By Gamal Ashor Ahmed - Bu · By Gamal Ashor Ahmed B.Sc Agricultural ... mildew disease in cucumber...
Transcript of By Gamal Ashor Ahmed - Bu · By Gamal Ashor Ahmed B.Sc Agricultural ... mildew disease in cucumber...
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
ب
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.
Review of Literature 5
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).
Review of Literature 6
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,
Review of Literature 7
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
Review of Literature 8
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.
Review of Literature 9
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.
Review of Literature 10
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
Review of Literature 11
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.
Review of Literature 12
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.
Review of Literature 13
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
Review of Literature 14
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
Review of Literature 15
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
Review of Literature 16
(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.
Review of Literature 17
(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
Review of Literature 18
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.
Review of Literature 19
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
Review of Literature 20
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
Review of Literature 21
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.
Review of Literature 22
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
Review of Literature 23
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
Review of Literature 24
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.
Review of Literature 25
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
Review of Literature 26
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
Review of Literature 27
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
Review of Literature 28
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
Review of Literature 29
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
Review of Literature 30
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.
Review of Literature 31
(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
Review of Literature 32
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
Review of Literature 33
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.
Review of Literature 34
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).
Materials and Methods 37
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.
Materials and Methods
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
Materials and Methods 39
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.
Materials and Methods
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.
Materials and Methods 41
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.
Materials and Methods
42
5.3. Induction of cucumber resistance against powdery mildew by
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.
5.4. Induction of cucumber resistance against powdery mildew by
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
Materials and Methods 43
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.
Two experiments (during spring and autumn 2003) were conducted
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.
Materials and Methods
44
6.3. Effect of spraying with phosphate salt (K2HPO4) 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 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.
Two experiments (during spring and autumn 2003) were conducted
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.
Materials and Methods 45
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.
Materials and Methods
46
6.8. Effect of spraying with biological control agent on controlling
powdery mildew disease in cucumber cv. Delta star (during
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
Materials and Methods 47
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
Materials and Methods
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.
Materials and Methods 49
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.
Materials and Methods
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
Materials and Methods 51
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)
Materials and Methods
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
Experimental Results 54
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
significantly reduced the percentage of conidia germination compared
with the control treatment. The high reduction was induced by clove oil at
Experimental Results 55
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
Experimental Results 56
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
Experimental Results 57
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
significantly reduced the percentage of conidia germination compared
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 +
Trichoderma filtrate 4.6 -88.30
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
Experimental Results 58
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
Experimental Results 59
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%
Experimental Results 60
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.
Table (8): Powdery mildew incidence in cucumber cv. Primo sprayed
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
Experimental Results 61
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.
Experimental Results 62
Table (9): Powdery mildew incidence in cucumber cv. Primo sprayed
with phosphate salt (K2HPO4) under greenhouse.
Efficacy
%
Disease
severity
Efficacy
%
Disease
percentage Treatment
-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.
Experimental Results 63
Table (10): Powdery mildew incidence in cucumber cv. Primo sprayed
with some biological agent, Propolis extract and their
combinations under greenhouse.
Efficacy
%
Disease
severity
Efficacy
%
Disease
percentage Treatment
-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
Experimental Results 64
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
Experimental Results 65
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
tested to study their efficiency against powdery mildew incidence and
severity.
Table (12): Powdery mildew incidence in cucumber cv. Primo sprayed
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
Experimental Results 66
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.
In two experiments (during spring and autumn 2003) foliar
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
seasons significantly reduced the percentage of powdery mildew
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
Experimental Results 67
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.
Table (13): Powdery mildew incidence in cucumber cv. Primo sprayed
with some plant oils 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
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
Experimental Results 68
4.3. Effect of spraying phosphate salt (K2HPO4) 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 phosphate salt (K2HPO4) at three concentrations was
tested to study their efficiency against powdery mildew incidence and
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.
Table (14): Powdery mildew incidence in cucumber cv. Primo sprayed
with phosphate salt (K2HPO4) under commercial protected
houses.
Mean Experiment2
(autumn 2003)
Experiment 1
(spring 2003) Treatment
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
Experimental Results 69
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.
In two experiments (during spring and autumn 2003) foliar
application with Bacillus subtilis, Trichoderma harzianm, propolis
extract and their combination were tested to study their efficiency
against powdery mildew incidence and severity.
Table (15): Powdery mildew incidence in cucumber cv. Primo sprayed
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
Experimental Results 70
The data in Table (15) indicate that, all tested treatment
significantly reduced the percentage of powdery mildew incidence and
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
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 (-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
Experimental Results 72
(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
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 (-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
powdery mildew disease in cucumber cv. Delta star (during
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
Experimental Results 75
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
powdery mildew disease in cucumber cv. Delta star (during
spring 2004).
