By Gamal Ashor Ahmed - Bu · By Gamal Ashor Ahmed B.Sc Agricultural ... mildew disease in cucumber cv. Primo under commercial protected ... and help that Prof. Dr. Abdou Mahdy Mohamed

USING PLANT EXTRACTS TO CONTROL POWDERY

MILDEW DISEASE THAT ATTACK CUCUMBER

PLANTS UNDER PROTECTED HOUSES

By

Gamal Ashor Ahmed B.Sc. Agricultural Sciences, 1998

Fac. Agric. Moshtohor, Zagazig Univ., Benha Branch

THESIS

Submitted in Partial Fulfillment of the

Requirements for

The Degree of

Master of Science

in (PLANT PATHOLOGY)

Agricultural Botany Department

(Plant Pathology)

Faculty of Agriculture, Moshtohor

Zagazig University, Benha Branch

2004

SUPERVISION COMMITTEE

Using Plant Extracts to Control Powdery Mildew Disease That Attack

Cucumber Plants Under Protected Houses

By

Gamal Ashor Ahmed B.Sc Agricultural Science, 1998

Fac. Agric. Moshtohor, Zagazig Univ., Benha Branch

This thesis for MSc. degree in Plant Pathology under the supervision of:

Prof. Dr. Abdou Mahdy Mohamed Mahdy Professor of Plant Pathology Vice- Dean for Community Development and Environmental Affairs Agric. Botany Dept., Fac. Agric., Moshtohor Zagazig Univ., Benha Branch

2. Prof. Dr. Mohamed Haroun abd-El- Mageed Professor of Plant Pathology Fungus and Pant Pathology Branch Agric. Botany Dept., Fac. Agric., Moshtohor Zagazig Univ., Benha Branch

3. Dr. Faten Mahmoud Abd-El-Latef Lectuer of Plant Pathology Fungus and Pant Pathology Branch Agric. Botany Dept., Fac. Agric., Moshtohor Zagazig Univ., Benha Branch

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CONTENTS

Page

1- INTRODUCTION ......................................................................................... 1

2- REVIEW OF LITERATURE .................................................................. 4

3- MATERIALS AND METHODS ............................................................. 35

4- EXPERIMENTAL RESULTS ................................................................. 53

1- Survey of cucumber diseases in protected houses. ................................ 53

2- Laboratory experiments: ......................................................................... 53

2.1.1. Effect of plant extracts on germination of Sphaerotheca fuliginea

conidia. ....................................................................................................... 53

2.1.2. Effect of some plant oils on germination of Sphaerotheca fuliginea

conidia. ....................................................................................................... 55

2.1.3. Effect of phosphate salt (K2HPO4) on germination of Sphaerotheca

fuliginea conidia. ........................................................................................ 56

2.1.4. Effect of some biological agent filtrate, propolis and their combination

on germination of Sphaerotheca fuliginea conidia. .................................. 57

2.1.5. Effect of UV, temperature and chloroform on germination of powdery

mildew spore. ............................................................................................. 58

3. Greenhouse experiments: .......................................................................... 59

3.1. Induction of cucumber resistance to powdery mildew by plant extracts. ...... 59

3.2. Induction of cucumber resistance to powdery mildew by plant oils. ............. 60

3.2. Induction of cucumber resistance to powdery mildew by phosphate salt

(K2HPO4). ....................................................................................................... 61

3.4. Induction of cucumber resistance to powdery mildew by biological agent

filtrate, propolis and their combination. ......................................................... 62

3.5. Effect of UV, temperature and chloroform on the infectivity powdery

mildew spores: ................................................................................................ 64

4. Commercial protected house studies: ....................................................... 65

4.1. Effect of spraying plant extracts on incidence and severity of powdery

mildew disease in cucumber cv. Primo under commercial protected houses.

........................................................................................................................ 65

4.2. Effect of spraying plant oils on incidence and severity of powdery mildew

disease in cucumber cv. Primo under commercial protected houses. ............ 66

4.3. Effect of spraying phosphate salt (K2HPO4) on incidence and severity of

powdery mildew disease in cucumber cv. Primo under commercial

protected houses. ............................................................................................ 68

4.4. Effect of spraying some biological agent, propolis and their combination

on incidence and severity of powdery mildew disease in cucumber cv.

Primo under commercial protected houses. ................................................... 69

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4.5. Effect of spraying with plant extracts on controlling powdery mildew

disease in cucumber cv. Delta star (during spring 2004). ............................... 70

4.6. Effect of spraying with plant oils on controlling powdery mildew disease

in cucumber cv. Delta star (during spring 2004). ........................................... 72

4.7. Effect of spraying with phosphate salt (K2HPO4) on controlling powdery

mildew disease in cucumber cv. Delta star (during spring 2004). .................. 72

4.8. Effect of spraying with biological control agent on controlling powdery

mildew disease in cucumber cv. Delta star (during spring 2004). .................. 75

4.9. Effect of spraying cucumber plants with ungerminated powdery mildew

spore on controlling powdery mildew disease in cucumber cv. Delta star

(during spring 2004). ...................................................................................... 77

5. Effect of tested treatment on some enzymes activites and lignin content........ 79

5.1. Effect of tested treatment on peroxidase (PO) activity: .................................. 79

5.2. Effect of tested treatment on polyphenoloxidase activity: .............................. 75

5.3. Effect of tested treatment on chitinase activity:.............................................. 82

5.4. Effect of tested treatments on lignin content of cucumber plants................... 85

6. Chemical analysis: ...................................................................................... 90

6.1. Effect of foliar spraying with plant extracts, oils and K2HPO4 on sugar

content of infected powdery mildew cucumber plants: .................................. 90

6.2. Effect of spraying plant extracts, oils and K2HPO4 on phenol content of

infected powdery mildew cucumber plants: ................................................... 93

6.3. Effect of spraying plant extracts, oils and K2HPO4 on total amino acid

content of infected powdery mildew cucumber plants: .................................. 93

6.4. Effect of some biological control agents’ filtrate on sugar content of

infected powdery mildew cucumber plants: ................................................... 96

6.5. Effect of some biological control agents’ filtrate on phenol content of

infected powdery mildew cucumber plants: ................................................. 100

6.6. Effect of some biological control agents’ filtrate on total amino acid

content of infected powdery mildew cucumber plants: ................................ 103

5- DISCUSSION ........................................................................................... 105

6- SUMMARY .............................................................................................. 128

7- REFERENCES ........................................................................................ 139

ARABIC SUMMARY .......................................................................................

ACKNOWLEDGMENT

Firstly my unlimited thanks to “Allah”

A word of gratitude is not enough towards the great effort

and help that Prof. Dr. Abdou Mahdy Mohamed Mahdy,

professor of Plant Pathology, Vice-Dean of Faculty for

community Development and Environmental Affairs, Faculty of

Agriculture at Moshtohor, Zagazig University, Benha Branch did

in the whole work. He has been always patient, helpful and kind

hearted. His advices are my guide in work and life. He gave me

his time and effort to introduce this thesis in the best form and it

was a pleasure to work under his supervision.

The author wishes to express his deepest gratitude and

indebtedness to the senior supervisor of the present work Prof. Dr.

Mohammad Haroun Abd-El-Mageed, Professor of Plant

Pathology, Agric. Botany Dept., Fac. Agric., Moshtohor, Zagazig

Univ. Benha Branch for his constructive supervision, valuable

advice, kind guidance and for his help in putting thesis in its final

form.

I’m also indebted to Dr. Mahmmad Al-Sayed Hafez

Lecturer of Plant Pathology, Agric. Botany Dept., Fac. Agric.,

Moshtohor, Zagazig Univ. Benha Branch, for continuous help

offered during the course of this investigation.

I’m also indebted to Dr. Faten Mahmoud

Abd-El-Latef Lecturer of Plant Pathology, Agric. Botany Dept.,

Fac. Agric., Moshtohor, Zagazig Univ. Benha Branch, for

continuous help offered during the course of this investigation.

At last but not least, I am indebted to all staff members

and my colleagues at Fungus and Plant Pathology Branch,

Department of Botany Faculty of Agriculture at Moshtohor,

Benha Branch, Zagazig University, for their help and

encouragement and to everyone helped this work to arise.

Finally, I would like to express my gratitude to my father, my

wife and my brother and sisters for their encouragement and

patience during preparing this investigation.

Introduction 1

INTRODUCTION

Cucumber (Cucumis sativus L.) is one of the most important

economical crops, which belongs to family cucurbitaceae. The economic

importance of this crop appears in both local consumption and exportation

purposes. Cucumber is grown either in the open field or under protected

houses. The purpose of growing crops under protected house conditions is

to extend their cropping season and to protect them from adverse

conditions as well as diseases and pests (Hanam et al., 1978).

The total cultivated area increased rapidly, especially in the

reclaimed lands. According to the recorded data, obtained from the

Department of Agriculture Economic Statistics, Ministry of Agriculture,

A.R.E. 2003, the cultivated area of cucumber in 2003 growing season

reached about 11881 feddan in open field which yielded 88575 ton fruits,

in addition to 13267 greenhouses, yielded about 44771 ton fruits.

Cucumber powdery mildew caused by Sphaerotheca fuliginea

(Schlectend: Fr.) Pollacci, (lbrahim et al., 1985; Elad et al., 1989;

Ahmed, 1995 and Abd-El–Sayed, 2000), or by Erysiphe cichoracearem

Dc (Mostafa et al., 1990).

The disease can cause damage to all plant parts including leaves,

stems and fruits and causing considerable reduction of quantity and quality

of cucumber yields. Disease control is generally achieved by the use of

fungicides (Reuveni et al., 1996). The fungicides resistant races of the

pathogen have been reported (Schepers, 1983; McGrath, 1991; O’Brien,

1994; McGrath and Staniszewska, 1996). As well as the side effects of

fungicides on human health and in the environment were recorded (Eckert

& Ogawa, 1988; Horst et al., 1992; Garcia, 1993 and Durmusogle et al.,

1997).

Biological control of the disease using biological agents, such as

Trichoderma spp., Bacillus subtilis, Ampelomyces quisqualis and

Introduction 2

Pseudomonas flourcent for controlling foliar pathogenic fungi was

recorded by many researchers (Minuto et al., 1991; Heijwegen, 1992;

Urquhart et al., 1994; Ahmed, 1995; Abo-foul et al., 1996; Dik et al.,

1998; Verhaar et al., 1996; Abd-El–Sayed, 2000).

Recently, plant extract, and vegetable oils, such as neem

(Azadirachta indica), (Reynoutria sachalinensis), ginger (Zingiber

officinale), garlic (Allium sativum), onion (Allium cepa), clove oil

(Syzygium aromaticum), nigella oil (Nigella sativa), Olive oil (Olea

europaea) and rapeseed oil have been used to control powdery mildew

fungi (Singh et al., 1991; Ahmed, 1995; Daayf et al., 1995; Volf and

Steinhouer, 1997; Abd-El-Sayed, 2000; Haroun, 2002 and Tohamy et

al., 2002).

Also, numerous of reports demonstrated that resistance could be

induced in number of plants by prior treatment with some chemical

substrates. Also, it has been reported that some phosphate salts induce

systemic resistance against various pathogens including powdery mildew

of cucumber (Reuveni et al., 1993 and Reuveni et al., 1995)

Thus, the present work was conducted to founding non-chemical

alternatives to reducing fungicides use in the control of cucumber powdery

mildew disease under protected houses as follows:

1- Evaluation the efficacy of some bio-control agents, including

antagonistic fungi, bacteria and propolis extract to control cucumber

powdery mildew disease.

2- Using some plant extracts as foliar spray before infestation to

reduce or control the cucumber powdery mildew disease.

3- Effect of plant essential and/or volatile oils diluted with water and

used as foliar spray.

4- Evaluation of the dipotassium phosphate (K2HPO4) salt, as an

inducer agent, to induce a resistance against powdery mildew pathogen.

Introduction 3

5- Effect of Topas-100 fungicide at the recommended dose, beside

the double and half recommended doses as foliar spray with all previous

treatments to compare their effects with of natural substances was done.

6- Ability of mature conidia of Sphaerotheca fuliginea which

previously subjected to some inhibitor treatments, such as UV-ray, heat,

chloroform, on germination and disease infection was studied beside the

ability of non-germinated spores to induce resistance.

Review of Literature 4

REVIEW OF LITERATURE

Causal of cucumber powdery mildew disease:

Cucumber powdery mildew disease is attributed to two fungal species,

i.e . Sphaerotheca fuliginea or Erysiphe cichoraceaeum, which are belonging

to two different genera in the family Erysiphaceae (Schlosser, 1972).

Many investigators reported that Sphaerotheca fuliginea is the

causal of cucumber powdery mildew disease ( Reifschmider et al., 1985;

El-Mahjoub and Romdhani, 1991; Pan and More, 1996; Askary et al.,

1997; Tajika et al., 1997 and Bardin et al., 1997).

Meanwhile, some researchers recorded Erysiphe cichoracearum as

the main causal of the disease (Neshev and Aleksandrova, 1977;

Mostafa et al., 1990 and El-Shami et al., 1995).

Clare (1964 ( reported that the causal fungus of cucurbits powdery

mildew did not forming perithecia in most parts of the world, but usually

had fibrosin bodies inside the conidia, therefore it was identified as

Sphaerotheca fuliginea

Abul-Hayia and Trabulsi (1981) reported that the causal fungus of

powdery mildew on squash, melon, watermelon and cucumber in Saudi

Arabia did not forming cleistothecia and identified as Sphaerotheca

fuliginea, not as Erysiphe cichoracearum.

El-Kazzaz (1981) surveyed the powdery mildew of cucurbits in

various localities in of Egypt. He reported that no cleistothecia were

observed and the disease was caused by Sphaerotheca fuliginea, not by

Erysiphe cichoracearum.

Lebeda (1983) observed the powdery mildew disease of cucumber at

37 localities in Czechoslovakia under open field and greenhouse conditions.


Identification trials of the casual fungus were based on conidial germination

mode and presence of fibrosin bodies, which recognized two different

pathogens, i.e. Erysiphe cichoracearum and Sphaerotheca fuliginea.

Mazzanti-De Castanon et al. (1987) decided that powdery mildew

of watermelon, melon and cucumber was caused by Sphaerotheca

fuliginea in Northeast Argentina. They mentioned that most reliable

characteristics for identification were the absence of appressoria, the

presence of fibrosin bodies.

El-Mahjoub and Romdhani (1991) reported that the casual agent

of powdery mildew on cucumber in Tunisia was Sphaerotheca fuliginea.

The fungus was identified according to the presence of fibrosin bodies in

conidia.

Ahmed (1995( reported that powdery mildew disease on cucumber

in Egypt caused by Sphaerotheca fuliginea not E. cichoracearum.

Abd-El-Sayed (2000) reported that the casual agent of powdery

mildew on cucumber in Egypt was Sphaerotheca fuliginea, not E.

cichoracearum.

Chemical control:

Abol-Wafa et al. (1976( mentioned that the systemic fungicide

Benlate at the rate of 0.05% was more effective in controlling E.

cichoracearum on cucumber than the non-systemic fungicide Karathane at

the rate of 0.04%.

Docea and Fratila (1979 ( reported that Topsin M-70

(thiopenatemethyle) and Benlate (benomyl) at the rate of 0.05%, were

recommended in Romania to control cucumber powdery mildew. Good

results were also obtained with 0.05% Morestan (quinomethionate).


Hassan and Berger (1980 ( demonstrated that the fungicides

Triforine and Ditalimfos were selected against cucumber powdery mildew

caused by Sphaerotheca fuliginea.

Paulus et al. (1980) stated that excellent control of Sphaerotheca

fuliginea was given by either Ciba-Geigy 64250 or 64251. Benlate

(benomyl)+ Manzate 200 (manzeb) gave intermediate control.

Kolbe (1981) mentioned that Bayleton (triadimefon) and Bycor

(bitrlanol) gave very good control to powdery mildew on field grown

cucumber varieties. Both fungicides increased the yield by 16%.

Cartia and Riva (1983) reported that Triadimefon, Biloxazol and

fenarimol, sprayed every 12 days on 2-month-old plants, significantly

reduced incidence of powdery mildew caused by E. cichoracearum and

Sphaerotheca fuliginea.

Jumayli (1985) reported that the Bupirimate Nimrod, Benlate and

Thiovit were best fungicides for controlling powdery mildew disease of

cucurbit under greenhouse conditions in Iraq.

Paulus et al. (1986) stated that Bayleton, Benlate and Phaltan were

currently registered for controlling powdery mildew of cucurbits. They

found that Bayleton provided excellent control in 1982 trial, but less

control was noticed in 1984 and 1985.

Charifi-Tehrani (1987) stated that good control of cucumber

powdery mildew under field and greenhouse conditions, was obtained with

one application of Sulphur (high dose) or Sulphur + Tridemorph (lower

doses) or two application of Tridemorph or Benomyl.

El-Desouky (1988) reported that the most effective fungicide for

controlling cucumber powdery mildew was Flandor, followed by Sumi-8,


Sisihane, Bayfidan, Bayleton and Afugan. The least effective fungicides

were Karathane and Sofril.

Nakayama et al. (1989) reported that powdery mildew of cucumber,

caused by Sphaerotheca fuliginea, was effectively controlled with

Trifluzole in Japan.

Mohamed et al. (1990) found that Flandor, Sumi-8, Sisthane,

Bayfidan, Byleton and Afugan were the most effective fungicides against

cucurbit powdery mildew, while Karathane and Sofril were the least

effective once.

Mostafa et al. (1990) reported that in field trials single applications

of Rubigan (fenarimol), Byleton (triadimefon) and Nimrod (bupirimate) at

recommended doses gave good control against E. cichoracearum, the

casual organism of cucumber powdery mildew. They concluded that

fungicide application at short intervals or unnecessarily high dosages

caused a sharp decline in disease control.

Ohtsuka et al. (1991) studied the efficacy of commercial fungicide

thiophanate-methyl, dimethirimol, triadinsfon, chinomethionat and

machine oil against 4 isolates of Sphaerotheca fuliginea, isolated from

diseased cucumber plants. They found that chinomethionat and machine

oil were effective to control all of the tested isolates.

Iqbal et al. (1994) mentioned that best control of E. cichoracearum,

in greenhouse was given by spraying pyrazophos, while binomiyl,

carbendazim and bupirimate were gave moderate control effect.

El-Shami et al. (1995) reported that Karathane (dinocap), Anvil

(hexaconazol), Alto 100 (cyproconazole), Topaz 100 (penconazole) and

Afugan (pyarophos) were the most effective fungicides against powdery


mildew of cucumber, while flowable sulfur, Benlate (benomyl), Dorado

(pyrifenox) and Sumi-8 (dinoconazole) were the least effective once.

Ahmed (1995) found that Karathane, Bayfidan 48%, Soril

(El-Shalk 98%) and Sumi-8 were the most effective fungicides against

cucurbit powdery mildew, while Kema-Z, Flandor and Benlate were the

least effective once.

Abd-El-Sayed (2000) reported that Afugan and Karathane were the

most effective fungicides against powdery mildew of cucumber, while

Soril were the least effective once.

The alternative chemical control:

(1) Plant extracts:

Singh and Singh (1981) found that garlic (Allium sativum) cloves,

onion (Allium cepa) bulbs and ginger (Zingiber officinale) rhizome volatile

compounds completely inhibited spore germination of Erysiphe polygoni.

Singh and Singh (1983) indicated that adequate control of powdery

mildew (Erysiphe polygoni DC.) of pea (Pisum sativum L.). could be

achieved with 3 sprays at 20-day intervals of ginger extract, garlic oil,

dinocap, wettable S or carbendazim.

Klingauf and Herger (1985) reported that barley seedlings were

sprayed with extracts from plants indigenous or naturalized 3 d before

inoculation with Erysiphe graminis f. sp. hordei gave the best results of

reduction the disease than after inoculation. They suggested that an

induced resistance mechanism in the plants might be involved.

Bosshard et al. (1987) reported that root extracts of Rumex

obtusifolius significantly reduced infection by Podosphaera leucotricha

(the casual of powdery mildew on apple) on apple seedlings in the

greenhouse, but applications in the field were less effective.


Herger et al. (1988) reported that applications of aqueous and

ethanolic leaf extracts of R. sachalinensis on cucumber plants under

commercial conditions suppressed the infection with E. cichoracearum +

Sphaerotheca fuliginea and increased chlorophyll content in cucumber crop.

Herger et al. (1989) reported that leaf extracts of Reynoutria

sachalinensis significantly reduced the incidence of powdery mildew

(Uncinula necator) on the grape. Disease development on treated plants

was much slower than on untreated controls.

Herger and Klingauf (1990) observed that the protective treatment

with aqueous and ethanolic extracts of fresh or dried leaf material of R.

sachalinensis controlled Podosphaera leucotricha on apples, Erysiphe

polyphaga on Begonia, Sphaerotheca fuliginea on cucumbers, Plasmopara

viticola on grapes, Uromyces phaseoli [U. appendiculatus] on Phaseolus

vulgaris and Uromyces dianthi on carnations. The fungi exhibited decreases

in sporulation and hyphal density, while the host plants exhibited delayed

senescence and increases in chlorophyll contents, ethylene production and

various enzyme activities were found in treated plants.

Singh et al. (1991) using ginger (Zingiber officinale) rhizome

extract, (fresh and stored at 5 and 15°C for 1 month) at 10 000, 15 000 and

20000 ppm. was used to control powdery mildew (caused by Erysiphe pisi)

of peas under field conditions. The highest dose of the fresh extract gave

the best control.

Bosshard (1992) reported that Leaf extracts of H. helix inhibited

conidial germination of Venturia inaequalis in vitro, and in controlled

conditions, were effective against scab (V. inaequalis) (1% cold water

extracts provided >90% control) and moderately effective against powdery

mildew (Podosphaera leucotricha) on apple seedlings.


Qvarnstrom (1992) stated that sprayed cucumber plants with 5%

emulsion of garlic extract at 7 days intervals, reduce the infection with E.

cichoracearum from 83% to 10% and from 85% to 2% during spring and

summer season, respectively. Meanwhile the application 0.2-1.0% extract

of Equisetum arvense, 3% liquid soap and horse manure extract were less

effective in preventing infection.

Rovesti et al. (1992) found that, aqueous neem kernel extract was as

effective as sulfur against Sphaerotheca fuliginea, Erysiphe graminis f.sp.

tritici and E. graminis f.sp. hordei on courgettes, wheat and barley,

respectively, when they were applied before or after artificial inoculation

with the causal pathogens. They found also that, neem extract gave

significant control on wheat rust caused by Puccinia recondita f.sp. tritici.

Reimers et al. (1993) found that, tomato, rose and cucumber

sprayed with ajoene, a compound derived from garlic (Allium sativum),

protected plants against Oidium lycopersicum, Sphaerotheca pannosa var.

rosae and S. fuliginea, respectively.

Steck and Schneider (1993) found that, spraying cucumber plants

with Reynoutria sachalinensis extract was active as control mean against

Sphaerotheca fuliginea, the causal pathogen of powdery mildew.

Dik and Staay (1994) reported that spraying the susceptible and

partially resistant cucumber cultivars with 2% solution of Milsana (a

product containing leaf extracts of Reynoutria sachalinensis) at the rate

1500 and 3000 liters/ha. All treatments reduced disease severity of

Sphaerotheca fuliginea in greenhouse cucumbers. Although a significant

yield increase was only obtained when the susceptible cultivar was sprayed

at 3000 liters/ha.

Ahmed (1995) reported that disease severity of cucurbita powdery

mildew was decreased by spraying with each one of the plant extracts


tested i.e. black cumin, henna, eucalyptus, margosa, santonica, thyme and

garlic. However, garlic and henna extracts were the most effective in

inhibiting disease infection by S. fuliginea.

Cheah et al. (1995) tested the effect of Reynoutria extracts on the

incidence of powdery mildew caused by Sphaerotheca fuliginea on squash.

The results indicated that the treatment was significantly reduced powdery

mildew. No phytotoxicity was observed on treated plants

Daayf et al. (1995) applied an aqueous formulation of conc. extracts

from leaves of the giant knotweed (Reynoutria sachalinensis) weekly at a

concn of 2% to control of powdery mildew (Sphaerotheca fuliginea) on

cucumber. The result showed that the treatment of milsana extract was

effective as benomyl. This treatment significantly reduced the severity of

powdery mildew compared with control plants.

Singh et al. (1995) found that complete inhibition of conidial

germination of (Erysiphe pisi) was observed when ajoene (a compound

derived from garlic) was used at 25 mg/litre. Spray at 1000 mg/litre gave

control of powdery mildew in a growth chamber.

Subrata-Biswas et al. (1995) recorded that extract of Adhatoda

zeylanica was most effective in decreasing the severity of powdery mildew

(Phyllactinia corylea) in mulberry followed by extracts of Azadirachta

indica, Launaea coromandelica [Lannea coromandelica] and Oxalis

corniculata.

Paik-SuBong et al. (1996) found that in tests using cucumber plants

grown in a polyethylene film house, 100% control of Sphaerotheca

fuliginea was obtained using a wettable powder (30% a.i.) formulation of

Rheum undulatum [R. rhababarum] extract.


