidence that Trilogy induced resistance in cucumber associated with increased extractable enzymatic activity. Accumulation of flavonoid compounds response to an eliciting treatment after infection creates incompatible interactions with powdery mildew.
INTRODUCTION
Cucumber (Cucumis sativus L.) and powdery mildew (Podosphaera xanthii, syn Sphaerotheca fuliginea Castagne; ([ Schlecht.] Fr.) Poll. is a highly valuable pathosystem providing insight into the interactions between plants and biotrophic phatogens (McNally et al., 2003). S. fuliginea has been subsequently renamed to Podosphaera xanthii (Castagne), the organism used in this work (Braun and Takamora, 2000)
Several effective synthetic fungicides are available to control this disease (Das, 1987). However, due to increasing awareness of the illeffects of synthetic pesticides on human and animal health and also to the agroecosystem, research efforts on alternative and more environmentally friendly methods of controlling pests and diseases have proliferated (Lyon et al., 1995). Beside biocontrol agents, the use of plant products in plant disease control seems to be a logical approach (Prithiviraj and Singh, 1995).
Many workers have screened extracts/products from higher plants for antifungal activity (Singh et al., 1990). For instance, leaf extract of Reynoutria sachalinensis induced resistance in long English cucumber against Sphaerotheca fuliginea through biochemical changes in the host plant (Daayf et al., 1995) and leaf extract of Azadirchata indica induced resistance in barely against leaf stripe pathogen caused by Drechslera graminea (Paul and Sharma, 2002).
During the past 40 years, several research groups have studied the defense reactions of cucumber plants attempting to unveil the underlying mechanisms to induced resistance (McNally et al., 2003; Remoero et al., 2004).
Biochemical and molecular-based techniques have revealed putative roles for pathogenesis-related and hypersensitive response proteins such as chitinase (Avdiushko et al., 1993), α and β-glucosidase (Avdiushko et al., 1993), lipoxygenase (Avdiushko et al., 1993), peroxidase (Avdiushko et al., 1993; Chen et al., 2000) and polyphenoloxidase (Avdiushko et al., 1993; Chen et al., 2000) in cucumber.
Biochemical studies examining disease-resistant plants have revealed the increased activities for key biosynthetic enzymes involved in the production of phenolic compounds including: phenyalanine ammonia lyase (Chen et al., 2000), More recently, phytochemical investigations of powdery mildew-resistant cucumber plants have led to identification of C-glycosyl flavonoids acting as phytoalexins, including the discovery of cucumerin A and B, two novel and structurally complex C-glycosyl flavonoids (McNally et al., 2003).
Three criteria were advanced for establishing the role of phytoalexins in disease resistance: (1) Phytoalexins must be associated with restriction of pathogen development and be conditioned by host-resistance genes; (2) Phytoalexins must accumulate to antimicrobial levels at infection site in resistant plants at the time pathogen development is stopped and (3) there must be evidence that phytoalexins are directly involved in defense and that this defensive role has a measurable benefit for the plant (Hammerschmidt, 1999).
In addition to modernizing phytoalexin theory by including data generated by more recent molecular-based techniques, these criteria introduced the concept of proximity of phytoalexins to the invading pathogen in planta as necessary evidence of their intervention within plant-pathogen interaction. Such reactions have been reported to occur as components of systemic acquired resistance in cucumber (Siegrist et al., 1994). As mentioned by Kowalewski (1993), Phenylalanine Ammonia Lyase (PAL) is the key enzyme of the phenylpropanoid pathway which converts phenylalanine to cinnamic acid, which is, in turn, modified by other enzymes resulting in highly antifungal phenolic including phytoalexins (PAL activity is induced by several factors such as pathogen ingress, insect damage and stress).
