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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
2
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
3
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
Fusarium wilt.
58
3.3 Laboratory studies: 59
3.3.1 Effect of antagonistic fungi on the linear growth of F. O. f.sp.
cucumerinum (FOC) in vitro.
59
3.3.2 Evaluating the effect of antagonistic fungi culture filtrates on the
linear growth and spore germination of F. O. f.sp. cucumerinum (FOC).
60
3.3.3 Effect of antagonistic bacteria in vitro against F. O. f.sp.
cucumerinum (FOC).
61
3.3.4 Evaluation of the effect of antagonistic bacteria culture filtrates on
the linear growth and spore germination of F. O. f.sp. cucumerinum (FOC).
62
3.3.5 Effect of different resistant inducing chemicals on the linear
growth and spore germination of F. O. f.sp. cucumerinum (FOC) in vitro.
65
3.4 Greenhouse experiments: - 67
3.4.1. Effect of treating cucumber seeds with some antagonistic fungi on
incidence with Fusarium wilt disease:
67
3.4.2. Effect of cucumber seeds treatment with cell suspension of
antagonistic bacterial isolates on incidence with Fusarium wilt.
68
3.4.3. Effect of treating cucumber seeds or treating soil with some
resistance inducing chemicals on incidence with Fusarium wilt.
69
3.5. Experiments of Commercial protected house: 72
3.5.1. Effect of cucumber seeds treatment with some antagonistic fungi
on incidence with Fusarium wilt disease under commercial protected house:
72
3.5.2. Effect of treating cucumber seeds with cell suspension antagonistic
bacterial isolates on incidence of Fusarium wilt disease under commercial
protected house:
74
3.5.3. Effect of treating cucumber seeds or treating soil with some
resistance inducing chemicals on incidence with Fusarium wilt disease.
75
77
4
3.6 Determination of enzymes activity, lignin content and peroxidase
isozyme:
3.6.1 Effect of treating cucumber seeds with spore suspension of
antagonistic fungus isolates in peroxidase activity in cucumber plants:
77
3.6.2 Effect of treating cucumber seeds with cell suspension of
antagonistic bacterial isolates in peroxidase activity in cucumber plants:
78
3.6.3 Effect of treating cucumber seeds with tested chemical compounds
on peroxidase activity in cucumber plants:
79
3.6.4 Effect of treating cucumber seeds with spore suspension of
antagonistic fungus isolates in Polyphenol-oxidase activity in cucumber
plants:
80
3.6.5 Effect of treating cucumber seeds with cell suspension of
antagonistic bacteria isolates in polyphenol-oxidase activity in cucumber
plants:
81
3.6.6 Effect of treatment of cucumber seeds with tested chemicals
compound in polyphenol-oxidase activity in cucumber plants:
82
3.6.7 Effect of treatment of cucumber seeds with spore suspension of
antagonistic fungal isolates on chitinase activity in cucumber plants:
83
3.6.8 Effect of treatment of cucumber seeds with cell suspension of
antagonistic bacterial isolates in chitinase activity in cucumber plants:
84
3.6.9 Effect of treatment of cucumber seeds with tested chemical
compounds on chitinase activity in cucumber plants:
85
3.6.10 Effect of treatment of cucumber seeds with spore suspension of
antagonistic fungal isolates on lignin content in cucumber plants:
86
3.6.11 Effect of treatment of cucumber seeds with cell suspension of
antagonistic bacterial isolates on lignin content in cucumber plants:
87
3.6.12 Effect of treatment of cucumber seeds with tested chemical
compounds on lignin content in cucumber plants:
88
3.6.13 Effect of treatment of cucumber seeds with spore suspension of
antagonistic fungal isolates on isozyme pattern of peroxidase in cucumber
plants:
89
3.6.14 Effect of treatment of cucumber seeds with spore suspension of
antagonistic bacterial isolates on isozyme pattern of peroxidase in cucumber
plants:
92
3.6.15 Effect of treatment of cucumber seeds with tested chemical
compounds on isozyme pattern of peroxidase in cucumber plants:
95
3.7 Chemical analysis of cucumber treated plants: 97
3.7.1 Effect of cucumber seeds treatment with spore suspension of
antagonistic fungal isolates on sugar content in cucumber plants:
97
3.7. 2 Effect of treatment of cucumber seeds with cell suspension of
antagonistic bacterial isolates on sugar content in cucumber plants.
98
3.7.3 Effect of treatment of cucumber seeds with tested chemicals
compound on sugar content in cucumber plants.
99
5
3.7.4 Effect of treatment of cucumber seeds with spore suspension of
antagonistic fungal isolates on phenol content in cucumber plants.
100
3.7.5 Effect of treatment of cucumber seeds with cell suspension of
antagonistic bacterial isolates on phenol content in cucumber plants
101
3.7.6 Effect of treating cucumber seeds with chemical compounds on
phenol content in cucumber plants:
102
3.7.7 Effect of cucumber seeds treatment with cell suspension of
antagonistic fungal isolates on amino acids content in cucumber plants:
103
3.7.8 Effect of cucumber seeds treatment with cell suspension of
antagonistic bacterial isolates in amino acids content in cucumber plants:
104
3.7.9 Effect of treating cucumber seeds with tested chemicals compound
in amino acids content in cucumber plants:
105
3.8 Anatomical studies: 106
3.8.1 Effect of cucumber seeds treatment with tested antagonistic fungal
isolates on the mean counts and measurements of certain histological features
of main cucumber root.
106
3.8.2 Effect of cucumber seeds treatment with tested antagonistic
bacterial isolates on the mean counts and measurements of certain histological
features of main cucumber root.
108
3.8.3 Effect of cucumber seeds treatment with tested chemical
compounds on the mean counts and measurements of certain histological
features of main cucumber root.
110
3.9 Effect of carrying the best antagonistic isolates of fungi and bacteria on
different carrier material on infection with Fusarium wilt.
112
3.9.1 Comparison between some different carrier materials of
antagonistic fungal isolates on cucumber seeds on infection with Fusarium
wilt.
112
3.92 Comparison between some different carrier materials of antagonistic
bacterial isolates on cucumber seeds on infection with Fusarium wilt.
113
4 DISCUSSION 114
CONCLUCTION 129
REFERENCES 132
APPENDIX 154
6
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
7
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.
8
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.
9
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.
10
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].
11
1.2 Inducing resistance by biotic agents:
Microbiological agents should not be used alone to control soil-borne pathogens
and nematodes. It has been observed that their use combined with other strategies
may help to provide the necessary control. Since manufacturing and registration of
microbiological agents are very expensive processes, they should be applied only in
high-value crops, which can pay back the investment of the application. The
advantages of the use of these agents are that they are non-toxic to humans, animals
and several useful organisms, do not normally cause pest resistance, and can be
applied effectively in integrated pest management (IPM) [26].
The first reports of induced resistance on cucurbits were the protection of
watermelon seedlings against Fusarium oxysporum by prior inoculation of their roots
with the pathogen of corn, Helminthosporium carbanum. Root of susceptible
watermelon seedlings were dipped into a spore suspension of H. carbanum prior to
transplanting into fusarium–infested soil. Resistance was characterized by a decrease
in the severity of wilt symptoms [27].
Systemic protection of cucumber against Colletotrichum lagenarium was
obtained by prior inoculation of cotyledons or the first true leaf with same the
pathogen [28].