Raj-Kishore et al. (1996) found that plant extract of lawsone,

menadione and eugenol gave 100% inhibition of conidial germination of

powdery mildew (E. polygoni) of opium poppy (Papaver somniferum) using

a conidial germination technique at the lowest concentration (250 ppm.).

Daayf et al. (1997) indicated that cucumber plants infected with

Sphaerotheca fuliginea produce elevated levels of phytoalexins in

response to treatment with Milsana (containing extracts of Reynoutria

sachalinensis).

Nikolov and Andreev (1997) found that spraying rose plants with

unrefined cotton-seed oil at rates of 0.5-2% reduced the powdery mildew

disease incidence by 81-93%, respectively.

Pasini et al. (1997a&b) found that weekly sprays of wine vinegar

and neem [Azadirachta indica] extract (FU-3 Trifolio M) provided good

control against Sphaerotheca pannosa var. rosae on roses and they found

that the aqueous formulation of concentrated extracts from leaves of

Reynoutria sachalinensis did not provide adequate control.

Singh and Prithiviraj (1997) studied the activity of neemazal

against pea powdery mildew in detached leaf and intact plant experiments.

Growth of Erysiphe pisi was significantly reduced and a hypersensitive

reaction was induced in the host. Pre-inoculation treatment with neemazal

gave more effective control than post-inoculation application. Neemazal

led to increasing phenylalanine ammonia lyase activity in pea leaves.

Amadioha (1998) reported that cold and hot water extracts of leaves

of pawpaw reduced the growth of Erysiphe cichoracearum in vitro and

reduced its incidence on capsicum plants. Cold water extracts were more

potent than hot water extracts, suggesting that the bioactive extract could

be heat sensitive.


Konstantinidou-Doltsinis and Schmitt (1998) studied the efficacy

of plant extracts from R. sachalinensis against powdery mildew in

greenhouse grown cucumbers compared with a commercial preparation of

R. sachalinensis (Milsana) and two fungicides (myclobutanil and sulfur).

Leaf extracts of R. sachalinensis efficacy of the resistance inducing

extracts from R. sachalinensis reached approx. 90% and was comparable

with that of fungicide treatments. R. sachalinensis extract applications

enhanced yield up to 49%.

Prithiviraj et al. (1998) evaluated the efficacy of ajoene, a

constituent of garlic (Allium sativum), and neemazal, a product of neem

(Azadirachta indica), in the field individually and also in combinations.

Both the products at different concentrations (Neemazal 50, 150, 250 ml-1;

ajoene 100, 500, 750 ml-1 and their combinations) reduced disease

intensity of powdery mildew of peas as compared to control. Yield

parameters were largely significant. Combined treatments were not better

than the individual applications.

Nikolov and Boneva (1999a) reported that plant extracts (from

Compositae [Asteraceae] and Umbelliferae [Apiaceae]) were effective in

the control of Sphaerotheca fuliginea and Sphaerotheca pannosa var.

rosae on rose and cucumber.

Nikolov and Boneva (1999b) reported that plant extracts (from

Compositae [Asteraceae] and Umbelliferae [Apiaceae]) were effective in

controlling Venturia inaequalis and Podosphaera leucotricha on apple

seedlings.

Singh et al. (1999) studied the efficacy of rhizome powders of two

medicinally important plants, Zingiber officinale and Acorus calamus, for

their efficacy against Erysiphe pisi in vitro, under laboratory and field

conditions. A. calamus powder at 50% concentration (w/w) prepared in


talc (magnesium silicate) was found to be highly effective. Appressorium

formation was significantly reduced by 62.6% 12 h after inoculation.

Additionally, a reduction in the colony growth, number of germ tubes and

haustoria was observed. Both the powders stopped disease development in

the growth chamber. Foliar treatment with a 50% (w/w) formulated

product from A. calamus and Z. officinale reduced the disease intensity

from 80% to 9.2% and 45.3%, respectively. Furthermore, numbers of

nodes, pods and seed weight were increased. Rhizome powder treatment

performed almost as well as commonly used fungicides (Sulfex [sulfur

0.2%] and Bavistin [carbendazim 0.1%]).

Sindhan et al. (1999) compared the efficacy of extracts from 10

plant species with Neemadol (a neem product) at 0.25, 0.50 and 1.0% and

Karathane [dinocap] at 0.1% for the control of powdery mildew of pea (cv.

Boune-villa) caused by Erysiphe polygoni. The results showed that most of

the plant extracts at 30% significantly reduced the disease in comparison

with the control. Neemadol and extracts of Azadirachta indica, Allium

cepa, Allium sativum and Zingiber officinale were highly effective and at

par with dinocap in reducing disease intensity. The efficacy of the extracts

increased with increasing concentration.

Abd-El-Sayed (2000) found that the foliar application of some plant

extracts (thyme, henna, eucalyptus and garlic) individually or mixed

decreased powdery mildew (S. fuliginea) intensity on cucumber than

control when used before or after inoculation.

Konstantinidou-Doltsinis and Tzempelikou (2000) applied

extracts of 69 native and introduced plant species in Greece on the first leaf

of young cucumber plants, 2-3 days before artificial inoculation with a

conidia suspension of Sphaerotheca fuliginea, in order to test their efficacy

against powdery mildew of cucumbers. Extracts of Cassia septentrionalis


and C. xfloribunda flowers and pods at the concentration of 2.5% (w/v)

highly reduced the percentage leaf coverage by the pathogen. It was shown

that all Cassia extracts were more effective against the pathogen. The

obtained data clearly show the antifungal properties of C. septentrionalis

and C. xfloribunda flower and pod extracts in the cucumber S. fuliginea

pathosystem.

Konstantinidou-Doltsinis et al. (2001) reported that weekly

applications of a new liquid formulation prepared from Fallopia

sachalinensis (formerly Reynoutria sachalinensis) called Milsana (VP99),

resulted in significant reduction of infection by Sphaerotheca fuliginea and

Uncinula necator, respectively. Increase in yield (fruit weight) after

induction of resistance amounted up to 29.5% in cucumber (Milsana 0.5%)

and up to 50.5% in raisin grapes (Milsana 1%).

Vidyasagar and Rajasab (2001) tested different concentrations of

leaf extracts (1, 5, 10, 15, 20, 25, 50 or 75%) of neem, parthenium and bulb

extract of garlic in a field experiment to assess their effect on conidial

germination of Phyllactinia corylea and powdery mildew disease

development on mulberry (cv. M5) leaves. All concentrations of garlic

bulb extract, neem and parthenium leaf extracts exhibited inhibitory effect

on conidial germination. Foliar spraying with neem (parthenium

hysterophorus) and garlic extracts on mulberry leaves significantly

reduced the percent disease index (PDI) from 50 to 5.8, 51 to 7.4 and 52 to

0.6 percent, respectively. Garlic extracts showed maximum effects in

disease control followed by neem and parthenium.

Apablaza et al. (2002) evaluated the antifungal activity of the

saponins obtained from extracts of quillay (Quillaja saponaria) against the

powdery mildew (Erysiphe cichoracearum and Sphaerotheca fuliginea) of

cucumber (Cucumis sativus) under greenhouse conditions and squash


(Cucurbita maxima) under field conditions. For cucumber, the extract QL

1000 (8% saponin) reached a maximum disease control of 51.8% with an

average of 37.9%, while QL Ultra (16% saponin) only gave 27.8% disease

control with an average of only 15.8%. For squash, QL 30B (4.37%

saponin) reached a maximum control of 52.2% with an average of 34.7%.

The lower and medium dosages of QL 30B, i.e. 32 and 400 ppm, gave 49

and 42% control, respectively.

Cohen et al. (2002) sprayed extracts of Inula vcosisa at 0.00,

0.00125, 0.250, 0.05, 0.10, 0.20, and 0.40% to cucumber plants after

inoculation with powdery mildew [Sphaerotheca fuliginea] pathogens.

Disease control was 50% with 0.05-0.10% of the extract, and 95-96% with

0.40% of the extract.

Nada (2002) found that spraying squash plants with some plant

extracts increased total phenolic contents of the leaves. The hot water

extract of blue gum, leek and thyme were the best treatments in decreasing

squash powdery mildew infection and increasing total phenols.

Schmitt (2002) found that plant extracts from R. sachalinensis

induced local resistance in a variety of crops and against different plant

pathogens. In cucumber, tomato and grape and in ornamentals, infection

with powdery mildew (Sphaerotheca fuliginea) or grey mould can be

reduced to a large degree by regular application of the inducer. Since

treatment with this extract leads to changes in plant metabolism and since

the effects are dependent on the plant.

Tohamy et al. (2002) reported that spraying cucumber plants with

plant extracts of garlic and neem at 2.5, 5 and 10% concentration gave

significant reduction in powdery mildew disease incidence and severity.


(2) Plant oils

Ohtsuka and Nakazawa (1991) observed on conidia of glass slides

were sprayed with machine oil emulsion morphological changes observed

by light microscopy. Cucumber cotyledons were inoculated with conidia

and sprayed for examination by scanning electron microscope. The oily

film trapped the conidia, and treated conidia and hyphae were deformed,

preventing germination and growth.

Ohtsuka et al. (1991) observed that ultrastructural alterations

induced by spraying inoculated cucumber seedlings with machine oil

suspension included deformation of the Sphaerotheca fuliginea hyphae,

separation of the hyphal plasma membrane and degeneration of the

cytoplasm, leading to death of the treated hyphae.

Haberle and Schlosser (1993) sprayed the upper side of leaves of

cucumber with a fine mist of Telmion, a product containing 85% rapeseed

oil, 1 d before and 2, 4 and 6 d after inoculation with Sphaerotheca

fuliginea. After 12 d incubation, counts of pustules per leaf showed the

protective treatment to give a significant (P <0.05) reduction in disease

severity (efficacy >90%), with the curative treatment applied 6 days after

inoculation having almost as high an efficacy. Pustule diameter decreased

by 28% and 55% after protective and curative treatments, respectively, and

the treatments gave significant reductions in numbers of conidia per

pustule and conidia per leaf

Ragupathi et al. (1994) reported that treatment with neem oil and

neem seed kernel extracts reduce incidence of Powdery mildew of

Abelmoschus esculentus caused by E. cichoracearum.

Cheah et al. (1995) studied the effect of olive oil and rapeseed oil on

the incidence of powdery mildew caused by Sphaerotheca fuliginea on


squash. And found that olive oil and rapeseed oil significantly reduced

powdery mildew. No phytotoxicity was observed on treated plants.

Collina (1996) evaluated the effect of mineral oil and rape oil on the

incidence of powdery mildew caused by Sphaerotheca fuliginea on squash.

The results indicated that all treatments were significantly reduced

powdery mildew.

Raj-Kishore et al. (1996) found that clocimum oil [Ocimum

gratissimum] and lemongrass oil [Cymbopogon spp.], gave 100%

inhibition of conidial germination of powdery mildew (E. polygoni) of

opium poppy (Papaver somniferum) using a conidial germination

technique at the lowest concentration (250 ppm).

Pasini et al. (1997a&b) found that weekly sprays of a canola oil

(Synertrol) and a petroleum oil (JMS Stylet-Oil) provided good control

against Sphaerotheca pannosa var. rosae on rose.

Fiume (1997) reported that sweet pepper (Capsicum annuum) plants

cultivated in greenhouses, sprayed with tetraconazole fungicide alone or

combined with neem oil was the most effective reduction in disease incidence

and disease severity of powdery mildew caused by Leveillula taurica.

Steinhauer and Besser (1997) found that extracted vegetable oils

have strongly reduced the formation of powdery mildew pustules and

pustule size when sprayed on cucumber plants at concentration 1%, one

day before and six days after inoculation with S. fuliginea.

Yohalem (1997) stated that management of grey mould (Botrytis

cinerea) and powdery mildews (Erysiphales) in tomatoes, cucumbers and

potted roses can be achieved when applied rapeseed oil amended with

either sodium bicarbonate or an emulsifier.


Azam et al. (1998) demonstrated that a rape oil derivative gave good

control of the important grapevine disease, powdery mildew sprays of the

rape oil derivative at rates of 2.0 and 5.0 ml (formulated product) per liter

prevented the development of foliar symptoms as effectively as either

wettable sulfur 2 g (formulated product) per litre or fenarimol 0.2 ml

(formulated product) per litre at label rates.

Nikolov (2000) investigated the efficacy of the new formulated

vegetable oil fungicide (mustard) from Cruciferae towards powdery

mildew (caused by Sphaerotheca fuliginea) and its effect on pollen

germination of cucumber under laboratory and greenhouse conditions. In

in vitro tests, the efficiency of mustard oil, applied at 0.1-15%, was

between 31-100% technical efficiency. In the greenhouse conditions, the

mustard oil at 1% showed higher activity than standards dinokap. Karatan

35 LS and moisten sulfur Tiovit 80 WP Mustard at concentrations higher

than 1% inhibit the germination of pollen tubes, and destroyed pollen cells.

Mustard at 0.1-0.7% did not show negative effect on pollen germination.

Nada (2002) reported that volatile oils as film on slides completely

prevented spore germination of Sphaerotheca fuliginea. Also, spraying

squash plants in greenhouse and field with eight essential oils as

preventative and curative treatments gave sufficient control to disease in

most cases. Thyme essential oil completely prevented the disease

incidence in the field.

Wojdyla (2002a) evaluated the efficacy of oils from rape, sunflower

seed (vegetable oil) and paraffin (Atpolan 80 EC) in controlling

Sphaerotheca pannosa var. rosae causing rose powdery mildew in the

greenhouse or in a plastic tunnel. The oils were applied curatively as plant

spray 4-times at 7-day-intervals at concentrations ranging from 0.25 to 4%.

All the oils controlled the fungal pathogen. The efficacy of the tested oils


increased with their concentrations. Paraffin oil was better in the control of

S. pannosa var. rosae than the vegetable oils.

Carneiro (2003) reported that neem oil was effective as the

fungicide normally used for controlling of tomato powdery mildew caused

by Oidium lycopersicum under greenhouse conditions.

Ko et al. (2003) found that plant oils canola oil, corn oil, grape seed

oil, peanut oil, safflower oil, soyabean oil or sunflower oil at 0.1% were

greatly reduced the severity of tomato powdery mildew caused by Oidium

neolycopersici. Sunflower oil was the most effective in the control of

powdery mildew. Scanning electron microscopy showed that control of

powdery mildew with sunflower oil resulted mainly from the inhibition of

conidial germination and suppression of mycelial growth of the pathogen.

(3) Biological control

Bosshard et al. (1987) found that spore suspensions and culture

filtrates of Chaetomium spp. reduced scab and powdery mildew infections

on apple seedlings in some experiments.

Heijwegen (1988) tested 17 mycoparasites, (19 isolates) for their

ability to control sporulation of Sphaerotheca fuliginea on cucumber

leaves in growth chamber experiments. More than half of the species

reduced the proportion of healthy conidiophores to <10%. Tilletiopsis

albescens gave the best control (almost 100%), followed by Ampelomyces

quisqualis. Certain disadvantageous characteristics possessed by several

species are discussed; as a result T. albescens, A. quisqualis and

Aphanocladium album were selected for use in greenhouse experiments.

Jarvis et al. (1989) reported that Sporothrix flocculosus and

Sporothrix. rugulosus colonized and killed Sphaerotheca fuliginea on leaf


discs of cucumber. The antagonists caused complete collapse of the

mycelium and conidia with no sign of hyphal invasion.

Verhaar and Hijwegen (1993) reported that an isolate of

Verticillium lecanii was highly antagonistic against Sphaerotheca

fuliginea the causal of cucumber powdery mildew.

Vozenilkova et al. (1995) applied Trichoderma harzianum T3 at 100

ml as suspensions of 105 spores/ml through watering to each plant. The

application of T. harzianum antagonists increased yield and inhibited

development of S. fuliginea.

Abo-Foul et al. (1996) investigated the biocontrol of cucumber

powdery mildew under greenhouse conditions. Verticillium lecanii and

Sporothrix rugulosa were applied to cucumber plants. Verticillium lecanii

reduced powdery mildew considerably on cucumber plants in comparison

with Sporothrix rugulosa.

Bettiol et al. (1997) reported that application of concentrated

metabolites of Bacillus subtilis 1 and 24 h before or after inoculation of

Sphaerotheca fuliginea (3 x 104 conidia/ml) reduced the number of lesions

on cucumber leaves by 90-99%.

Pasini et al. (1997a&b) found that weekly sprays of the

mycoparasite Ampelomyces quisqualis (AQ10 Biofungicide), provided

good control against Sphaerotheca pannosa var. rosae on roses.

Verhaar et al. (1997) studied the effect of timing of the application

with Verticillium lecanii, on cucumber powdery mildew (caused by

Sphaerotheca fuliginea). The timing of application with V. lecanii is

important to achieve good control.


Vogt and Buchenauer (1997) reported that single soil drench or

seed treatment with fluorescent Pseudomonas strains (BS8651) reduced the

severity of powdery mildew caused by Sphaerotheca fuliginea.

Askary et al. (1998) tested the antagonistic effect of three strains of

Verticillium lecanii against Sphaerotheca fuliginea the causal of cucumber

powdery mildew. They stated that strain 198499 gave the best result in

controlling the disease under greenhouse condition.

Dik et al. (1998) applied Ampelomyces quisqualis, Verticillium lecanii

and Sporothrix flocculosa as a biocontrol against S. fuliginea on susceptible

and a partially resistant cucumber cultivars. They found that Sporothrix

flocculosa gave the best disease control followed by Verticillium lecanii

meanwhile Ampelomyces quisqualis had no effect on the pathogen.

Application with S. flocculosa reduced disease in the partially resistant

cultivar to the same level as a treatment in which the fungicides bupirimate

and imazalil. Yields in the treatment with S. flocculosa were not significantly

different from those in the fungicide treatment.

Elad et al. (1998) stated that Trichoderma harzianum T39 spray (as

TRICHODEX) reduced severity Sphaerotheca fusca (the casual of

powdery mildew in greenhouse cucumber) by up to 97% in younger leaves

but its efficacy declined to 18-55% control in older leaves. On the other

hand applying Ampelomyces quisqualis (AQ10) achieved up to 98% of

control of S. fusca and it retained significant control capability on older

leaves. They suggested that the mode of action of T. harzianum T39 in

powdery mildew control was induced resistance, not mycoparasitism or

antibiotic action.

Elad et al. (1999) indicated that the application of T. harzianum T39

conidia to the root zone of plants resulted in the reduction of foliar grey


mould, white mould and powdery mildews. The modes of action of T.

harzianum T39 are competition with the pathogen for nutrients and space,

suppression of hydrolytic enzymes of the pathogen and induced host

resistance.

El-Hafiz Mohamed (1999) mentioned that some mutants of

Tilletiopsis washingtonensis reduced powdery mildew infection caused by

Sphaerotheca fuliginea on greenhouse cucumber. Scanning electron

microscopy of treated mildewed leaves indicated that hyphae appeared

shrunken and collapsed in comparison with the turgid hyphae on untreated

plants.

Schmitt et al. (1999) compared the antifungal activity of Bacillus

brevis and its antifungal metabolite, gramicidin S against powdery mildew

of cucumbers caused by S. fuliginea. In in vivo studies on cucumber plants

Bacillus brevis cultures reduced the disease intensity of S. fuliginea

significantly when applied one day before or after inoculation, with the

latter showing stronger effects (average of 40 % efficacy). In vitro studies

with conidia of S. fuliginea revealed that the antifungal metabolite

gramicidin S inhibited conidial germination by around 80 %. The results

indicate that Bacillus brevis has the potential to be used as a biocontrol

agent against S. fuliginea and other plant pathogens.

Seddon and Schmitt (1999) recorded that both the bacterial

biological control agent Bacillus brevis and plant extracts from Reynoutria

sachalinensis have been shown to control Botrytis cinerea (grey mould)

and Sphaerotheca fuliginea (powdery mildew), respectively. That act

directly against B. cinerea conidial germination and to some extent

mycelial growth. Plant extracts of R. sachalinensis act indirectly via

inducing resistance in the plant. B. brevis also inhibited S. fuliginea in vitro

and in vivo. Studies with S. fuliginea indicate that integrated biological


control with high efficacy is achievable with a combination of the 2

biocontrols used at lower levels than when used separately.

Elad (2000) reported that the biocontrol agent Trichoderma

harzianum isolate T39 controls the foliar pathogens, Botrytis cinerea,

Pseudoperonospora cubensis, Sclerotinia sclerotiorum and Sphaerotheca

fusca (syn. S. fuliginea) in cucumber under commercial greenhouse

conditions. Involvement of locally and systemically induced resistance has

been demonstrated. Cells of the biocontrol agent applied to the roots, and

dead cells applied to the leaves of cucumber plants induced control of

powdery mildew. A combination of several modes of action is responsible

for biocontrol. They found that biocontrol agent has the potential to

degrade cell-wall polymers, such as chitin.

Seddon et al. (2000) observed that Brevibacillus brevis (formerly

Bacillus brevis) inhibits a range of fungal plant pathogens in vitro

including Botrytis cinerea, Sphaerotheca fuliginea and Pythium ultimum.

Bacillus brevis has two modes of antagonism: the antifungal metabolite,

gramicidin S, and a biosurfactant that reduces periods of surface wetness.

Romero et al. (2001) studied that the biological controls abilities of

two mycoparasitic fungi, Acremonium alternatum and Verticillium lecanii,

against cucurbita powdery mildew by in vitro assays on detached melon.

They suggested that both mycoparasites, when applied in early curative

treatments, are interesting for biological control of melon powdery mildew.

Brand et al. (2002) studied the interaction between L. taurica and

the biological control agents Trichoderma harzianum T39 (TRICHODEX)

and Ampelomyces quisqualis (AQ10). The biological control agents were

more effective in disease control.


Lima et al. (2002) evaluated the antagonistic activity of the yeasts

Rhodotorula glutinis, Cryptococcus laurentii and Aureobasidium pullulans

against powdery mildew of cucurbits (Sphaerotheca fusca; syn. S. fuliginea).

The antagonists significantly reduced the disease incidence on leaves,

showing an activity comparable to that of the fungicide penconazole.

Wojdyla (2002b) found that under greenhouse conditions, Bacillus

polymyxa [Paenibacillus polymyxa] strongly inhibited the spread of

powdery mildew on rose, caused by S. pannosa var. rosae. In vivo

experiments garlic juice and mineral oil decreased the disease.

El-Desouky (2004) evaluated Telletiopsis pallescens as a biocontrol

agent against powdery mildew (Sphaerotheca fuliginea) on squash and

cucumber. The results showed that spore suspension or culture filtrate of T.

pallescens provided complete control of powdery mildew on both squash and

cucumber plants. Both hosts treated with a spore suspension or culture filtrate

had a significant reduction in the severity of powdery mildew infection

compared with plants treated with distilled water or those untreated. Also, the

density of S. fuliginea conidia was significantly reduced.

(4) Phosphate salts

Descalzo et al. (1990) compared the efficacy of various inducers of

systemic resistance against three diseases of cucumber. Dibasic and

tribasic phosphate and oxalic acid were tested as resistance inducers

against cucumber anthracnose, gummy stem/leaf blight and powdery

mildew caused by Sphaerotheca fuliginea on field cucumber cultivars

under laboratory conditions. All treatments were ineffective against

powdery mildew under simulated commercial greenhouse conditions.

Reuveni et al. (1993) sprayed the upper surface of the first true leaf

of cucumber plants with 100mM solutions of K2HPO4, KH2PO4, Na4P2O7


and Na2PO4 2 hours before inoculation with a conidial suspension of

Sphaerotheca fuliginea. They observed that a single spray of any of these

solutions induced systemic protection to powdery mildew in leaves 2 and 3.

Application of Na2HPO4 had little or no effect however, a mixture of

KH2PO4 and Na2HPO4 sprayed on leaf 1 markedly induced systemic

resistance on leaves 2 and 3. Spraying K2HPO4 on leaf 1 at the same

concentration at 96, 48 and 2 hours before inoculation induced 74, 76 and

96%, respectively, of systemic protection in the number of powdery

mildew pustules per plant compared with plants sprayed with water.

Inductions with K2HPO4 or KH2PO4 were consistently the most effective

for inducing systemic protection.

Gamil (1995) stated that foliar spraying of squash plant with CoSO4

at the 1st true leaf stage induced resistance of pot-grown squash plants to

natural infection by S. fuliginea. Infection decreased with increase in spray

concentration from 0.025 to 0.1mM. Sprays of K2HPO4 were most

effective at 6mM. Cobalt sulfate treatment reduced peroxidase and

polyphenol oxidase activity in detached squash leaves after inoculation.

Potassium phosphate decreased polyphenol oxidase activity but increased

peroxidase in detached leaves 48 hours after inoculation.

Reuveni and Reuveni (1995) recorded that foliar sprays of 0.025M

and 0.04M solutions of K2HPO4 and KH2PO4 + KOH (both plus Triton

X-100) and commercial systemic fungicides inhibited development of

powdery mildew fungi on fruit clusters, flower clusters, fruits and leaves of

field-grown grapevines, mango and nectarine. The effectiveness of

phosphates in controlling powdery mildew on berries of chardonnay

grapevines was similar to that of the systemic fungicide pyrifenox (Dorado

480 EC). However, the systemic fungicides diniconazole (Marit 12.5%

WP), myclobutanil (Sisthane 12E) and penconazole (Ophir), were more


effective in controlling the disease on inflorescences of mango and fruits of

nectarine, respectively, than either phosphate. The inhibitory effectiveness

of phosphate salts makes them useful as ‘biocompatible’ fungicides and

ideal foliar fertilizers for field application for disease control.