This study was carried out to explore the potential of Trilogy, for its effect against cucumber powdery mildew. However, in vitro experiments, carried out herein, on the development of P. xanthii on cucumber leaves and its control by Trilogy indicate that the latter induces resistance in cucmber against powdery mildew and stimulates the production of fungitoxic phenolic compounds in cucumber. The bulk of the fungitoxic activity was found in phenolics in their aglycone form. The presence of these aglycones appeared to correlate with the prophylactic properties of the products. Accordingly, the present work was expanded to determine whether these compounds were phytoalexins and could play a role in the resistance induced by Trilogy in cucumber.
MATERIALS AND METHODS
Plants
Seeds of cucumber (Cucumis sativus L., c.v. Corona) were sown in
plastic pots (9 cm diameter) containing unsterilized garden soil and maintained
in a growth chamber (12 h of light/12 h of darkness, 24°C day/20°C night).
Source of Inoculum
Inoculum for the trails was obtained from three to four week old cucumber
plants artificially inoculated with Podosphaera xanthii (Syn. Sphaerotheca
fuliginea, Castagne, ([ Schlecht.] Fr.) Poll. grown in a green chamber at
20-24°C in a 12 h photo period. Inoculum from fresh pustules was collected
by removing the conidia with a brush and water. Conidial suspensions were adjusted
to 3000 conidia mL-1 and sprayed immediately mainly on the upper
leaf surface of the tested plants with hand sprayer.
Trilogy
Trilogy was provided by CERTIS USA, LIC. (CERTIS Crop, Protection). It,
a commercial product, contains 70% hydrophobic extract of Neem oil and the remaining
30% is other inert ingredients, including 2% by weight total limonoids, which
includes azadiractin and 6 or 7 additional compounds.
Assessment of Powdery Mildew
The prepared spore suspension was sprayed on the leaves of 20 days old cucumber
plants and the development of powdery mildew was assessed at 1 week after treatment.
Disease severity was estimated by calculating percentage of infected leaf area
for all leaves on each plant and the mean for each treatment. A visual estimate
for % cover of the whole plant was recorded. Careful attention was given to
maintaining consistency throughout the work. This method was used by Paloukidou
(2005). Plants were treated with 4 Trilogy concentrations, i.e., 0.5, 1, 1.5
and 2%, one day before inoculation (protective doses) and six days after inoculation
(curative doses). Four replicates were used for each treatment giving 16 plants
in total and 4 plants for control sprayed with sterile distilled water.
Effect of Trilogy on Conidium Germination of P. xanthii on Cucumber
Leaves
Cucumber seeds were sown in plastic pots (9 cm diameter) containing unsterilized
garden soil maintained a growth chamber. Twenty-day old plants were used for
the experiment. Second nodal leaves from the bottom were excised with sharp
scissors. The leaves were then arranged on filter paper towels, with the upper
surfaces facing up and inoculated with conidia of P. xanthii by tapping
heavily infested leaves over them. The leaves were then transferred to Petri
dishes containing Trilogy solution (0.5, 1, 1.5 or 2%) prepared in 2% sucrose
in distilled water (Yarwood, 1964). The control treatment contained 2% aqueous
sucrose solution only. Nine replicates for each treatment giving 36 leaves in
total, each leaf put separately in a Petri dish. The Petri dishes with the leaves
were incubated in growth chamber at 20°C with a 12/12 h photoperiod. The
incubation started at the onset of dark cycle. Three leaves representing each
treatment were removed at 24 h intervals for three days and fixed immediately
in ethyl alcohol acetic acid (3: 1). The fixation was done as described by Carver
and Adaigbe (1990). Observations on germination and appressorium formation were
made 24 h after inoculation on 100 conidia on each replicate leaf. Observations
on percentage of conidia with multiple germ tubes were indicated. The establishment
of a functional primary haustorium, were made 24 and 48 h post treatment, on
at least 100 conidia per replicate. Hypersensitive host reactions were observed
in the cells just below well developed appressoria which had failed to produce
further germ tubes, 72 h after inoculation. At least 100 observations were made
per treatment and the percentage of the attacked host cells showing a hypersensitive
response was calculated. For calculation of the frequency and distribution of
germlings with multiple germ tubes, 250 germinated conidia were measured.