When tomato seedlings were screened simultaneously for resistance to the
vascular pathogen Verticillium dahliae and Fusarium oxysporum f.sp. lycopersici
(FOL), progenies resistant to FOL also exhibited resistance to V. dahliae even though
these progenies were genetically susceptible to the latter pathogen [29].
Similar to cucumbers and melons, inoculation of one leaf of watermelon with
spores of the anthracnose fungus also resulted in systemic protection of the entire
plant against subsequent infection by the same pathogen under both greenhouse and
field condition [30].
Symptoms of verticillium wilt in cucumber was reduced by treating the roots
with culture filtrates of the fungus or by spraying the two basal leaves with a
suspension of Verticillium albo-atrum conidia [31].
The protection of cucumber from the anthracnose (caused by Colletorichum
lagenarium) by prior inoculation with an avirulent isolate of F. oxysporum f.sp.
cucumerinum was systemic. F. oxysporum f.sp. cucumerinum was detected primarily
in the root and hypocotyls, but not at the leaf infection site or in the stem [32].
The growth of both F. oxysporum and M. phaseolina the causal of wilt and
charcoal rot of sesame was inhibited by Trichoderma sp. isolated from rhizosphere
region of sesame [33].
Resistance to cucumber wilt (caused by F. oxysporum f.sp. cucumerinum) was
induced in cucumber plants growing on a mineral agar medium by inoculation of the
medium with F. oxysporum formae speciales nonpathogenic on cucumber and by leaf
infection with Colletotrichum lagenarium or tobacco necrosis virus (TNV).
Resistance was not induced against the disease in cucumber plants growing in
synthetic soil mixture in a greenhouse, by any of the tested fungi when challenge
followed induction by 3 days or less. Resistance was induced by foliar infection with
C. lagenarium or TNV, but not F. oxysporum f.sp. melonis when the interval was
increased to 7 days [34].
12
The incidence of Fusarium wilt disease in glasshouse on cucumbers grown in
naturally infested soil was reduced by amendments of ground crab shell, the effect
appearing after c. 30 d. The population of the causal fungus was reduced in amended
natural soil. In amended autoclaved soil incidence was not reduced and the
population was increased. The populations of total fungi, bacteria and actinomycetes
were increased in both amended soils. Actinomycetes antagonistic to F. oxysporum
f.sp. cucumerinum and Gram- bacteria were more abundant in the natural amended
soil. Production of CO2 in natural soil was accelerated by crab shell amendment and
the pH in amended soil became alkaline [35].
Two closely related cucurbit fusarium wilt, F.oxysprum f.sp. cucumerirum
(FOC) pathogenic to cucumber, and F. oxysporum f.sp. melonis (FOM) pathogenic
to muskmelon, were evaluated for their protect watermelon from avirulent race of F.
oxysporum f.sp. niveum (FON). FOC protected 2-week old watermelon seedlings. No
wilt was observed in FOC-treated seedling 8 week after challenge, while FOM and
water-treated seedlings had 33% and 50% wilt respectively [36].
Growth of T. viride was various in the dual culture. Trichoderma spp was an
effective hyperparasite, penetration and coiling Fusarium oxysporum around hyphae.
Trichoderma glaucom produced effective metabolites, while, T. album caused lysis
and inhibited the pathogen [37].
Isolates of Trichoderma spp., T. viride, T. harzianum and T. koningii were
inoculated into the growing medium at a concentration of 4.6 x 108
propagules/g of
dray soil, and assessed for the control of Verticillium dahliae and Fusarium
oxysporum in greenhouse. All the isolates reduced disease incidence [38].
F. oxysporum f.sp. cucumerinum causes severe damage to cucumber under
glasshouse conditions. The disease was suppressed 30-35% by application of organic
matter; the best results were obtained with mushroom compost and chicken manure.
Disease occurrence was delayed in the presence of organic amendments in nonsterile
soil compared with that under sterile conditions. At 30 d after inoculation, the
numbers of actinomycetes, fungi and bacteria were greater and F. oxysporum
decreased in organically amended soils compared with natural control soils [39].
The use of Trichoderma spp. led to a decrease in symptoms caused by
Verticillium dahliae and Fusarium oxysporum in greenhouse [40].
Fluorescent pseudomonads and nonpathogenic isolates of F. oxysporum were
effective in inducing suppressiveness to Fusarium wilt of cucumber (F. o. f.sp.
cucumerinum) when added to soil together (pH 6.7) but ineffective when added
separately. Suppressiveness by such combination treatments was enhanced in nearly
neutral (pH 6.7) to alkaline soils (pH 8.1), in comparison with acid soil (pH 5.5). Strs
of fluorescent pseudomonads reduced the germination of chlamydospores of
nonpathogenic and pathogenic isolates of F. oxysporum in the rhizospheres of
cucumber plants. Population densities of fluorescent pseudomonads increased
significantly in the rhizosphere of cucumber in the presence of a nonpathogenic
isolate of F. oxysporum in soil of pH 8.1. It is hypothesized that the activity of
fluorescent pseudomonads and their siderophore production are enhanced by
increased root exudates induced by relatively high population densities of
nonpathogenic isolates of F. oxysporum. This, in turn, leads to competition for iron,
13
which is essential for successful germination of the pathogen and penetration of the
host [41].
Mutants of Pseudomonas putida (Agg−) that lack the ability to agglutinate with
components present in washes of bean and cucumber roots showed limited potential
to protect cucumber plants against Fusarium oxysporum f. sp. cucumerinum.
However, a higher level of protection was observed against Fusarium wilt in
cucumber plants coinoculated with the parental bacterium (Agg+), which was
agglutinable. The Agg− mutants did not colonize the roots of cucumber plants as
extensively as the Agg+ parental isolate did. In competition experiments involving
bean roots inoculated with a mixture of Agg+ and Agg
− bacteria, the Agg
+ strains
colonized roots to a greater extent than the Agg− cells did. These data suggest that the
Agg+ phenotype provides additional interactions that aid in the beneficial character of
P. putida [42].
4 Trichoderma isolates (2 isolates Trichoderma harzianum and 2 isolates
Trichoderma viride) were highly antagonistic to Fusarium oxysporum f.sp. fragariae.
In dual culture, T. harzianum parasitized F. o. f.sp. fragariae and inhibited mycelial
growth, the processes of mycoparasitism including coiling round and attachment to
host hyphae, microconidia and macroconidia and penetration into the hyphae or
breaking the septa of hyphae and conidia. T. viride produced non-volatile antibiotics
inhibiting growth of F. oxysporum f.sp. fragariae but its antagonistic effect in vitro
was relatively low [43].
The antagonistic activities of 56 isolates of Gliocladium virens from cucumber
and 9 of Trichoderma harzianum from strawberry fields against Fusarium oxysporum
in vitro were evaluated. Isolate G C27 of G. virens strongly inhibited mycelial growth
by means of non-volatile antibiotics which were unaffected by the N source in the
medium. The strongly mycoparasitic T. harzianum T42, however, had low antibiotic
activity which was increased when ammonium tartarate or NH4NO3 was present. The
addition of chitin, a cell wall preparation of the pathogen, cellulose or various organic
supplements had no effect on the antibiotic activity of T42. Effects of G C27 on
mycelial growth and conidial germination were not affected by the cell wall
preparation but were inhibited by wheat bran or malt. In pot tests incorporation of G
C27 or T42 cultures on wheat bran media into sterilized soil infected with pathogen
was much more effective in reducing the incidence of wilt disease caused by F.
oxysporum than application of conidial suspension without an organic food base. The
wheat bran culture of G C27 with or without inorganic nutrients decreased disease
incidence by 54 – 59% compared with the control without antagonist. The application
of T42 + inorganic nutrients also decreased wilt by 52 – 59%, but without nutrients
the decrease was only 18% [44].