Reuveni et al. (1995) found that a single spray of 0.1 M solution of

phosphate (K2HPO4, KH2PO4, NH4H2PO4) or potassium (KCl, KNO3,

K2SO4) salts on the upper surface of the first true leaf of cucumber, before

inoculation with Sphaerotheca fuliginea induced systemic protection of

powdery mildew on leaves 2-5 up to 94%. The protection on the upper

leaves remained efficient up to 25 days after inoculation regardless of the

high concentration of challenge inoculum of S. fuliginea. Post-inoculum

application of phosphate on the first leaf induced systemic protection

against powdery mildew on upper leaves, even when sprayed 4 days after

inoculation. It is concluded that the efficiency of induction of systemic

protection and curative properties of phosphate and potassium fertilizers

can be considered for disease control in the field.

Collina (1996) reported that monopotassium phosphate reduced the

percentage area of leaves infected by Sphaerotheca fuliginea compared

with that of untreated plants.

Mosa (1997) examined the effect of various potassium phosphate

salts, applied as foliar spray treatments, for controlling powdery mildew of

cucumber (Sphaerotheca fuliginea). Cucumber plants were treated with

aqueous solutions (25 or 50mM) of KH2PO4, K2HPO4 and K3PO4, either 2

days before or 3 days after inoculation. All phosphate salts reduced

powdery mildew development on cucumber. The most effective treatments

were K2HPO4 and K3PO4 showing both protective and curative effects

against S. fuliginea infection. Resistance in the second true leaf of

cucumber to powdery mildew was induced following treatment of the first


true leaf with K2HPO4, K3PO4 and KH2PO4, respectively. Powdery

mildew infection was significantly reduced by 92% when the plants were

treated with 50mM K2HPO4, 3 days after inoculation. Production of

conidia was greatly reduced on phosphate treated leaves. K2HPO4

treatment caused a remarkable increase of peroxidase activity in both

infected and non-infected control plants. K2HPO4 treatment caused

significant inhibition of length of secondary hyphae, density of surface

hyphae and conidiophore and conidial production. These results provide

further evidence that phosphate salts could induce resistance and provide a

curative effect against cucumber powdery mildew.

Pasini et al. (1997a&b) found that weekly sprays of the mineral salt

KH2PO4, of potassium salts provided good control against Sphaerotheca

pannosa var. rosae on roses,

Abd-El-Kareem (1998) reported that spraying cucumber plants

with phosphate (K2HPO4) at concentration 100 mM/L reduce the severity

of cucumber powdery mildew (S. fuliginea) and significantly increase fruit

yield by 178.5% compared with plants treated with distilled water.

Orober et al. (1998) recorded that foliar application of phosphate

induced systemic acquired resistance (SAR) in cucumber )Cucumis sativus)

against anthracnose (Colletotrichum lagenarium [C. orbiculare]), and

powdery mildew (Sphaerotheca fuliginea). The maximum levels of

protection were 97% and 56%, respectively. Effective SAR induction with

phosphate salts was strictly dependent on the formation of necrotic lesions

on the treated leaves. Localized cell death on the inducer leaves associated

with the generation of reactive oxygen species (oxidative burst) was

detected within 48h after treatment. In phosphate treated plants an

accumulation of salicylic acid (SA) was measured. Besides a high content

in the treated leaves a significant accumulation at a lower level was also


evident in the systemically protected leaves. As a further consequence of

phosphate application, activities of typical defense-related enzymes like

peroxidase (POX) and polyphenoloxidase (PPO) increased in all parts of

the induced plants. It is assumed that phosphate as an abiotic SAR inducing

agent triggers local and systemic defense mechanisms in cucumber plants

by the same mode of action as a necrotising pathogen.

Reuveni et al. (1998) found that the foliar spray of 1% (w/v)

solution of mono-potassium phosphate (MKP) (KH2PO4) on the upper

surfaces of lower leaves of greenhouse-grown peppers (Capsicum)

induced local and systemic control of Leveillula taurica. This protection

was expressed by a reduction in the leaf area covered with sporulating

colonies, and in conidial production on leaf tissue, 24 or 48 hours

post-treatment when MKP was applied on the lower leaves of plants that

had been exposed to the source of inoculum. Foliar application of MKP,

initiated before or after exposure to heavily diseased plants as the source of

inoculum, was effective in controlling powdery mildew. The efficacy of

MKP was compared with a sterol-inhibiting systemic fungicide. Both

treatments significantly inhibited powdery mildew compared with

non-treated controls. Phosphate solutions were not phytotoxic to plant

tissues when compared with the fungicide treatment. It is suggested that

MKP may be applied as an alternative practice for the control of powdery

mildew in peppers.

Ehret et al. (2002) reported that foliar applications of a number of

inorganic fertilizer salts were found to significantly reduce powdery mildew

[Erysiphe orontii] on greenhouse tomato (Lycopersicon esculentum) leaves.

In a series of single-application experiments, the foliar applications, each

with 0.1% surfactant, were applied to the third and fourth leaves of young

tomato plants 24 hours before inoculation with an atomized application of


mildew conidia. Control treatments consisted of a water application and a

water plus surfactant application. Powdery mildew colonies were counted

7–10 days later. Surfactant alone significantly reduced mildew colony

numbers. CaCl2, Ca(NO3)2, and K2HPO4 reduced colony counts compared

with the surfactant alone. Surfactant alone was not as effective as in the

single-application treatments, often having no effect. All the Ca-salt

treatments that were effective in the single-application series were effective

as multiple applications. Repeated applications of combinations of Ca salts

were often just as effective as applications of elemental sulfur (S), KCl,

MgSO4 and K2HPO4 also significantly reduced mildew counts with multiple

applications. This study did not attempt to explain the differences or

similarities in efficacy of the salts tested; both osmotic (concentration) and

specific-ion effects could play a role.

Mosa (2002) determined the efficacy of 25 or 50mM monobasic,

dibasic and tribasic potassium phosphate as pre- or post-inoculation foliar

sprays in controlling powdery mildew caused by Erysiphe betae in

sugarbeet. All treatments reduced incidence of powdery mildew compared

to the control. Potassium phosphate salts at 25mM recorded higher crop

protection compared to 50mM. Potassium phosphate monobasic and

dibasic at 25mM exhibited curative and protective effects against E. beta.

Single foliar spraying of 25 or 50mM monobasic and dibasic potassium

phosphate salts on the lower leaves induced systemic resistance in the

upper leaves. Peroxidase activity was higher in treated than untreated

sugarbeet plants.

El-Habbak (2003) reported that spraying squash plants with

phosphate (KH2PO4) reduce the severity of squash powdery mildew (S.

fuliginea) compared with plants treated with distilled water.


(5) Propolis activity:

La-Torre et al. (1990) mentioned that, the alcoholic solutions of

propolis exhibited fungicidal activity against Botrytis cinerca, and the

effect was proportional to the concentration of propolis.

AbdulSalam (1995) studied the bioactivity of five concentrations

(0-800 ppm) of propolis ethanol extracts (PEE) against ten soil borne fungi

(Fusarium solani, F. monilform, F. oxysporum, F. xylairoides, Diplodia

phoenicis, Rhizoctonia solani, Alternaria alternata, Botrytis sp.

Helminthosporium sp. and Curvularia lunata). The fungi were isolated

from date palm and other plants. The results indicated that, the growth

diameter of the tested fungi decreased significantly with each increase in

PEE concentration. The higher concentration of PEE (800 ppm) was more

effective than lower concentrations against all the tested fungi. The

greatest decrease in growth diameter was observed in F. solani, Botrvtis sp.,

C. lunata and Helminthosporium sp.

Garibaldi et al. (1995) reported that propolis showed moderate

effect against the powdery mildew disease (Sphaerotheca fuliginea) in

zucchini.

Giuseppe Lima et al. (1998) reported that propolis (0.5% w/v)

showed a high antifungal activity, particularly against B. cinerea in vitro

and significantly reduced the infections caused by B. cinerea and/or P.

expansum in vivo.

(6) Cross protection:

Mahmoud et al. (1995) revealed that spraying faba been plants with

non viable spores of Botrytis fabae led to significant protection of faba

been plants against chocolate spot disease. They also found that, spores

were killed by instant subjection to hot water (80-90癈 ) were more


effective in controlling the disease compared with spores killed by

autoclaving at 121°C for 10 minutes.

Attiatalla et al. (1998) reported that Fusarium spp. which was

non-pathogen to tomato acted as effective inhibitor to tomato wilt

pathogen Fusarium exysporum f.sp. lycopersici in vitro and under

greenhouse condition.

Abd-El-Kareem (1998) reported that spraying cucumber plant with

Fusarium oxysporum f.sp. niveum, which was non pathogen to cucumber

showed significant effect in reducing the powdery mildew (Sphaerotheca

fuliginea) disease incidence.

Abd-El-Moneim (2001) reported that spraying cucumber plants

with powdery mildew spores killed with UV for 30 minutes were more

effective in inducing resistance to powdery mildew (Sphaerotheca

fuliginea) compared with spores killed by heat 90癈 for 10 minutes or 1 ml

chloroform/L.

(7) Combination between different control agents

Horst et al. (1992) reported that powdery mildew (caused by

Sphaerotheca pannosa var. rosae) and black spot (caused by Diplocarpon

rosae) were significantly controlled by weekly sprays of 0.063M aqueous

solution of sodium bicarbonate plus 1.0% (v/v) Sunspray ultrafine spray

oil on Rosa spp.

Steinhauer and Besser (1997) studied the effect of spraying

cucumber plants with formulated vegetable oils at a concentration of 1%

one day before and six days after inoculation with Sphaerotheca fuliginea,

respectively. He observed strongly reduce in the formation of pustules and


pustule size. The addition of 0.2% sodium bicarbonate slightly increased

the effect on the powdery mildew in curative treatments.

Verhaar et al. (1999) found that V. lecanii formulated with arachid

oil showed significantly better control of cucumber powdery mildew

(Sphaerotheca fuliginea) than without. A concentration of 0.5% arachid oil

was somewhat toxic to mildew but 0.05% was not.

Casulli et al. (2000) found that both sodium bicarbonate (0.5%) and

mineral oil (1%) proved to be effective in keeping infections of powdery

mildew, caused by Sphaerotheca fuliginea, grown under glasshouse

conditions and artificially inoculated with S. fuliginea under control. These

compounds showed a remarkable effectiveness when used in combination.

The best results were achieved when the plants were treated after infection

but before the disease appeared.

Singh et al. (2000) found that seed bacterization by Pseudomonas

fluorescens and P. aeruginosa alone and in combination with aerial spray

of their cell suspensions or Neemazal, a product of neem (Azadirachta

indica), at different concentrations controlled powdery mildew (Erysiphe

pisi) of pea through induced resistance in pea. A combination of seed

bacterization with either aerial spray of bacterial cell suspensions or

Neemazal was more effective in controlling the disease than seed

bacterization alone. Bacterization by both bacteria and aerial spray of

Neemazal increased the dry weight of aerial parts, number of nodes and

pods as well as seed weight of pea plants.

Konstantinidou-Doltsinis et al. (2002) investigated the improvement

of the effectiveness of Milsana (an extract from Reynoutria sachalinensis)

against cucumber powdery mildew (Sphaerotheca fuliginea) and grey mould

(Botrytis cinerea), when combined with other control methods. Cucumber

plants were treated with either Milsana, Pseudozyma flocculosa (syn.


Sporothrix flocculosa) or Brevibacillus brevis as independent or combined

treatments. All treatments with Milsana significantly reduced powdery

mildew severity in all trials. Milsana alone or in combination with P.

flocculosa increased the number and weight of harvested fruits. The three

control agents generally reduced powdery mildew.

Allan et al. (2003) evaluated the efficacy of Brevibacillus brevis

against powdery mildew of cucumber caused by Sphaerotheca fusca.

Brevibacillus brevis was evaluated singly and in combination with a plant

extract of Reynoutria sachalinensis that induces resistance mainly against

powdery mildew. Brevibacillus brevis reduced the disease. Significant and

increased control of S. fusca was achieved using the Brevibacillus brevis

and R. sachalinensis combination.

El-Gamal (2003) reported that spraying cucumber plants with

Sacchromyces cerevisiae as single treatment or combined with potassium

phosphate as (KH2PO4 or K2HPO4 at 50 mM) reduce cucumber powdery

mildew (Sphaerotheca fuliginea). The most effective treatments were

KH2PO4 and K2HPO4 at 50 mM combined with Sacchromyces cerevisiae

which reduce cucumber powdery mildew by 68.4 and 63.2% and increased

fruit yield by 63.8 and 57.1% respectively.

Materials and Methods 35

MATERIALS AND METHODS

1- Survey of cucumber diseases under commercial protected

house conditions:

Diseases of cucumber plants grown under protected house

conditions in two different locations at Kalubia Governorate, Tukh and

Kaha were recorded, in spring season 2003 to determining the most

important of diseases that attack cucumber plants.

2. Assessment of cucumber disease

2.1. Powdery mildew

Powdery mildew scale from 0 to 5 according Descalzo et al.(1990)

with slight modification was used to assessment the disease, where 0 = no

powdery mildew colony, 1 = 1-25, 2 = 26-50, 3 = 51-75, 4 = 76-100 and

5 = more than 100 colonies/leaf.

Disease severity (%) = value)rating(highest leaves) no. (Total

(100) category) ratingin leaves (no. no.) rating[

Percentage of disease = No. of infected leaves

X 100 Total number of leaves

2.2. Root diseases and virus infection

Fusarium wilt, root-rot, root knot nematode and virus infection

were recorded as percentage of diseased plants as follows:

Percentage of diseased plants = No. of diseased plants

X 100 Total number of plants

3. Tested treatments:

3.1. Plant extracts

Three plants i.e. garlic (Allium sativum) bulb, cloves (Syzygium

aromaticum) dry flowers and withania (Withania somniferum) leaves

were used. 100 g of each plant material were cut, placed in a blender in

Materials and Methods

36

sterilized distilled water at the ratio of 1:1 w/v and blended for 10

minutes. The plant material residues were filtered through cheesecloth.

The filtrates were centrifuged at 3000 rpm for 10 minutes and separated

to obtain the extract. The extracts were kept under freezing (-20C) until

used (Abd-El-Sayed, 2000).

Garlic extract was used at concentration 20, 10 and 5%, clove

extract was used at concentration 10, 5 and 2.5% while withania extract

was used at concentration 50, 25 and 12.5%.

3.2. Plant oils:

Four different oils i.e. clove oil (Syzygium aromaticum), nigella oil

(Nigella sativa), olive oil (Olea europaea) and rocket oil (Eruca sativa)

were obtained from the market and used at different concentrations.

Clove oil was used at concentration 10, 5 and 2.5%, nigella oil,

olive oil and rocket oil were used at concentration 8, 4 and 2%. The tested

oils were diluted with sterilized distilled water.

3.3. Biological control agent and propolis extract:

Trichoderma harzianum and Bacillus subtilis were obtained from

Biological Control Res. Dept. Agric., Res. Center Giza, Egypt.

Trichoderma harzianum was grown on gliotoxin fermentation medium

(GFM) under complete darkness just to stimulate toxin production (Abd-

El-Moity and Shatla, 1981) for 9 days, Meanwhile bacteria Bacillus

subtilis was grown on nutrient agar (NG) broth for 48 hours. whereas

culture filtrates of Trichoderma and Bacillus were collected. The obtained

filtrates were centrifuged for 15 minutes at 4000 r.p.m. to separate the

fungal or bacterial growth, The concentration of T. harzianum spore

suspension was adjusted to 3x107 spore/ml, meanwhile the concentration

of B. subtilis cell suspension was adjusted to 3x107 cell/ml (Abd-El-

Moneim 2001).


Water extract of propolis was prepared using five grams of the

specimens were mixed with 100 ml of deionized water and the water

level marked on the tubes, then shaken at 95°C for 2 h, and cooling to

room temperature, water was added to the marked level and the contents

centrifuged to obtain the supernatant. Propolis extract was used at

concentration 5g/liter.

The tested biological agents and their culture filtrates as well as

propolis extract were used singly or in combination as follows:

Propolis extract, Trichoderma filtrate, Trichoderma spore

suspension, Bacillus filtrate, Bacillus cell suspension, propolis extract +

Trichoderma filtrate, propolis extract + Trichoderma spore suspension,

propolis extract + Bacillus filtrate, propolis extract + Bacillus cell

suspension, Trichoderma filtrate + Bacillus filtrate, Trichoderma spore

suspension + Bacillus cell suspension, propolis extract + Trichoderma

filtrate + Bacillus filtrate and propolis extract + Trichoderma spore

suspension + Bacillus cell suspension.

3.4. Phosphate was tested with solution of K2HPO4 at concentrations 50,

75 and 100 mM/L. ( 8.7g/L, 13.05g/L and 17.4g/L) as foliar spray.

3.5. Topas-100(R)

(10.0% penconazole “w/v” [(R,S-1-(2-(2,4-

dichlorophenyl) -Q pentyl)-1H-1,2,4-triazole]) was used as fungicidal

compared treatment and tested at concentrations 12.5cm3/100L,

25cm3/100 and 50cm

3/100L..

(R) = Registered trade mark of NOVARTIS Limited, Switzerland.

Manufactured by NOVARTIS AGROEGYPT, local record number 295.

4. Laboratory experiments:

These experiments were conducted under laboratory conditions

at the Agric. Bot. Dept., Pl. Pathol. Branch, Fac. Agric., Moshtohor

Zagazig Univ., Benha Branch.


38

4. Conidial germination:

4.1. Effect of plant extracts on germination of Sphaerotheca fuliginea

conidia.

According to the methods described by Nair et al. (1962), Powdery

mildew conidia of Sphaerotheca fuliginea (Schltdl.) Pollacci, were

harvested from only young leaves of cucumber. To avoid the presence of old

conidia, lesions were gently shaken first by a glass rod to discard any old

conidia presented on such young leaves. The new conidia, which formed on

conidiophores after four to six hours, were spread on dry clean glass slides

previously received 0.1 ml. From each one of the previously prepared plant

extracts garlic extract at concentration 20, 10 and 5%, clove extract at

concentration 10, 5 and 2.5% withania extract at concentration 50, 25 and

12.5% and Topas-100 at 12.5 ppm, 25 ppm and 50 ppm. However glass

slides prepared with sterilized distilled water were served as a control

treatment. These conidia were examined microscopically to determine the

uniformity of distribution and the number of spores that had germinated in

Situ. This percentage was used as a correction factor to determine the actual

conidial germination. Each slide was placed on a U-shaped glass rod in a

moist chamber made up of sterile Petri dish lined with filter paper saturated

with sterile distilled water. Petri dishes were incubated at 251.5C (Awad

et al., 1990) for 24 hours before examination. Three slides were used as

replicates for each particular treatment. The percentage of germination was

based on counts of 300 conidia.

Percentage of germination = No. of germinated spores

X 100 Total number of spores

The percentage of treatment efficiency in the reduction of powdery

mildew severity was calculated using the following equation:

Efficacy = Treatment - control

X 100 control


4.2. Effect of plant oils on germination of Sphaerotheca fuliginea.

The same technique in (4.1.1) was followed to evaluate the effect

of clove oil at concentration 10, 5 and 2.5, nigella oil, olive oil and rocket

oil at concentration 8, 4 and 2% on germination of Sphaerotheca

fuliginea conidiospores.

4.3. Effect of phosphate salt (K2HPO4) on germination of

Sphaerotheca fuliginea.

Phosphate with solution of K2HPO4 at concentration 50, 75 and

100 mM/L were used to evaluate their effect on germination of

Sphaerotheca fuliginea conidia as mentioned in (4.1.1).

4.4. Effect of some biological agents and thier filtrates, propolis

extract and their combination on germination of Sphaerotheca

fuliginea conidia.

Propolis extract, Trichoderma filtrate, Bacillus filtrate, propolis

extract + Trichoderma filtrate, propolis extract + Bacillus filtrate,

Trichoderma filtrate + Bacillus filtrate and propolis extract +

Trichoderma filtrate + Bacillus filtrate were used to evaluate their effect

on germination of Sphaerotheca fuliginea conidia as mentioned in (4.1.1).

4.5. Effect of UV, temperature and chloroform on powdery

mildew spores germination.

Conidia of Sphaerotheca fuliginea were harvested from only young

leaves of cucumber. To avoid the presence of old conidia, lesions were

gently shaken first by a glass rod to discard any old conidia presented on

such young leaves. The new conidia, which formed on conidiophores

after four to six hours, were collected. Spore suspensions was prepared by

adding sterile saline solution 0.5% to spore and adjusted to contain 6x107

spore/ml. Spore suspensions were subjected to three different treatments

either UV, temperature or chloroform. UV was used for 10, 20 and 30

minutes at wavelength of 1200 nm at 30 cm apart form the treated spores.


40

Temperature of 50, 70 and 90°C were used for 10 minutes. Chloroform

was used at 0.3, 0.5 and 1.0 ml/L/ 30 min. one ml of each spore

suspension was placed on dry clean glass slides. Each slide was placed on

a U-shaped glass rod in a moist chamber made up of sterile Petri dish

lined with filter paper saturated with sterile distilled water. Petri dishes

were incubated at 251.5C for 24 hours before examination. Three

replicates were used for each particular treatment. The percentage of

germination was based on counts of 300 conidia

5. Greenhouse experiments:

These experiments were conducted under greenhouse

conditions at the Agric. Bot. Dept., Pl. Pathol. Branch, Fac.

Agric., Moshtohor, Zagazig Univ., Benha Branch.

5.1. Induction of cucumber resistance against powdery mildew

by plant extracts.

Plant extracts induction of the systemic resistance was performed

at seedling stage (14 days after sowing) by spraying the upper surface of

the first two true leaves (Strobel and Kuc, 1995) with one of the

following aqueous extract 2 days before challenge inoculation by conidia

of the powdery mildew fungus. Garlic extract at concentration 20, 10 and

5%, clove extract at concentration 10, 5 and 2.5 and withania extract at

concentration 50, 25 and 12.5%.

For comparison with the tested resistance-inducers, spraying with

the fungicide Topas-100 at (12.5cm3/100L, 25cm

3/100L, and

50cm3/100L) and spraying with tap water were used in control treatments.

Spraying was done by covering the upper surface of true 1st and 2

nd leaf

by a given tested aqueous solution. Three replicates were used for each

treatment.


Challenge inoculation

Inoculation was accomplished by shaking powdery mildew heavily

diseased cucumber plants over the treated plants at a height of about

30cm. Inoculated plants were incubated on glasshouse benches until

disease assessment was undertaken. Inoculation was done 2 days after

foliar application with resistance-inducers (Strobel and Kuc, 1995).

Disease assessment:

Seven days after challenge inoculation, powdery mildew disease

development - as affected by the different tested treatment - was

evaluated as follows:

Disease Severity: Each leaf was rated on an increasing powdery

mildew scale of Descalzo et al. (1990) with a slight modification where:

0 = no mildew, 1 = 1-25, 2 = 26-50, 3 = 51-75, 4 = 76-100 and 5 = more

than 100 colonies/leaf. Then, disease-rating scores were converted to a

leaf damage percent (disease severity) using the equation suggested by

Townsend and Heuberger (1943) as follows:

Disease severity (%) = value)rating(highest leaves) no. (Total

(100) category) ratingin leaves (no. no.) rating[

The percentage of treatment efficiency in the reduction of powdery

mildew severity was calculated using the following equation:

Efficiency = Treatment - Control

X 100 Control

5.2. Induction of cucumber resistance against powdery mildew by

plant oils.

Efficacy of clove oil at concentration 10, 5 and 2.5, nigella oil, olive oil

and Rocket oil at concentration 8, 4 and 2% in induction the systemic

resistance were studied as mentioned before.


42


phosphate salt (K2HPO4).

Efficacy of Phosphate K2HPO4 at concentration 50, 75 and 100

mM/L for induction the systemic resistance was studied as mentioned

before.


biological control agent.

Efficacy of biological agents individually or in combination with

propolis extract was studied as follows: Trichoderma filtrate,

Trichoderma spore suspension, Bacillus filtrate, Bacillus cell suspension,

propolis extract + Trichoderma filtrate, propolis extract + Trichoderma

spore suspension, propolis extract + Bacillus filtrate, propolis extract +

Bacillus cell suspension, Trichoderma filtrate + Bacillus filtrate,

Trichoderma spore suspension + Bacillus cell suspension, propolis

extract + Trichoderma filtrate + Bacillus filtrate and propolis extract +

Trichoderma spore suspension + Bacillus cell suspension in induction the

systemic resistance were studied as mentioned before.

5.5. Effect of UV, temperature and chloroform on powdery mildew

spores infection activity.

Cucumber leaves showing intensive powdery mildew symptoms,

were collected form greenhouses. Spore of powdery mildew were

collected form infected leaves using smooth brush. Spore suspension was

prepared by adding sterile saline solution 0.5% to spore and adjusted to

contain 6x107 spore/ml. Spore suspension were subjected to three

different treatments either UV, temperature or chloroform. UV was used

for 10, 20 and 30 minutes at wavelength of 1200 nm at 30 cm apart form

the treated spore suspensions. Temperature of 50, 70 and 90°C were used

for 10 minutes. Chloroform was used at 0.3, 0.5 and 1.0 ml/L. Different

treated spores were used to spray cucumber plants just to examine their

viability. Six cucumber plants 4 weeks old were used for each treatment.

