Induction and Expression of Systemic Resistance to Downy Mildew Disease in Cucumber by Elicitors

8 Egypt. J. Phytopathol., Vol. 36, No.1-2, pp. 95-111 (2008)

Induction and Expression of SystemicResistance to Downy Mildew Disease inCucumber by ElicitorsS. Farouk*; K.M. Ghoneem** and Abeer A. Ali**

* Agric. Botany Dept., Fac. Agric. Mansoura Univ., Egypt.** Mycol. Res. and Plant Dis. Surv. Dept., Plant Pathol. Res.

Inst., Agric. Res. Centre, Giza, Egypt.

ost of elicitors are cheap, available, easy to use andenvironmentally safe, beside their ability to induce resistance in

many plants against pathogens attack. In this connection, exogenouslyfoliar application of chitosan (CHI), salicylic acid (SA) and methyljasmonate (MeJA) proved to be effective in reducing downy mildew(caused by Pseudoperonospora cubensis) occurrence (DO) andseverity (DS) in cucumber plants under field conditions. Most ofconcentrations used of the tested elicitors significantly increasedcucumber growth parameters (plant height, no. of branches, shoot dryweight and leaf area) and physiological aspects (nitrogen, potassium,phosphorus, calcium, total phenols and photosynthetic pigments) inshoot as well as yield and its quality. CHI at 0.05% and SA at 100mg/lwere the most effective in this concern. Application of elicitorsincreased leaf anatomical characters, i.e. thickness of leaf blade,thickness of palisade and spongy parenchyma as well as thickness ofmidrib region and dimension of vascular bundles. Results indicate thatCHI and SA can be used in the field as a means of protection againstcucumber downy mildew as well as improving its growth and yield inaddition to decrease of the fungicides used.

Keywords: Chitosan, cucumber, downy mildew, induced resistance,methyl jasmonate, Pseudoperonospora cubensis andsalicylic acid.

Cucumber (Cucumis sativa L.) is one of the most important and popularvegetable crops allover the world and in Egypt as well. The crop is mainly cultivatedduring the summer season in open fields, while it could be grown in two growingseasons, i.e., autumn and spring under plastic house conditions. Downy mildewcaused by Pseudoperonospora cubensis (Berk. et Curt.) Rost. is a major disease ofcucurbits in temperate regions of the world that considerably reduces the productionand greatly affects both yield quantity and quality (Abd EL-Kereem, 1998).

There are many chemical products available for controlling the disease, but inview of their undesired secondary effects and the fact that cucumber has a relativelyprolonged harvesting season, there is an increasing interest in finding and using newnatural products which are more reliable and less dangerous for the environment(Haggag, 2002).

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Egypt. J. Phytopathol., Vol. 36, No. 1-2 (2008)

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Phenotypically, systemic resistance is manifested as protection which is longlasting and active against a broad spectrum of plant pathogens. The induced state iscorroborated by an increase in production of a range of defense related products likepathogenesis related proteins, phytoalexins and signaling compounds (Heil andBostock, 2002). This induced systemic resistance is triggered by a number ofchemicals such as methyl jasmonate (MeJA) and salicylic acid (SA) (Yao & Tian,2004 and Amin et al., 2007) or biologically active oligoglucosides, the so-calledelicitors (Benhamou et al., 1994) which have been shown to induce systemicresistance in many crops like tomato, tobacco, pea, maize, cotton, rice, potato andother vegetables against viruses, fungi and bacteria (Oostendorp et al., 2001).

Among the most promising bioactive oligosaccharides is CHI which hasattracted attention because of its unique biological properties, including inhibitoryeffect on the growth of various pathogenic fungi and its ability to be a potent elicitorof plant defense reactions (Prapagdee et al., 2007). CHI has been reported to protecttomato, cucumber, pea, melon, strawberry, lettuce and pearl millet against powderyand downy mildews as well as other diseases (Sharathchandra et al., 2004).