T. harzianum, Gliocladium roseum or Chaetomium globosum added to soil as
granules or used as seed dressing. All formulations were more effective for
controlling S. rolfsii, R. solani, M. phaseolina and Pythium ultimum the causals of
seed cotton and seedling diseases than adding the same antagonists as soil drench,
[45].
Two Trichoderma oureoviride and two T. harzianum isolates from organic
composts were tested for antagonism of Fusarium oxysporum and Verticillium
14
dahliae in vitro. Microscopic examination indicated that hyphae from both T.
aureoviride isolates grew and coiled around the hyphae of F. oxysporum. Non–
volatile compounds released by both T. harzianum isolates growing on cellophane
discs over malt agar significantly inhibited growth of F. oxysporum and V. dahliae,
[46].
In soil-less culture of vegetables and flowers in greenhouses, Fusarium diseases
may induce severe damage. Under these growing conditions, biological control can
be achieved by application of selected strains of fluorescent Pseudomonas or non-
pathogenic F. oxysporum. A total of 74 strains of fluorescent Pseudomonas were
tested for their ability to reduce the incidence of Fusarium wilt of flax when applied
either alone or in association with one preselected non-pathogenic strain of F.
oxysporum (Fo47). Four classes were established, based on the effect of bacteria on
disease severity, on their own or in association with Fo47. Most of the strains did not
modify the percentage of wilted plants. However 10.8% of them, although having no
effect on their own, significantly improved the control attributable to Fo47. One of
these bacterial strains (C7) was selected for further experiments. Two trials
conducted under commercial-type conditions demonstrated the effectiveness of the
association of the bacterial str. C7 with the non-pathogenic F. str. Fo47 to control
Fusarium crown and root rot of tomato, even when each antagonistic microorganism
was not efficient by itself. The yields were not significantly different in the protected
plots in comparison with the healthy control [47].
Bacillus megaterium and Trichoderma spp. especially T. harzianum were the
best antagonistic agents against F. oxysporum f.sp. sesame [48].
Chaetomium isolates from soil reduced the development of wilt caused by
Verticillium dahliae and were antagonistic against the pathogen in dual culture. From
the culture filtrate of the most antagonistic isolate, identified as C. globosum, 2 active
substances were obtained by silica gel column chromatography and HPLC. The
major one, identified as Cheatomium globosum A, completely inhibited spore
germination of V. dahliae at 32µg/ml. was also active against V. albo-atrum and
Rhizoctonia solani [49].
Acremonium sp. Trichoderma sp. and Chaetomium globosum inhibited
Verticillium dahliae development on PDA the most effectively; reducing the radial
colony growth by 65-76%. Alveophoma sp., and Trichoderma sp. colonized 100 and
20% of Verticillium dahliae mycelium; respectively and C. globosum produced
antibiotic substances inhibitory to V. dahliae growth; producing 1.1-0.7 cm diam.
Haloes [50].
Biological control agents G. virens G872B and P. putida Pf3 were compatible
with each other and successfully colonized cucumber rhizospheres, which contributed
to a long-term inhibition of cucumber Fusarium wilt (F. oxysporum f.sp.
cucumerinum). G872B colonized successfully on the cucumber root system
irrespective of the introduction of Pf3. Pf3 colonized well in the rhizosphere
regardless of the presence of G872B. Individual strains effectively suppressed
cucumber wilt up to 56 d after transplanting. A combined treatment of G872B and
Pf3 provided long-term protection of - 80 d with the efficacy greater than that
obtained by any individual strains under greenhouse conditions. It is suggested that
15
the colonization of the biological control agents in the rhizosphere could be
correlated directly to Fusarium wilt-suppressive potentials [51].
Plant growth-promoting rhizobacteria (PGPR) strains 89B-27 (Pseudomonas
putida) and 90-166 (Serratia marcescens) were tested for their ability to induce
systemic resistance against Fusarium wilt, a vascular disease of cucumber, using a
split-root assay. PGPR strains and F. oxysporum f.sp. cucumerinum were inoculated
on separate halves of roots of cucumber seedlings at the same time and then planted
in separate pots. Both PGPR strains induced systemic resistance against F.
oxysporum f.sp. cucumerinum as expressed by delayed disease symptom
development and reduced number of dead plants after PGPR treatments compared
with the nonbacterized, F. oxysporum f.sp. cucumerinum inoculated controls 5 weeks
after inoculation. F. oxysporum f.sp. cucumerinum was recovered from lower stems 2
weeks after root inoculation and from the 1st, 2nd and 3rd petioles 5 weeks after
inoculation in the nonbacterized control. In contrast, F. oxysporum f.sp. cucumerinum
was isolated from stems of plants treated with PGPR only 4 weeks after inoculation
and from the 1st petiole 5 weeks after inoculation, indicating that PGPR treatment
reduced spread of the pathogen. Movement of PGPR in cucumber split-root systems
was monitored with a bioluminescent derivative of 89B-27, strain L211, that was
detected with a charge-coupled device camera. Strain L211 provided protection
against F. oxysporum f.sp. cucumerinum at levels similar to the wild type PGPR
strain. L211 colonized cucumber roots up to 5 weeks after root inoculation and was
not detected inside stems or petioles. The bacterium showed only limited movement
within inoculated pots and did not move to the pots in which the pathogen was
inoculated, demonstrating that the PGPR and pathogen remained spatially separated.
It is concluded that the 2 PGPR strains induced resistance systemically in cucumber
against Fusarium wilt [52].
Soil solarization in a plastic tunnel was tested in combination with antagonistic
Trichoderma harzianum and Fusarium spp. as seed inoculants during experiments
carried out in the Italian Riviera during 1991-92. The application of polyethylene
mulching alone allowed significant control of Rhizoctonia solani on bean [Phaseolus
vulgaris], Pythium ultimum on cucumber and Fusarium oxysporum f.sp. basilicum on
basil [Ocimum basilicum]. The biological control agents were effective on P.
vulgaris, but not on cucumber or basil when sown in non-solarized soils. The
integration of solar heating and antagonistic microorganisms did not generally
provide a significantly different level of control from solarization alone. However,
although not statistically significant, differences between such an integrated approach
and the use of solarization alone were observed in the reduction of disease on the 3
crops during both 1991 and 1992. The effectiveness of integrated control strategies
which employ solar heating and biocontrol inoculants are briefly discussed with
respect to marginally suitable conditions for solarization found in southern Europe
and to the possibility of reducing the mulching period [53].
Trichoderma harzianum suppressed Fusarium wilt caused by Fusarium
oxysporum f.sp. fragariae in strawberries. The wheat bran or rice straw culture of T.
harzianum suppressed disease incidence more effectively than the other culture
substrates. T. harzianum cultured on wheat bran or rice decreased disease incidence
16
to 68% of the control. A conidial suspension of T. harzianum alone or a suspension
mixed with crab shell also reduced disease incidence. T. harzianum was highly
effective in controlling disease in acidic soil (pH 3.5 – 5.5). Disease incidence and
population density of F. o. f.sp. fragariae decreased in sandy loam soil inoculated
with T. harzianum. There were no similar effects on inoculated loam soil [54].
A strain of P. fluorescens, prepared on different carrier materials as seed or soil
treatments, reduced Fusarium wilt of watermelon [55].