Six plants were sprayed with non treated powdery mildew spore


suspension as control treatment. Treated cucumber plants were then

covered with plastic bags to prevent any other outside source of infection

reach to the treated plants. All plants were kept under plastic bags under

greenhouse condition for seven days. Percentages of disease incidence

and disease severity were assayed as mentioned before.

6. Commercial protected house studies:

These experiments were conducted under commercial protected

house conditions belongs to Ministry of Agric., Tukh, Kalubia

6.1. Effect of spraying with plant extracts on incidence and

severity of powdery mildew disease in cucumber cv. Primo.

Two experiments (during spring and autumn 2003) were conducted

to evaluate the effect of spraying cucumber plants with plant extracts

on incidence and severity of powdery mildew under protected

houses. Cucumber plants cv. Primo 4 week old were sprayed with one of

the following extracts; garlic extract at concentration 20, 10 and 5%,

clove extract at concentration 10, 5 and 2.5% and withania extract at

concentration 50, 25 and 12.5%. Plants were sprayed with tap water or

Topas-100 at 12.5 cm3/100L, 25 cm

3/100L, and 50 cm

3/100L were served

as control treatments. Spraying with plant extracts were applied weekly.

Four spraying were applied. Three plants were used as replicates for each

treatment and control one (ten leaves for each plant). Percentages of

disease incidence and disease severity were assayed after week from last

spraying as mentioned before.

6.2. Effect of spraying with plant oils on incidence and severity

of powdery mildew disease in cucumber cv. Primo.


to evaluate the effect of spraying cucumber plants with plant oils on

incidence and severity of powdery mildew. Effect of plant oils clove

oil at concentration 10, 5 and 2.5%, nigella oil, olive oil and rocket oil at

concentration 8, 4 and 2% on percentages of disease incidence and

severity of powdery mildew were studied as mentioned before.


44

6.3. Effect of spraying with phosphate salt (K2HPO4) on

incidence and severity of powdery mildew disease in

cucumber cv. Primo.


to evaluate the effect of spraying cucumber plants with Phosphate salt

(K2HPO4) on incidence and severity of powdery mildew. Efficacy of

phosphate K2HPO4 at concentration 50, 75 and 100 mM/L on percentages

of disease incidence and severity of powdery mildew was studied as

mentioned before.

6.4. Effect of spraying with biological control agents on incidence

and severity of powdery mildew disease in cucumber cv.

Primo.


to evaluate the effect of spraying cucumber plants with biological

agents on incidence and severity of powdery mildew under protected

houses. Efficacy of biological agent propolis extract, Trichoderma

filtrate, Trichoderma spore suspension, Bacillus filtrate, Bacillus cell

suspension, propolis extract + Trichoderma filtrate, propolis extract +

Trichoderma spore suspension Trichoderma spore suspension, propolis

extract + Bacillus filtrate, propolis extract + Bacillus cell suspension,

Trichoderma filtrate + Bacillus filtrate, Trichoderma spore suspension +

Bacillus cell suspension, propolis extract + Trichoderma filtrate +

Bacillus filtrate and propolis extract + Trichoderma spore suspension +

Bacillus cell suspension on percentages of disease incidence and severity

of powdery mildew were studied as mentioned before.


6.5. Effect of spraying with plant extracts on controlling

powdery mildew disease in cucumber cv. Delta star (during

spring 2004).

Cucumber plants cv. Delta star 4 week old were sprayed with one of

the following extracts; garlic extract at concentration 20%, clove extract at

concentration 10% and withania extract at concentration 50%. Plants were

sprayed with tap water and Topas-100 at 50cm3/100L as control treatments.

Spraying with plant extracts were applied weekly. Three plants were used

as replicates for each treatment and control one (ten leaves for each

plant). Percentages of disease incidence, disease severity, average number

of fruit/plant and average weight of fruit/plant were measured to evaluate the

effect of plant extracts and control.

6.6. Effect of spraying with plant oils on controlling powdery

mildew disease in cucumber cv. Delta star (during spring

2004).

Efficacy of spraying cucumber plants with clove oil at

concentration 10%, nigella oil, olive oil and rocket oil at concentration

8% on controlling powdery mildew disease were studied as mentioned

before.

6.7. Effect of spraying with phosphate salt (K2HPO4) on

controlling powdery mildew disease in cucumber cv. Delta

star (during spring 2004).

Efficacy of phosphate K2HPO4 at concentration 100 mM/L on

controlling powdery mildew disease was studied as mentioned before.


46

6.8. Effect of spraying with biological control agent on controlling


spring 2004).

Efficacy of biological agents, propolis extract, Trichoderma

filtrate, Bacillus filtrate, propolis extract + Trichoderma filtrate, propolis

extract + Bacillus filtrate, Trichoderma filtrate + Bacillus filtrate and

propolis extract + Trichoderma filtrate + Bacillus filtrate on controlling

powdery mildew disease were studied as mentioned before.

6.9. Effect of spraying cucumber plants with ungerminated

powdery mildew spore suspension on controlling powdery

mildew disease in cucumber cv. Delta star (during spring

2004).

Effect of spraying powdery mildew killed spore by UV for 30

minutes or heat 90°C for 10 minutes or 1 ml chloroform/L on inducing

cucumber resistant to powdery mildew disease were studied as mentioned

before.

7. Determination of enzymes activity and lignin content:

The effect of selected inducers i.e. Garlic extract at 20%, clove

extract at 10%, clove oil at 10%, olive oil at 8%, propolis extract +

Trichoderma filtrate, Trichoderma filtrate + Bacillus filtrate, propolis

extract + Trichoderma filtrate + Bacillus filtrate, K2HPO4 at 100 mM/L.

and Topas-100 at 50cm3/100L. in addition to untreated control treatment

on peroxidase, polyphenol-oxidase and chitinase activity were

determined. Cucumber seed cv. Primo were planted in pots 20 cm

containing sandy, loam and peat-moss (1:1:1, v/v/v). Induction of the

systemic resistance was performed at seedling stage (14) days after

sowing) by spraying the upper surface of the first two true leaf challenge


with powdery mildew was done 2 days after Induction (Strobel and Kuc,

1995).

The whole plants were taken as samples before challenge and 1, 3,

5, 10 days after challenge.

7.1. Extraction of enzymes:

Samples was ground with 0.2 M Tris HCl buffer (pH 7.8)

containing 14 mM -mercaptoethanol at the rate 1/3 w/v. The extracts

were centrifuged at 10,000 rpm for 20 min at 4°C. The supernatant was

used to determine enzyme activities (Tuzun et al. 1989).

7.2. Peroxidase assay:

Peroxidase activity was determined according to the method

described by Allam and Hollis (1972), The cuvette contained 0.5 ml. 0.1

M potassium phosphate buffer at pH 7.0 + 0.3 ml of enzyme extract + 0.3

ml 0.05 M pyrogallol + 0.1 ml 1.0% H2O2 and distilled water to bring

cuvette contents to 3.0 ml. The reaction mixture incubated at 25°C for 15

minutes, then the reaction were inactivated by adding 0.5 ml. of 5.0%

(v/v) H2SO4 (Kar and Mishra, 1976). Peroxides activity was expressed

as the increase in absorbance at 425nm/gram fresh weigh/15 minutes.

7.3. Polyphenoloxidase assay:

The polyphenoloxidase activity was determined according to the

method described by Matta and Dimond (1963). The reaction mixture

contained 0.2 ml enzyme extract, 1.0 ml of 0.2 M sodium phosphate

buffer at pH 7.0 and 1.0 ml 10-3

M catechol and complete with distilled

water up to 6.0 ml. The reaction mixture was incubated for 30 minutes at

30°C. Polyphenoloxidase activity was expressed as the increase in

absorbance at 420nm/g fresh weigh/30 min.

7.4. Chitinase assay:

The determination was carried out according to the method of

Monreal and Reese, (1969), 1 ml of 1% colloidal chitin in 0.05 M citrate


48

phosphate buffer (pH 6.6) in test tubes, 1ml of enzyme extract was added

and mixed by shaking. Tubes were kept in a water bath at 37°C for 60

minutes, then cooled and centrifuged before assaying. Reducing sugar

was determined in 1ml of the supernatant by dinitrosalicylic acid (DNS).

Optical density was determined at 540nm. Chitinase activity was

expressed as mM N-acetylglucose amine equivalent released / gram fresh

weigh tissue / 60 minutes.

The substrate colloidal chitin was prepared from chitin powder

according to the method described by Ried and Ogryd-Ziak (1981).

Twenty five grams of chitin was milled, suspended in 250ml of 85%

phosphoric acid (H3PO4) and stored at 4°C for 24 h, then blended in 2

litre of distilled water and the suspension was centrifuged. The washing

procedure was repeated twice. The colloidal chitin suspension in the final

wash was adjusted to pH 7.0 with (1 N) NaOH, separated by

centrifugation and the pelted colloidal chitin was stored at 4°C.

7.5. Determination of lignin content:

Effect of selected inducers i.e. Garlic extract at 20%, clove extract

at 10%, clove oil at 10%, olive oil at 8%, propolis extract + Trichoderma

filtrate, Trichoderma filtrate + Bacillus filtrate, propolis extract +

Trichoderma filtrate + Bacillus filtrate, K2HPO4 at 100 mM/L and Topas-

100 at 50cm3/100L were tested to study their effects on lignin content in

cucumber plants. Induction and challenge with powdery mildew were

carried out as mentioned before. Samples were taken after 10 days of

challenge (Abd-El-Kareem, 1998).

The determination was carried out according to the method of

Bjorkman (1956). Five gram of dried cucumber tissue was extracted in a

soxhlet apparatus with acetone-water (9:1) and the organic solvent was

evaporated under reduced pressure at 70°C. After that, the aqueous

mixture was acidified with diluted HCl until pH 2 and the precipitated

lignin was filtered and washed with a small amount of water. The lignin

was dried at 70°C for 12 h.


8. Chemical analysis of cucumber treated plants:

Cucumber plants cv. Primo were grown under natural infection by

powdery mildew, were sprayed with (Garlic extract at 20%, clove extract

at 10% withania extract at 50 %, clove oil at 10%, nigella oil, olive oil

and rocket oil at 8% K2HPO4 at 100 mM/L, propolis extract, Trichoderma

filtrate, Bacillus filtrate, propolis extract + Trichoderma filtrate, propolis

extract + Bacillus filtrate, Trichoderma filtrate + Bacillus filtrate and

propolis extract + Trichoderma filtrate + Bacillus filtrate , Topas-100 at

50 ppm and control treatment (water only). Samples for chemical analysis

were taken 1 day after treatment (Daayf et al. 1995). Extraction from

cucumber leaves was prepared as follows:

Samples of 2 g of cucumber leaves from each treatment cut into

small portions. These portions were immediately placed in 50 ml of 95%

ethanol in brown bottles and kept in darkness at room temperature for one

month then homogenized in sterile mortar as recommended by Bozarth

and Diener (1963). The resultant homogenate was filtered through filter

paper. The residue was thoroughly washed with 80% ethanol. The

ethanolic extracts were air dried at room temperature till near dryness and

then were quantitatively transferred to 10 ml 50% isopropanol, and used

for chemical analysis of sugars, phenols and amino acids as follows:

8.1. Determination of sugar content:

Total and reducing sugars were determined spectrophotometrically

with picric acid as described by Thomas and Dutcher (1924). The sugar

content was calculated as mg glucose from standard curve prepared for

glucose. The following two solutions were used for the determination of

the total soluble and reducing sugars.


50

Picrate-picric solution:

Thirty six grams of picric acid were added to 500 ml of a 1%

solution of sodium hydroxide in one liter flask, 400 ml of hot water were

added and the mixture was shaken occasionally until the picric acid was

dissolved, and after wards, it was cooled and diluted to one liter.

Sodium carbonate solution:

Twenty grams of sodium carbonate were dissolved in 100 ml of

distilled water.

For determination of total soluble sugars, 0.5 ml of a given sample

was placed in 70 ml test tube, containing 5 ml of distilled water plus 4 ml

picrate-picric solution and then the mixture was boiled for 10 minutes, on

a water bath. After cooling, one ml of sodium carbonate was added and

the mixture was boiled again for 10 minutes, then cooled and completed

to 50 ml with distilled water. The optical density of the developed color

was measured by using spectrophotometer (SPECTRONIC 20-D) in the

presence of a blank at 540 nm.

The above technique was applied also for determination of

reducing sugars except that picrate-picric acid and sodium carbonate were

added together at the same time and boiled only for 10 minutes.

Total and reducing sugars concentrations were calculated as

milligrams of glucose per one gram fresh weight according to a standard

curve of glucose. However, the non-reducing sugars were determined as

the difference between the total and reducing sugars.

8.2. Determination of phenolic compounds:

Phenolic compounds were determined using the colorimetric

method of analysis described by Bary and Thorpe (1954). Phenol

reagent (Folin-Ciocalteu reagent) was prepared by boiling a mixture of

100 g of sodium tungestate, 25 g of sodium molybdate, 700 ml of

distilled water, 50 ml of 85% phosphoric acid and 100 ml of concentrated

hydrochloric acid under reflux for 10 hours in a water bath. Then 150 g of


lithium sulphate, 50 ml of distilled water and a few drops of bromine was

added to the mixture and boiled again for 15 minutes without a reflex

condenser to remove excess bromine, then cooled, diluted to 1 liter with

distilled water and filtered.

The free phenols were determined as follows, one ml of the phenol

reagent and 5 ml of a 20% solution of sodium carbonate were added to

the isopropanol sample (0.2 ml) and diluted to 10 ml with warm water,

(30-35°C). The mixture was let to stand for 20 minutes and read using

spectrophotometer (SPECTRONIC 20-D) at 520 nm against a reagent

blank.

For total phenols determination, 10 drops of concentrated

hydrochloric acid were added to the isopropanol sample (0.2ml) in a test

tube, heated rapidly to boiling over a free flame, with provision for

condensation. Then the tubes were placed in a boiling water bath for 10

minutes. After cooling 1ml of the reagent and 2.5 ml of 20% Na2CO3

were added to each tube. The mixture was diluted to 50 ml with distilled

water, and after 20 minutes was determined using spectrophotometer

(SPECTRONIC 20-D) at 520 nm against a reagent blank.

The total and free phenol contents were calculated for each

treatment as milligrams of catechol per one-gram fresh weight according

to standard curve of catechol. The conjugated phenols were determined

by subtracting the free phenols from the total phenols.

8.3. Determination of total amino acid:

Total amino acid was determined using the method of analysis

described by Muting and Kaiser (1963). The ethanolic extract (0.1 ml)

was placed into tube containing 1.5 ml. of ethanol/acetone mixture (1:1

v/v). 0.1 of pH 6.5 phosphate buffer and 2.0 ml. of 0.5% ninhydrin

solution in n-butanol. The tube was placed in boiling water bath for 10

minutes, then immediately cooled in ice water and the mixture volume

was made up to 10 ml. with absalut methanol. The developed colour was

measured at 580 nm using spectrophotometer (SPECTRONIC 20-D)


52

against a reagent blank. Data were obtained referring to standard pure

glycine curve.

Statistical analyses:

Statistical analyses of all the previously designed experiments have

been carried out according to the procedures (ANOVA) reported by

Snedecor and Cochran (1989). Treatment means were compared by the

least significant difference test “ L.S.D ” at 5% level of probability.

Experimental Results 53

EXPERIMENTAL RESULTS

1- Survey of cucumber diseases in protected houses.

Survey was carried out in Tukh and Kaha greenhouses in spring

season 2003 for identifying and determining the important diseases that

attack cucumber plants.

The data in Table (1) indicate that powdery mildew disease is the

most important disease of cucumber grown in protected houses. That

observed in both surveyed locations. Wilt occupied the second order in its

importance; meanwhile, virus infection recorded the third order and root-

rot and root–knot nematode, occupied the least order.

Table (1): Survey of foliar and soil borne disease of cucumber

plants in protected houses.

Soil borne diseases Foliar diseaseCultivarLocation

Total

%

Root –knot

nematodeWiltRoot- rot

Virus

disease

Powdery

mildew

10.672.136.402.1346.6312.60Delta

starTukh

7.661.534.601.536.3415.60dp 16215.250.0012.203.053.0024.00Sina 1Kaha

2. Effect of the tested treatments on germination of Sphaerotheca

fuliginea conidia.:

2.1. Effect of plant extracts on germination of Sphaerotheca fuliginea

conidia.

The efficacy of water extract of three plants on percentage of

Sphaerotheca fuliginea spores germination was studied under laboratory

condition.

The data in Table (2) reveal that all tested plant extracts

significantly reduced the percentage of conidia germination compared

with the control treatment (water only). The high reduction was induced


by garlic extract at concentration 20% (88.13 %) and clove extract at

concentration 10% (84.74 %) less than control treatment. While the

Topas-100 at concentrations 12.5cm3/100L, 25cm

3/100L and withania

extract at concentration 12.5% were the least effective treatments. Garlic

extract, clove extract and withania extract were effective more than

Topas-100 fungicide. Generally the reduction in spores germination were

significantly increased by increasing concentration of the extracts.

Table (2): Effect of some plant extracts on spore germination of

Sphaerotheca fuliginea conidia “in vitro”.

Treatments Conidia

Germination (%) Efficacy

Clove extract

10%

5%

2.5%

6.0

12.0

17.0

-84.74

-69.49

-56.78

Garlic extract

20%

10%

5%

4.67

6.0

10.64

-88.13

-84.74

-72.95

Withania extract

50%

25%

12.5%

12.0

14.67

18.0

-69.49

-62.70

-54.23

Topas-100

50cm3/100L

25cm3/100L

12.5cm3/100L

17.33

19.33

22.67

-56.00

-50.85

-27.10

Control 39.33

Treatment

L.S.D. at 5% 3.141

2.2. Effect of some plant oils on germination of Sphaerotheca

fuliginea conidia.

The efficacy of four plants oils on percentage of Sphaerotheca

fuliginea spores germination was studied under laboratory condition.

The results in Table (3) indicate that, all tested plant oils


with the control treatment. The high reduction was induced by clove oil at


concentration 10% (89.83 %), nigella oil at concentration 8% (74.57 %)

and olive oil at concentration 8% (72.87 %) less than control treatment.

While the Topas-100 at concentrations 12.5cm3/100L, 25cm

3/100L and

olive oil at concentration 2% were the least effective treatments. Clove

oil, olive oil, nigella oil, and rocket oil were effective more than Topas-

100 fungicide. Generally the reduction in spores germination were

significantly increased by increasing concentration of the plant oils.

Table (3): Effect of some plant oils on spore germination of

Sphaerotheca fuliginea “in vitro”.

Treatments Conidia Germination

(%) Efficacy

Clove oil

10%

5%

2.5%

4

6

8.67

-89.83

-84.74

-77.96

Olive oil

8%

4%

2%

10.67

14.67

20

-72.87

-62.70

-49.15

Rocket oil

8%

4%

2%

12.0

14.0

19.33

-69.49

-64.40

-50.85

Nigella oil

8%

4%

2%

10.0

12.0

14.67

-74.57

-69.49

-62.70

Topas-100

50cm3/100L

25cm3/100L

12.5cm3/100L

17.33

19.33

22.67

-56.01

-50.85

-27.10

Control 39.33

Treatment

L.S.D. at 5% 4.981


2.3. Effect of phosphate salt solution (K2HPO4) on germination of

Sphaerotheca fuliginea conidia.

The efficacy of phosphate salt (K2HPO4) on percentage of

Sphaerotheca fuliginea spores germination was studied under laboratory

condition.

The data in Table (4) show that, phosphate salt (K2HPO4) as well

as Topas-100 significantly reduced the percentage of conidia germination

compared with the control. The high reduction was induced by phosphate

salt (64.40 %) at concentration 100 mM/L less than control. While the

Topas-100 at concentrations 12.5cm3/100L, 25cm

3/100L and phosphate

salt (K2HPO4) at concentration 50 mM/L were the least effective

treatments. Generally the reduction in spores germination were

significantly increased by increasing concentration of phosphate salt.

Phosphate salt at concentration 100 mM/L was effective more than

Topas-100 fungicide.

Table (4): Effect of phosphate salt (K2HPO4) on spore germination of

Sphaerotheca fuliginea “in vitro”.

Treatments Conidia

Germination (%) Efficacy

Phosphate salt

100 mM/L

75 mM/L

50 mM/L

14

16.67

20.67

-64.40

-57.62

-52.55

Topas-100

50cm3/100L

25cm3/100L

12.5cm3/100L

17.33

19.33

22.67

-56.01

-50.85

-27.10

Control 39.33

Treatment

L.S.D. at 5% 2.575


2.4. Effect of some biological agent filtrate, propolis extract and their

combination on spores germination of Sphaerotheca fuliginea.

The efficacy of, propolis extract, Trichoderma filtrate and Bacillus

filtrate and their combination on percentage of Sphaerotheca fuliginea

spores germination was studied under laboratory condition.

The data in Table (5) indicate that, all tested treatments


with the control. The high reduction was induced by propolis extract +

Bacillus filtrate + Trichoderma filtrate (88.30 %), Bacillus filtrate +

Trichoderma filtrate (85.52 %) and propolis extract + Trichoderma

filtrate (83.98 %) less than control treatment. While the Topas-100

concentrations 12.5cm3/100L and 25cm

3/100L were the least effective

treatments.

Propolis extract, Trichoderma filtrate, Bacillus filtrate and their

combination were effective more than Topas-100 fungicide.

Table (5): Effect of some biological agent filtrates, propolis extract and

their combination on spores germination of Sphaerotheca

fuliginea “in vitro”.

Biological agent filtrate Conidia

Germination(%)

Efficacy

%

Propolis extract 6.7 -82.96

Trichoderma filtrate 7.3 -81.44

Bacillus filtrate 10.0 -74.57

Propolis extract + Trichoderma filtrate 6.3 -83.98

Propolis extract + Bacillus filtrate 7.3 -81.44

Bacillus filtrate + Trichoderma filtrate 5.3 -85.52

Propolis extract + Bacillus filtrate +


Topas-100

50cm3/100L

25cm3/100L

12.5cm3/100L

17.3

19.33

22.67

-56.01

-50.85

-27.10

Control 39.33

Treatment

L.S.D. at 5% 3.035


2.5. Effect of UV, temperature and chloroform on germination

of powdery mildew spores.

The results in Table (6) show that treated powdery mildew spores

suspension with UV for 30 minutes or temperature 90°C for 10 minutes or

add chloroform 1.0 ml/L completely inhabited spore germination. Treated

powdery mildew spores with UV for 20 minutes or temperature 70°C for 10

minutes or adding chloroform at 0.5 ml/L spores suspension reduced the

percentage of conidia germination from 40.33% in control treatment to

12.67, 13.33 and 14.67% respectively. While powdery mildew spores

treated with UV for 10 minutes or temperature 50°C for 10 minutes or

adding chloroform at 0.3 ml/L spore suspension reduced the percentage of

conidia germination from 40.33% in control treatment to 20.33, 20.67 and

22.00% respectively.

Table (6): Effect of UV, temperature and chloroform on powdery mildew

spores infection spores germination.

Treatment Conidia

Germination (%) Efficacy %

UV. 10 minutes

20 minutes

30 minutes

20.33

12.67

0.00

-49.60

-68.58

-100.0

Temperature 50C

70C

90C

20.67

13.33

0.00

-48.75

-66.95

-100.0

Chloroform 0.3 ml/L

0.5 ml/L

1.0 ml/L

22.00

14.67

0.00

-45.45

-63.63

-100.0

Control 40.33

Treatment

L.S.D. at 5% 1.706


3. Greenhouse studies:

3.1. Induction of cucumber resistance to powdery mildew infection

by some plant extracts.

Foliar application with water extract of three plants at three

concentrations were tested to study their efficiency against powdery

mildew incidence and severity.

Table (7): Powdery mildew incidence in cucumber cv. Primo sprayed

with some plant extracts under greenhouse.

Efficacy% Disease

severity Efficacy%

Disease

percentage Treatment

-57.20

-42.80

-28.53

3.33

4.45

5.56

-39.97

-19.99

0.0

16.67

22.22

27.77

Clove extract

10 %

5 %

2.5 %

-85.73

-57.20

-28.53

1.11

3.33

5.56

-60.0

-39.97

-19.99

11.11

16.67

22.22

Garlic extract

20 %

10 %

5 %

-57.20

-42.80

-14.27

3.33

4.45

6.67

-60.0

-39.97

0.0

11.11

16.67

27.77

Withania extract

50 %

25 %

12.5 %

-71.47

-42.80

-28.53

2.22

4.45

5.56

- 60.0

-39.97

- 19.98

11.11

16.67

22.22

Topas-100

50 cm3/100L

25 cm3/100L

12.5 cm3/100L

7.78 27.77 Control

Disease severity

L.S.D. at 5% 4.896

The data in Table (7) show that, all tested plant extracts

significantly reduced the percentage of powdery mildew incidence and

severity compared with the control. The high reduction in disease severity

was induced by garlic extract at 20% (-85.73%) followed by Topas-100 at

50 cm3/100L (-71.47%) and clove extract at 10% & withania extract at

50% (-57.20%). While the withania extract at concentration 12.5% were

the least effective treatments. Garlic extract at 20%, clove extract at 10%


and withania extract at 50% were more effective than Topas-100

fungicide at 25 cm3/100L. Generally the reduction in powdery mildew

incidence and severity were significantly increased by increasing

concentration of the extracts tested.

