Kazakh National Agrarian University

151 Pages·2013·3.87 MB·English

Kazakh National Agrarian University

UDC. 63.632.4.631.234 On manuscript rights

GAMAL ASHOUR AHMED MOHAMED

Induction Resistance of Cucumber Plants (Cucumis sativus L.) Against
Fusarium Wilt Disease under Protected Houses Conditions

Dissertation submitted in fulfillment of the requirements for the academic degree
Doctor of Philosophy (Ph.D.) specialty: 6D081100 − Plant protection and quarantine

Supervision committee

Director of the institute, Professor,
Academician of the Kazakh
National Academy.RK
Prof. Dr. Sagitov A. O.

Professor of Plant Pathology, Agric.
Botany Dept., Fac. Agric.,
Moshtohor, Benha Univ. АRЕ.
Prof. Dr. A. M. M. Mahdy

Republic of Kazakhstan
Almaty 2011
This work was carried out at Kazakh National Agrarian University and Institute of
plant protection and quarantine

Supervision committee Director of the institute, Professor, Academician
of the Kazakh National Academy.RK
Prof. Dr. Sagitov A. O.

Professor of Plant Pathology, Agric. Botany Dept.,
Fac. Agric., Moshtohor, Benha Univ. АRЕ
Prof. Dr. A. M. M. Mahdy

Reviewers Doctor of Agricultural Sciences
Prof. Sarbaev A.T.

Doctor of Agricultural Sciences
Prof. Bayrakimov S.I.

Defense and discussion will take place on «3» Augast, 2011 at 1000 o'clock at session
of state certifying commission, KazNAU, address: 050010, Almaty, abai 8, at the
Kazakh National Agrarian University

The dissertation is available in library of KazNAU, address: 050010, Almaty,
Abai 8