Trichoderma viride 9 mutants obtained by using different mutagenic agents in
vitro were evaluated for their relative efficacy against M. phaseolina in terms of
antibiotic production. The cell free culture filtrate of mutant M1 showed the highest
in vitro inhibition of mycelial growth (54.4%) and sclerotial germination (75.8%) of
M. phaseolina followed by M3 and they were on par with each other. The mutants
M3 and M8 produced maximum volatiles in vitro as evidenced by the retardation in
growth of M. phaseolina which registered a growth of 10 mm after 48 h of incubation
while in the control the growth was 90 mm [56].
The rhizosphere competence was higher in soils of pH 5.0 and 6.0 than in soils
of pH 7.0 when cucumber seeds were treated with Gliocladium virens (G872B) and
Trichoderma harzianum (T12MT). Mycelial growth of Pythium ultimum and
Rhizoctonia solani was strongly inhibited by culture filtrates of G872B and T12MT
grown for 4 days at acidic condition of pH 4.5 and 6.0. Rhizosphere competence of
Pseudomonas putida (Pf3), G872B and T12MT were also influenced by salt
concentration in soil. That of Pf3 was high in soils with a high salinity level of EC 2.0
(mS/cm). That of G872B was consistent throughout the wide range of EC (from 0.5
to 1.5), suggesting that its salt tolerance and that of T12MT decreased with an
increase in the EC level. Treatment of G872B and T12MT significantly reduced the
incidence of Fusarium wilt of cucumber in soils of EC 0.5 and 1.0, but less effective
in higher salinity soils of 1.5 and 2.0. Correspondingly, disease incidence was lower
in soil with low EC level of 0.5 and 1.0 than in soil with high EC level of 1.5 and 2.0,
[57].
Mycelial growth of F. oxysporum was inhibited more than M. phaseolina by the
antagonistic fungi. Trichoderma spp. particularly T. viride was the most effective in
this regard followed by Gliocladium penicilloides and Chaetomium bostrycoides. T.
harzianum followed by C. bostrycoides were the best for reducing root-rot and/or wilt
disease incidence on sesame and increased percentage of healthy plants compared
with other antagonistic fungi [58].
Trichoderma viride, T. koningii, T. harzianum, Gliocladium virens and G.
catenulatum showed the greatest potential in controlling the growth of Fusarium
oxysporum and Rhizoctonia solani on grasses [59].
Investigate the process of infection and cytological structural changes in
susceptible and resistant cucumber cultivar seedling roots inoculated with F.
oxysporum f.sp. cucumerinum showed that, Fusarium spores germinated as infecting
hyphae which may penetrate the host epidermal cells, pass through the cortical cells,
and then enter into the xylem vessels. After infection by the pathogen, some
occlusions such as wall-coatings, tyloses and brown materials were formed in the
vessels. The various reactions of occlusions were earlier and more extensive in the
17
resistant cultivar than in the susceptible one. Tyloses were formed by elongated
parenchyma cells and secreted from pits. A parenchyma cell could produce tylose
simultaneously. It is concluded that the function of these occlusions is to prevent
pathogen growth [60].
Gliocladium virens and Trichoderma hamatum significantly reduced wilt of
tomatoes, watermelons and muskmelons compared with controls by (30-60%
reduction) [61].
Trichoderma spp., Gliocladium virens, Pseudomonas fluorescens, Burkholderia
cepacia and others. Specific non-pathogenic isolates of F. oxysporum and F. solani
collected from a Fusarium wilt-suppressive soil were the most effective antagonists
in controlling wilt of tomato caused by Fusarium oxysporum, providing significant
and consistent disease control (50 to 80% reduction of disease incidence) in several
repeated tests. These isolates were also equally effective in controlling Fusarium wilt
diseases of other crops, including watermelon and muskmelon. Other organisms,
including isolates of G. virens, T. hamatum, P. fluorescens and B. cepacia, also
significantly reduced Fusarium wilt compared with disease controls (30 to 65%
reduction), but were not as consistently effective as the non-pathogenic Fusarium
isolates. Commercially available biocontrol products containing G. virens and T.
harzianum (SoilGard and RootShield, respectively) also effectively reduced disease
(62 to 68% reduction) when granules were incorporated into potting medium at 0.2%
(wt/vol). Several fungal and bacterial isolates collected from the roots and
rhizosphere of tomato plants also significantly reduced Fusarium wilt of tomato, but
were no more effective than other previously identified biocontrol strains.
Combinations of antagonists, including multiple Fusarium isolates, Fusarium with
bacteria and Fusarium with other fungi, also reduced disease but did not provide
significantly better control than the non-pathogenic Fusarium antagonists alone [62].
Assessed the inhibitory effects of 2 beneficial Bacillus subtilis isolates,
Gliocladium roseum and 6 Trichoderma spp. against Pythium ultimum and Fusarium
oxysporum f.sp cucumerinum in vitro and in vivo revealed that, The isolates of
Trichoderma spp. and the 2 isolates of Bacillus showed antagonistic effects against
the pathogens. In a greenhouse experiment, B. subtilis (isolates 1 and 2), T. viride
(isolates 1 and 3) and T. harzianum significantly suppressed basal stem rot caused by
Pythium by 50, 56.7, 46.7, 40 and 46.7%, respectively, whereas, B. subtilis (isolates 1
and 2), T. viride (isolate 2) and T. harzianum (isolates 5 and 6) suppressed Fusarium
wilt by 26.7, 33.3, 33.3, 33.3 and 26.3%, respectively [63].
Forty isolates of exospore-forming actinomycetes and endospore-forming
bacteria (20 isolates each) were randomly isolated from the rhizosphere soil of a
healthy cucumber plant. Among these isolates, 8 actinomycetes and 6 spore-forming
bacterial isolates exhibited antagonistic activities against F. oxysporum. One isolate
of actinomycetes and another one of endospore-forming bacteria, which showed the
highest antagonistic activities against the pathogenic fungus were selected and
identified as Streptomyces spp. and Bacillus mycoides, respectively. Inoculation of
cucumber plants, grown in Fusarium-infected soils with any of the antagonistic
microorganisms (Streptomycin spp. or B. mycoides), resulted in a marked reduction in
total count of fungi in the rhizosphere soils and much lower percentages of diseased
18
plants as compared with the uninoculated ones. Higher antifungal activities were
achieved by application of these microorganisms in an immobilized form
(encapsulated in sodium alginate beads) than in the case of using free cells. With
application of mixed inocula of these 2 antagonistic microorganisms in free or
immobilized states, lower antifungal activities were observed, than when each was
used separately. This was attributed to the antagonistic activity of Streptomyces spp.
against B. mycoides. The lowest number of diseased cucumber plants was achieved
when immobilized Streptomyces spp. was applied to plants grown in Fusarium-
infected soils [64].
Two chitinolytic bacterial strains, Paenibacillus sp. 300 and Streptomyces sp.
385, suppressed Fusarium wilt of cucumber (Cucumis sativus caused by Fusarium
oxysporum f.sp. cucumerinum in nonsterile, soilless potting medium. A mixture of
the two strains in a ratio of 1:1 or 4:1 gave significantly (P 0.05) better control of the
disease than each of the strains used individually or than mixtures in other ratios.
Several formulations were tested, and a zeolite-based, chitosan-amended formulation
(ZAC) provided the best protection against the disease. Dose-response studies
indicated that the threshold dose of 6 g of formulation per kilogram of potting
medium was required for significant (P 0.001) suppression of the disease. This dose
was optimum for maintaining high rhizosphere population densities of chitinolytic
bacteria (log 8.1 to log 9.3 CFU/g dry weight of potting medium), which were
required for the control of Fusarium wilt [65].