Jasmonic acid is found in many plant species and is involved in regulatingdiverse plant functions, including plant resistance to pathogens (Creelman & Mullet,1997). Jasmonic acid and (MeJA) are produced by the plant after pathogen invasionand results in increased production of compounds involved in resistance (Yao &Tian, 2004). SA is now considered as a hormone-like substance which plays animportant role in stimulating adventitious root development (Kling and Meyer,1983), showing a herbicidal effect (Shettel and Blake, 1983), providing resistanceagainst pathogens (De Gara, 2003), and modifying the quality and quantity ofproteins (Çanaki, 2003). During the last 20 years this substance has drawn theattention of researchers because of its ability to induce systemic acquired resistance(SAR) in plants.

The objective of this study is to evaluate the efficacy of chitosan (CHI), salicylicacid (SA) and methyl jasmonate (MeJA) as elicitors in controlling cucumber downymildew disease, under field conditions. Also, effects of the tested elicitors onphenolic compounds, photosynthetic pigments, some probable defense mechanismsand the plant parameters and yield.

M a t e r i a l s a n d M e t h o d s

1. Elicitors and cucumber seeds:CHI and SA were obtained from Sigma Chemicals Co. (St. Louis, MO, USA),

meanwhile MeJA was obtained from Servo Co. (Bulgaria). Cucumber seeds of thesusceptible cultivar Concord, hybrid to downy mildew pathogen were obtained fromEl-Mers Company, Egypt.

2. Effect of elicitors on downy mildew incidence and severity under field conditions:Two field experiments were carried out under natural infection at the

Experimental Farm of Tag El-Ezz, Agric. Res. Station, Dakahliya, Egypt, during thetwo growing seasons of 2006 and 2007. In addition to the fungicide Ridomil GoldPlus 50% (150g/100 Liter H2O), CHI at 0.025, 0.050 and 0.075%; SA at 50, 100 and150 mg/l and MeJA at “10-5, 5x10-5 and 10-4 M" were used as foliar treatments.

INDUCTION AND EXPRESSION OF SYSTEMIC RESISTANCE TO …..


97

Seeds were sown on the 10th and the 15th of April 2006 and 2007 seasons,respectively. All agricultural practices were carried out according to therecommendations of the Ministry of Agricultures, Egypt. Complete randomizedblock design with four replicates for each treatment was used. Each replicateconsisted of 50 plants spaced at 50 cm apart on both sides of row. The plants fromeach assigned treatment were sprayed with individual elicitors twice at 30 and 50days after sowing till dripping as well as tape water (check treatment) or fungicide.

Disease severity (DS) and occurrence (DO) were measured at 60 and 90 daysafter sowing. At 100 days, the growth characters, photosynthetic pigments andphenols content were determined in the third upper leaf, while carbohydrates as wellas nitrogen, potassium, calcium and phosphorous content were determined in theshoots. At harvesting, cucumber yield (kg/feddan) as well as the fruit contents ofascorbic acid, protein, carbohydrates, potassium and phosphorous were determined.

A. Disease assessment:The plants were rated for both DO and DS, the former as the presence or absence

of disease (percentage of infected leaves on the plant) and the latter as the severitypercentage of disease damage. DS was measured according to Reuveni (1983) usingcolor index and infected area. The color index was calculated as follows:

0= no symptoms 1= greenish 2- yellowish 3- yellow 4= brown

The infected area index was measured as follows: 1= symptoms on 25% or less2= >25-50 % 3= >50-100% of leaf area.

Multiplication of the color and infected area indices for each leaf yields a valueof disease severity. Reduction percentage in either DI or DS was determinedaccording to the equation as follows:

Reduction% = {(Infection in control% - Infection in treatment %) / Infection incontrol %} X100.

B. Growth parameters:Ten cucumber plants were randomly selected from the middle part of each plot,

leaving two rows from each side to avoid border effects. Plant height, number ofbranches, shoot dry weight and leaf area were determined.