Estimation of Protein Concentration in Intercellular Fluid
Cucumber leaves were floated on aqueous Trilogy (1, 1.5 and 2%) and distilled
water (control) in 9 cm glass Petri dishes with adaxial surface in contact
with the extract or distilled water. The leaves were incubated for 24, 48, 72
an 96 h; five replicates were taken for each treatment and control. After each
incubation period, leaves were removed and blotted dry. Approximately 1 g of
these leaves was used for collecting the intercellular fluid using the method
described by DeWit et al. (1986) and protein concentration in the collected
fluid was estimated by Bradfords (1976) method.
Treatment of Plants for Estimation of Enzymes
Twenty-day old cucumber plants were sprayed to run off with different concentrations
of Trilogy, i.e., 1, 1.5 and 2%. The plants were then placed in a growth chamber
at 20-24°C in 12 h photoperiod. Leaves from control and treatment plants
were sampled after 24, 48 and 72 and 96 h of treatment for estimation the activity
of phenylalanine ammonia lyase (PAL) and Tyrosine Ammonia Lyase (TAL) enzymes
as well as total phenol content. PAL and TAL activity was assayed by the method
described by Havir and Hanson (1970).
Estimation of Total Phenol Contents
Total phenols in the ethanol extract were estimated by the method described
by Bray and Thrope (1954).
Statistical Analysis
The effect of treatments on intercellular protein contents, PAL and TAL
activity and total phenol contents of cucumber leaves were analyzed by analysis
of variance (ANOVA).
Elicitation of Induced Resistance
To determine the purpose of this experiment, 3 treatments were created:
plants elicited with Trilogy (1%) and inoculated with P. xanthii (E+
I+), non elicited inoculated plants (E- I+)
and control plants, i.e., non inoculated and non-elicited (E- I-).
Treatments, E- I+ and E+ I+ were
inoculated with P. xanthii after the emergence of the fourth real leaf.
Inoculation was achieved by shaking the leaves of heavy diseased cucumber plants,
kept in the nursery, over E+ I+ and E- I+
plants. When the infection covered 2% of the leaf surface (10 days after inoculation),
the elicited treatment (E+ I+) plants were sprayed with
Trilogy (1% V/V sprayed until run-off) once a week all over the duration of
the experiment (3 weeks). Non-elicited treatments, E- I+
and E- I-, received an equal volume of water applied at
the same frequency.
Extraction and Identification of Phytoalexins
Approximately 100 g (fresh wt.) of leaf tissue were harvested from the areas
of leaves harboring powdery mildew colonies for E+ I+
and E- I+ plants using number 13 cork borer. The modified
extraction method of Dercks and Buchenauer (1986) was adopted, as described
by Daayf et al. (1995), allowing for the determination of free and glycoside-linked
phenolics. The frozen dried foliar material was homogenized with 80% methanol
at 10 mL g-1, protected from oxidation by replacing oxygen with nitrogen
and eliminating light and extracted for 48 h on rotatory shaker. After extraction,
the methanolic homogenate was filtered and the residue was washed with 20 mL
of 80% methanol. According to McNally et al. (2002), cholorphyll, carotenoids,
lipids and waxes were removed by partitioning against light petroleum ether
three times (Fraction, 1). The methanolic fraction containing the phenolic constituents
was evaporated at 38°C and the aqueous residue was partitioned three times
with 30 mL of anhydrous ethyl ether. Free phenolic compounds were found in the
ether fraction (Fraction, 11). The aqueous function was diluted with an equal
volume of 4NHCl and acid hydrolysis was performed for 90 min in a water bath
at 100°C. After cooling, the hydrolysate was partitioned against anhydrous
ethyl ether (FIII) to recover aglycones and the aglycone-containing ether fraction
was evaporated, resuspended in methanol and finely subjected to HPLC analysis.