A strain of Trichoderma viride with high antagonistic potential against
Rhizoctonia solani and Sclerotium rolfsii [Corticium rolfsii] in dual culture was
isolated from soil .It was formulated in talc powder as a biofungicide. The antagonist,
T. viride, survived in the formulation with 28X108 colony forming units even after
four months of storage. Coating seeds with different doses of the biofungicide
increased germination of seeds, seedling root and shoot lengths in cotton, okra and
sunflower. Maximum germination was recorded when seeds were treated with
biofungicide at 25 g/kg seeds. Treating seeds with high doses of biofungicide (50 or
100 g/kg seeds) did not inhibit germination. Seed treatment followed by soil
application of the biofungicide significantly reduced plant mortality caused by root
pathogens and increased yield compared to chemical fungicides and untreated
controls. In soil inoculated with Rhizoctonia solani, application of biofungicide at 5
and 2.5 kg/ha significantly reduced plant mortality and increased yield in cotton, okra
and sunflower [66].
Pseudomonas aureofaciens (=P. chlororaphis) strain 63-28 is a biocontrol agent
active against many soil-borne fungal plant pathogens and shows antifungal activity
in culture assays. 3-(1-Hexenyl)-5-methyl-2-(5H)-furanone was isolated from culture
filtrates of this bacterium. The purified furanone showed antifungal activity against
Pythium ultimum, Fusarium solani, Fusarium oxysporum, and Thielaviopsis basicola.
The ED50S for spore germination of these fungi were 45, 54, 56, and 25 µ g/ml
respectively. The compound also inhibited the germ tube growth of Rhizoctonia
solani growing from microsclerotia, with an ED50 of 61 µ g/ml. This volatile
antifungal furanone has structural similarity to other antifungal furanones produced
by actinomycetes (Streptomyces spp.), fungi (Trichoderma harzianum), and higher
19
plants (Pulsatilla and Ranuculus spp.) [67].
Soaking sesame seeds in ascorbic and salicylic acid (at 5 mM) for 12 h before
sowning and then treated with ascorbic and salicylic acid 15 days after sowing
resulted in the best control against F.oxysporum f.sp. sesami compared to Benlate
[68].
Inoculation of root tips of chickpea by P. fluorescens, 2,6-dichloroisonicotinic
acid, and acetylsalicylic acid induced systemic resistance against charcoal rot.
Disease was 33 to 55.5% higher in control plants than in plants inoculated with
chemical inducers or P. fluorescens [69].
To develop efficient biopesticides that could be used as components for
integrated pest management programmes, a multidisciplinary approach was adopted.
For soilborne plant pathogens, attention was focused in protection of spermosphere or
rhizosphere by combining seed priming with a strain of Trichoderma koningii, and by
adding a suspension of the antagonist twice into the soil. This strategy resulted in
82% and 62% protection against Rhizoctonia solani in bean and tomato, respectively,
and 70% protection against Fusarium oxysporum f.sp lycopersici in tomato. Conidia
of T. koningii produced by solid state fermentation were formulated as granules for
soil application under field conditions, which can also be dispersed as a suspension.
For the control of Sclerotium cepivorum in onion more than 60% protection was
obtained by using native isolates of Trichoderma sp., Clonostachys sp. and Beauveria
sp. [70].
Antagonistic bacteria isolated from livestock manures and soils, coded as 94-I,
94-II, 94-III, 96-II, 98-I and 98-II, exhibited fungistatic activity against cucumber
fusarium wilt (Fusarium oxysporum f.sp. cucumerinum). In suspended spore culture,
these bacteria exhibited 48.29, 0.00, 60.69, 39.28, 61.29 and 72.32% inhibition of F.
oxysporum f.sp. cucumerinum spore germination. Moreover, some spores and hyphae
of the pathogen became deformed when the pathogen was grown with the
antagonistic bacteria. The relative inhibition rates of these bacteria against F.
oxysporum f.sp. cucumerinum in Petri dishes were 74.52, 83.27, 90.60, 88.59, 89.35
and 94.30%, respectively, while the relative inhibition rates of their metabolites
against the pathogen were 33.21, 55.22, 62.84, 46.57, 47.61 and 70.34%, respectively
[71].
Roots of cucumber plants treated with Five fungal isolates ( Trichoderma
,Fusarium ,Penicillium ,Phoma and a sterile fungus) these fungal isolates using
barley grain inocula (BGI), mycelial inocula (MI) or culture filtrate (CF). Most
isolate/inoculum form combinations significantly reduced anthracnose disease
(pathogen, Colletotrichum orbiculare) except BGI of Trichoderma. These fungal
isolates were also evaluated for induction of systemic resistance against bacterial
angular leaf spot and Fusarium wilt by treatment with BGI. Penicillium, Phoma and
the sterile fungus significantly reduced the disease incidence of bacterial angular leaf
spot. Phoma and sterile fungus protected plants significantly against Fusarium wilt.
Roots treated with CFs of these fungal isolates induced lignification at
Colletotrichum penetration points indicating the presence of an elicitor in the CFs
[72].
20
Saprophytic bacteria from the genus Pseudomonas are important in biological
control, as the biopreparations that restricted the development of soilborne plant
pathogens. The biotic and abiotic factors involved in the rhizosphere competence of
bacteria, traits essential for induction of the plant resistance and factors involved in
antagonism of PGPR strains against soilborne plant pathogens are important for the
success of biological control [73].
Introduction of bacterial and fungal biological control agents offers a promising
alternative to manage soilborne diseases. The combination of bacterial and fungal
antagonists could be a useful method to enhance biological control activity.
Fluorescent pseudomonads are established biological control agents against several
soilborne pathogens. Trichoderma strains are well known for their biological control
activity against several plant pathogens through chitinase production such as the ECH
42 endochitinase and the NAGl N-acetyl-beta-glucosaminidase [74].
Biotic and abiotic elements of the soil environment contribute to
suppressiveness; however, most defined systems have identified biological elements
as primary factors in disease suppression. Many soils possess similarities with regard
to microorganisms involved in disease suppression, while other attributes are unique
to specific pathogen-suppressive soil systems. The organisms' operative in pathogen
suppression does so via diverse mechanisms including competition for nutrients,
antibiosis, and induction of host resistance. Non-pathogenic Fusarium spp. and
fluorescent Pseudomonas spp. play a critical role in naturally occurring soils that are
suppressive to Fusarium wilt [75].
The soil fungus Trichoderma atroviride, a mycoparasite, responds to a number
of external stimuli. A number of soil isolates are being studied because of their ability
to antagonize soilborne plant pathogens. These Trichoderma species are often
referred to as biological control fungi. During the initial stages of this interfungal
interaction, T. atroviride responds to the presence of the host by coiling around the
host hyphae. In the presence of a fungal host, T. atroviride produces hydrolytic
enzymes and coils around the host hyphae [76].