3.2. Induction of cucumber resistance to powdery mildew mildew

infection by some plant oils.

Foliar application with four plant oils at three concentrations were

tested to study their efficiency against powdery mildew incidence and

severity.


with some plant oils under greenhouse.

Efficacy% Disease

severity Efficacy% Disease percentage Treatment

-85.73

-57.20

-42.80

1.11

3.33

4.45

-60.0

-39.97

-39.97

11.11

16.67

16.67

Clove oil 10 %

5 %

2.5 %

-100.0

-85.73

-57.20

0.0

1.11

3.33

-100.0

-79.98

-39.97

0.0

5.56

16.67

Olive oil 8 %

4 %

2 %

-85.73

-71.47

-57.20

1.11

2.22

3.33

-79.98

-60.0

-39.97

5.56

11.11

16.67

Rocket oil 8 %

4 %

2 %

-85.73

-57.20

-42.93

1.11

3.33

4.44

-79.98

-39.97

-19.98

5.56

16.67

22.22

Nigella oil 8 %

4 %

2 %

-71.47

-42.80

-28.53

2.22

4.45

5.56

- 60.0

-39.97

- 19.98

11.11

16.67

22.22

Topas-100 50 cm

3/100L

25 cm3/100L

12.5 cm3/100L

7.78 27.77 Control

Disease severity

L.S.D. at 5% 3.294

The results in Table (8) reveal that all tested plant oils significantly

reduced the percentage of powdery mildew incidence and severity


compared with the control treatment. Olive oil at 8% completely

prevented powdery mildew incidence. The high reduction was induced by

olive oil at 4%, rocket oil at 8%, nigella oil at 8% and clove oil at 10% (-

85.73%) followed by Topas-100 at 50 cm3/100L (-71.47%). While the

Topas-100 at 12.5 cm3/100L was the least effective treatment. Olive oil,

rocket oil, nigella oil and clove oil were more effective than Topas-100

fungicide. Generally the reduction in powdery mildew incidence and

severity were significantly increased by increasing concentration of the

oils tested.

3.3. Induction of cucumber resistance to powdery mildew infection

by phosphate salt (K2HPO4).

Foliar application with phosphate salt (K2HPO4) at three

concentrations was tested to study their efficiency against powdery

mildew incidence and severity.

The data in Table (9) indicate that, phosphate salt (K2HPO4) as

well as Topas-100 significantly reduced the percentage of powdery

mildew incidence and severity compared with the control treatment. The

high reduction was induced by Topas-100 at concentration 50 cm3/100L

(-71.47%) and phosphate salt (K2HPO4) at concentration 100 mM/L (-

57.20%). While the Topas-100 12.5cm3/100L and phosphate salt

(K2HPO4) at concentration 50 mM/L were the least effective treatments.

K2HPO4) at concentration 100 mM/L more effective than Topas-100

fungicide at concentration 25 cm3/100L. Generally the reduction in

powdery mildew incidence and severity significantly increased by

increasing concentration of phosphate salt.



with phosphate salt (K2HPO4) under greenhouse.

Efficacy

%

Disease

severity

Efficacy

%

Disease


-57.20

-42.80

-28.53

3.33

4.45

5.56

-60.0

-19.98

-19.98

16.67

22.22

22.22

Phosphate salt

100 mM/L

75 mM/L

50 mM/L

-71.47

-42.80

-28.53

2.22

4.45

5.56

- 60.0

-39.97

- 19.98

11.11

16.67

22.22

Topas-100

50 cm3/100L

25 cm3/100L

12.5 cm3/100L

7.78 27.77 Control

Disease severity

L.S.D. at 5% 3.420

3.4. Induction of cucumber resistance to powdery mildew mildew

infection by some biological agent filtrate, propolis extract and

their combinations.

Foliar application with Bacillus subtilis, Trichoderma harzianm,

propolis extract and their combination were tested to study their

efficiency against powdery mildew incidence and severity.

The data in Table (10) show that all tested treatments significantly

reduced the percentage of powdery mildew incidence and severity compared

with the control treatment. Propolis extract + Bacillus filtrate + Trichoderma

filtrate completely prevented powdery mildew incidence. The high reduction

was induced by Trichoderma filtrate, propolis extract + Trichoderma

filtrate, propolis extract + Bacillus filtrate and Trichoderma filtrate +

Bacillus filtrate (-85.73%). While the Topas-100 12.5cm3/100L,

Trichoderma spore suspension and Bacillus cell suspension were the least

effective treatment. Propolis extract, Bacillus filtrate, Trichoderma filtrate

and their combination were more effective than Topas-100 fungicide.



with some biological agent, Propolis extract and their

combinations under greenhouse.

Efficacy

%

Disease

severity

Efficacy

%

Disease


-57.203.33-39.9716.67Propolis extract

-85.731.11-79.985.56Trichoderma filtrate

-35.735.0-9.9725Trichoderma spore suspension

-57.203.33-39.9716.67Bacillus filtrate

-35.735.0-9.9725Bacillus cell suspension

-85.731.11-79.985.56Propolis extract + Trichoderma

filtrate

-57.203.33-60.011.11Propolis extract +Trichoderma

spore suspension

-85.731.11-79.985.56Propolis extract + Bacillus

filtrate

-57.203.33-39.9716.67Propolis extract + Bacillus cell

suspension

-85.731.11-79.985.56Bacillus filtrate +

Trichoderma filtrate

-42.804.45-19.9822.22Trichoderma spore suspension +

Bacillus cell suspension

-100.00.0-100.00.0Propolis extract + Bacillus

filtrate + Trichoderma filtrate

-57.203.33-39.9716.67Propolis extract + Trichoderma

spore suspension + Bacillus cell

suspension

-71.47

-42.80

-28.53

2.22

4.45

5.56

- 60.0

-39.97

- 19.98

11.11

16.67

22.22

Topas-100

50 cm3/100L

25 cm3/100L

12.5 cm3/100L

7.78 27.77 Control

Disease severity

L.S.D. at 5% 3.17


3.5. Effect of UV, temperature and chloroform on powdery mildew

spores infection activity:

The data in Table (11) reveal that the spores subjected to UV effect

for 10 minutes or temperature 50°C for 10 minutes or add chloroform at

the rate of 0.3 ml/L reduction disease severity from 20.00% in control

treatment to 9.33, 10.67 and 10.67% respectively. Treated powdery

mildew spores with UV for 20 minutes or temperature 70°C for 10

minutes or add chloroform 0.5 ml/L spore suspension reduced the

percentage of disease severity from 20.00% in control treatment to 6.67,

8.00 and 9.33% respectively. The data also reveal that increasing time of

revelation to UV, temperature or concentration of chloroform led to

increase efficacy of the treatment in reducing the percentage of disease

incidence. Treated powdery mildew spores with UV for 30 minutes or

temperature 90°C for 10 minutes or add chloroform 1ml/L spore

suspension completely inhabited all spores of powdery mildew and no

disease was developed.

Table (11): Effect of UV, temperature and chloroform on powdery

mildew spores infection activity.

Treatment Disease

percentage

Disease

severity

UV. 10 minutes

20 minutes

30 minutes

46.67

33.33

0.00

9.33

6.67

0.00

Temperature

50C

70C

90C

53.33

40.00

0.00

10.67

8.00

0.00

Chloroform 0.3 ml/L

0.5 ml/L

1.0 ml/L

46.67

40.00

0.00

10.67

9.33

0.00

Control 73.33 20.00

Treatment

L.S.D. at 5% 4.299


4. Commercial protected house studies:

4.1. Effect of spraying plant extracts on incidence and severity of

powdery mildew disease in cucumber cv. Primo under

commercial protected houses.

In two experiments (during spring and autumn 2003) foliar

application with water extract of three plants at three concentrations were


severity.


with some plant extracts under commercial protected

houses.

Mean Experiment 2

(autumn 2003)

Experiment 1

(spring 2003) Treatment

Disease

severity

Disease

percentage

Disease

severity

Disease

percentage

Disease

severity

Disease

percentage

3.67

4.67

7.33

15

18.33

28.34

3.33

4.0

7.33

13.33

13.33

26.67

4.0

5.33

7.33

16.67

23.33

30.0

Clove extract

10 %

5 %

2.5 %

2.67

4.0

6.34

11.67

16.67

25

2.67

4.0

6.67

10.0

20.0

26.67

2.67

4.0

6.0

13.33

13.33

23.33

Garlic extract

20 %

10 %

5 %

4.67

6.34

7.0

18.34

23.34

23.33

5.33

6.67

7.33

20.0

26.67

23.33

4.0

6.0

6.67

16.67

20.0

23.33

Withania extract

50 %

25 %

12.5 %

4.34

5

5.67

13.33

16.67

21.67

4.0

4.67

5.33

13.33

16.67

23.33

4.67

5.33

6.0

13.33

16.67

20.0

Topas-100

50 cm3/100L

25 cm3/100L

12.5 cm3/100L

15.67 48.34 14.67 46.67 16.67 50.0 Control

L.S.D. at 5%

Exp.1

3.349

Exp.2

4.227


The data in Table (12) reveal that all tested plant extracts at both

seasons significantly reduced the percentage of powdery mildew

incidence and severity compared with the control.

Garlic extract at concentration 20% was the most effective in reducing

disease severity during both seasons (average 2.67%) followed by clove

extract at concentration 10% (average 3.67%), garlic extract at concentration

10% (average 4%), Topas-100 at concentration 50 cm3/100L (average

4.34%), and withania extract at concentration 50% (average 4.67%)

compared with control (average 15.67%). While the clove extract at

concentration 2.5% and withania extract at concentration 12.5% were the

least effective treatments at both seasons. Garlic extract at 20% and clove

extract at 10% were more effective than Topas-100 fungicide. Generally

increasing concentration of the extracts tested significantly increased the

reduction in powdery mildew incidence and severity.

4.2. Effect of spraying plant oils on incidence and severity of powdery

mildew disease in cucumber cv. Primo under commercial

protected houses.


application with four plant oils at three concentrations were tested to

study their efficiency against powdery mildew incidence and severity.

The data in Table (13) reveal that all tested plant oils at both


incidence and severity compared with the control.

Clove oil at concentration 10% and olive oil at concentration 8%

were the most effective treatments in reducing disease severity during both

seasons (average 2.67%) followed by nigella oil at concentration 8%

(average 4%) and Topas-100 at concentration 50 cm3/100L & rocket oil

at concentration 8% (average 4.34%) compared with control (average

15.67%). While the nigella oil at concentration 2% and rocket oil at


concentration 2% were the least effective treatment at both seasons. Olive

oil, rocket oil, nigella oil and clove oil were more effective than Topas-

100 fungicide. Generally increasing concentration of the plant oils tested

significantly increased the reduction in powdery mildew incidence and

severity.


with some plant oils under commercial protected houses.

Mean Experiment 2

(autumn 2003)

Experiment 1


Disease

severity

Disease

percentage

Disease

severity

Disease

percentage

Disease

severity

Disease

percentage

2.67

4.67

5.67

13.33

20.0

21.67

2.67

4.0

5.33

13.33

20.0

20.0

2.67

5.33

6.0

13.33

20.0

23.33

Clove oil 10 %

5 %

2.5 %

2.67

4.34

5.67

11.67

16.67

21.67

2.67

4.0

5.33

10.0

13.33

20.0

2.67

4.67

6.0

13.33

20.0

23.33

Olive oil 8 %

4 %

2 %

4.34

5.0

6.0

18.34

21.67

23.33

4.0

4.67

6.0

16.67

20.0

23.33

4.67

5.33

6.0

20.0

23.33

23.33

Rocket oil 8 %

4 %

2 %

4.0

5.0

6.34

13.33

18.34

23.33

4.0

5.33

6.67

13.33

20.0

23.33

4.0

4.67

6.0

13.33

16.67

23.33

Nigella oil 8 %

4 %

2 %

4.34

5

5.67

13.33

16.67

21.67

4.0

4.67

5.33

13.33

16.67

23.33

4.67

5.33

6.0

13.33

16.67

20.0

Topas-100

50 cm3/100L

25 cm3/100L

12.5 cm3/100L

15.67 48.34 14.67 46.67 16.67 50.0 Control

L.S.D. at 5%

Exp.1

3.520

Exp.2

3.557


4.3. Effect of spraying phosphate salt (K2HPO4) on incidence and

severity of powdery mildew disease in cucumber cv. Primo

under commercial protected houses.


application with phosphate salt (K2HPO4) at three concentrations was


severity.

The data in Table (14) indicate that, Phosphate salt (K2HPO4) at

concentration 100 mM/L was the most effective treatment in mean of both

seasons by (average 3%) followed by Phosphate salt (K2HPO4) at

concentration 75 mM/L (average 4%) and Topas-100 at concentration 50

cm3/100L (average 4.34 %) compared with control (average 15.67%). While

the Topas-100 at concentration 12.5cm3/100L was the least effective

treatments. K2HPO4) at concentration 100 mM/L was more effective than

Topas-100 fungicide. Generally the reduction in powdery mildew incidence

and severity significantly increased by increasing concentration of

phosphate salt.


with phosphate salt (K2HPO4) under commercial protected

houses.

Mean Experiment2

(autumn 2003)

Experiment 1


Disease

severity

Disease

percentage

Disease

severity

Disease

percentage

Disease

severity

Disease

percentage

3.0

4.0

5.0

13.33

16.67

20.0

2.67

4.0

4.67

13.33

16.67

20.0

3.33

4.0

5.33

13.33

16.67

20.0

Phosphate salt 100 mM/L

75 mM/L

50 mM/L

4.34

5

5.67

13.33

16.67

21.67

4.0

4.67

5.33

13.33

16.67

23.33

4.67

5.33

6.0

13.33

16.67

20.0

Topas-100 50 cm

3/100L

25 cm3/100L

12.5 cm3/100L

15.67 48.34 14.67 46.67 16.67 50.0 Control

L.S.D. at 5% 3.443 3.090


4.4. Effect of spraying some biological agent, Propolis extract and their

combination on incidence and severity of powdery mildew disease

in cucumber cv. Primo under commercial protected houses.


application with Bacillus subtilis, Trichoderma harzianm, propolis

extract and their combination were tested to study their efficiency

against powdery mildew incidence and severity.


with some biological agent, Propolis extract and their

combination under commercial protected houses.

Mean Experiment 2

(autumn 2003)

Experiment 1

(spring 2003) Treatment Disease

severity

Disease

percentage

Disease

severity

Disease

percentage

Disease

severity

Disease

percentage

3.34 10.0 2.67 10.0 4.0 10.0 Propolis extract 2.34 15.0 2.0 13.33 2.67 10.0 Trichoderma filtrate

4.34 15.0 4.0 13.33 4.67 16.67 Trichoderma spore suspension

2.34 10.0 2.0 10.00 2.67 16.67 Bacillus filtrate 5.0 18.34 4.67 16.67 5.33 20.0 Bacillus cell suspension

2.0 15.0 2.0 10.0 2.0 30.0 Propolis extract + Trichoderma

filtrate

5.0 16.67 5.33 20.0 4.67 13.33 Propolis extract +Trichoderma

spore suspension

3.0 11.67 2.67 10.0 3.33 13.33 Propolis extract + Bacillus

filtrate

5.33 20.0 5.33 20.0 5.33 20.0 Propolis extract + Bacillus

filtrate

2.0 8.34 2.0 10.0 2.0 6.67 Bacillus filtrate + Trichoderma

filtrate

7.0 23.33 6.67 23.33 7.33 23.33 Trichoderma spore suspension +

Bacillus cell suspension

1.67 8.34 2.0 10 1.33 6.67 Propolis extract + Bacillus

filtrate + Trichoderma filtrate

6.0 20.0 6.0 20 6 20.0

Propolis extract + Trichoderma

spore suspension + Bacillus cell

suspension

4.34

5

5.67

13.33

16.67

21.67

4.0

4.67

5.33

13.33

16.67

23.33

4.67

5.33

6.0

13.33

16.67

20.0

Topas-100

50 cm3/100L

25 cm3/100L

12.5 cm3/100L

15.67 48.34 14.67 46.67 16.67 50.0 Control

L.S.D. at 5% Exp.1

2.728

Exp.2

2.743


The data in Table (15) indicate that, all tested treatment


severity compared with the control treatment.

The combination of propolis extract, Bacillus filtrate and

Trichoderma filtrate was the most effective treatment in mean of both

seasons by (average 1.67%) followed by propolis extract + Trichoderma

filtrate & Bacillus filtrate + Trichoderma filtrate (average 2%) and

Bacillus filtrate & Trichoderma filtrate (average 2.34%). While the least

effective treatments were Trichoderma spore suspension + Bacillus cell

suspension, propolis extract + Trichoderma spore suspension + Bacillus

cell suspension and the Topas-100 12.5cm3/100L. Propolis extract,

Bacillus filtrate, Trichoderma filtrate and their combination were more

effective than Topas-100 fungicide.

4.5. Effect of spraying with plant extracts on controlling powdery

mildew disease in cucumber cv. Delta star (during spring 2004).

Cucumber plants cv. Delta star 4 week old were sprayed with one of

the following extracts; garlic extract at concentration 20%, clove extract at

concentration 10% and withania extract at concentration 50%. Plants were

sprayed with tap water and Topas-100 at 50 cm3/100L as control treatments.

The data in Table (16) indicate that, all tested plant extracts


severity compared with the control treatment. The high reduction in

disease severity was induced by garlic extract (-45.27%) followed by

clove extract (-39.62%), Topas-100 (-37.75%) and withania extract (-

26.43%). Also, all tested plant extracts increased the fruit number/plant

and fruit weight/plant. The highest increased in fruit number/plant and

fruit weight/plant was induced by garlic extract (31.26% and 35.84%)

followed by clove extract (25.02%and 28.01%), withania extract (18.75%

and 21.38%) and Topas-100



(13.39% and 13.55%) respectively over control. Garlic extract at

20%, and clove extract at 10% more effective than Topas-100 fungicide.

Withania extract at 50% increasing yield more than Topas-100 fungicide.

4.6. Effect of spraying with plant oils on controlling powdery mildew

disease in cucumber cv. Delta star (during spring 2004).

Cucumber plants cv. Delta star 4 week old were sprayed with one

of the following Plant oils; clove oil at concentration 10%, nigella oil,

olive oil and rocket oil at concentration 8%. Plants were sprayed with tap

water and Topas-100 at 50 cm3/100L as control treatments.

The data in Table (17) indicate that, all tested plant significantly


compared with the control treatment. The high reduction in disease

severity was induced by olive oil (-67.91%) followed by clove oil (-

64.18%), nigella oil (-60.38%), rocket oil (-47.20%) and Topas-100 (-

37.75%). Also, all tested plant oils increased the fruit number/plant and

fruit weight/plant. The highest increased in fruit number/plant and fruit

weight/plant was induced by clove oil (37.50% and 37.65%) followed by

olive oil (26.79% and 28.31%), nigella oil (21.43% and 24.40%), rocket

oil (13.39% and 15.96%) and Topas-100 (13.39% and 13.55%)

respectively. Olive oil, nigella oil, rocket oil and clove oil were more

effective than Topas-100 fungicide.

4.7. Effect of spraying with phosphate salt (K2HPO4) on controlling


spring 2004).

Cucumber plants cv. Delta star 4 week old was sprayed with

phosphate salt 100 mM/L. plants were sprayed with tap water and Topas-

100 at 50 cm3/100L as control treatments.

The data in Table (18) indicate that, phosphate salt 100 mM/L as well

as Topas-100 significantly reduction the percentage of powdery mildew


Tab17


Tab18


incidence and severity compared with the control treatment. Phosphate

salt reduced disease severity by 35.88% and Topas-100 reduced disease

severity by 37.75 less than control.

Phosphate salt and Topas-100 increased the fruit number/plant and

fruit weight/plant by (14.31%, 16.87% and (13.39% and 13.55%)

respectively over control.

4.8. Effect of spraying with biological control agent on controlling


spring 2004).


propolis extract, Trichoderma filtrate, Bacillus filtrate and their

combination. Plants were sprayed with tap water and Topas-100 at 50

cm3/100L as control treatments.



severity compared with the control treatment. The high reduction in

disease severity was induced by propolis extract + Trichoderma filtrate +

Bacillus filtrate (-69.84%) followed by Trichoderma filtrate +

Bacillus filtrate (-60.38%), propolis extract + Trichoderma filtrate &

propolis extract + Bacillus filtrate (-58.52%) and Bacillus filtrate (-

54.73%). Also, all tested treatments increased the fruit number/plant and

fruit weight/plant. The highest increased in fruit number/plant and fruit

weight/plant was induced by propolis extract + Trichoderma filtrate +

Bacillus filtrate (43.83% and 44.00%) followed by propolis extract +

Trichoderma filtrate (38.41% and 40.06%) and Bacillus filtrate +

Trichoderma filtrate (37.50% and 39.17%) respectively over control.

Propolis extract, Trichoderma filtrate, Bacillus filtrate and their

combination among propolis extract alone more effective than Topas-100

fungicide.


Tab19


4.9. Effect of spraying cucumber plants with non-germinated

powdery mildew spore on controlling powdery mildew

disease in cucumber cv. Delta star (during spring 2004).


powdery mildew killed spore by UV for 30 minutes either temperature

90°C for 10 minutes or 1 ml chloroform/L. Plants were sprayed with tap

water and Topas-100 at 50 cm3/100L as control treatments.



severity compared with the control treatment. UV, temperature,

chloroform and Topas-100 reduce disease severity by 69.84%, 62.25%,

54.73% and 37.75% respectively less than control. Also, all tested

treatments increased the fruit number/plant and fruit weight/plant. The

highest increased in number fruit/plant and weight fruit/plant was induced

by UV (25.02% and 25.00%) followed by temperature (18.75% and

19.88%) respectively over control.



5. Effect of the tested treatments on some enzyme activities and

lignin content:

5.1. Effect of the tested treatments on peroxidase activity:

The results in Table (21) and Fig. (1) reveal that, all treatments

significantly increased peroxidase activity compared with control

treatment in all times. The highest activity of peroxidase was induced

before inoculation, one day and three days after inoculation by propolis

extract +Trichoderma filtrate (231.48, 176.33 and 225.23%) followed by

propolis extract + Trichoderma filtrate + Bacillus filtrate (230.00, 175.12

and 176.00%). After five days from inoculation the highest activity of

peroxidase was induced by propolis extract +Trichoderma filtrate

(219.83%) followed by propolis extract + Trichoderma filtrate + Bacillus

filtrate (181.43%), phosphate salt (K2HPO4) (143.23%) and garlic extract

(142.71%) over control. While, after ten days from inoculation the

activity of peroxidase decreased nevertheless increased over control, in

this respect the highest activity was noticed by propolis extract +

Trichoderma filtrate + Bacillus filtrate (115.35%) followed by phosphate

salt (K2HPO4), (94.10%) and garlic extract (92.37%).


Tab21


Figure (1): Activity of peroxidase enzyme in cucumber plants as

affected by the tested foliar spray treatments and their

interaction with powdery mildew subsequent inoculation.

0

5

10

15

20

25

30

35

Before inocultion After one day After 3 days After 5 days After 10 days

Clove extract 10% Garlic extract 20%

Clove oil 10% olive oil 8%

Propolis +Trichoderma filtrate Trichoderma filtrate +Bacillus filtrate

Propolis + Trichoderma filtrate + Bacillus filtrate Phosphate salt 100 mM/L

Topas 50cm3/100L Control


5.2. Effect of the tested treatments on polyphenoloxidase activity:

The results in Table (22) and Fig. (2) show that, all treatments

significantly increased polyphenoloxidase activity compared with control

in all times. The highest activity before inoculation was induced by

propolis extract + Trichoderma filtrate (364.46%) followed by olive oil

(284.30%) and Topas-100 (282.65%). The highest activity after one day

from inoculation was induced by propolis extract + Trichoderma filtrate

(397.12%) followed by clove extract (360.43%) and olive oil (238.13%).

The highest activity after three days from inoculation was induced by

propolis extract +Trichoderma filtrate (324.19%) followed by

Trichoderma filtrate + Bacillus filtrate (320.43%), and clove extract

(275.58%) over control. After five days from inoculation the highest

activity was induced by propolis extract +Trichoderma filtrate + Bacillus

filtrate (229.04%) followed by propolis extract + Trichoderma filtrate

(219.85%) and clove extract (215.44%) over control. While, after ten

days from inoculation the activity of polyphenoloxidase decreased

nevertheless increased over control, in this respect the highest activity

was noticed by garlic extract (342.65%) followed by propolis extract +

Trichoderma filtrate + Bacillus filtrate (295.59%) and Trichoderma

filtrate + Bacillus filtrate (294.12%) over control.