C. Physiological determination:In the 2nd season, the total phenolic compounds were determined according to the

method of Singleton and Rossi (1965) using Folin-Ciocalteau reagent.Photosynthetic pigments were extracted from the 3rd upper leaf by methanol(Robinson and Britz, 2000), and determined spectrophotometrically and calculatedby the equation introduced by Mackinney (1941). In shoots and fruits,determinations of total nitrogen by microkjeldahl methods and Potassium usingflame photometer (Kalra, 1998), phosphorous using ammonium molybdate andascorbic acid (Cooper, 1977), and Calcium using versenate methods (Richards,1954), were carried out. Total carbohydrates and ascorbic acid content in fruits wereestimated using the anthrone method and 2.6-dichlorophenol indophenol,respectively, as described by Sadasivam and Manickam (1996). Total protein wasextracted from the fruit and estimated by the method of Bradford (1976).

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D. Leaf structure:In the 2nd season also, small pieces from the midrib region of the 3rd upper leaf

(second season) were taken 100 days after sowing. The samples were killed andfixed in formalin-acetic-alcohol for 48 h, then dehydrated in a series of ethanol andembedded in paraffin wax (52-54oC melting points). Sections were made at 15-17µm thick using rotary microtome, stained with erythrosin/crystal violet and mountedin Canada balsam (Johansen, 1940). The selected sections were examinedmicroscopically for determining the anatomical changes in leaves.

Statistical analysis:Data were subjected to Analysis of Variance (ANOVA) and means separation

were adjusted by the Multiple Comparison test (Norman and Streiner, 2003) usingthe statistical computer program "MSTAT-C" at 5% level of significance. Duncan(1955) was used to compare between means.

R e s u l t s

1. Disease incidence and disease severity:The present data (Table 1 and Fig. 1) of the two growing seasons show that the

application of the used elicitors on cucumber plants significantly reduced downymildew occurrence (DO) and severity (DS) as compared to the check treatment.However, at 60 and 90 days after sowing, CHI at 0.025% followed by SA at100mg/L showed superiority in reducing DS but less than the fungicide (74.17 &69.4 and 68.56 & 62.68% reduction, respectively) in the first season and 71.1 & 69.1and 67.29 & 61.63% reduction, in the second season, respectively. MeJA came inthe second rank in reducing DS during the growth intervals of the first season (66.11& 57.50% reduction, respectively) and second season (63.93 and 59.76% reduction,respectively). In all cases, the tested fungicide (Ridomil Gold Plus 50%) gave thebest disease control during the two growing seasons. Generally, the severity ofdowny mildew disease increased during the growth period up to 90 days fromsowing.

Table 1. Effect of elicitors on downy mildew severity on cucumber plants at 60and 90 days after sowing

Treatment Conc.1st season (2006) 2nd season (2007)

60 day(DS)

Reduction(%)

90 day(DS)

Reduction(%)

60 day(DS)

Reduction(%)

90 day(DS)

Reduction(%)

Chitosan(%)

0.025 1.57d* 74.17b 3.53f 68.56b 1.9c 71.08ab 3.93c 67.29b0.050 2.01b-d 66.49b-d 4.66d-f 58.50b-d 2.13bc 67.58ab 4.77bc 61.06bc0.075 2.53b 58.38d 6.11bc 45.59ef 2.77bc 57.84b 6.27b 48.82c

Salicylic acid(mg/l)

50 2.62b 56.90d 6.37ef 43.27f 2.03bc 67.58ab 5.37bc 56.16bc100 1.86cd 69.40bc 4.19ef 62.68bc 2.13bc 69.10ab 4.7bc 61.63bc150 2.42bc 60.19cd 5.84b-c 47.99d-f 3.03b 53.88b 6.3b 48.57c

Methyl jasmonate(M)

10-5 2.47bc 59.37cd 5.95b-c 47.00d-f 2.85bc 56.62b 6.32b 48.41c5×10-5 2.06b-d 66.11b-d 4.81c-f 57.16c-e 2.37bc 63.93b 4.93bc 59.76bc

10-4 2.26bc 62.82cd 5.38b-e 52.09c-f 2.6bc 60.43b 6.05b 50.61cFungicide (g/l) 1.5 0.67e 89.98a 1.83g 83.70a 0.8d 87.82a 1.75d 85.71a

Check 0.0 6.08a - 11.23a - 6.57a - 12.25a -*Values followed by the same letter(s) in each column do not differ significantly (p ≤0.05).