Ph.D. student G. A. Ahmed
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Content
Subject Page
ABBREVIATIONS 6
INTRODUCTION 7
1 REVIEW OF LITERATURES 10
1.1 Important of the disease: 10
1.2 Inducing resistance by biotic agents: 11
1.3 Inducing resistance by abiotic agents 28
1.4 Physiological aspects of defense reaction: 33
1.5 Anatomical features of immunized plants: 40
2 MATERIALS AND METHODS 43
3 EXPERIMENTAL RESULTS 57
3.1Isolation of the causal fungi 57
3.2 Pathological studies 57
3.2.1 Pathogenicity tests and inoculum densities. 57
3.2.2 Host range of F. oxysporum: 58
3.2.3 Susceptibility of commercial cucumber cultivars to infection with 58
Fusarium wilt.
3.3 Laboratory studies: 59
3.3.1 Effect of antagonistic fungi on the linear growth of F. O. f.sp. 59
cucumerinum (FOC) in vitro.
3.3.2 Evaluating the effect of antagonistic fungi culture filtrates on the 60
linear growth and spore germination of F. O. f.sp. cucumerinum (FOC).
3.3.3 Effect of antagonistic bacteria in vitro against F. O. f.sp. 61
cucumerinum (FOC).
3.3.4 Evaluation of the effect of antagonistic bacteria culture filtrates on 62
the linear growth and spore germination of F. O. f.sp. cucumerinum (FOC).
3.3.5 Effect of different resistant inducing chemicals on the linear 65
growth and spore germination of F. O. f.sp. cucumerinum (FOC) in vitro.
3.4 Greenhouse experiments: - 67
3.4.1. Effect of treating cucumber seeds with some antagonistic fungi on 67
incidence with Fusarium wilt disease:
3.4.2. Effect of cucumber seeds treatment with cell suspension of 68
antagonistic bacterial isolates on incidence with Fusarium wilt.
3.4.3. Effect of treating cucumber seeds or treating soil with some 69
resistance inducing chemicals on incidence with Fusarium wilt.
3.5. Experiments of Commercial protected house: 72
3.5.1. Effect of cucumber seeds treatment with some antagonistic fungi 72
on incidence with Fusarium wilt disease under commercial protected house:
3.5.2. Effect of treating cucumber seeds with cell suspension antagonistic 74
bacterial isolates on incidence of Fusarium wilt disease under commercial
protected house:
3.5.3. Effect of treating cucumber seeds or treating soil with some 75
resistance inducing chemicals on incidence with Fusarium wilt disease.
77
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3.6 Determination of enzymes activity, lignin content and peroxidase
isozyme:
3.6.1 Effect of treating cucumber seeds with spore suspension of 77
antagonistic fungus isolates in peroxidase activity in cucumber plants:
3.6.2 Effect of treating cucumber seeds with cell suspension of 78
antagonistic bacterial isolates in peroxidase activity in cucumber plants:
3.6.3 Effect of treating cucumber seeds with tested chemical compounds 79
on peroxidase activity in cucumber plants:
3.6.4 Effect of treating cucumber seeds with spore suspension of 80
antagonistic fungus isolates in Polyphenol-oxidase activity in cucumber
plants:
3.6.5 Effect of treating cucumber seeds with cell suspension of 81
antagonistic bacteria isolates in polyphenol-oxidase activity in cucumber
plants:
3.6.6 Effect of treatment of cucumber seeds with tested chemicals 82
compound in polyphenol-oxidase activity in cucumber plants:
3.6.7 Effect of treatment of cucumber seeds with spore suspension of 83
antagonistic fungal isolates on chitinase activity in cucumber plants:
3.6.8 Effect of treatment of cucumber seeds with cell suspension of 84
antagonistic bacterial isolates in chitinase activity in cucumber plants:
3.6.9 Effect of treatment of cucumber seeds with tested chemical 85
compounds on chitinase activity in cucumber plants:
3.6.10 Effect of treatment of cucumber seeds with spore suspension of 86
antagonistic fungal isolates on lignin content in cucumber plants:
3.6.11 Effect of treatment of cucumber seeds with cell suspension of 87
antagonistic bacterial isolates on lignin content in cucumber plants:
3.6.12 Effect of treatment of cucumber seeds with tested chemical 88
compounds on lignin content in cucumber plants:
3.6.13 Effect of treatment of cucumber seeds with spore suspension of 89
antagonistic fungal isolates on isozyme pattern of peroxidase in cucumber
plants:
3.6.14 Effect of treatment of cucumber seeds with spore suspension of 92
antagonistic bacterial isolates on isozyme pattern of peroxidase in cucumber
plants:
3.6.15 Effect of treatment of cucumber seeds with tested chemical 95
compounds on isozyme pattern of peroxidase in cucumber plants:
3.7 Chemical analysis of cucumber treated plants: 97
3.7.1 Effect of cucumber seeds treatment with spore suspension of 97
antagonistic fungal isolates on sugar content in cucumber plants:
3.7. 2 Effect of treatment of cucumber seeds with cell suspension of 98
antagonistic bacterial isolates on sugar content in cucumber plants.
3.7.3 Effect of treatment of cucumber seeds with tested chemicals 99
compound on sugar content in cucumber plants.
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3.7.4 Effect of treatment of cucumber seeds with spore suspension of 100
antagonistic fungal isolates on phenol content in cucumber plants.
3.7.5 Effect of treatment of cucumber seeds with cell suspension of 101
antagonistic bacterial isolates on phenol content in cucumber plants
3.7.6 Effect of treating cucumber seeds with chemical compounds on 102
phenol content in cucumber plants:
3.7.7 Effect of cucumber seeds treatment with cell suspension of 103
antagonistic fungal isolates on amino acids content in cucumber plants:
3.7.8 Effect of cucumber seeds treatment with cell suspension of 104
antagonistic bacterial isolates in amino acids content in cucumber plants:
3.7.9 Effect of treating cucumber seeds with tested chemicals compound 105
in amino acids content in cucumber plants:
3.8 Anatomical studies: 106
3.8.1 Effect of cucumber seeds treatment with tested antagonistic fungal 106
isolates on the mean counts and measurements of certain histological features
of main cucumber root.
3.8.2 Effect of cucumber seeds treatment with tested antagonistic 108
bacterial isolates on the mean counts and measurements of certain histological
features of main cucumber root.
3.8.3 Effect of cucumber seeds treatment with tested chemical 110
compounds on the mean counts and measurements of certain histological
features of main cucumber root.
3.9 Effect of carrying the best antagonistic isolates of fungi and bacteria on 112
different carrier material on infection with Fusarium wilt.
3.9.1 Comparison between some different carrier materials of 112
antagonistic fungal isolates on cucumber seeds on infection with Fusarium
wilt.
3.92 Comparison between some different carrier materials of antagonistic 113
bacterial isolates on cucumber seeds on infection with Fusarium wilt.
4 DISCUSSION 114
CONCLUCTION 129
REFERENCES 132
APPENDIX 154