At least 15 HPLC runs from different samples were carried out to properly assess
the presence of compounds. All compounds were visualized with photodiode array
(PDA) detector (Water Co. Ltd., Montréal, Quc Canada) at 272 nm wavelength
known to discriminate flavonoid compound in cucumber (Fawe et al., 1998).
RESULTS
Effect of Trilogy on the Incidence of Powdery Mildew
Plants were treated with Trilogy one day before inoculation or six days
after inoculation (Table 1). In both cases the pustule number
on all treated plants was significantly reduced as compared with the untreated
check. The protective treatment was generally more effective than the curative
treatment. Even at the lowest Trilogy concentration (0.5%), the pustule number
was reduced by more than 83.5% following pre-inoculation treatment, while the
efficacy was 56.2% after post-inoculation treatment.
At the highest concentration of Trilogy, i.e., 2% pre-inoculation treatment showed 94.7% efficacy, while after the protective treatment, it recorded 85.9%.
After protective treatments (one day before) the pustules were 7.0-7.5 mm in diameter which was comparable with the untreated check (15-20 mm), the shape of the pustules was circular. After curative treatments (6 days after) the pustule size was 2-3 mm, some pustules were not circular. Trilogy elicited plants inoculated with P. xanthii (E+ I+) maintained a low level of infection that never exceeded 5% for the duration of the experiment (3 weeks). By contrast, the level of infection for non elicited inoculated plants (E- I+) progressed rapidly and covered approximately 80% of the leaf surfaces.
Effect of Trilogy on Conidial Germination of P. xanthii on Cucumber
Leaves
Trilogy was not able statistically to affect the conidial germination or
appressorium formation of P. xanthii on leaves fixed 24 h after treatment
and inoculation (Table 2). However, a significant reduction
in the number of germlings producing multiple germ tubes was observed at 24
and 48 h post treatment. In the control (2% sucrose solution), 87.9% of conidia
formed multiple germ tubes 48 h post inoculation, while only 3.7% did so in
the treatment with 2% of Trilogy (Table 3).
There was a significant (p<0.05) effect of Trilogy on colony growth-number of germ tubes and number of haustoria per colony (Table 4). A maximum colony length of 600 μm was recorded in the control, while colony length was only 120 μm following treatment with 1.5% of Trilogy. The effect of Trilogy in reducing the number of haustoria appeared to be dose dependent.
Trilogy greatly affected the host cell response to P. xanthii ingress,
as indicated by the hypersensitive browning of cell associated with cytoplasmic
granulation below appressoria and failure of the fungus to produce multiple
germ tubes. The maximum response was observed at 1%, in which more than 3 times
as many hypersensitive cells were found compared to the control (Fig.
1). Trilogy also, affected the development of P. xanthii, as indicated
by the number of germ tubes compared with the control. Most conidia had four
germ tubes in control, while in all the treatments conidia with a single germ
tube predominated, although a few had multiple germ tubes. The frequency of
multiple germ tubes was decreased with increasing Trilogy concentration up to
2% (Table 4).
| Table 1: | Effect of pre- and post-inoculation treatments with Trilogy against P. xanthii on cucumber at one week after inoculation |
| *Same letter(s) means no significant difference in the students t-test (p = 0.05) | |
| Table 2: | Effect of Trilogy on conidial germination and appressorium formation by P. xanthii on cucumber leaves 24 h after treatment and inoculation |
| *Differences in percentage germination and appressorium formation were not statistically significant (p>0.05) by student t-test | |
| Table 3: | Effect of Trilogy on multiple germ tube formation by P. xanthii conidia 24 and 48 h after treatment and inoculation |
| In columns, values with the same superscript letter do not differ significantly p<0.05 by student's t-test | |
| Table 4: | Effect of Trilogy on germling development of P. xanthii on cucumber leaves 72 h after treatment |
| *Based on at least 100 independent observations (± standard deviation) | |
| Fig. 1: | Percentage of attacked cucumber epidermal cells showing hypersensitive browning in cucumber leaves treated with different concentrations of Trilogy and fixed 72 h after inoculation. Bars capped with the same letter do not differ significantly (p<0.05) |
Intercellular Protein Concentration of Leaves
Compared with the control, the protein concentration in the intercellular
fluid of leaves treated with Trilogy was higher (Fig. 2).