99

Fig. 1: Incidence of downy mildew (%) at 60 and 90 days after sowing asaffected by the tested elicitors during two successive seasons (2006 and2007).

L.S.D at 0.05= 8.79 for 1st season after 60 day, 12.85 for 1st season after 90 day, 8.76 for 2nd

season after 60 day and 14.25 for 2nd season after 90 day.

Data in Fig. (1) indicate that using salicylic at the concentration of 100 mg/l wasthe most effective treatment in reducing DO (about 50% reduction in both seasons)which came second after the fungicide at 60 days after sowing, meanwhile, at 90days from sowing the exogenous application of 100 mg/l SA gave the highestreduction (45.25 and 55.1%) of DO in 1st and 2nd seasons, respectively, compared toother treatments including the fungicide. In general, the application of anyconcentration of the tested substances significantly reduced both DS & DO duringthe growth period of cucumber plants.

2. Growth parameters:The tested elicitors significantly affected the growth of cucumber plants (Table 2).

In both seasons, maximum values of each of plant height (136.33 & 139.1 cm),number of branches (6.66 & 7.03 cm), shoot dry weight (35.68 & 37.15 g) as well asleaves area per plant (658.34 & 663.9 cm2) were determined in the treatment where0.050% CHI was applied. The lowest values of plant height (96 & 93.13 cm), no. ofbranches (4 & 4.58), shoot dry weight (17.59 & 15.67g) as well as leaves area perplant (396.03 & 389.93 cm2) were found at 10-4M of MeJA treatment.

3. Photosynthetic pigment and total carbohydrates contents:Data (Table 3) show that photosynthetic pigments were increased significantly, in

most cases, due to application of the tested elicitors as well as fungicide compared tothe check treatment. This increase in photosynthetic pigments contents wasaccompanied with an increase in total carbohydrates contents in cucumber shoot.CHI at 0.05 % and SA at 100 mg/l were the most effective treatments in this respect.

S. FAROUK et al.


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Table 2. Cucumber growth as affected by elicitors under natural infection bydowny mildew 100 days after sowing

Treatment

Con

cent

ratio

n 1st season (2006) 2nd season (2007)

Plan

t hei

ght

(cm

)

No

ofbr

anch

es

Shoo

t dra

yw

eigh

t (g)

Lea

f ar

ea(c

m2 )

Plan

t hei

ght

(cm

)

No

ofbr

anch

es

Shoo

t dra

yw

eigh

t(g

pla

nt-1

)

Lea

f ar

ea(c

m2 )

Chitosan(%)

0.025 126.33b* 5.33b 28.60c 533.13bc 122.73bc 5.9bc 29.23c 535.07bc0.050 136.33a 6.66a 35.68a 658.34a 139.1a 7.03a 37.15a 663.9a0.075 125.67b 5.0bc 31.59b 531.41bc 129.6b 5.67cd 32.13b 540.17bc


50 100.0d 4.0c 24.48de 450.87cd 104.87de 4.87ef 27.8c 459.33de100 131.6ab 6.33a 29.97c 608.22ab 129.77b 5.6c-e 23.0de 597.5ab150 111.33c 4.66bc 25.83d 493.60c 105.1de 4.73f 23.88de 508.7cd

Methyljasmonate

(M)

10-5 96.00d 4.0c 17.59g 396.03d 93.13f 4.58f 15.67f 389.93e5×10-5 128.67b 6.33a 25.68d 599.26ab 126.1bc 6.58ab 28.81c 595.53ab

10-4 112.33c 5.17b 23.78f 497.64c 108.27d 4.87ef 23.85de 499.1cdFungicide (g/l) 1.5 96.33d 4.0c 21.78f 449.90cd 97.67f 3.73g 21.75e 451.67de

Check 0.0 96.33d 4.0c 21.78f 449.90cd 97.67f 3.73g 21.75e 451.67de* Values followed by the same letter(s) in each column do not differ significantly (p ≤0.05).