0

1

2

3

4

5

6

7

8

9

Before

inoculation

after one day after 3 days after 5 days after 10 days

Clove extract 10%Garlic extract 20%Clove oil 10%olive oil 8%Propolis +Trichoderma filtrateTrichoderma filtrate +Bacillus filtratePropolis + Trichoderma filtrate + Bacillus filtratePhosphate salt 100 mM/LTopas 50cm3/100LControl

Figure (2): Activity of polyphenoloxidase enzyme in cucumber plants

as affected by the tested foliar spray treatments and their

interaction with powdery mildew subsequent inoculation.


5.3. Effect of the tested treatments on chitinase activity:

The data in Table (23) and Fig. (3) indicate that, all treatments

significantly increased chitinase activity compared with control treatment

in all times. The highest activity of chitinase before inoculation was

induced by propolis extract + Trichoderma filtrate (468.42%) followed by

olive oil (444.74%) and propolis extract +Trichoderma filtrate + Bacillus

filtrate (392.10%) over control. The highest activity of chitinase after one

day from inoculation was induced by propolis extract + Trichoderma

filtrate (692.50%) followed by Trichoderma filtrate + Bacillus filtrate

(637.50%) and clove extract (557.50%) over control. The highest activity

of chitinase after three days from inoculation was induced by propolis

extract +Trichoderma filtrate (517.14%) followed by Trichoderma filtrate

+ Bacillus filtrate (465.71%), and propolis extract +Trichoderma filtrate

+ Bacillus filtrate (350.00 %) over control. After five days from

inoculation the highest activity of chitinase was induced by propolis

extract +Trichoderma filtrate (573.97%) followed by Trichoderma filtrate

+ Bacillus filtrate (458.90 %) and clove extract (439.73%) over control.

While, after ten days from inoculation the activity of chitinase decreased

nevertheless increased over control, in this respect the highest activity

was noticed by propolis extract +Trichoderma filtrate (435.56%)

followed by olive oil (168.89%) and Trichoderma filtrate + Bacillus

filtrate (164.44%) over control.



0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Before

inoculation

after one day after 3 days after 5 days after 10 days

Clove extract 10%

Garlic extract 20%

Clove oil 10%

olive oil 8%

Propolis +Trichoderma filtrate

Trichoderma filtrate +Bacillus filtrate

Propolis + Trichoderma filtrate + Bacillus filtrate


Topas 50cm3/100L

Control

Figure (3): Activity of chitinase enzyme in cucumber plants as affected

by the tested foliar spray treatments and their interaction with

powdery mildew subsequent inoculation.


5.4. Effect of the tested treatments on lignin content of cucumber

plants:

The data in Table (24) and Fig. (4) reveal that, all treatments

significantly increased lignin content compared with control treatment. The

most effective treatment were garlic extract, propolis extract +Trichoderma

filtrate, phosphate salt (K2HPO4), propolis extract + Trichoderma filtrate +

Bacillus filtrate and clove extract which increased total lignin by 92.11,

82.90, 71.58, 56.84 and 52.36% respectively over control.

Table (24): Lignin content in cucumber plants as affected by the

tested treatments and powdery mildew inoculation:

Treatment Lignin weight

(mg/1g) Increase %

Clove extract

10% 290 52.36

Garlic extract

20% 365 92.11

Clove oil

10% 347.5 18.42

olive oil

8% 206.3 8.58

Propolis extract +Trichoderma

filtrate 298 82.90

Trichoderma filtrate +Bacillus

filtrate 225 25.68

Propolis extract +

Trichoderma filtrate + Bacillus

filtrate

238.8 56.84

Phosphate salt

100 mM/L 326 71.58

Topas-100

50cm3/100L205 7.9

Control 190

Treatment

L.S.D. at 5% 4.280


0

50

100

150

200

250

300

350

400

mg

/g

Lignin content

Clove extract 10%

Garlic extract 20%

Clove oil 10%

Olive oil 8%


Trichoderma filtrate +Bacillus filtrate

Propolis + Trichoderma filtrate + Bacillus filtrate


Topas 50cm3/100L

Control

Figure (4): Lignin content in cucumber plants as affected by the tested

treatments and powdery mildew inoculation:


6. Chemical analysis:

6.1. Effect of foliar spraying with plant extracts & oils and K2HPO4

on sugar content of powdery mildew infected cucumber

plants:

The data in Table (25) and Fig. (5) indicate that sugars content was

significantly affected by the tested treatments. All treatments decreased

the reducing sugars except Topas-100. The highest decrease was induced

by clove oil & nigella oil (54.11%) followed by olive oil, withania

extract, clove extract, rocket oil, garlic extract and phosphate salt

(K2HPO4) decreased the reducing sugars by 41.10, 37.68, 29.47, 26.32,

19.79 and 1.68%, respectively less than the control. While, Topas-100

increased it by 63.79% over control treatment.

All treatments increased the non-reducing sugars except olive oil

and withania extract decreased it. The highest increase was induced by

garlic extract, clove extract, Topas-100, rocket oil, nigella oil, phosphate

salt (K2HPO4) and clove oil increased it by 280.85, 232.00, 100.00, 97.87,

82.98, 65.96 and 17.02% respectively over control. While olive oil and

withania extract decreased it by 34.04%, respectively less than the

control.

As for the total sugars, Topas-100, withania extract, garlic extract,

and phosphate salt (K2HPO4) increased the total sugars by 68.2, 37.36,

7.28 and 4.41% respectively over control. While clove oil, nigella oil,

olive oil, rocket oil and clove extract decreased it by 47.70, 41.76, 40.42,

15.13 and 5.94%, respectively less than control treatment.


Table (25): Effect of spraying plant extracts and oils, and K2PO4 on sugar

content of powdery mildew infected cucumber plants as mg

/1 g fresh weight.

Treatments

Sugar content Efficacy %

Reducing

sugars

Non

reducing

sugars

Total sugars Reducing

sugars

Non

reducing

sugars

Total

sugars

Clove extract 10% 3.35 1.56 4.91 -29.47 232.00 -5.94

Garlic extract 20% 3.81 1.79 5.60 -19.97 280.85 7.28

Withania extract 50%

2.96 0.31 3.27 -37.68 -34.04 -37.36

Clove oil 10%

2.18 0.55 2.73 -54.11 17.02 -47.7

Olive oil 8%

2.80 0.31 3.11 -41.10 -34.04 -40.42

Rocket oil 8%

3.50 0.93 4.43 -26.32 97.87 -15.13

Nigella oil 8%

2.18 0.86 3.04 -54.11 82.98 -41.76


4.67 0.78 5.45 -1.68 65.96 4.41

Topase 50cm3/100L

7.78 0.94 8.78 63.79 100.00 68.2

Control 4.75 0.47 5.22

Reducing sugars Non reducing sugars Total sugars

L.S.D. at 5% 0.7516 0.1627 0.7398


0

1

2

3

4

5

6

7

8

9

mg

/g

reducing sugar Non-reducing sugar Total sugar

Clove extract 10% Garlic extract 20%

Withania extract 50% Clove oil 10%

Olive oil 8% Rocket oil 8%

Nigella oil 8% Phosphate salt 100 mM/L

Topas 50cm3/100L Control

Figure (5): Effect of spraying plant extracts & oils and K2HPO4 on sugar

content of infected powdery mildew cucumber plants as mg /1

g fresh weight.


6.2. Effect of spraying plant extracts & oils and K2HPO4 on phenol

content of infected powdery mildew cucumber plants:

The results in Table (26) and Fig. (6) reveal that, the free,

conjugated and total phenols were affected significantly by the tested

treatments. All tested treatment increased the free phenols. The highest

increase in the free phenols was induced by Topas-100 (326.88%)

followed by K2HPO4 (289.96%) and clove oil (205.73%).

As for the total phenols all tested treatments increased the total

phenols. The highest increase in the total phenols was induced by Topas-

100 (97.33%) followed by K2HPO4 (66.27%) and clove oil (56.80%).

While treatments were differed in the effect on the conjugated

phenols, Topas-100, garlic extract and clove oil increased it by 18.17,

7.29 and 5.44% respectively over control. While, withania extract, nigella

oil, clove extract, rocket oil, phosphate salt (K2HPO4) and olive oil

induced the highest decrease by 45.49, 31.00, 27.32, 18.17, 10.88 and

7.29 % respectively less than control.

6.3.Effect of spraying plant extracts & oils and K2HPO4 on total

amino acid content of infected powdery mildew cucumber

plants:


significantly decreased the total amino acid content. The highest decrease

induced by olive oil (-94.55%) followed by clove extract (-85.19%),

garlic extract (-78.70%) and withania extract (-77.92%).


Table (26): Effect of spraying plant extracts & oils and K2HPO4 on

phenol content of infected powdery mildew cucumber plants

as mg/1 g fresh weight.

Treatments

Phenol content Efficacy %

Free

phenols Conjugated

phenols

Total

phenols

Free

phenols Conjugated

phenols

Total

phenols

Clove extract

10% 7.65 5.88 13.53 174.19 -27.32 24.36

Garlic extract

20% 7.50 8.68 16.18 168.8 7.29 48.71

Withania extract 50% 7.21 4.41 11.62 158.42 -45.49 6.80

Clove oil

10% 8.53 8.53 17.06 205.73 5.44 56.80

Olive oil

8% 5.15 7.50 12.65 84.59 -7.29 16.27

Rocket oil

8% 7.35 6.62 13.97 163.44 -18.17 28.40

Nigella oil

8% 7.79 5.59 13.38 179.2 -31.00 22.98

phosphate salt

100 mM/L 10.88 7.21 18.09 289.96 -10.88 66.27

Topas-100

50cm3/100L 11.91 9.56 21.47 326.88 18.17 97.33

Control 2.79 8.09 10.88

Free

phenols

Conjugated

phenols Total phenols

L.S.D. at 5% 0.8137 0.8101 0.7917


0

5

10

15

20

25

mg

/g

Free phenols Conjugated phenols Total phenols

Clove extract 10%

Garlic extract 20%


Clove oil 10%

Olive oil 8%

Rocket oil 8%

Nigella oil 8%

Phosphate salt 100mM/L

Topas 50cm3/100L

Control

Figure (6): Effect of spraying plant extracts & oils and K2HPO4 on

phenol content of infected powdery mildew cucumber plants

as mg/1 g fresh weight.


Table (27): Effect of spraying Plant extracts & oils and K2HPO4 on total


plants as mg/g fresh weight.

Treatments Total amino

acids content Efficacy %

Clove extract

10% 0.57 -85.19

Garlic extract 20% 0.82 -78.70

Withania extract 50% 0.85 -77.92

Clove oil 10% 1.75 -54.55

Olive oil 8% 0.21 -94.55

Rocket oil 8% 1.97 -48.83

Nigella oil 8% 1.43 -62.86

phosphate salt

100 mM/L 2.34 -39.22

Topas-100

50cm3/100L

1.64 -57.40

Control 3.85

Treatment

L.S.D. at 5% 0.7258

6.4. Effect of some biological control agents’ filtrate on sugar content

of infected powdery mildew cucumber plants:

The data in Table (28) and Fig. (8) reveal that, sugars content was

significantly affected by the tested treatments. All treatments decreased

the reducing sugars except Topas-100. The highest decrease was induced

by propolis extract + Bacillus filtrate (56.84 %), propolis extract +

Bacillus filtrate + Trichoderma filtrate (55.79 %) and Bacillus filtrate

(54.11 %) less than the control. While Topas-100 increased it by 63.79 %

over control treatment.


0

0.5

1

1.5

2

2.5

3

3.5

4m

g/g

Total amino acids content

Clove extract10%

Garlic extract 20%


Clove oil 10%

Olive oil 8%

Rocket oil 8%

Nigella oil 8%

phosphate salt 100 mM/L

Topas 50cm3/100L

Control

Figure (7): Effect of spraying Plant extracts & oils and K2HPO4 on total


plants as mg /1 g fresh weight.

As for the non-reducing sugars, Trichoderma filtrate, Topas-100,

Bacillus filtrate and Bacillus filtrate + Trichoderma filtrate increased the

non-reducing sugars by 397.87, 100.0, 17.02 and 17.02% over control.

While propolis extract + Bacillus filtrate + Trichoderma filtrate,

propolis extract +Trichoderma filtrate, propolis extract and propolis

extract + Bacillus filtrate decreased it by 85.11, 51.10, 34.04 and 23.40%,

respectively less than control treatment.

All treatments decreased total sugars except Topas-100 and

Trichoderma filtrate increase it. Propolis extract + Bacillus filtrate +

Trichoderma filtrate, propolis extract + Bacillus filtrate, propolis extract


+Trichoderma filtrate and Bacillus filtrate induced the highest decrease by

58.43, 53.83, 47.90 and 47.70% respectively less than the control. While

Topas-100 and Trichoderma filtrate increase it by 68.20 and 26.82%

respectively over control.

Table (28): Effect of some biological control agents filtrate on sugar

content of infected powdery mildew cucumber plants as mg

/1 g fresh weight.

Treatments

Sugar content Efficacy %

Reducing sugars

Non reducing

sugars

Total sugars

Reducing sugars

Non reducing

sugars

Total sugars

Propolis extract 2.80 0.31 3.11 -41.1 -34.04 -40.42

Trichoderma filtrate 4.28 2.34 6.62 -9.9 397.87 26.82

Bacillus filtrate 2.18 0.55 2.73 -54.11 17.02 -47.70

Propolis extract

+Trichoderma filtrate 2.49 0.23 2.72 -47.58 -51.1 -47.90

Propolis extract + Bacillus

filtrate 2.05 0.36 2.41 -56.84 -23.40 -53.83

Bacillus filtrate +

Trichoderma filtrate 2.96 0.55 3.51 -37.68 17.02 -32.76


filtrate + Trichoderma

filtrate

2.10 0.07 2.17 -55.79 -85.11 -58.43

Topas-100

50cm3/100L 7.78 0.94 8.78 63.79 100.00 68.2

Control 4.75 0.47 5.22

Reducing

sugars Non reducing

sugars

Total sugars

L.S.D. at 5% 0.7760 0.1896 0.7875


0

1

2

3

4

5

6

7

8

9

mg

/g

Reducing sugars Non reducing sugars Total sugars

Propolis extract


Bacillus filtrate


Propolis + Bacillus filtrate

Bacillus filtrate + Trichoderma filtrate

Propolis + Bacillus filtrate + Trichoderma filtrate

Topas 50cm3/100L

Control

Figure (8): Effect of some biological control agents filtrate on sugar

content of infected powdery mildew cucumber plants as mg

/1 g fresh weight.


6.5. Effect of some biological control agents’ filtrate on phenol

content of infected powdery mildew cucumber plants:

The data in Table (29) and Fig. (9) show that, the free, conjugated

and total phenols was affected significantly by the tested treatments

compared with control. All tested treatments increased the free phenols.

The highest increase in the free phenols was induced by Bacillus filtrate +

Trichoderma filtrate (342.65%) followed by Topas-100 (326.88%) and

propolis extract + Bacillus filtrate (300.7%) over control.

As for the total phenols, all tested treatments increased the total

phenols. The highest increase in the total phenols was induced by Topas-

100 (97.33%) followed by Bacillus filtrate + Trichoderma filtrate

(71.6%) and propolis extract + Bacillus filtrate (48.71%) over control.

While treatments were differed in the effect on the conjugated

phenols, Trichoderma filtrate and Topas-100 increased the conjugated

phenols by 32.76 and 18.17% respectively over control. Propolis extract

+ Bacillus filtrate + Trichoderma filtrate, Bacillus filtrate, propolis extract

+ Bacillus filtrate, propolis extract + Trichoderma filtrate and Bacillus

filtrate + Trichoderma filtrate decreaseD the conjugated phenols by

60.00, 41.78, 38.20, 25.46 and 21.88% respectively less than control. On

the other hand, propolis extract did not affect on the conjugated phenols

and gave equal value (8.09 mg/1 g) with control treatment.


Table (29): Effect of some biological control agents filtrate on phenol

content of infected powdery mildew cucumber plants as

mg/g fresh weight.

Treatments

phenol content Efficacy %

Free

phenols

Conjugated

phenols

Total

phenols

Free

phenols

Conjugated

phenols

Total

phenols

Propolis extract 7.21 8.09 15.30 158.42 00.00 40.63

Trichoderma filtrate 10.59 10.74 21.33 279.57 32.76 96.1

Bacillus filtrate 6.62 4.71 11.33 137.28 -41.78 4.14

Propolis extract +

Trichoderma filtrate 6.47 6.03 12.50 131.90 -25.46 14.89

Propolis extract +

Bacillus filtrate 11.18 5.00 16.18 300.70 -38.2 48.71

Bacillus filtrate +

Trichoderma filtrate 12.35 6.32 18.67 342.65 -21.88 71.60

Propolis extract +

Bacillus filtrate +


8.97 3.24 12.21 221.51 -60.00 12.22

Topas-100

50cm3/100L 11.91 9.56 21.47 326.88 18.17 97.33

Control 2.79 8.09 10.88


L.S.D. at 5% 0.8706 0.7683 0.8462


0

5

10

15

20

25m

g/g


Propolis


Bacillus filtrate

Propolis + Trichoderma filtrate




Topas 50cm3/100L

Control

Figure (9): Effect of some biological control agents filtrate on phenol

content of infected powdery mildew cucumber plants as

mg/g fresh weight.


6.6. Effect of some biological control agents’ filtrate on total amino

acid content of infected powdery mildew cucumber plants:


significantly decreased the total amino acid content. The highest decrease

induced by Bacillus filtrate + Trichoderma filtrate (-95.84%) followed by

Trichoderma filtrate (-89.10%), propolis extract + Bacillus filtrate (-

88.00%) and propolis extract + Trichoderma filtrate (-86.75%).

Table (30): Effect of some biological control agents filtrate on total


plants as mg/1g fresh weight.

Treatments Total amino

acid contents Efficacy %

Propolis extract 1.10 -71.43


Bacillus filtrate 1.40 -63.64

Propolis extract +



filtrate 0.46 -88.00

Bacillus filtrate +



filtrate + Trichoderma

filtrate

1.18 -69.34

Topas-100

50cm3/100L

1.64 -57.40

Control 3.85

Treatment

L.S.D. at 5% 0.7663


0

0.5

1

1.5

2

2.5

3

3.5

4m

g/g

Total amino acid contents

Propolis


Bacillus filtrate

Propolis + Trichoderma filtrate




Topas 50cm3/100L

Control

Figure (10): Effect of some biological control agents filtrate on total


plants as mg/g fresh weight.

Discussion 105

DISCUSSION

Cucumber (Cucumis sativus L.) is one of the important

economically crops. This belongs to family cucurbitaceae. Cucumber is

grown either in the open field or under protected houses. The total

cultivated area increased rapidly, especially in the reclaimed lands.

Cucumber plants are attacked with several diseases as downy

mildew disease, powdery mildew, root-rot disease, wilt disease, root –knot

nematode, bacterial diseases and viral diseases.


(Schlectend: Fr.) Pollacci, is considered one of the most economically

important and widespread diseases on cucumber in Egypt (El-Desouky,

1988; El-Shami et al., 1995; Mosa, 1997 and Abd-El–Sayed, 2000).

The disease causes damage to all plant parts including leaves, stems

and fruits especially under protected houses conditions and causing

considerable reduction of quantity and quality of cucumber yields.

The chemical fungicides have been used for along time as the main

strategy for control in order manage these obligate fungal diseases and

subsequently increase yield production (Ghawande, 1989; El-Naggar,

1997 and Nada, 2002), on the other hand the fungicides resistant races of

the pathogen have been reported by (O’Brien, 1994 and McGrath &

Staniszewska, 1996). As well as the side effects of fungicides on human

health (Eckert & Ogawa, 1988 and Durmusoglu et al., 1997) and the

environment (Horst et al., 1992 and Garcia, 1993). Therefore

development of nontoxic alternative to chemical fungicides would be

useful in reducing the undesirable effects of their uses.

Several plant extracts and plant oils were found to be effective in

controlling obligate diseases including powdery mildew all over the world

(Achimu & Schlosser, 1992; Rovesti et al., 1992; Singh et al., 1992;

Ahmed, 1995; El-Naggar, 1997; Amadioha, 1998; Abdel-Megid et al.,

Discussion106

2001; Sallam Minaas, 2001; Haroun, 2002 and Nada, 2002). As well as

Biological control of powdery mildew disease has been reported by many

researchers (Minuto et al., 1991; Heijwegen, 1992; Urquhart et al.,

1994; Ahmed, 1995; Abo-foul et al., 1996; Dik et al., 1998; Verhaar et

al., 1998; Abd-El–Sayed, 2000).

The presented work is investigating the effect of three plants,

extracts garlic (Allium sativum), cloves (Syzygium aromaticum) and

withania (Withania somniferum) at three concentrations on spore

germination of Sphaerotheca fuliginea. The results indicate that, all tested

plant extracts significantly reduced the percentage of conidia germination

compared with Topas-100 fungicide and the control treatment. The high

reduction was induced by garlic extract followed by clove extract and

withania more than Topas-100 fungicide and the control treatment. The

effect of plant extracts might be mainly due to the inhibitory effects of the

antifungal compounds in the extracts on germination of the fungal spores,

since some of these extracts in preventive treatment completely prevented

infection. This is in agreement with the observance of (Daayf et al. 1995,

El-Naggar, 1997; Singh et al. 1999 and Haroun, 2002). In this respect,

Ahmed and Agnihotri (1977) found germ tube abnormalities, followed by

lysis and disintegrated in germinated fungal spores after they were treated

with a plant extract. Singh and Singh (1981), found that garlic (Allium

sativum) cloves, onion (Allium cepa) bulbs and ginger (Zingiber officinale)

rhizome had volatile compounds completely inhibited spore germination

of Erysiphe polygoni. Ahmed (1995) found complete inhibition in spore

germination of Sphaerotheca fuliginea the causal pathogen of cucumber

powdery mildew by using garlic, thyme and henna plant extracts at 100%

and blue gum, marjoram and chamomile at 50% concentration. Singh et al.

(1995) found that complete inhibition of conidial germination of (Erysiphe

pisi) was observed when ajoene (a compound derived from garlic) was

Discussion 107

used at 25 mg/litre. Seddon and Schmitt (1999) mentioned that plant

extracts of R. sachalinensis have been shown to inhibit spore germination

of Sphaerotheca fuliginea.

Evaluation the effect of clove oil, nigella oil, olive oil and rocket oil

at three concentrations on spore germination of Sphaerotheca fuliginea

conidia reveal that, all tested plant oils significantly reduced the percentage

of conidia germination compared with the control. The high reduction was

observed by clove oil, nigella oil and olive oil more than Topas-100

fungicide and the control treatment. The present results are in agreement

with Dubey and Dwivedi (1991) who mentioned that the difference in the

behavior of the essential oils might due to either their volatility or

composition. Similar results were obtained by Raj-Kishore et al. (1996).

They found that clocimum oil [Ocimum gratissimum] and lemongrass oil

[Cymbopogon spp.], gave 100% inhibition of conidial germination of

powdery mildew (E. polygoni) of opium poppy (Papaver somniferum)

using a conidial germination technique at the lowest conc. (250 ppm).

Nada (2002) reported that volatile oils as film on slides completely

prevented spore germination of Sphaerotheca fuliginea.

The reduction in spore germination was significantly increased by

increasing concentration of the plant extracts and plant oils. Increasing the

reduction in spore germination by increasing concentration of the extracts

might be mainly due to the high concentration of the antifungal compounds,

which were presented in the water extracts. In this respect, Shetty et al.,

(1989) stated that the difference in activity between the extracts might be

due to variation in the concentration and composition of antifungal

compounds in the different plants. Similar results were obtained by

Abd-El-Megid et al., (2001) who stated that downy mildew disease

incidence and severity were decreased by increasing concentration of some

plant extracts (Eucalyptus and Rosemary) sprayed on onion plants.

Discussion108

Evaluation the effects of phosphate salt (K2HPO4) at three

concentrations on germination of Sphaerotheca fuliginea conidiospores

indicated that, phosphate salt (K2HPO4) significantly reduced the

percentage of conidia germination. Generally, the reduction in spore

germination was significantly increased by increasing concentration of

phosphate salt. In this respect, El-Habbak (2003) reported that phosphate

salt (KH2PO4) reduced conidial germination of Sphaerotheca fuliginea the

causal organism of powdery mildew in squash by 82.1% compared with

control.

Evaluation the effect of propolis extract, Trichoderma filtrate,

Bacillus filtrate and their combinations on spore germination of

Sphaerotheca fuliginea conidia showed that, all tested treatments

significantly reduced the percentage of conidia germination more than

Topas-100 fungicide and the control treatment. The high reduction was

induced by propolis extract + Bacillus filtrate + Trichoderma filtrate,

Bacillus filtrate + Trichoderma filtrate and propolis extract + Trichoderma

filtrate compared with control treatment. Many authors also recorded the

antagonistic effect of Trichoderma sp. against pathogenic fungi. Dennis

and Webster (1971) reported that Trichoderma spp. produced the

antibiotic "Trichodermol". This antibiotic can inhibit the growth of several

fungi. Ahmed (1995) tested the effect of some fungal filtrates on spore

germination of S. fuliginea. He found that T. harzianum and T. viride had

the most antagonistic effect on spore germination of the pathogen. Many

authors also recorded the antagonistic effect of B. subtilis. Pusey and

Wilson (1984) reported that B. subtilis exerted a heat stable antibiotic

interfering with spore germination, or early germ tube development of

stone fruit brown root pathogen. Schmitt et al. (1999) reported that the

antifungal metabolite gramicidin S of Bacillus brevis inhibited conidial

germination of S. fuliginea by around 80 % compared with control.