Intercellular fluids from 1.5% treated leaves had maximum protein concentration
(p<0.01) followed by concentrations, estimated in leaves treated with 1 and
2% of Trilogy (p<0.05). There was an increase in intercellular fluid protein
content with longer incubation periods in Trilogy treatment, while intercellular
fluid from leaves floated on water did not increase with prolonged incubation
(Fig. 2).
| Fig. 2: | Effect of Trilogy treatment on protein content of intercellular fluids. Control (♦) ; 1% (■) ; 1.5% (x) and 2% (▲) Triology |
| Fig. 3: | Effect of Trilogy treatment on phenylalanine ammonia-lyase activity in cucumber leaves. The vertical bars represent standard diviation, Control (♦) ; 1% (■) ; 1.5% (x) and 2% (▲) of Triology |
| Fig. 4: | Effect of Trilogy treatment on TAL activity in cucumber leaves.
The vertical bars represent standard diviation, Control ( |
Phenylalanine Ammonia Lyase and Tyrosine Ammonia Lyase Activity
The activity of both, PAL and TAL was higher in Trilogy- treated cucumber
leaves (Fig. 3 and 4). However, maximum
activity of both the enzymes was recorded in the leaves treated with 1.5% followed
by 1 and 2% treatments (p<0.05).
| Fig. 5: | Effect of Trilogy treatment on total phenol contents of cucumber leaves. Control (♦), 1% (■), 1.5% (x) and 2% (▲) of Triology |
PAL and TAL activities were increased in all Trilogy-treated leaves with either peaking at 48 h (1.5%) or at 72 h (1 and 2%) (Fig. 3 and 4).
Total Phenol Content
It is evident from Fig. 5 that 1 and 2% Trilogy had no
significant effect on the total phenol content of cucumber leaves p>0.05.
However, 1.5% dilution of Trilogy could significantly increased phenols of cucumber
leaves (p>0.01). Maximum phenol content in cucumber leaves treated with 1.5%
of Trilogy was recorded after 48 h of treatment.
Identification of Phytoalexins
HPLC analysis of extracts prepared from carefully selected leaf tissues
of control (E- I-), inoculated plants (E- I+)
and elicited inoculated plants (E+ I+) harvested 48 h
post the first eliciting Trilogy application revealed important differences
among the treatments for the hydrolyzed extract (Fig. 6).
For inoculated plants, E- I+, the presence of the pathogenic fungus P. xanthii alone induced the production of at least 12 identifiable compounds within the leaves of (E- I+) cucumber plants (listed in order of increasing elution time): Caffeic acid, isoorientin, orientin, p-coumaric acid, isovitexin, vitexin, sinapinic acid, ferulic acid, vitexin-6 (4-hydroxy-l-ethylbenzene) or cucumeric A, isovitexin 8 (4-hydroxy-l-ethylbenzene) or cucumeric B, p-came and trans cinnanic acid (Fig. 6B), compared with control (E- I-) which have 5 common identified phenolic compounds;p-coumaric acid, isovitexin, vitexin, sinapinic acid and p-came (Fig. 6A).
These same compounds were detected within the hydrolyzed leaf extracts of Trilogy-elicited
inoculated plants (E+ I+), albeit in much greater quantities
with the exception of caffeic and ferulic acid (Fig. 6C).