Cucumber plants cv. Delta star 4 week old was sprayed with
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.
The data in Table (19) indicate that, all tested treatments
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 (-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.
Experimental Results 77
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).
Cucumber plants cv. Delta star 4 week old was sprayed with
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.
The data in Table (20) indicate 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 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.
Experimental Results 79
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%).
Experimental Results 81
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
Experimental Results 82
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.
Experimental Results 84
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.
Experimental Results 85
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.
Experimental Results 87
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
Phosphate salt 100 mM/L
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.
Experimental Results 88
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
Experimental Results 89
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%
Propolis +Trichoderma filtrate
Trichoderma filtrate +Bacillus filtrate
Propolis + Trichoderma filtrate + Bacillus filtrate
Phosphate salt 100 mM/L
Topas 50cm3/100L
Control
Figure (4): Lignin content in cucumber plants as affected by the tested
treatments and powdery mildew inoculation:
Experimental Results 90
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.
Experimental Results 91
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
Phosphate salt 100 mM/L
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
Experimental Results 92
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.
Experimental Results 93
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:
The data in Table (27) and Fig. (7) indicate that, all treatments
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%).
Experimental Results 94
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
Experimental Results 95
0
5
10
15
20
25
mg
/g
Free phenols Conjugated phenols Total phenols
Clove extract 10%
Garlic extract 20%
Withania extract 50%
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.
Experimental Results 96
Table (27): Effect of spraying Plant extracts & oils and K2HPO4 on total
amino acid content of infected powdery mildew cucumber
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.
Experimental Results 97
0
0.5
1
1.5
2
2.5
3
3.5
4m
g/g
Total amino acids content
Clove extract10%
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 (7): Effect of spraying Plant extracts & oils and K2HPO4 on total
amino acid content of infected powdery mildew cucumber
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
Experimental Results 98
+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
Propolis extract + Bacillus
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
Experimental Results 99
0
1
2
3
4
5
6
7
8
9
mg
/g
Reducing sugars Non reducing sugars Total sugars
Propolis extract
Trichoderma filtrate
Bacillus filtrate
Propolis +Trichoderma 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.
Experimental Results 100
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.
Experimental Results 101
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 +
Trichoderma 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
Free phenols Conjugated phenols Total phenols
L.S.D. at 5% 0.8706 0.7683 0.8462
Experimental Results 102
0
5
10
15
20
25m
g/g
Free phenols Conjugated phenols Total phenols
Propolis
Trichoderma filtrate
Bacillus filtrate
Propolis + Trichoderma filtrate
Propolis + Bacillus filtrate
Bacillus filtrate + Trichoderma filtrate
Propolis + Bacillus filtrate + 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.
Experimental Results 103
6.6. Effect of some biological control agents’ filtrate on total amino
acid content of infected powdery mildew cucumber plants:
The data in Table (30) and Fig. (10) indicate that, all treatments
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
amino acid content of infected powdery mildew cucumber
plants as mg/1g fresh weight.
Treatments Total amino
acid contents Efficacy %
Propolis extract 1.10 -71.43
Trichoderma filtrate 0.42 -89.10
Bacillus filtrate 1.40 -63.64
Propolis extract +
Trichoderma filtrate 0.51 -86.75
Propolis extract + Bacillus
filtrate 0.46 -88.00
Bacillus filtrate +
Trichoderma filtrate 0.16 -95.84
Propolis extract + Bacillus
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
Experimental Results 104
0
0.5
1
1.5
2
2.5
3
3.5
4m
g/g
Total amino acid contents
Propolis
Trichoderma filtrate
Bacillus filtrate
Propolis + Trichoderma filtrate
Propolis + Bacillus filtrate
Bacillus filtrate + Trichoderma filtrate
Propolis + Bacillus filtrate + Trichoderma filtrate
Topas 50cm3/100L
Control
Figure (10): Effect of some biological control agents filtrate on total
amino acid content of infected powdery mildew cucumber
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.
Cucumber powdery mildew caused by Sphaerotheca fuliginea
(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
significantly reduced the percentage of powdery mildew incidence and
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)
reduced the percentage of powdery mildew incidence and severity
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
extract + Trichoderma filtrate, Trichoderma filtrate + Bacillus filtrate,
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.
Cucumber powdery mildew caused by Sphaerotheca fuliginea
(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
seasons significantly reduced the percentage of powdery mildew
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
significantly reduced the percentage of powdery mildew incidence and
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
significantly reduced the percentage of powdery mildew incidence and
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
extract + Trichoderma filtrate, Trichoderma filtrate + Bacillus filtrate,
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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