Sugarbeet seeds coating with two biological antagonists, namely Trichoderma
harzianum and Gliocladium virens, was evaluated against damping off of seedlings,
caused by four soilborne plant pathogens, namely Pythium aphanidermatum,
Rhizoctonia solani, Rhizoctonia bataticola [Macrophomina phaseolina] and
Sclerotium rolfsii [Athelia rolfsii], and compared with fungitoxicant mixtures in
reducing the pre- and post-emergence mortality. Seed coating with all combination of
fungitoxicants gave better results in reducing the seedling mortality compared to
dipping of seeds in aqueous suspensions of combinations of fungitoxicants. Seed
coating with T. harzianum gave better results in reducing the disease compared with
G. virens [77].
Applying Penicillium oxalicum at a rate of approx. 106-10
7 CFU/g in seedbed
substrate and rhizosphere before transplanting tomato plants was effective in
controlling of fusarium and verticillium wilt of tomato, and that formulation of P.
oxalicum has a substantial influence on its efficacy [78].
Certain fluorescent pseudomonads can protect plants from soil-borne pathogens,
and it is important to understand how these biocontrol agents survive in soil [79].
21
Trichoderma harzianum were effective in inhibiting Fusarium oxysporum f.sp.
ciceri fungal growth in vitro ( the causal of chickpea wilt) [80].
Selected isolates of Pseudomonas fluorescens (Pf1-94, Pf4-92, Pf12-94, Pf151-
94 and Pf179-94) and chemical resistance inducers (salicylic acid, acetylsalicylic
acid, DL-norvaline, indole-3-carbinol, and lichenan) for growth promotion and
induced systemic resistance against Fusarium wilt of chickpea (cv. JG-62) were
examined. A significant increase in shoot and root length was observed in P.
fluorescens treated plants. The isolates of P. fluorescens systemically induced
resistance against Fusarium wilt of chickpea caused by F. oxysporum f.sp. ciceri
(FocRs1), and significantly (P=0.05) reduced the wilt disease by 26-50% compared to
the control. Varied degree of protection against Fusarium wilt was recorded with
chemical inducers. The reduction in disease was more pronounced when chemical
inducers were applied with P. fluorescens. Among chemical inducers, SA showed the
highest protection of chickpea seedlings against wilting. Fifty two- to 64% reduction
of wilting was observed in soil treated with isolate Pf4-92 along with chemical
inducers. A significant (P=0.05; r=-0.946) negative correlation was observed in
concentration of salicylic acid and mycelial growth of FocRs1 and at a concentration
of 2000 micro g ml-1 mycelial growth was completely arrested. Exogenously
supplied SA also stimulated systemic resistance against wilt and reduced the disease
severity by 23 and 43% in the plants treated with 40 and 80 micro g ml-1 of SA
through root application. All the isolates of P. fluorescens produced SA in synthetic
medium and in root tissues. HPLC analysis indicated that Pf4-92 produced
comparatively more SA than the other isolates. 1700 to 2000 eta g SA g-1 fresh root
was detected from the application site of root after one day of bacterization whereas,
the amount of SA at distant site ranged between 400-500 eta g. After three days of
bacterization the SA level decreased and was found more or less equal at both the
detection sites [81].
Induced resistance in tomato plants against Fusarium oxysporum f. sp.
lycopersici and/or Verticillium dahliae by Penicillium oxalicum was related to renew
or prolonged cambial activity that led to the formation of additional secondary xylem
in Penicillium oxalicum-treated plants. Penicillium oxalicum reduced disease in
different cultivars of tomato, with different degrees of susceptibility/resistance to F.
oxysporum f.sp. lycopersici. The application of a conidial suspension of Penicillium
oxalicum by watering the tomato seedlings in seedbeds 7 days before transplanting
resulted in a variable reduction in Fusarium wilt ranging from 20 to 80% in growth
chamber and greenhouse experiments. Disease suppression was maintained for 60-
100 days after inoculation with the pathogen in the greenhouse. Repeated application
of Penicillium oxalicum prolonged the duration of control of fusarium wilt especially
when disease incidence was high, although the timing of repeated applications of
Penicillium oxalicum did not affect the efficacy of control. Penicillium oxalicum may
be effective for biological control of other tomato disease such as Botrytis cinerea,
Phytophthora parasitica [Phytophthora nicotianae var. parasitica], Phytophthora
infestans, Verticillium dahliae, Verticillium spp. and the viruses CMV and ToMV in
experimental glasshouse experiments. In addition Penicillium oxalicum demonstrated
control of the vascular wilt caused by the most common pathogens that invade the
22
plant vascular system of tomato, the soil-borne fungi V. dahliae and F. oxysporum f.
sp. lycopersici in field experiments under natural soil infestation. Penicillium
oxalicum demonstrated a good potential for development as a commercial biocontrol
agent [82].
The effect of treating seed of chickpea (Cicer arietinum) cv. BG 256 with
commercial formulations (2 g/kg seed) of Trichoderma harzianum and Pseudomonas
fluorescens, alone and in combination, to control wilt, Fusarium oxysporum f. sp.
ciceri [Fusarium oxysporum f.sp. ciceris] was studied. On untreated control plants,
wilting was observed and significantly decreased dry weight and the yield of
chickpea by 20 and 18%, respectively. On chickpea without wilt, treatment with P.
fluorescens improved the yield by 36% and T. harzianum+P. fluorescens by 25%.
Both biofungicides suppressed wilt severity, the most effective being T.
harzianum+P. fluorescens (66%). Carbendazim reduced wilt severity by 51%. On
chickpea inoculated with the wilt, yield increased by 39% with P. fluorescens, by
33% with T. harzianum+P. fluorescens, by 44% with T. harzianum, and by 20% with
carbendazim compared with the inoculated control. The soil population of F.
oxysporum f.sp. ciceris (cfu/g soil) in untreated plots increased during the first 2
months, but in the biofungicide/fungicide treated plots, it gradually and significantly
decreased during the 4 months of the crop season. The greatest decrease in the soil
population of F. oxysporum f.sp. ciceris occurred with T. harzianum or T.
harzianum+P. fluorescens, followed by P. fluorescens and carbendazim. The
rhizosphere population of the bioagents increased significantly in plots where wilt
populations decreased. The greatest increase in the population of the bioagents was
recorded for T. harzianum (108-120%), followed by P. fluorescens (65-119%) in the
combined treatment, compared with the pre-plant control (December). When the
bioagents were applied alone, the population of T. harzianum increased by 71-96%
and P. fluorescens by 46-103% [83].
Assess efficacy of an integrated management strategy for Fusarium wilt of
chickpea that combined the choice of sowing date, use of partially resistant chickpea
genotypes, and seed and soil treatments with biocontrol agents Bacillus megaterium
RGAF 51, B. subtilis GB03, nonpathogenic F. oxysporum Fo 90105, and
Pseudomonas fluorescens RG 26. Advancing the sowing date from early spring to
winter significantly delayed disease onset, reduced the final disease intensity (amount
of disease in a microplot that combines disease incidence and severity, expressed as a
percentage of the maximum possible amount of disease in that microplot), and
increased chickpea seed yield [84].
Compared between cucumber plants induced with either plant growth-promoting
rhizobacteria (PGPR) or chemicals. Inoculation with PGPR strains Serratia
marcescens (90-166) and Pseudomonas fluorescens (89B61) induced systemic
protection in the aerial part of cucumber plants against the anthracnose pathogen
Colletotrichum orbiculare. Disease development was significantly reduced in these
plants compared to control plants that were not inoculated with the PGPR strains.