For instance, p-coumaric acid, p-came and especially the C glycosyl flavonoid
compounds isoorientin, orientin, isovitexin, vitexin, cucumerin A and B were
systematically more concentrated within the leaves of elicited inoculated plants
(E+ I+) compared with non elicited inoculated plants (E-
I+) (Fig. 6B and C). Of particular interest, the
C glycosylflavones cucumerin A and B (compounds 9, 10) were dominant within
the leaves of E+ I+ plants compared with E-
I+ plants (Fig. 6B and C). Based
on area under-curve calculations, there was approximately 30 times more cucumerin
A and B within leaves of E+ I+ plants compared to E-
I+ plants.
| Fig. 6: | HPLC analysis (272 nm) of hydrolyzed cucumber leaf extracts prepared from, A- control plants (E- I-), B- inoculated plants (E- I+) and C- elicited/inoculated plants (E+ I+) harvested 48 h after the first eliciting Trilogy® treatment. Identified compounds are numbered in order increasing elution time of: (1) caffeic acid, (2) isorientin, (3) orientin p-coumaric acid, (5) isovitexin, (6) vitexin,(7) sinapinic acid,(8) ferulir acid, (9) cucumerin A, (10) cucumerin B, (11) p-came, (12) trnas cinnamic acid |
DISCUSSION
Pre-inoculation treatments of cucumber with formulated Trilogy had a good efficiency against powdery mildew. At a concentration of 0.5% Trilogy, the pustule number was reduced by ~ 83.5%. At the highest applied concentration, i.e., 2% the efficiency of Trilogy ranged between 56.2-85.9%, dose dependent.
The effect of Trilogy on powdery mildew development on intact cucumber plants showed that the pre-inoculation treatment with Trilogy was significantly more effective than post-inoculation application.
To explain these differences, two modes of action can be proposed. In the pre-inoculation treatment, the oil component irrespective of Trilogy ingredients forms a barrier on the cucumber leaves. Most of the conidia on the leaf cannot develop, only few pustules are formed. The pustules produced are of normal size. This is in line with the observation by Häberle and Schlösser (1993), who used Telmion, formulated rape seed oil, against powdery mildew of cucumber. The oil component, irrespective of neem ingredient seems to be most important for a protection against powdery mildew in the very sensitive stage of conidia germination.
In the post-inoculation at the stage of the powdery mildew fungus has already established itself on the leaves, sometimes young developing pustules were seen. Compounds with fungicidal activity in Trilogy can stop further development of the mildew pustules. The pustules that still develop are smaller in size and the form is not circular. It can introduce systemic acquired resistance.
Rovesti et al. (1992) reduced the affected leaf surface with S. fuliginea, Erysiphe graminis f. sp. tritici and E. graminis f. sp. hordei to 6.8, 0 and 25%, respectively by a postinoculation treatment (four days after inoculation) with aqueous neem seed exract. Phytophthora infestans, Cercospora beticola and Septoria apiicola were not affected by a treatment one day before inoculation.
The curative effect of formulated neem products against powdery mildew of cucumber is most important for a practical use of these extracts in green houses. It seems to be possible to control developing powdery mildew epidemic at stage where first pustules become visible. The recommended concentration should be 1% Trilogy. In addition to its good protective effect, Triology is distinguished by a good curative effect against powdery mildew of cucumber even at rather low concentration.
Secondary growth parameters of the fungus, like the number of germ tubes emerging from a conidium was greatly affected by Trilogy, whereas primary germ tube formation and appressorium development appeared unaffected. The host tissues reacted hypersensitively in the form of browning of cell following treatment with this chemical. Tissue browning in rice plants following treatment with 2, 2-dichloro-3-dimethyl-cyclopropane carboxylic (DCP) in response to picolinic acid (The Pathotoxin of Pyricularia oryzae) has also been reported (Langcake and Wickins, 1975).