Table 3. Effect of different concentrations of elicitors on photosyntheticpigments and total carbohydrates in cucumber plants grown undernatural infection by downy mildew, in the 2nd season (2007)

Treatment

Con

cent

ratio

n

Photosynthetic pigments content mg/100gfresh weight

Tot

alca

rboh

ydra

tes

(mg/

g D

W)

Chl

. a

Chl

. b

Tot

al C

hl.

Car

oten

oids

Chitosan(%)

0.025 32.70b* 8.00d 40.70b-d 7.50b 12.41d0.050 41.20a 8.60d 49.80a 8.90a 15.87a0.075 23.90d-f 12.40a-c 36.30d-f 6.10cd 11.41f


50 21.30ef 13.50a 34.80ef 5.50d 10.29h100 30.70bc 13.60a 44.30b 8.20ab 15.13b150 22.90d-f 13.20ab 36.10ef 6.00cd 10.96g

Methyljasmonate

(M)

10-5 20.50f 12.00a-c 32.50f 4.50e 9.29j5×10-5 30.10bc 11.20bc 41.30bc 8.00ab 14.42c

10-4 26.70cd 10.70c 37.40c-d 6.60c 11.86eFungicide (g/l) 1.5 22.20ef 13.00ab 35.20ef 5.90cd 10.56gh

Check 0.0 25.40de 7.60d 33.00ef 5.30de 9.78I* Values followed by the same letter(s) in each column do not differ significantly (p ≤0.05).



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4. Total phenols:Illustrated data in Fig. (2) show that total soluble phenols in cucumber leaves

increased significantly with exogenous application of elicitors. SA was the mosteffective especially at 150mg/l followed by CHI at 0.05% and the fungicideRidomil.

Fig. 2: Phenol content of cucumber plants in response to application of differentelicitors.

5. Nitrogen, potassium phosphorus and calcium content:Data presented in Table (4) indicate that application of elicitors increased

significantly the content of nitrogen, potassium, phosphorous and calcium incucumber shoots as compared to control or fungicide treatments. CHI at 0.05% gavethe highest values of nitrogen and calcium as compared to the check treatment.

6. Leaf structure:Data presented in Table (5) and Fig. (3) show that, except for the two

concentrations of MeJA, the tested elicitors caused an increase in the thickness ofmidrib region in cucumber leaf specially CHI at 0.050% which gave the highestvalues. This increase is mainly due to increasing the dimensions of the mainvascular bundle (length and width) as in Table (5). However, data showed thatapplication of MeJA especially at low and medium concentrations decreased thevascular bundle dimensions. Concerning thickness of leaf blade, application ofelicitors or fungicide increased the thickness of leaf blade; this increment is due to acorresponding increase in the thickness of palisade and spongy parenchyma as wellas epidermis. In most cases CHI at 0.05% gave the best result in increasing leafblade thickness.

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Table 4. Nitrogen, Potassium, Phosphorus and Calcium contents in cucumberplants as affected by elicitors under natural infection by downymildew in the 2nd season (2007)

Treatment

Con

cent

rati

on

Nm

g/g

dry

wt

Pm

g/g

dry

wt

Km

g/g

dry

wt

Ca

mg/

g dr

y w

t

Chitosan(%)

0.025 31.47cd* 2.59c 14.53d 2.35d0.050 41.19a 2.93a 18.25a 3.73a0.075 30.93cd 2.53cd 13.99de 2.19de


50 30.15cd 2.41d 13.50de 1.80f100 37.82b 2.77b 16.52b 3.33b150 30.69cd 2.51cd 13.78de 2.00ef

Methyljasmonate

(M)

10-5 22.40e 2.18f 11.79f 1.39g5×10-5 33.40c 2.70b 15.34c 2.70c

10-4 27.90d 2.44d 13.56de 2.04efFungicide (g/l) 1.5 30.33cd 2.47d 13.60de 1.83f

Check 0.0 27.87d 2.28e 13.02e 1.76f* Values followed by the same letter(s) in each column do not differ significantly (p ≤0.05).