Inoculation with the PGPR strains caused no visible toxicity, necrosis, or other
morphological changes. Induction with DL-3-aminobutyric acid (BABA) or amino
salicylic acid (ASA) also significantly reduced disease development. Soil drench with
23
10 mM BABA and 1.0 mM ASA induced resistance in cucumber leaves without any
toxicity to the plants. Higher concentrations of ASA (up to 10 mM) were phytotoxic,
resulting in plant stunting and blighted appearance of leaves. Cytological studies
using fluorescent microscopy revealed a higher frequency of autofluorescent
epidermal cells, which are related to accumulation of phenolic compounds, at the
sites of fungal penetration in plants induced with PGPR and challenged by the
pathogen. Neither spore-germination rate nor formation of appressoria was affected
by PGPR treatments. In contrast, both BABA and ASA significantly reduced spore-
germination rate and appressoria formation, while there were no differences from
controls in the frequency of autofluorescent epidermal cells at the sites of fungal
penetration. Our findings suggest that PGPR and chemical inducers cause different
plant responses during induced resistance [85].
Trichoderma viride reduced the growth of F. oxysporum f.sp. sesami by 83.18%
whereas Trichoderma harzianum reduced the growth by 79.54% after 7 days of
incubation. A strong antibiotic activity was recorded in T. harzianum and T. viride
against F. oxysporum f.sp. sesami. A combined treatment of the antagonists in soil
and seed significantly controlled wilt incidence in sesame [86].
An isolate of Gliocladium virens from disease affected soil in a commercial
tomato greenhouse proved highly antagonistic to Fusarium oxysporum f.sp.
lycopersici, together with an isolate of the nematophagus fungus Verticillium
chlamydosporium. Significant disease control was obtained when young mycelial
preparation (on a food-base culture) of the G. virens together with V.
chlamydosporium was applied in potting medium. Similar results were observed
when a Trichoderma harzianum isolate was treated in combination with the V.
chlamydosporium isolate. Most promising, in terms of minimizing the Fusarium wilt
of tomato incidence, was also the effect of the bacteria associated with
entomopathogenic nematodes (Steinernema spp.), Pseudomonas oryzihabitans and
Xenorhabdus nematophilus [87].
Trichoderma sp. strain T97 had strong competitive dominance against 7
pathogenic fungi including Fusarium solani f.sp. pisi, Botrytis cinerea, Verticillium
dahliae, Fusarium oxysporum f.sp. cucumerinum, Gaeumannomyces graminis [G.
graminis var. graminis], Bipolaris sorokiniana [Cochliobolus sativus] and
Rhizoctonia solani. Microscopic observation illustrated that T97 parasitized R.
solani, G. graminis and B. cinerea obviously by coiling or penetrating into their
hyphae. The infected pathogen hyphae were distorted, contracted or broken. It was
also demonstrated that the hyphae of F. solani f.sp. pisi were lysed and the tip of
Sclerotinia sclerotiorum hyphae get swollen and became dark. The soilborn diseases
including aubergine Verticillium wilt and Sclerotinia blight, cucumber Fusarium wilt
and Sclerotinia rot and pea root rot, were controlled with efficiency of 66-81% by
soil treatment with T97 (0.6% (w/w)) before sowing. By spraying with spore
suspension (108 cfu/ml) of T97, the control efficiency for grey mould of hot pepper,
cucumber and tomato in greenhouse corresponded to that of the fungicide
procimidone (50%, WP) [88].
The impact of inoculation of cucumber at the germination stage with Glomus
etunicatum BEG168 on plant yield and incidence of Fusarium oxysporum f.sp.
24
cucumerinum inoculated 28 days after the start of the experiment was investigated.
Inoculation with the AM fungus decreased both disease incidence and disease index.
Mycorrhizal inoculation also increased P concentrations in the cucumber seedlings.
The mycorrhizal seedlings had higher concentrations of proline and polyphenol
oxidase activity but lower malondialdehyde than non-mycorrhizal seedlings,
indicating that AM inoculation may have protected membrane permeability and
reduced the extent of the damage caused by F. oxysporum. The results indicate that
the mycorrhizal fungus may influence plant secondary metabolites and increase
resistance to wilt disease in cucumber seedlings and may therefore have some
potential as a biological control agent [89].
Among 3 bioagents (Trichoderma viride, Gliocladium virens and T. harzianum,
seed treatment with T. viride was found highly effective against chickpea wilt incited
by Fusarium oxysporum f.sp. ciceris and giving 77.8% control [90].
Nine isolates of Trichoderma spp., i.e. T. harzianum (PDBCTH-10, THB-9
THB-10), T. viride (PDBCTV-32, TV-97 TVA-7), T. virens [Gliocladium virens]
(PDBCTVS-12, PDBCTVS-13), and T. hamatum (TH-138), were tested for their
ability to inhibit soilborne fungal pathogens of chickpea, viz. Rhizoctonia solani,
Sclerotium rolfsii [Corticium rolfsii] and Fusarium oxysporum f.sp. ciceris, under
both in vitro and in vivo conditions. Laboratory evaluation of Trichoderma isolates by
dual-culture test, inverted plate technique and poisoned food technique revealed T.
harzianum PDBCTH 10 to be more inhibitory against R. solani and S. rolfsii
followed by T. viride PDBCTV 32 and T. virens PDBCTVs 12, whereas T. virens
PDBCTVs 12 was found to inhibit Fusarium oxysporum f.sp. ciceris to a greater
extent than other isolates. Pot culture evaluations under greenhouse conditions using
T. harzianum PDBCTH 10, T. viride PDBCTV 32 and T. virens PDBCTVs 12
revealed T. harzianum PDBCTH 10 to be an effective biological control agent against
rhizoctonia root rot and sclerotium collar rot whereas T. virens PDBCTVs 12 was
found effective against Fusarium wilt. Further, in addition to biological control of
soil borne fungal pathogens seed inoculation of Trichoderma spp. also found to
increase growth and yield of chickpea under greenhouse conditions [91].
T. harzianum and T. viride showed the maximum growth inhibition of Fusarium
oxysporum f.sp. pisi the causal of Fusarium wilt of pea in vitro and suppressed the
disease under field conditions. T. viride and T. harzianum resulted in increased seed
germination, decreased disease incidence to 22.8 and 25.5% with an AUDPC of 4.16
and 4.60 and had minimum apparent infection rate of 0.046 and 0.046, respectively,
compared to 0.063 in the control treatment [92].
Biological control of cucumber Fusarium wilt with Trichoderma viride T23 was
detected through bioassay, and its induction of several defense enzymes in cucumber
was examined. Treatments with conidiospores and chlamydospores of T. viride T23
on cucumber seedlings reduced the disease index of Fusarium wilt from 33.69 to
13.12 and 10.28, respectively [93].
Trichoderma harzianum, T. viride and T. virens (Gliocladium virens) inhibited
the mycelial growth of F. oxysporum f.sp. tuberosi. The antagonism included lysis
and dissolution of the host cytoplasm and/or transformation into cords and/or coiling
around pathogen hyphae. Moreover, substrate application of Trichoderma species
25
(108
spores/ml) before inoculation by F. oxysporum f.sp. tuberosi controlled
Fusarium wilt of potato plants compared with the non-inoculated plants and
untreated-inoculated plants. This approach may be beneficial for biological control in
F. oxysporum f.sp. tuberosi and could allow protecting plants from this pathogen
[94].
New biofungicides, antagonistic activity of soil Actinomycetes isolates against
Fusarium oxysporum f.sp. melonis causes root rot and Fusarium wilt. Streptomyces
olivaceus strain 115 showed anti-fusarium activity both in vitro and in vivo
experiments. The active strain was grown in aqueous media on rotary shakers to
monitor activity versus time and prepare active dry crude for further biological and
physical studies. Antifungal activity was of fungistatic type on the pathogen mycelia.