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ABBREVIATIONS

IPM: Integrated pest management
FOL: Fusarium oxysporum f.sp. lycopersici
FOC: Fusarium oxysporum f.sp. cucumerinum
FOM: Fusarium oxysporum f.sp. melonis
FON : Fusarium oxysporum f.sp. niveum
PGPR: Plant growth-promoting rhizobacteria
EC : Soil electrical conductivity
BABA: DL-3-aminobutyric acid
ASA : Amino salicylic acid
BHA : Butylated hydroxyanisol
DMSO: Dimethyl sulfoxidants
ABA : Aminobutyric acid
PS: Potassium salicylate
OA: Oxalic acid
SA : Salicylic acid
AA: Ascorbic acid
IAA: Indole acetic acid
IBA: Indole butyric acid
SAR: Systemic acquired resistance
ISR: Induced systemic resistance
JA: Jasmonic acid
ETL: Ethylene
PAL: Phenylalanin ammoialysae
PO: Peroxidase
PPO: Polyphenol oxidase
CAT: Catalase
INA: 2,6-dichloroisonicotinic acid
BTH: Benzothiadiazole S-methyl ester
EBL: 24-epibrassinolide

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INTRODUCTION
Significance of work: 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 [1].
Cucumber plants are affected by several fungal pathogens, and Fusarium
oxysporum Schlechtend.:Fr. is among the most important [2]. The causal agent of wilt
disease in cucumber Fusarium oxysporum f. sp. cucumerinum is economically
important wilting pathogen of cucumber and causing significant yield losses in
greenhouse cucumber.
Concerns about impacts of agrichemicals on water quality and food safety have
led to enhance research aimed at developing alternative approaches for managing
crop diseases [3].
Cucumber plants are liable to be attacked by several pathogens causing powdery
mildew, anthracnose, root-rot and wilt diseases. These diseases are difficult to be
controlled and consequently caused high losses in fruit yield and quality in many
parts of the world [4]. Induced resistance is a promising technique for controlling
plant diseases in about 26 crops including cereals, cucurbits, legumes and
solanaceous plants [5]. Disease resistance can be induced by pre-treating plants
with a number of biotic and abiotic agents which alter disease reaction to
subsequent challenge inoculation [6]. Many reports exist in the literature about
chemicals, plant extracts and microbes with resistance inducing activity [7, 8].
Biological control of Fusarium wilts of numerous crops by application of
antagonistic fungi and bacteria isolated from suppressive soils has been accomplished
during the last two decades all over the world [9, 10, 11, 12].
The purpose and objectives of the research work.
The present study aimed to use biotic and abiotic agents to induce resistance of
Fusarium wilt of cucumber and study their mechanism of action on biochemical
indicators and anatomical changes in cucumber plants. Production the most effective
biotic and abiotic agents in commercial products as alternatives to reducing use of
fungicides in the control of cucumber Fusarium wilt disease under protected houses.
To achieve this purpose it was necessary to achieve the following objectives:
1. Isolation and identification of causative wilt fungus of cucumber plants under
protected houses.
2. Testing the pathogenicity of isolated wilt fungi.
3. Studying the effect of some bio-control agents and resistance-inducers against the
selected wilt fungus in laboratory.
4. Evaluation cucumber hybrids for the resistance to Fusarium wilt under
greenhouses.
5. Studying the efficiency of selected bio-control agents and resistance-inducers on
inducing cucumber plants resistance against Fusarium wilt fungi under green houses.
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6. Evaluation the effect of selected control agents )Trichoderma, Cheatomium,
Penicillium, Bacillus, Pseudomonas and Serratia) on inducing cucumber plants
resistance against Fusarium wilt fungi under protected houses.
7. Studying the expressive indicators of resistance in treated plants as (phenols, lytic
enzymes, oxidative enzymes, Iso-enzymes, lignifications in treated plant roots and
anatomical changes in treated cucumber plants). Production the most effective bio-
control agents in commercial products and test their effects against wilt disease under
protected houses.
The dissertation work was carried out in 2008-2011 at Kazakh National
Agrarian University, Kazakh scientific-research institute of plant protection and
quarantine and Agriculture faculty, university Benha, Egypt.