Discussion 109

Giuseppe Lima et al. (1998) reported that propolis extract (0.5% w/v)

showed a high antifungal activity, particularly against B. cinerea in vitro.

The combination between propolis extract, Trichoderma filtrate and

Bacillus filtrate gave high inhibition.

Evaluation the effect of three plants extracts garlic (Allium sativum),

cloves (Syzygium aromaticum) and withania (Withania somniferum) at

three concentrations in induction cucumber resistance to powdery mildew

under greenhouse conditions revealed that all tested treatments

significantly reduced the percentage of powdery mildew disease incidence

and severity compared with the control treatment. The high reduction was

induced by garlic extract followed by Topas-100 and clove extract and

withania extract.

On the other hand the results obtained under protected houses

(during spring and autumn 2003) revealed that garlic extract was the most

effective treatment in the mean of both seasons followed by clove extract,

Topas-100 and withania extract compared with control.

The more effective concentration of plant extracts in previous

studies was evaluated to their effect on the percentage of powdery mildew

incidence, severity, The fruit number/plant and fruit weight/plant under

protected houses at (spring 2004). The results indicated that, all tested

plant extracts significantly reduced the percentage of powdery mildew

incidence and severity compared with the control treatment. The high

reduction in disease severity was induced by garlic extract followed by

clove extract, Topas-100 and withania extract. Also, all tested plant

extracts significantly increased the fruit number/plant and fruit

weight/plant. The highest increase in fruit number/plant and fruit

weight/plant was induced by garlic extract followed by clove extract,

withania extract and Topas-100 respectively.

Discussion110

The inhibitory effect may be attributed to the formation of a physical

barrier (Ziv, 1983), which prevent fungal penetration and reduced disease

incidence. Also, it formed a continuous membrane that permits diffusion of

oxygen and carbon dioxide but inhibits the passage of water and promotes

a healthy physiological state of the plant (Han, 1990). Similar results were

obtained by Herger et al. (1988) who reported that weekly applications of

aqueous extract solution of Reynoutria sachalinensis (Milsana) leaf

suppressed the infection of E. cichoracearum and S. fuliginea and

increased the chlorophyll in cucumber leaves. Ahmed (1995) reported that

Garlic and/or Henna extracts were the most effective in inhibiting disease

infection of S. fuliginea. The reduction of disease intensity may be due to

the increment in chlorophyll content in cucumber plants, which was

reduced as a result to powdery mildew infection. Also, the accumulation of

fungitoxic phenolic compounds in cucumber infected leaves treated with

(Milsana) supported the hypothesis that the bioinducer may act indirectly

by inducing plant defense reaction (Daayf et al., 1995). Furthermore,

Daayf et al. (1997) found that cucumber plants produced elevated levels of

phytoalexins in response to an eliciting treatment with Milsana after

infection with S. fuliginea. Raj-Kishore et al. (1996) reported that eugenol

was effective compound in controlling powdery mildew (E. polygoni)

which gave 100% inhibition at the lowest concentration (250 ppm).

Abd-El-Sayed (2000) found that the foliar application of some plant

extracts (thyme, henna, eucalyptus and garlic) individually or mixed

decreased powdery mildew (S. fuliginea) intensity on cucumber than

control when used before or after inoculation. One week after extract

application, the disease intensity was highly decreased by increasing

concentrations of all treatments 3 days before or 5 days after inoculation.

Abdel-Megid et al. (2001) who stated that downy mildew disease

Discussion 111

incidence and severity were increased by increasing concentration of some

plant extracts (Eucalyptus and Rosemary) sprayed on onion plants.

In the present study, reduction in the disease incidence with plant

extracts might be due to: (1) altering the physiology and biochemistry of

plants through augmented phenolic levels, (2) increasing enzymes activity

as peroxidase, polyphenol-oxidase and chitinase (3) Increasing the barrier

defense through increasing the cell-wall lignification (4) altering the

physiology of plant through decreasing sugars content and total amino acid.

In this respect, Natarajan and Lalithakumari (1987) found significant

inhibition in respiration (oxygen uptake) in fungal cells resulted in a

general reduction of total protein, DNA and R-NA. Milsana flulssing

(commercial product of the plant extract) as preventive treatment

effectively controlled powdery mildew (S, fuliginea) of cucumber and the

mode of action appeared to involve the induction of plant defense

responses (Herger and Klingauf, 1990), and particulary phenolics (Daayf

el al., 1997) were among the defense molecules found to be enhanced by

this product. Wurms et al. (1999) provided evidence on the appearance of

local resistance from that prophylactic compounds resulted in collapse of

powdery mildew mycelia and hustoria. Also, scanning micrography of the

wheat leaves treated with plant extracts (chamomile and lemon grass)

revealed peculiar morphological changes of the rust fungus; Puccinia

recondita f.sp. tritici (Sallam Minaas, 2001) appeared as excessive

branching and a distinguished elongation of germ tubes, though the evident

failure of appresoria development.

Moreover, control of powdery mildew of cucumber by spraying

with garlic extract (20%), clove extract (10%) and withania extract (50%)

was always comparable or over to the fungicide Topas-100. These results

are in agreement with those obtained on downy and powdery mildews by

Rovesti et al. (1992) (neem extract = Sulfur), Ahmed (1995) (garlic

Discussion112

extract = Top Cop), Daayf et al. (1995) (giant knotweed extract; Milsana

flussing, as. commercial product = Benomyl), Abdel-Megid et al. (2001)

(black cumin extract = Ridomil plus), Haroun (2002) (garlic extract (20%)

and clove extract effective more than Bayfidan (E.C) Rubigan (E.C)

Thiophate-14 (W.P) Sumi-8 (E.C)) and Nada (2002) (thyme, blue gum,

leek and marjoram = Rubigan).

Evaluation the effect of plant oils i.e. clove oil, nigella oil, olive oil

and rocket oil at three concentrations in induction cucumber resistance to

powdery mildew under greenhouse conditions showed that all tested

treatments significantly reduced the percentage of powdery mildew

disease incidence and severity compared with the control treatment. Olive

oil at concentration 8% completely prevented powdery mildew incidence.

The high reduction was induced by olive oil (4%), rocket oil (8%), nigella

oil (8%) and clove oil (10%) followed by Topas-100. On the other hand,

the results obtained under protected houses (during spring and autumn

2003) revealed that all tested plant oils in the mean of both seasons


severity compared with the control treatment. Clove oil and olive oil were

the most effective treatments at both seasons followed by nigella oil,

Topas-100 and rocket oil compared with control. The more effective

concentration of plant oils in previous studies was evaluated to their effect

on the percentage of powdery mildew incidence, severity the fruit

number/plant and fruit weight /plant under protected houses at (spring

2004). The results indicated that, all tested plant oils significantly reduced

the percentage of powdery mildew incidence and severity compared with

the control treatment. The high reduction in disease severity was induced

by olive oil followed by clove oil, nigella oil, rocket oil, and Topas-100.

Also, all tested plant oils significantly increased the fruit number/plant and

fruit weight/plant. The highest increased in the fruit number/plant and fruit

Discussion 113

weight/plant was induced by clove oil, followed by olive oil, nigella oil,

rocket oil and Topas-100 respectively. These results in agreement with

those obtained by Haberle and Schlosser (1993) who sprayed the upper

side of leaves of cucumber with a fine mist of Telmion, a product

containing 85% rapeseed oil, 1 d before and 2, 4 and 6 d after inoculation

with Sphaerotheca fuliginea. After 12 d incubation, counts of pustules per

leaf showed the protective treatment to give a significant (P <0.05)

reduction in disease severity (efficacy >90%), with the curative treatment

applied 6 d after inoculation having almost as high an efficacy. Pustule

diam. decreased by 28% and 55% after protective and curative treatments,

respectively, and the treatments gave significant reductions in numbers of

conidia per pustule and conidia per leaf. Nada (2002) reported that volatile

oils as film on slides completely prevented spore germination of

Sphaerotheca fuliginea. Also, spraying squash plants in greenhouse and

field with eight essential oils as preventative and curative treatments gave

sufficient control to disease in most cases. Thyme essential oil completely

prevented the disease incidence in the field. Ko et al. (2003) found that

plant oils i.e. canola oil, corn oil, grape seed oil, peanut oil, safflower oil,

soya bean oil or sunflower oil at 0.1% were greatly reduced the severity of

tomato powdery mildew caused by Oidium neolycopersici. Sunflower oil

was the most effective in the control of powdery mildew. Scanning

electron microscopy showed that control of powdery mildew with

sunflower oil resulted mainly from the inhibition of conidial germination

and suppression of mycelial growth of the pathogen. The high fungicidal

activities of the essential oils tested might be due to their high capabilities

to damaging structure and function of the fungal cell wall as well as

disturbing the physiology of the enzymatic bioactivity. In this respect, the

fungal growth damages caused by the essential oils might be due to their

capabilities to penetrate into the fungal cells (Saksena & Tripathi, 1987;

Discussion114

Wilson et al., 1987; Zambonelli et al., 1996 and Jaspal & Tripathi, 1999)

and to cause alternations in fungal metabolism by their mutagenic

activities (Zani et al., 1991). Also, the fungicidal activities of the essential

oils might be due to increase in the permeability of the fungal cell as well

as inhibition in the fungal detoxification enzymes of the antifungal oil

substances. The essential oils are also capable to affect respiration of the

fungal cell (oxygen uptake) and having toxic substances acting as

antisporulation compounds (Inouye et al., 1988). In the present study,

reduction in the disease incidence with plant extracts might be due to:

alternation in physiology and biochestiry activities of plant through (1)

increasing phenolic levels, (2) increasing enzymes activity as peroxidase,

polyphenol-oxidase and chitinase (3) Increasing the barrier defense

through increasing the cell-wall lignifection (4) altering the physiology of

plant through decreasing sugars content and total amino acid. However,

the harmful effects on fungi were restricted in: (a) partial or complete

inhibition on spore germination, sporulation or mycelial growth (Reuveni

et al., 1984; Saksena & Tripathi, 1985; Nachman et al., 1994 and

El-Shazly, 2000) (b) alternation in physiology and biochestiry activities of

the fungal cells (Zani et al., 1991; Favaron et al., 1993; Zedan et al.,

1994 and Zambonelli et al., 1996).

Moreover, control of powdery mildew of cucumber by spraying

with olive oil (8%), rocket oil (8%), nigella oil (8%) and clove oil (10%)

was always comparable or over to that of the fungicide Topas-100. These

results in agreement with those obtained on downy and powdery mildew

by Singh et al. (1983) (ginger extract and garlic oil = dinocap, wettable S

or carbendazim), Haroun (2002) (Clove oil) effective more than Delmite

(L.), Microthiol-80 (W.G), Golf (E.C), Sumi-8 (E.C), Flint (W.G),

Bayfidan (E.C), Domark (E.C), Topas-200 (W.P), Thiophate-14 (W.P),

Rubigan (E.C) and Bellkute (W.P) and (Nigella oil effective more than

Discussion 115

Thiophate-14 (W.P), Microthiol-80 (W.G) and Delmite (L.)), Nada (2002)

(thyme oil = Rubigan) and Azam et al. (1998) (rape oil = sulfur and

fenarimol).

Evaluation the effect of Phosphate K2HPO4 at concentration 50, 75

and 100 mM for induction the systemic resistance to powdery mildew

under greenhouse conditions revealed that phosphate salt (K2HPO4)


compared with the control. The high reduction was induced by phosphate

salt (K2HPO4) at concentration 100 mM/L. On the other hand the results

obtained under protected houses (during spring and autumn 2003) revealed

that phosphate salt (K2HPO4) at both seasons significantly reduced the

percentage of powdery mildew incidence and severity compared with the

control treatment. Phosphate salt (K2HPO4) at concentration 100 mM/L the

most effective treatment in the mean of both seasons followed by

phosphate salt (K2HPO4) at concentration 75 mM/L and Topas-100 at

concentration 50 ml/100L compared with control. Phosphate salt (K2HPO4)

at concentration 100 mM/L, the most effective treatment in previous

studies was evaluated to their effect on the percentage of powdery mildew

incidence, severity, the fruit number/plant and fruit weight/plant under

protected houses at (spring 2004). The results indicated that, phosphate salt

(K2HPO4) at concentration 100 mM/L significantly reduced the percentage

of powdery mildew incidence and severity compared with the control

treatment. The high reduction in disease severity was induced by

phosphate salt followed by Topas-100. Also, phosphate salt increased the

fruit number/plant and fruit weight/plant. The presented results are in

agreement with Abd-El-Kareem (1998) who reported that spraying

cucumber plants with phosphate (K2HPO4) at concentration 100 mM/L

reduced the severity of cucumber powdery mildew (S. fuliginea) and

significantly increased fruit yield by 178.5% compared with plants treated

Discussion116

with distilled water. Also the same results were obtained by (Reuveni et al.,

1995) who reported that semple compounds such as phosphate and

potassium salts were effectively suppressed and controlled powdery

mildew development on cucumber plants. This suppression was a direct

effect of phosphate on the mycelia and conidia which were collapsed, and

the direct effect on encourage of antagonistic phylloplane organisms.

Furthermore, there are host metabolites such as phytoalexins production

(Yoshikawa, 1978 and Hargreaves, 1979). Also, increased accumulation

of peroxidase in phosphate treated leaves is strongly indicate the possible

role or peroxidase in defense mechanism (Gamil 1995; Reuveni et al.,

1995 and Abd-El-Sayed, 2000). Mosa (1997) examined the effect of

various potassium phosphate salts applied as foliar spray treatments, for

controlling powdery mildew of cucumber (Sphaerotheca fuliginea). He

reported that the most effective treatments were K2HPO4 and K3PO4

showing both protective and curative effects against S. fuliginea infection.

Resistance in the second true leaf of cucumber to powdery mildew was

induced following treatment of the first true leaf with K2HPO4, K3PO4 and

KH2PO4, respectively. Powdery mildew infection was significantly

reduced by 92% when the plants were treated with 50mM K2HPO4, 3 days

after inoculation.

Evaluation the effect of propolis extracts, Trichoderma filtrate

Bacillus filtrate and their combinations for induction cucumber resistance

to powdery mildew under greenhouse conditions was done. Results

indicated that all tested treatments significantly reduced the percentage of

powdery mildew disease incidence and severity compared with the control

treatment. Propolis extract + Bacillus filtrate + Trichoderma filtrate

completely prevented powdery mildew incidence. The high reduction was

induced by Trichoderma filtrate, propolis extract + Trichoderma filtrate,

propolis extract + Bacillus filtrate and Trichoderma filtrate + Bacillus

Discussion 117

filtrate. On the other hand the results obtained under protected houses

(during spring and autumn 2003) revealed that all tested biological agents

at both seasons significantly reduced the percentage of powdery mildew

incidence and severity compared with the control treatment. Propolis

extract + Bacillus filtrate + Trichoderma filtrate the most effective

treatment in the mean of both seasons followed by propolis extract +

Trichoderma filtrate & Trichoderma filtrate + Bacillus filtrate and Bacillus

filtrate & Trichoderma filtrate. The more effective treatments in previous

studies evaluated to their effect on the percentage of powdery mildew

incidence, severity, the fruit number/plant and fruit weight/plant under

protected houses at (spring 2004). The results indicated that, all biological

agent tested significantly reduced the percentage of powdery mildew

incidence and severity compared with the control treatment. The high

reduction in disease severity was induced by propolis extract +

Trichoderma filtrate + Bacillus filtrate followed by Trichoderma filtrate +

Bacillus filtrate, propolis extract + Trichoderma filtrate & propolis extract

+ Bacillus filtrate and Bacillus filtrate. Also, all tested treatments increased

the fruit number/plant and fruit weight/plant. The highest increased in the

fruit number/plant and fruit weight/plant was induced by propolis extract +

Trichoderma filtrate + Bacillus filtrate followed by propolis extract +

Trichoderma filtrate and Trichoderma filtrate + Bacillus filtrate. This high

potentiality in antagonism might be due to that Trichoderma spp. act

through different mechanisms including mycoparasitism (Abd EI-Moity

and Shatla, 1981; Martin and Hancock, 1987 and Benhamou & Chet,

1993), also, through production of antifungal substances (Turner, 1971

and Hayes, 1992). Trichoderma spp. also act through production of

destructive enzymes i.e. chitinase (Ab-El-Moity, 1981; Paderes et al.,

1992 and Bolar et al., 2000).

Discussion118

Bacillus subtilis also showed considerable effect in controlling

powdery mildew. This might be due to that, this bacterium produces more

antibiotics (Bacteriocin and subtilisin) which act as inhibitors to

pathogenic fungi (Ferreira et al., 1991; Asaka & Shoda, 1996 and

Farahat, 1998), in addition to this action, B. subtilis also grows very fast

and occupies the court of infection and consumes all available nutrients.

These actions prevent pathogen spores to reach susceptible tissues. Also,

its effect might be due to competition for spaces or nutrients (Wolk and

Sorkar, 1994).

The combination between propolis extract, Trichoderma filtrate,

Bacillus filtrate gave high effect, the effect of mixture can be explained in

the light of work carried out by Abd El-Moity (1985) who stated that this

synergistic effect might be due to complementary effect between different

isolates included in the mixture.This means that, one isolate produced

antifungal substance (Hayes, 1992), whereas the second isolate has high

potentialities in mycoparasitism (Abd El-Moity, 1981 and Martin &

Hancock, 1987), while the third one induce plant resistant (Elad el at.,

1998 and Bolar et al., 2000).

All antagonists significantly reduced the percentage of disease

incidence or disease severity and increased cucumber yield compared with

control treatment. This might be due to that, all used antagonists have

different effect against pathogenic fungi. These effects might be due to

direct mycoparasitism as in some T. harzianum as previously mentioned or

affect through enzyme and/or antifungal substances (Turner, 1971 and

Padares et al., 1992) or also stimulate resistant in the host (Elad et al.,

1998, Howell et al., 2000 and Bolar et al., 2000). In this respect, Elad et al.

(1999) indicated that the application of T. harzianum T39 conidia to the

root zone of plants resulted in the reduction of foliar grey mould, white

mould and powdery mildews. The modes of action of T. harzianum T39

Discussion 119

are competition with the pathogen for nutrients and space, suppression of

hydrolytic enzymes of the pathogen and induced host resistance. Schmitt

et al. (1999) reported that in vivo studies on cucumber plants Bacillus

brevis cultures reduced the disease intensity of S. fuliginea significantly

when applied one day before or after inoculation, with the latter showing

stronger effects (average of 40 % efficacy). In vitro studies with conidia of

S. fuliginea revealed that the antifungal metabolite gramicidin S inhibited

conidial germination by around 80 %. The results indicate that B. brevis

has the potential to be used as a biocontrol agent against S. fuliginea and

other plant pathogens. Elad (2000) reported that the bio-control agent

Trichoderma harzianum isolate T39 controls the powdery mildew

Sphaerotheca fusca (syn. S. fuliginea) in cucumber under commercial

greenhouse conditions. Involvement of locally and systemically induced

resistance has been demonstrated. Cells of the Trichoderma harzianum

applied to the roots, and dead cells applied to the leaves of cucumber plants

induced control of powdery mildew. A combination of several modes of

action is responsible for biocontrol. They found that bio-control agent has

the potential to degrade cell-wall polymers, such as chitin.

Evaluation the effect of UV, temperature and chloroform on

powdery mildew spores germination revealed that treated powdery mildew

spores suspension with UV for 30 minutes or temperature 90癈 for 10

minutes or add Chloroform 1.0 ml/L to spore suspension completely

inhibited spore germination. Data also revealed that increasing time of

revelation to UV, temperature or concentration of chloroform led to

increase efficacy of the treatment in reducing the percentage of

conidiospores germination.

Evaluation the effect of UV, temperature and chloroform on

powdery mildew spores infectivity showed that the most effective

Discussion120

treatments which completely inhibited all spores of powdery mildew and

no disease was developed had been recorded when spores were subjected

to one of the following treatments: UV for 30 minutes or 90C for 10

minutes and 1ml chloroform /L for 30 minutes. Data also revealed that

increasing time of revelation to UV, temperature or concentration of

chloroform /ml led to increase efficacy of the treatment in reducing the

percentage of disease incidence.

The ungeminated spore exposed to UV for 30 minutes, temperature

90癈 for 10 minutes or treated with 1ml chloroform/L. were used for

inducing cucumber resistant to powdery mildew disease. The results

indicated that all tested treatments significantly reduced the percentage of

powdery mildew incidence and severity compared with the control

treatment. UV, temperature, chloroform and Topas-100 reduced disease

severity respectively. Also, all tested treatments increased the fruit

number/plant and fruit weight/plant. The highest increase in fruit

number/plant and fruit weight/plant was induced by UV followed by

temperature and chloroform respectively. This could be explained in the

light of fact, that UV works on the enzyme and never destroys the outer

layer of spores Abd-El-Moneim (2001). As a result, spores were killed

without any changes in the structure of outer layer. Consequently, these

killed spores occupied the same site of infection and block these sites

against viable pathogenic spores. On the contrary, using heat or chloroform

lead to changes in this outer layer of spores due to agglutination (effect of

heat) (Robert, 1975), or dissolving lypoprotein layer in the outer layer

(James, 1982). Due to these changes the killed spores could not occupy the

same sites of infection, consequently give chance to viable pathogenic

spore to reach the proper sites, germinate, invade and cause disease. In this

respect, competition for site suitable for infection process between

Discussion 121

avirulant and virulent pathogens was recorded (Kerr, 1980; Moor &

Cooksey, 1981; Cooksey & Moor, 1982a; 1982b and Garibaldi et al.,

1992). Also it was recorded that avirulant isolate stimulate production of

some substances which resist invasion of pathogenic germ tube

(Mahmoud et al., 1995).

The pervious results in agreement with Abd-El-Moneim (2001)

who reported that spraying cucumber plants with powdery mildew spores

which had been killed with UV for 30 minutes were more effective in

inducing resistance to powdery mildew (Sphaerotheca fuliginea)

compared with spores killed by heat 90 癈 for 10 minutes or 1 ml

chloroform/L.

The present work evaluated the effect of the best resistance inducers

i.e. garlic extract at 20%, clove extract at 10%, olive oil at 8%, propolis

extract + Trichoderma filtrate, Trichoderma filtrate + Bacillus filtrate,

propolis extract + Trichoderma filtrate + Bacillus filtrate, K2HPO4 at 100

mM. and Topas-100 at 50 ml/100L each one alone and their combination

with powdery mildew disease on peroxidase, polyphenol-oxidase and

chitinase enzymes activity. Results indicated that all tested treatments

significantly increased the activity of all enzyme tested. The high value of

all enzymes obtained after five days from inoculation with powdery

mildew spores. While the highest increase in peroxidase activity compared

with control was induced by propolis extract + Trichoderma filtrate before

inoculation. On the other hand, the highest increase in polyphenol-oxidase

and chitinase was induced by propolis extract + Trichoderma filtrate after

one day from inoculation. Many plant enzymes are involved in defense

reaction against plant pathogen. These included oxidative enzymes such as

peroxidase and polyphenoloxidase which catalase the formation of lignin

and other oxidative phenols that contribute to formation of defense barriers

for reinforcing the cell structure (Avdiushko et al., 1993). Enzyme activity

Discussion122

played an important role in plant disease resistance through increasing

plant defense mechanisms that are considered the main tool of varietal

resistance (Takuo et al., 1993).

The present results concerning the increase in peroxidase,

polyphenol-oxidase and chitinase enzymes activity are in agreement with

those reported by Matta et al. (1988); Yurina et al. (1993) Mosa (1997);

Reuveni et al. (1997); Abd-El-Kareem (1998) and El-Habbak (2003).

In this respect, Smith and Hammerschmidt (1988) found that induced

resistance in cucurbit plants accompanied by a marked increase in

intercellular peroxidase isozymes. Induced resistance in cucumber plants

with K2HPO4 increased the activity of peroxidase and chitinase enzymes

(Irving and Kuc 1990) and -1.3-glucanase (Avdiushko et al., 1993).

Induced resistance in cucumber plants with acetylsalicylic acid (aspirin)

led to an increase activity of chitinase, -l,3-glucanase, peroxidase

polyphenol-oxidase and phenylalanine ammonia.reported (Schneider and

Ullrich, 1994).

Similar results were obtained by Yurina et al. (1993); who found

that peroxidase activity was related to resistance and tolerance against

powdery and downy mildew. Resistance or tolerant varieties of cucurbits

had higher activity of the enzyme than susceptible ones. Orober et al.

(1998) recorded that the foliar application of phosphate induced systemic

acquired resistance in cucumber against powdery mildew (Sphaerotheca

fuliginea). As a further consequence of phosphate application, activities of

typical defense-related enzymes like peroxidase and polyphenoloxidase

increased in all parts of the induced plants. Roth et al. (2000) found that

application of aqueous solutions of an extract of Lychnis viscaria seeds in

concentrations from 0.5 to 10 mg/l (dry weight of extract) resulted in an

enhanced resistance of cucumber to viral and fungal pathogens of up to

36% compared with water-treated control plants. After treatment and

Discussion 123

inoculation with powdery mildew a stimulation of different PR-proteins

(approx. + 20% for peroxidase, + 30% for chitinase and up to + 68% for

-1,3-glucanase) in cucumber was found.