Table 5. Structural changes in cucumber leaves as affected by three elicitors orRidomil Gold Plus under natural infection by downy mildew in the 2nd

season (2007)

Treatment

Con

cent

rati

on

Thi

ckne

ssof

leaf

blad

e

Thi

ckne

ssof

upp

erep

ider

mis

thic

knes

sof

palis

ade

Thi

ckne

ssof

Spon

gy

Thi

ckne

ssof

low

erep

ider

mis

Thi

ckne

ssof

mir

ib Main vascularbundle dimensions

Length Width

μ % μ % μ % μ % μ % μ % μ % μ %

Chitosan(%)

0.025 84 123 3 150 32 100 47 142 2 200 328 134 112 121 52 100

0.050 88 129 5 250 44 137 37 112 2 200 480 196 200 217 144 276

0.075 56 83 5 250 28 875 22 66.6 1 100 384 157 136 100 184 284

Salicylicacid

(mg/l)

50 64 95 3 150 20 625 39 118 2 200 392 160 96 104 52 100

100 72 105 4 200 40 125 28 84.8 2 200 436 178 144 156 88 115

150 80 117 3 150 28 875 47 142 2 200 340 139 100 108 56 107

Methyljasmonate

(M)

10-5 72 105 3 150 36 112 31 93.9 2 200 252 103 84 91.3 44 84.6

5×10-5 72 111 2 150 40 125 29 96.9 1 100 246 100.8 72 78.2 48 92.3

10-4 72 105 2 100 40 125 28 84.8 2 200 244 100 64 69.5 64 123

Fungicide 1.5 g/l 68 100 3 150 36 112 27 81.8 2 200 316 129 116 126 64 123

Check 0.0 68 100 2 100 32 100 33 100 1 100 244 100 92 100 52 100



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Fig. 3: Structural changes in cucumber leaves as affected by three elicitors orRidomil Gold Plus 50% under natural infection by downy mildew(1, Check; 2, Fungicide; 3, SA at 100 mg/l; 4, MeJA at 5×10-5, 5, CHI at0.050%)

S. FAROUK et al.


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7. Yield and fruits quality:Data in Table (6) show that ascorbic, carbohydrate, protein, potassium and

phosphorus contents were significantly enhanced with foliar application by elicitorscompared to negative and positive check. Clearly, CHI at 0.05%, SA at 100mg/l andMeJA at 5x10-5M showed equal significant increase in the yield of cucumber plants,being 11.13, 10.79 and 10.21 ton fed.-1 in the first season and 11.52, 10.84 and 10.63ton fed.-1 in the second season, compared to the check treatment, being 6.543 and5.63 ton fed-1, respectively.

Table 6. Yield and fruit quality of cucumber plants as affected by elicitorsunder natural infection by downy mildew

Treatment

Con

cent

ratio

n

Yie

ld(t

on/f

edda

n)

Prot

ein

g/10

0g

Car

bohy

drat

eg/

100g

Asc

orbi

c ac

id(m

g/g

fres

h w

t)

K(m

g/g

fres

h w

t)

P(m

g/g

fres

h w

t)

1st

season2nd

season

Chitosan(%)

0.025 9.21b* 9.61bc 0.943cd 3.52c 13.65c 165.3bc 27.67c

0.050 11.13a 11.52a 1.070 a 3.86a 15.30a 178.7a 30.33a

0.075 8.88b-d 9.06c 0.926c-e 3.48c 13.28d 158.0c 27.33c

Salicylicacid

(mg/l)

50 7.87d 7.49de 0.876d-f 3.15ef 11.26g 133.3c 24.00e

100 10.79a 10.84a 1.023b 3.67b 14.41b 175.7ab 29.00b

150 8.75b-d 8.44cd 0.900d-f 3.35d 12.52e 155.0c 26.33d

Methyljasmonate

(M)

10-5 6.27e 6.52ef 0.773g 2.96g 10.04i 117.7f 20.33g5 x10-5 10.21a 10.63ab 0.970c 3.55c 13.85c 173.3ab 28.33bc

10-4 9.05bc 9.58bc 0.871ef 3.25e 11.95f 145.5d 24.33eFungicide

(g/l)1.5 8.14cd 8.74c 0.890d-f 3.21e 11.70f 140.7de 25.67a

Check 0.0 6.54e 5.63f 0.853f 3.09f 10.55h 124.3f 22.00f* Values followed by the same letter(s) in each column do not differ significantly (p ≤0.05).