Scientific novelty: For the first time in Kazakhstan studied the use of biotic and
abiotic agents to induce resistance of cucumber plants to Fusarium wilt. And study
the mechanism of their effect on biochemical parameters of cucumber plants.
Studying the possibility to use biotic agents to induce resistance of cucumber
against Fusarium revealed that, many of biotic isolates can used to induce resistance
of cucumber against Fusarium and also abiotic agent as methods to control.
The important results of the research that, production of biotic agents in commercial
products which we can depend on this products, antioxidants and chemical inducers
to control of Fusarium wilt disease that attack cucumber plants under greenhouses
and reducing the use of fungicides because the side effects and hazards of fungicides
on human health and in the environment. The results of this dissertation are of
great importance and would be necessary to conduct further research work on
using commercial products that produced, antioxidants and chemical inducers to
control of different diseases of many vegetable plants that produced under protected
houses.
Theoretical value and practical applications of research:
The dissertation paper findings can be used as:
- A lecture material for general and special courses in "Plant pathology",
"Biological control of plant diseases", " New trends in controlling of plant pathology
in protected houses" and "Dynamics of plant resistance to diseases" in higher
educational institutions for plant pathology;
- A material for production bio-control agents in commercial products.
- The results of this dissertation provide base information and a system which is
necessary to conduct further studies related to the induction resistance to plant
diseases.
Statements submitted for defense:
1. The selection of the most effective biotic and abiotic inducers against fusarium wilt
in the laboratory.
2. Evaluation of selected biotic and abiotic inducers in the greenhouse and in
protected house conditions.
3. Study of the mechanism of action of selected biotic and abiotic agents on
biochemical indicators and anatomical changes in cucumber plants.
4. Evaluation the effectiveness of biotic agents that made in commercial products.

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Approbation of the work: The main findings and results of the dissertation
were reported and discussed in the following international/state conferences:
- Republican Scientific-theoretical conference «Seyfullinskie reading 5» in Astana,
Republic of Kazakhstan, 23-25 April, 2009.
- Biological Diversity and Sustainable Development of Nature and Society
conference, in Almaty, Republic of Kazakhstan, 12-13 May, 2009.
- The XIII. Czech and Slovak Conference of Plant Protection, Brno Czech Republic,
2-4 September 2009.
- Republican Scientific-theoretical conference «Seyfullinskie reading 6» in Astana,
Republic of Kazakhstan, 22-23 April, 2010.
- International scientific-practical conference "Introduction, conservation of
biodiversity and the use of plants, Bishkek, Kyrgyztan Republic from 7- 9 September 2010.
- Industrial and innovative development of agroindustrial complex: current state and
perspectives Almaty Kazakhstan Republic from 22 -23 October 2010.
Publications:
18 articles and abstracts based on the data related to the dissertation were
published in journals and proceedings of international and state conferences. 9
articles were published in local editions and rate journals of far abroad:
“Инновацинное развитие аграрной науки в исследованиях молодых ученых
конференц (казнау)", Almaty, Kazakhstan, 2010; «International scientific-practical
conference "Introduction, conservation of biodiversity and the use of plants» 2p.,
Bishkek, Kyrgyztan, 2010; «Industrial and innovative development of agroindustrial
complex: current state and perspectives» Almaty, Kazakhstan, 2010; "Journal of Life
Sciences" IF (3.30), USA, 2010; "Исследование результаты" 2p., Almaty,
Kazakhstan, 2011, and "Annals of Agricultural Science" 2p., Moshtohor, Egypt,
2011.
Structure and volume of the dissertation.
The dissertation includes introduction, literature review, materials and research
methods, research results and discussion, conclusion, references, and the summaries
in English, Kazakh, Russian and Arabic languages, pointing at 308 resources. The
text covers 154 pages. The work contains 37 Figures and 43 Tables.