From the results, it is suggested that treatment of cucumber leaves with Trilogy induced an increase in the activity of PAL and TAL enzymes which led to an increased phenol biosynthesis rendering the leaves resistant to P. xanthii.
An increase in the concentration of PAL and TAL enzymes is reflected by enhanced protein levels in intercellular fluid of treated cucumber leaves. Increased activities of PAL and TAL, the key enzymes in phenol biosynthesis by neem products have also been demonstrated earlier by Singh and Prithiviraj (1997). These defensed enzymes were selected because they represent parts of different physiological host response to pathogen. PAL activity is required for biosynthesis of phenyl propanoids, which are required for penetration and all death responses to pathogen in barely to powdery mildew (Shiraishi et al., 1995).
In this study, Trilogy elicited plants inoculated with P. xanthii (E+ I+) maintained a low level of infection that never exceeded 5% for the duration of the experiment (3 weeks). In contrast, the level of infection for non elicited inoculated plants (E- I+) progressed rapidly and covered approximately 80% of the leaf surfaces. These results are consistent with those obtained by Fofana et al. (2002) who examined the prophylactic properties of Milsana.
The production of phytoalexin compounds has in recent times been proposed to explain induced resistance for cucumber on the basis of correlative evidence (Fawe et al., 1998). McNally et al. (2003) established a clear link between the production of these compounds, viz., C-glycosyl flavonoids and induced resistance against powdery mildew. During the present studies, phytochemical analyses of targeted leaf tissues harboring fungal colonies eliminated the effects of whole leaf extraction and led to the identification of six structurally related C-glycosyl flavonoid phytoalexins, i.e., vitexin, isovitexin, orientin, isoorientin, cucumerin A and B. At the same time, phenolic acids involved in lignification, such as sinapinic acid, were detected in approximately equal concentration within the leaves of E- I+, E+ I+ plants, thereby negating this process as an important form of resistance against powdery mildew in cucumber. In fact, high concentration of ferulic and caffeic acids were detected within the leaves of E- I+ plants.
Expectedly, higher concentration of p-coumaric acid and its derivative, p-came, were observed within E+ I+ plants, since these phenolic acids are biosynthetic precursors of all six identified C-glycosy/flavone phytoalexins (Burbulis and Winkel-Shirly, 1999). The accumulation of approximately 30 times more cucumerin A and B within the leaves of E+ I+ compared to E- I+ is consistent with the increase in phytoalexin concentration. This agreed with the previous studies reported by Essenberg et al. (1992). Moreover, the induction of C-glycosyl falvonoids as phytoalexins and the existence of p-coumaric acid and its methyl ester (p-came) within E+ I+ plants supports the previous studies that associated these compounds with induced resistance against powdery mildew in cucumber (Fawe, 1997).
Phytoalexin synthesis was triggered by the combination of an eliciting treatment and fungal penetration and seemed to be closely synchronized with germination of fungal conidia. Moreover, elicited plants became sensitized to the presence of the pathogen by producing phytoalexins more rapidly in response to subsequent infection.
Based on the chemical analyses, flavonoid (glycosylates and aglycones) compounds were found to accumulate in larger quantities in elicited plants than in controls. These findings are particularly interesting since they suggest that the increased activity of flavonoid biosynthetic enzymes following elicitation leads to a strong accumulation of defensive compounds in elicited cucumber, highlighting the correlation between phytoalexin accumulation and induced resistance. These results are in agreement with those mentioned by Fawe et al. (1998) who described the presence of fungitoxic flavonoids in cucumber tissues following an eliciting treatment. Previously, flavonoid phytoalexins had been found only in a few plant species such as sorghum, rice and barley (Christensen et al., 1998).
In conclusion, resistance of cucumber plants to powdery mildew following an eliciting treatment with Trilogy appears to be associated with accumulation of the flavonoid biosynthetic enzymes. Both flavonoids and phenolic acids have specific role in the complex defence system of cucumber.