D i s c u s s i o n

Plants respond to pathogen attack or elicitor treatments by activating a widevariety of protective mechanisms designed to prevent pathogen replication andspreading (Malolepsza and Rózalaska, 2005). The defense mechanisms include thefast production of reactive oxygen species (De Gara et al., 2003); alterations in thecell wall constitution; accumulation of antimicrobial secondary metabolites knownas phytoalexins (Agrios, 2005); activation and/or synthesis of defense peptides andproteins (Castro and Fontes, 2005). In various plant species, resistance can beinduced with elicitors such as SA, MeJA and CHI against a wide range of pathogens(Yao and Tian, 2004; Sharathchandra et al., 2004 and Amin et al., 2007).



105

In the present study, it was found that the treatment of cucumber plants withthese inducers efficiently reduces susceptibility to downy mildew caused byP. cubensis under field conditions, in a way similar to that of Ridomil Gold Plus50%, which is a standard fungicide. Applications of CHI at 0.05% and SA at100mg/l were the most effective in this concern.

There are numerous reports concerning the protective effects of CHI againstpathogen infection in a range of crops (Sharathchandra et al., 2004 and Romanazziet al., 2006). The reduction of DS and DO in cucumber plants by CHI applicationmay be referred to its propriety as a hydrophobic material, thus creating a low waterpotential in infected leaves which prevented spore germination, infection and growthwhen applied before infection as well as, its ability to reduce esterase secretion bypathogens (Hsieh and Huang, 1999). Southerton and Deverall (1990) added thatapplication of elicitors like CHI resulted in accumulation of osmiophilic masses inthe intercellular spaces of reacting host cells. So, the invading fungal cells coated bythis osmiophilic material causing frequently pronounced disorganization such asplasmalemma retraction thus preventing pathogen penetration. Another explanationmay be referred to the role of elicitors in induction of local and/or systemic acquiredresistance (SAR) in cucumber treated plants against invasion of the pathogen. In thisconcern, SAR was reported in a number of crops due to CHI application(Bhaskarareddy et al., 1999 and Barka et al., 2004). Several reports indicated alsothat application of elicitors as CHI, SA or MeJA stimulates the accumulation ofsignal molecule as jasmonic acid, SA, hydrogen peroxide, reactive oxygen speciesand protein kinases, all of which plays crucial role in intracellular signalingpathways (Atia et al., 2005). A pioneering study demonstrated that the application ofexogenous SA or CHI induces the synthesis of pathogenesis related proteins andpartial resistance to pathogens such as β-1,3-glucanase, chitinase and PR proteins(Raucher et al., 1999 and Atia et al., 2005).

Exogenous applications of JA, MeJA and SA have been shown to movesystemically through plants, resulting in the expression of a set of defense genes thatare activated by pathogen infection (Lu et al., 2006). The development of acquiredresistance by SA may be attributed at least partly, to the SA-induced phenylalaninammonia lyase (PAL) gene expression and activation (Wen et al., 2005).

Phenolic compounds may be an important group of secondary metabolitesinvolved in resistance to pathogens. The present results indicate that elicitors,especially SA stimulated the accumulation of soluble phenolic compounds incucumber leaves. This effect might be due to the impact of these substances onenzymatic activity and translocation of the metabolites to cucumber plant. Theseresults are in agreement with those obtained by Shehata et al. (2001) who reportedthat phenols content in maize leaves was significantly increased by acetyl salicylicacid application (20 and 40 μM). Also, Amin et al. (2007) showed that foliarapplication of SA at the concentration of 50 and 100 mg/l caused significant increaseconstituents of onion bulb from total phenols, soluble sugars, total free amino acidsand total indoles. Some of the phenolics act as antioxidants and induce resistance;epicatechin and diene are examples (Prusky et al., 1994). This accumulation ofphenolic compounds in leaves may be due to inhibition of catalase activity, which in

S. FAROUK et al.


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turn induces PAL gene expression and synthesis of phenolic compounds (Vermerrisand Nicholson, 2006). Normally, phenol metabolism is activated in plants reactingto pathogens (Ellard-Ivery and Douglas, 1996). This is due not only to themechanical role that phenolics play in cell walls, but also to their anti fungalproperties (Harborne, 1991). Yet total phenols have long been considered asimportant defense-related compounds whose levels are naturally high in resistantvarieties of many crops (Gogoi et al., 2001).