Evaluation of the effect of selected inducers i.e. (Garlic extract at

20%, clove extract at 10%, clove oil at 10%, olive oil at 8%, propolis


propolis extract + Trichoderma filtrate + Bacillus filtrate, K2HPO4 at 100

mM. and Topas-100 at 50 ml/100L) on lignin content in cucumber plants

indicated that all tested treatments significantly increased the lignin

content. Lignification play its role as defense mechanisms, increasing the

mechanical resistance of the host cell wall, restricting the diffusion of

pathotoxins and nutrients and inhibiting growth of the pathogens by the

action of toxic lignin precursors and lignifications of the pathogen (Kuc,

1982). Dean and Kuc (1987) reported that more rapid lignifications

following challenge in protected leaves of immunized plants as compared

with leaves from control plants. The rate of lignifications increased more

rapidly in immunized plants as compared with control plants, after

wounding by pricking the leaf with a pin. Thus immunization may

sensitize cucumber to respond rapidly in response to injury as well as

infection. Rapid lignification in resistant or immunized cucumber plants

after penetration by Cladosporium cucumenmim or Colletotrichum

lagenarium and fungal mycelia of both pathogens were lignified in the

presence of confiferyl, hydrogen peroxide and peroxidase prepared from

immunized cucumber leaves (Hammerschmidt and Kuc, 1982). Spraying

cucumber plants with K2HPO4 at 100 mM before ten days from inoculation

with powdery mildew increased lignin content by 65% compared with

control (Abd-El-Kareem, 1998). Induced resistance in various plants is

correlated with enhancement of chitinase activity and -1,3-glucanase

Discussion124

enzymes which hydrolyses hyphal cell wall of pathogenic fungi

(Abd-El-Kareem et al., 2002 and El-Gamal, 2003).

Evaluation the effect of spraying cucumber plants with abiotic

selected treatments (clove extract, garlic extract, withania extract, clove oil,

olive oil, nigella oil, rocket oil and K2HPO4 as well as Topas-100) on sugar

content indicated that, all treatments significantly decreased the reducing

sugars except Topas-100. The highest decrease was induced by clove oil &

nigella oil followed by olive oil, withania extract, clove extract, rocket oil,

garlic extract and phosphate salt (K2HPO4) respectively. All treatments

increased the non-reducing sugars except olive oil and withania extract

decreases it. The highest increase was induced by garlic extract, clove

extract, Topas-100, rocket oil, nigella oil, phosphate salt (K2HPO4) and

clove oil respectively. Topas-100, withania extract, garlic extract, and

phosphate salt (K2HPO4) increased the total sugars and clove oil, while

nigella oil, olive oil, rocket oil and clove extract decreased it.

On the other hand, the effect of spraying cucumber plants with biotic

selected treatments (propolis extract, Bacillus filtrate, Trichoderma filtrate

and their combination as well as Topas-100) on sugar content revealed that

all treatments significantly decreased the reducing sugars and total sugars

while Trichoderma filtrate increased total sugars.

Whereas Trichoderma filtrate, Bacillus filtrate and their

combination increased the non-reducing sugars, propolis extract and his

mixture with the same treatments decreased it. These results are in

agreement with the fact that the powdery mildew could represent as a “high

sugar disease” (Horsfall and Dimond, 1957). Resistance to powdery

mildew was positively correlated with a low sugar content in the leaves of

resistant cultivars (Helal et al., 1978; El-Shanawani et al.1990 and

Mohamed, 1994), while high sugar content in the susceptible cultivars

was reported by (Omar (1977), Helal et al. (1978) and Farahat (1980).

Discussion 125

Growth of powdery mildews is favored by a high carbohydrate level of

their hosts (Yarwood, 1957). Similar results were obtained by (Awad,

2000) who reported that sugars tended to increase the susceptibility of

detached leaves to fungal parasites by providing an extra source of energy

for the invader.

Evaluation of the effect of spraying cucumber plants with abiotic

selected treatments on phenol content indicated that, all tested treatments

increased the free phenols and the total phenols and the highest increase

was induced by Topas-100 followed by K2HPO4 and clove oil.

Whereas all treatments were differed in their effects on the

conjugated phenols, some treatments significantly decreased the

conjugated phenols except Topas-100, garlic extract and clove oil while

others such as withania extract, nigella oil, clove extract, rocket oil,

phosphate salt (K2HPO4) and clove oil reported the highest decrease

respectively.

On the other hand, the effect of spraying cucumber plants with biotic

selected treatments on phenol content indicated that, all tested treatments

significantly increased the free phenols and total phenols. The highest

increase in the free phenols was induced by Bacillus filtrate + Trichoderma

filtrate followed by Topas-100 and propolis extract + Bacillus filtrate. As

refer to the total phenols highest increase was induced by Topas-100

followed by Bacillus filtrate + Trichoderma filtrate and propolis extract +

Bacillus filtrate.

The effect on the conjugated phenols was applied by Trichoderma

filtrate and Topas-100 which increased the conjugated phenols while

propolis extract + Bacillus filtrate + Trichoderma filtrate, Bacillus filtrate,

propolis extract + Bacillus filtrate, propolis extract + Trichoderma filtrate

and Bacillus filtrate + Trichoderma filtrate decreased them respectively.

Discussion126

Also, propolis extract did not affect the conjugated phenols and gave equal

value with control treatment.

This increase in the total phenol levels gave surely an increase in the

capability of plants to defense against disease infection process and disease

development, The present results concerning the increase in total phenol

contents are in agreement with those reported by Daayf et al., (1995 &

1997). They reported that milsana flussing (a concentrated extract from

leaves of Reynoutria sachalinensis) stimulated the production of

fungitoxic phenolic compounds as a result of elicitation with the extract

and this being particularly evident when the cucumber plant was stressed

by the pathogen of powdery mildew (S. fuliginea). Moreover, the amounts

of these compounds in treated plants were nearly five times the level found

in the control plants. Since the role of secondary metabolic substances,

such as phenolic compounds, on disease resistance mechanisms are well

known (Kalaichelvan and Nagarajan, 1992). Moreover, the toxic

phenolic compounds in plant cells acting through: (1) the structure of bond

form with cell wall components of plant tissues (Mahadevan and Sridhar,

1986), (2) enhance host resistant by stimulating host defense mechanisms

(Subba Rao et al., 1988), (3) prevent the extent of fungal growth in plant

tissues (Soni et al., 1992) and (4) penetrate the microorganisms and cause

considerable damage to the cell metabolisms (Kalaichelvan and

Elangovan, 1995). On the other hand, the efficiency of these extracts in

controlling mildews was similar to those reported by Cheah et al. (1995);

Collina (1996); Daayf et al., (1995 & 1997); Pasini et al. (1997 a&b);

Abd-El-Sayed (2000) and Haroun (2002). In this respect, Helal et al.

(1978) reported that the resistant cucumber variety “Poinsett” contained

higher amounts of performed phenols, which hinder infection with E.

cichoracearum. Abdel-Sattar et al. (1985) indicated that, free phenols

content was higher in “Yomaki” the highly- resistant cucumber variety to

Discussion 127

E. cichoracearum than its respective content in “Beit-alpha”, the highly

susceptible one. Mostafa (1986) showed that powdery mildew host

resistance was due to phenolic compounds with markedly fungistatic

properties presented before infection. Nada (2002) found that spraying

squash plants with some plant extracts increased total phenolic contents of

the leaves. The hot water extract of blue gum, leek and thyme were the best

treatments in decreasing squash powdery mildew infection and increasing

total phenols.

Spraying cucumber plants with abiotic and biotic selected

treatments and their effects on total amino acid content were studied, the

results indicated that all tested treatments significantly decreased the total

amino acid content. The treatments decreased the total amino acid content

as a result to decreasing the powdery mildew disease severity. Similar

results was observed on resistance variety compared with susceptible

variety plants against powdery mildew disease which in agreement with

Farahat (1980) who reported that powdery mildew infected pea leaves

contained higher content of free amino acids especially in the highly

susceptible variety. El-Shanawani et al. (1990) indicated that the highly

susceptible variety of cucumber contained higher amounts of total free

amino acids in healthy leaves than the highly resistant one. While S.

fuliginea infection increased total free amino acids in both varieties. The

increase in total free amino acids was more pronounced in the highly

susceptible variety than in the highly resistant one. Awad et al. (1990) and

Haroun (2002) reported that the highly susceptible tomato cvs. contained

higher contents of free amino acids than the least susceptible ones. In this

respect, El-Kafrawy (1997) found that the highest susceptible cultivars of

pepper contained higher amounts of total free amino acids in healthy

leaves than the least susceptible cultivars.

Summary 128

SUMMERY

Cucumber (Cucumis sativus L.) is one of the most important

economically crops, which belongs to family cucurbitaceae. The economic

importance of this crop appears in both local consumption and exportation

purposes. Cucumber is grown either in the open field or under protected

houses. The cultivated area of cucumber in 2003 growing season reached

about 11881 feddan in open field which yielded 88575 ton fruits, in

addition to 13267 greenhouses, yielded about 44771 ton fruits.


(Schlectend: Fr.) Pollacci. This disease can cause damage to all plant parts

including leaves, stems and fruits especially under protected house

conditions and causing considerable reduction of quantity and quality of

cucumber yields. Disease control is generally achieved by the use of

fungicide.

Thus, the present work was conducted to founding alternative

non-chemical methods to fungicides and reducing fungicides use to control

of cucumber powdery mildew disease under protected houses.

The obtained results from present studies can be summarized as

follows:-

1. Survey of cucumber diseases in protected houses revealed that, powdery

mildew disease was important disease of cucumber grown in

protected houses that observed in both surveyed locations. Wilt

occupied the second order in its importance; meanwhile, virus

infection recorded the third order.

2. Conidial germination was significantly reduced by all plant extracts. The

high reduction was induced by garlic extract at concentration 20%

Summary 129

(88.13%) and clove extract at concentration 10% (84.74%) less than

control.

3. All plant oils significantly reduced conidial germination. The high

reduction was induced by clove oil at concentration 10% (89.83%),

nigella oil at concentration 8% (74.57%) and olive oil at

concentration 8% (72.87%) less than control.

4. Phosphate salt (K2HPO4) was significantly reduced conidial germination.

The high reduction was induced by phosphate salt at concentration

100 mM/L (64.40%) less than control.

5. Conidial germination was significantly reduced by Bacillus filtrate,

Trichoderma filtrate, propolis extract and their combination. The high

reduction was induced by propolis extract + Bacillus filtrate +

Trichoderma filtrate (88.30%), Bacillus filtrate + Trichoderma

filtrate (85.52%) and propolis extract + Trichoderma filtrate (83.98%)

less than control.

6. Induction of cucumber resistance to powdery mildew by plant extracts

proved that all tested plant extracts significantly reduced the

percentage of powdery mildew incidence and severity. The high

reduction was induced by garlic extract 20% (85.73%) and clove

extract 10% & withania extract 50% (57.20%).

7. Induction of cucumber resistance to powdery mildew by plant oils

revealed that all tested plant oils significantly reduced the percentage

of powdery mildew incidence and severity. Olive oil at concentration

8% completely prevented powdery mildew incidence. The high

reduction was induced by olive oil 4%, rocket oil 8%, nigella oil 8%

and clove oil 10% (85.73%).

Summary 130

8. Induction of cucumber resistance to powdery mildew by phosphate salt

(K2HPO4) exhibited that phosphate salt (K2HPO4) significantly

reduced the percentage of powdery mildew incidence and severity. The

high reduction was induced by Topas-100 at concentration 50

cm3/100L (71.47%) and phosphate salt (K2HPO4) at concentration 100

mM/L (57.20%).

9. Induction of cucumber resistance to powdery mildew by biological

agent filtrate, propolis extract and their combination revealed that all

tested treatments significantly reduced the percentage of powdery

mildew incidence and severity. Propolis extract + Bacillus filtrate +

Trichoderma filtrate completely prevented powdery mildew incidence.

The high reduction was induced by Trichoderma filtrate, propolis

extract + Trichoderma filtrate, propolis extract + Bacillus filtrate and

Trichoderma filtrate + Bacillus filtrate (85.73%).

10. Spraying with plant extracts on incidence and severity of powdery

mildew disease in cucumber cv. Primo (during spring and autumn 2003)

were studied. The result showed that all tested plant extracts at both


incidence and severity. Garlic extract at 20% was the most effective

treatment at both seasons (2.76%) followed by clove extract at 10%

(3.67%), garlic extract at 10% (4%) and withania extract at 50%

(4.67%) compared with control (15.67%). Generally increasing

concentration of the plant extract tested significantly increased the

reduction in powdery mildew incidence and severity.

11. Spraying of plant oils on incidence and severity of powdery mildew

disease in cucumber cv. Primo (during spring and autumn 2003) was

studied. Results revealed that all tested plant oils at both seasons


Summary 131

severity. Clove oil at 10%, and olive oil at 8% were the most effective

treatments at both seasons by (2.67%) followed by nigella oil at 8%

(4%) and rocket oil at 8% by (4.34%) compared with control (15.67%).

12. Study of spraying phosphate salt (K2HPO4) on incidence and severity

of powdery mildew disease in cucumber cv. Primo (during spring and

autumn 2003) was studied. The results showed that in two experiments

phosphate salt (K2HPO4) at both seasons significantly reduced the

percentage of powdery mildew incidence and severity. Phosphate salt

(K2HPO4) at concentration 100 mM/L was the most effective treatment

at both seasons followed by (3%) Phosphate salt (K2HPO4) at

concentration 75 mM/L (4%).

13. Spraying some biological agents, propolis extract and their

combination on incidence and severity of powdery mildew disease in

cucumber cv. Primo (during spring and autumn 2003) was studied. The

results revealed that, all tested treatments significantly reduced the

percentage of powdery mildew incidence and severity. Propolis extract

+ Bacillus filtrate + Trichoderma filtrate were the most effective

treatments at both seasons by (1.67%) followed by propolis extract +

Trichoderma filtrate & Trichoderma filtrate + Bacillus filtrate and

Bacillus filtrate & Trichoderma filtrate (2%).

14. Evaluation of spraying with plant extracts on controlling powdery

mildew disease in cucumber cv. Delta star showed that, all tested plant

extracts significantly reduced the percentage of powdery mildew

incidence and severity. The high reduction in disease severity was

induced by garlic extract (45.27%) followed by clove extract (39.60%)

and withania extract (26.43%). Also, all tested plant extracts

significantly increased the number fruits/plant and weight fruits/plant.

The highest increase in number of fruits/plant and weight fruits/plant

Summary 132

was induced by garlic extract (31.26 and 35.84%) followed by clove

extract (52.02 and 28.01%) and withania extract (18.75 and 21.38%)

compared with control.

15. Spraying with plant oils on controlling powdery mildew disease in

cucumber cv. Delta star revealed that, all tested plant oils significantly

reduced the percentage of powdery mildew incidence and severity. The

high reduction in disease severity was induced by olive oil (67.91%)

followed by clove oil (64.18%), nigella oil (60.38%) and rocket oil

(47.20%). Also, all tested plant oils significantly increased the number

fruits/plant and weight fruits/plant. The highest increase in number

fruits/plant and weight fruits/plant was induced by clove oil (37.50 and

37.65%) followed by olive oil (26.79 and 28.30%) , nigella oil (21.43

and 24.40%) and rocket oil (13.39 and 15.96%) compared with control.

16. Spraying cucumber plants cv. Delta star with phosphate salt (K2HPO4)

to control powdery mildew disease showed that, Phosphate salt 100

mM/L as well as Topas-100 significantly reduced the percentage of

powdery mildew incidence and severity. Phosphate salt and Topas-100

reduced disease severity by 35.88 and 37.55% compared with control.

Also, phosphate salt and Topas-100 increased the number fruits/plant

and weight fruits/plant (14.31and 16.18%) and (13.39 and 13.55%)

compared with control.

17. Spraying cucumber plants cv. Delta star with biological control agent

to control powdery mildew disease showed that all tested treatments


severity. The high reduction in disease severity was induced by

propolis extract + Trichoderma filtrate + Bacillus filtrate (69.84%)

followed by Trichoderma filtrate + Bacillus filtrate (60.38%), propolis

extract + Trichoderma filtrate & propolis extract + Bacillus filtrate

Summary 133

(58.52%) and Bacillus filtrate (54.73%). Also, increased number and

weight of fruits/plant. The highest increase in number and weight

fruits/plant was induced by propolis extract + Trichoderma filtrate +

Bacillus filtrate (43.83 and 44.00%) followed by propolis extract +

Trichoderma filtrate (38.41and 40.06%) and Trichoderma filtrate +

Bacillus filtrate (37.50 and 39.17%) compared with control.

18. Treatment of powdery mildew spore suspension with UV for 30

minutes or temperature 90°C for 10 minutes or adding chloroform 1.0

ml/L spore suspension inhibited the spore germination completely.

While treatment of powdery mildew spores with UV for 20 minutes or

temperature 70°C for 10 minutes or adding chloroform 0.5 ml/L spore

suspension reduced the percentage of conidia germination from

40.33% in control to 12.76, 13.33 and 14.67% respectively.

19. Treatment of powdery mildew spores by using UV, temperature and

chloroform revealed that increasing time of exposure to UV,

temperature or increasing amount of chloroform led to increase

efficacy of the treatment. The most effective treatments which

complete inhibited all spores of powdery mildew and no disease was

developed had been recorded when spores were subjected to one of the

following treatments: UV for 30 minutes, 90C for 10 minutes and 1ml

chloroform /L.

20. Spraying cucumber cv. Delta star with powdery mildew with

non-germinated spore by UV, temperature, chloroform to induce

resistance led to reduce powdery mildew disease severity by 69.84,

62.25 and 54.73% less than control in addition increased the number

and weight of fruits/plant. The highest increase in number and weight

fruits/plant was induced by UV (25.02 and 25.00%) and temperature

(18.75 and 19.88%) compared with control.

Summary 134

21. Evaluation of the effect of the best resistance inducers i.e. garlic extract

at 20%, clove extract at 10%, clove oil at 10%, olive oil at 8%, propolis


propolis extract + Trichoderma filtrate + Bacillus filtrate, K2HPO4 at

100 mM. and Topas-100 at 50 ml/100L in addition to untreated control

on peroxidase (PO), polyphenol-oxidase (PPO), chitinase activity and

lignin content was showed following points.

1. Peroxidase (PO) activity:

All treatments significantly increased PO activity compared with

control in all times. The highest value of peroxidase enzymes obtained

after five days from inoculation with powdery mildew spores, while the

highest increased in peroxidase activity compared with control was

induced by propolis extract + Trichoderma filtrate (231.48%) before

inoculation with powdery mildew spores.

2. Polyphenol-oxidase (PPO) activity:

All treatments significantly increased PPO activity compared with

control in all times. The highest value of (PPO) enzymes obtained after

five days from inoculation with powdery mildew spores while the highest

increase in (PPO) activity compared with control was induced by propolis

extract + Trichoderma filtrate (397.12%) after one day from inoculation

with powdery mildew spores.

3. Chitinase activity:

All treatments significantly increased chitinase activity compared with

control in all times. The highest value of chitinase enzymes obtained after

five days from inoculation with powdery mildew spores. While the highest

increased in chitinase activity compared with control was induced by

propolis extract + Trichoderma filtrate was induced by propolis extract +

Summary 135

Trichoderma filtrate (692.50%) after one day from inoculation with

powdery mildew spores.

4. Lignin content:

All treatments significantly increased lignin content. The most

effective treatment were garlic extract, propolis extract +Trichoderma

filtrate, phosphate salt (K2HPO4), propolis extract + Trichoderma filtrate +

Bacillus filtrate and clove extract which they increased total lignin by

92.11, 82.90, 71.58, 56.84 and 52.36% respectively over control.

22. Study effect of foliar spraying with plant extracts, plant oils, phosphate

salt and the fungicide Topas-100 on sugar content in cucumber plants

revealed that, the reducing sugars were significantly decreased by all

treatments except Topas-100. The highest decrease was induced by

clove oil & nigella oil (54.11%) followed by olive oil, withania

extract, clove extract, rocket oil, garlic extract and phosphate salt

(K2HPO4) decreased the reducing sugars by 41.10, 37.68, 29.47,

26.32, 19.79 and 1.68%, respectively less than the control, while

Topas-100 increased it by 63.79% over control.

All treatments increased the non-reducing sugars except olive oil

and withania extract decreased it. The highest increase was induced by

garlic extract, clove extract, Topas-100, rocket oil, nigella oil, phosphate

salt (K2HPO4) and clove oil increased it by 280.85, 232.00, 100.00, 97.87,

82.98, 65.96 and 17.02% compared with control, while olive oil and

withania extract decreased it by 34.04% less than the control. On the other

hand, Topas-100, withania extract, garlic extract, and phosphate salt

(K2HPO4) increased the total sugars by 68.2, 37.36, 7.28 and 4.41% over

control, while clove oil, nigella oil, olive oil, rocket oil and clove extract

decreased it by 47.70, 41.76, 40.42, 15.13 and 5.94% less than control.

Summary 136

23. Spraying of cucumber plants with plant extracts, plant oils, phosphate

salt and Topas-100 showed that, all tested treatments increased the free

phenols. The highest increase in the free phenols was induced by

Topas-100 (326.88%) followed by K2HPO4 (289.96%) and clove oil

(205.73%) over control.

As for all tested treatments the highest increase in the total phenols

was induced by Topas-100 (97.33%) followed by K2HPO4 66.27%) and

clove oil (56.80%). Meanwhile all treatments decreased the conjugated

phenols except Topas-100, garlic extract and clove oil. In this respect,

withania extract, nigella oil, clove extract, rocket oil, phosphate

salt(K2HPO4) and olive oil induced high decrease by 45.49, 31.00, 27.32,

18.17, 10.88 and 7.29% less than control treatment. While, Topas-100,

garlic extract and clove oil increased it by 18.17, 7.29 and 5.44% over

control.

24. Spraying of cucumber plants with plant extracts, plant oils, phosphate

salt and Topas-100 revealed that, all treatments significantly decreased

the total amino acid content. Olive oil, clove extract, garlic extract and

withania extract were induced the highest decrease by 94.55, 85.19,

78.70 and 77.92%.

25. Study the effect of some biological control agents’ filtrate and propolis

extract compared with fungicide on sugar content of cucumber plants

proved that all treatments decreased the reducing sugars except

Topas-100 compared with control. The highest decrease was induced

by propolis extract + Bacillus filtrate, propolis extract + Bacillus

filtrate + Trichoderma filtrate and Bacillus filtrate 54.84% while,

Topas-100 increased it by 63.79%.

As for the non-reducing sugars, Trichoderma filtrate, Topas-100,

Bacillus filtrate and Bacillus filtrate + Trichoderma filtrate increased the

Summary 137

non-reducing sugars by 397.87, 100.0, 17.02 and 17.02% compared with

control. While propolis extract + Bacillus filtrate + Trichoderma filtrate,

propolis extract +Trichoderma filtrate, propolis extract and propolis

extract + Bacillus filtrate decreased it by 85.11, 51.10, 34.04 and 23.40%.

All treatments decreased total sugars except Topas-100 and

Trichoderma filtrate increase it compared with control. Propolis extract +

Bacillus filtrate + Trichoderma filtrate, propolis extract + Bacillus filtrate,

propolis extract +Trichoderma filtrate and Bacillus filtrate induced the

highest decrease by 58.43, 53.83, 47.90 and 47.70% while Topas-100 and

Trichoderma filtrate increased it by 68.20 and 26.82%..

26. Study the effect of some biological control agents’ filtrate on phenol

content of cucumber plants showed that all tested treatments increased

the free phenols. The highest increase in the free phenols was induced

by Bacillus filtrate + Trichoderma filtrate (342.65%) followed by

Topas-100 (326.88%) and propolis extract + Bacillus filtrate (300.7%)

over control. All tested treatments increased the total phenols. The

highest increase in the total phenols was induced by Topas-100

(97.33%) followed by Bacillus filtrate + Trichoderma filtrate (71.6%)

and propolis + Bacillus filtrate (48.71%) over control.

Trichoderma filtrate and Topas-100 increased the conjugated

phenols over control. On the contrary propolis extract + Bacillus filtrate +

Trichoderma filtrate, Bacillus filtrate, propolis extract + Bacillus filtrate,

propolis extract + Trichoderma filtrate and Bacillus filtrate + Trichoderma

filtrate decreased the conjugated phenols by 60.00, 41.78, 38.20, 25.46 and

21.88% less than control treatment. While, propolis extract did not effect

on the conjugated phenols.

27. Evaluation the effect of some biological control agents’ filtrate and

propolis extract compared with fungicide on total amino acid content

Summary 138

of cucumber plants exhibited that all tested treatments decreased the

total amino acid content compared with control. The highest decrease

induced by Bacillus filtrate + Trichoderma filtrate (95.84%) followed

by Trichoderma filtrate (89.10%) and propolis extract + Bacillus

filtrate (88.00%).

References 139

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By Gamal Ashor Ahmed - Bu · By Gamal Ashor Ahmed B.Sc Agricultural ... mildew disease in cucumber cv. Primo under commercial protected ... and help that Prof. Dr. Abdou Mahdy Mohamed