The author expresses his gratitude to his supervisors, professor, academician of the
Kazakh National academy A.O. Sagitova and Professor A.M.M. Mahdi Professor of
Plant Pathology, Fac. Agric., Moshtohor, Benha Univ. АRЕ for valuable advice and
assistance in research, and also all the staff of "the kazakh research institute for the
plant protection and quarantine."
I thank all the staff of Agriculture faculty, university Benha, Egypt.

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1 REVIEW OF LITERATURE

1.1 Important of the disease:
Root and stem rot, caused by Fusarium oxysporum Schlechtend.:Fr. f.sp.
radicis-cucumerinum [2], is a disease of cucumber (Cucumis sativus L.) which was
recorded for the first time in Crete, Greece in 1989 and thereafter in Canada in 1994,
in France in 1998, in Spain in 1999, and in China in 2000 causing significant yield
losses in greenhouse crops [13, 14]. At present, it is the most destructive disease of
green-house grown cucumber in Crete and Peloponnese, Greece [15, 16, 17].
Symptomology and disease development have been described by [15, 18]. Infected
roots, crown, and stem tissues are rotted and contain mycelia and spore masses of the
pathogen [18].Fusarium oxysporum f.sp. cucumerinum, the agent of cucumber wilt
[19].
Among the 62 cultivars tested for resistance to F. oxysporum f.sp.
cucumerinum during 1988-90 in the Sichuan province of China, none proved immune
but one was highly resistant (Da Bai Huang Gua). A further 20 were classed as
resistant. Cultivars with white or whitish yellow skin were more resistant than those
with green skin [20].
Greenhouse cucumber plants infected with Fusarium oxysporum showed the
following symptoms, root and stem rot was increased in frequency and severity.
Affected plants wilted at the fruit-bearing stage, especially at temperatures over 27
degrees C, and mycelial growth and orange spore masses developed on the crown and
stem, [18].
Reactions of 25 cucumber cultivars ranged from highly susceptible to
moderately resistant; the widely-grown long English cultivars Flamingo, Mustang,
and Serami were all highly susceptible to wilt ( the causal fungus is Fusarium
oxysporum forma specialis radicis-cucumerinum) [18].
Cucumbers (Cucumis sativus cv. Albatros) in several commercial glasshouses
exhibited symptoms of wilt, yellowing and necrotic streaks on the stems. Internal,
vascular discoloration in infected plants extended from the base of the stem upward
[21].
Plants that are grown in greenhouses may be attacked by a number of plant
pathogenic fungi. This way of plant production is very specific due to
characteristically temperature conditions, as well as air and soil humidity, which are
usually very favourable for development of plant pathogenic fungi [22].
Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum (FOC), is
one of the major diseases in cucumber (Cucumis sativus) production [23]
Fusarium wilt caused by Fusarium oxysporum f.sp. cucumerinum is one of the
most devastating diseases in cucumber production worldwide [24].
Fusarium oxysporum f.sp. cucumerinum is a destructive pathogen on cucumber
( Cucumis sativus L.) seedlings and the causal organism of crown and root rot of
cucumber plants [25].

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