Foliar application of elicitors in both season showed in most cases a significantincrease in cucumber growth parameters, especially CHI at 0.050% and SA at100mg/l. These increases may be attributed to elicitors effect on physiologicalprocesses in plant such as ion uptake, cell elongation, cell division, enzymaticactivation and protein synthesis. In this concern, low SA doses enhanced growth ofwheat (Shakirova et al., 2003), pea (Farouk, 2005) and onion (Amin et al., 2007)plants. Jasmonic acid is a final product of the enzymatic oxidation of unsaturatedfatty acids and lipoxygenase is a pivotal enzyme in this pathway. This compound,defined as a natural plant growth regulator, was found to be active in manyphysiological systems (Vick and Zimmermann, 1987).

The application of elicitors increased the total chlorophyll content of thecucumber plants. This increment may be due to stimulating pigment formation andenhancing the efficacy of photosynthetic apparatus with a better potential forresistance and decrease in photophosphorylation rate usually occurring afterinfection (Amaresh and Bhatt, 1998). Elicitors were found to increase potassiumcontent, which may increase the number of chloroplasts per cell, number of cells perleaf and consequently leaf area (Possingham, 1980). This is in harmony with thework carried out by Farouk (2005) which indicated that application of either MeJAor SA increased significantly photosynthetic pigments content. Moreover, SAproved to decrease ethylene production and subsequently increased chlorophyll(Leslie and Romani, 1986), and activated the synthesis of carotenoids which protectchlorophyll from oxidation and finally increased chlorophyll content as reported inthis study.

Results also proved that application of tested elicitors increased significantlycation contents i.e., N, P, K and Ca in cucumber shoot which reflect to increasingplant growth and plant resistance to the pathogen due to its roles in plantmetabolism, such as promoting the development of thicker outer walls and stabilityof plant membrane in epidermal cells, thus preventing disease attack. Moreover,increasing of nitrogen in plant stimulate synthesis of phenols and lignin contentwhich are parts of the defense system of plants against infection (Marschner, 1995).

The increase in cucumber yield may be due to the role of elicitors in stimulationof physiological processes which reflect on improving vegetative growth thatfollowed by active translocation of the photoassimilates from source to sink incucumber plant due to increasing leaf blade thickness as well as dimensions ofvascular bundles. In this concern, SA might be regulating plant growth by increasingenzyme activity as α-amylase and nitrate reductase, which accelerate the sugartranslocation from the leaves to developing fruits (Sharma et al., 1986). Moreover,



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application of all elicitors leads to an increase in potassium content, which enhancephotosynthetic pigments and capacity, and subsequently dry matter accumulation(Umar and Bansal, 1995). In addition, application of SA inhibits ethylene productionleading to increasing fruit number and consequently increasing fruit yield per plant(Leslie and Romani, 1986).

In conclusion, it may be possible to, in certain cases, replace conventionalchemical fungicides with any of the three elicitors especially CHI at 0.05% due to itssafety for human and environment and thus providing both economical andecological efficacy.

A c k n o w l e d g e m e n t s

The researchers would like to thank Prof Dr. I. Fedina (Bulgaria) for providingmethyl jasmonate. Also, thank for Prof. M.R.A. Shehata, Plant Pathol. Dept., Fac.Agric., Alexandria Univ. and Prof. M.N. Helali, Agric. Botany Dept., Fac. Agric.,Mansoura Univ. for helpful insights and critical reviews of the manuscript.

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(Received 07/09/2008;in revised form 03/11/2008)



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ششبقة في مجملها تشجع .

CHISA

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Induction and Expression of Systemic Resistance to Downy Mildew Disease in Cucumber by Elicitors