From the results of our studies, it is clear that usage of Streptomyces olivaceus strain
115 as a biofungistatic natural product applied as an amendment in greenhouse soil
mix will lead to inhibition or reduction of the pathogen effects [95].
The combination of several PGPRs could be more effective than individual
strains as a horticultural product. LS213 is a product formed by a combination of two
PGPRs, Bacillus subtilis strain GB03 (a growth-promoting agent), B.
amyloliquefaciens strain IN937a (an inducer of systemic resistance) and chitosan.
The aim of this work is to establish if the combination of three PGPR, B.
licheniformis CECT 5106, Pseudomonas fluorescens CECT 5398 and
Chryseobacterium balustinum CECT 5399 with LS213 would have a synergistic
effect on growth promotion and biocontrol on tomato and pepper against Fusarium
wilt and Rhizoctonia damping off. When individual rhizobacterium and the LS213
were put together, the biometric parameters were higher than with individual
rhizobacterium both in tomato and pepper, revealing a synergistic effect on growth
promotion, being the most effective combination that of B. licheniformis and LS213.
When P. fluorescens CECT 5398 was applied alone, it gave good results, which
could be due to the production of siderophores by this strain. Biocontrol results also
indicate that those treatments that combined LS213 and each of the bacteria
(Treatments: T7 and T8) gave significantly higher percentages of healthy plants for
both tomato (T7: 65%) and pepper (T7: 75% and T8: 70%) than the LS213 alone
(45% of healthy plants for tomato and 60% for pepper) three weeks after pathogen
attack. The effects in pepper were more marked than in tomato. The best treatment in
biocontrol was the combination of P. fluorescens and LS213. In summary, the
combination of microorganisms' gives better results probably due to the different
mechanisms used [96].
Pseudomonas fluorescens strain WCS 417, known for its ability to suppress
fusarium wilt diseases (WCS 417), reduced Fusarium wilt of banana incidence by
87.4%. These isolates should be further evaluated for potential application in the
field, independently and in combination [97].
Two potential biocontrol agents namely T. harzianum and T. viride, applied by
different methods i.e. dry mix of wheat bran culture (placement), spore suspension
dip, slurry prepared in 20 percent sugar, Bavistin (0.1% dip)+slurry, were conducted
both under storage as well as in the field. In storage experiment with these BCAs,
Bavistin (0.1% dip) + slurry method of application of T. harzianum and T. viride
26
separately resulted in minimum (8.9 and 11.1%) corm rot incidence followed by
slurry method (11.2 and 13.3%) alone. The slurry method of application of these
BCAs also supported maximum population counts (148.7x104 and 130.00 x104
cfu/g) after three months of storage. Carbendazim (0.1% dip) + slurry treatment
combination was also very effective in in-vivo in managing the Fusarium wilt and
promoting the growth of gladiolus. In a separate field trial, T. harzianum in
comparison to T. viride particularly with soil placement method resulted in minimum
disease incidence and improved growth of gladiolus [98].
Pseudomonas fluorescens strain LRB3W1 inhibited the mycelial growth of
Fusarium oxysporum f. sp. lycopersici and suppressed the Fusarium wilt of tomato.
The chemical fungicide, benomyl, did not suppress the disease incidence at low
concentrations. However, the disease incidence was decreased by the combined
application of benomyl at low concentrations with strain LRB3W1. Combined
application of benomyl with the bacterium was more effective than treatment with the
bacterium alone. The survival of strain LRB3W1 was not influenced by the presence
of benomyl. This combined use of the biocontrol bacterium, strain LRB3W1, and a
fungicide, benomyl, should be an attractive approach for suppressing tomato wilt
[99].
Evaluating the effect of biocontrol bacterial strains of Paenibacillus polymyxa
BRF-1 and Bacillus subtilis BRF-2 on spore germination and mycelium growth of
two vegetable pathogenic germ, Fusarium oxysporum f. sp. cumerinum and Fusarium
oxysporum f. sp lycopersici. showed that, 80% inhibition rate of pathogenic germ
spore germination by metabolic materials of the two strains with 5 dilution. 40%-80%
inhibition rates are also observed in mycelium growth of two pathogenic germ with 2
and 5 times dilution of metabolic materials of BRF-1 and BRF-2, which are the most
significant difference from the control. Pot experimental results indicate that bacterial
suspension or metabolic solution of BRF-1 and BRF-2 not only effectively controls
Fusarium wilt disease of cucumber and tomato, but also significantly promote
seedling growth [100].
One strain of bacteria, which was isolated from Pacifigorgia sp. in south China
sea, showed inhibitory activity against Fusarium oxysporum f.sp. cucumerinum.
Laboratory simulation experiments showed that the strain colonized in soil and
sterilized soil in high density and promoted the growth of cucumber seedlings. In
order to investigate the biological control role and growth-promoting effects of the
strain on cucumber under field conditions, pot experiments were conducted in China.
The results showed that the strain was effective against cucumber Fusarium wilt,
promoted cucumber growth, increased the chlorophyll content and enhanced the
cucumber yield [101].
Trichoderma harzianum T-h-30 had obvious growth-promoting effects on
vegetables and could significantly improve vegetables plant height, length of root,
yield, and economic properties. All of the data showed a good control efficacy of
Trichoderma harzianum T-h-30 on cucumber Fusarium wilt disease and powdery
mildew. It also had obvious biological control effect and decreased the infection ratio
of virus disease caused by CeMV on celery without any injury on vegetables [102].
27
Evaluate effect of organic fertilizer application either with or without
antagonistic bacteria (Bacillus subtilis SQR-5 and Paenibacillus polymyxa SQR-21)
on the control of Fusarium oxysporum f. sp. Cucumerinum J. H. Owen wilt disease
in cucumber.revaeled that The incidence of Fusarium wilt disease was 5.3-13.5% for
cucumber plants treated with bioorganic fertilizer, while it was 30.3-51% in controls
(only with organic fertilizer). Higher yields and lower disease incidences were
observed in the dry season when compared with the wet season for both types of
organic fertilizer treatments. Biolog analysis showed a significant change in soil
bacterial composition and activity after bioorganic fertilizer application. The numbers
of colony forming units of F. oxysporum f. sp. Cucumerinum. Owen for bioorganic-
fertilizer-treated soils were significantly decreased compared with control. Scanning
electron micrographs of cucumber basal stems showed a presence of mycelia-like
mini strands accompanied by an amorphous substance within the xylem vessels. This
amorphous substance and mini strands were richer in calcium and phosphorus but
had low carbon and oxygen than the living mycelia [103].
The antagonists Trichoderma viride,Gliocladium virens,Enterobacter cloacae,
saprofitic Pythium olygandrum can be use for seed treatment, soil introduction,
pouring and sprays of plants for control of same important diseases on vegetable
crops in protected facilities.. The application of the biological products on base
Trichoderma viride and Gliocladium virens with titre 2.10 conidia per g at
consumption rate 4 g/m before transplanting of pepper in the field decreased
Verticillium wilt development in pepper by 42-59% and increased of yield by 26-
42%. Effect of Trichoderma viride application against the development of
Verticillium wilt on tomato in hydroponics is 40%. Efficacy of bio-products on base
Trichoderma viride for application in period of planting in rate 4 g/m and 0.1%
"Poliversum" ( Pythium oligandrum solution for seed treatment and spray on the
vegetation for control of Fusarium wilt on greenhouse cucumber up 70%. Effect of
the bacterial preparation application depends on the degree of the powdery mildew
and downy mildew attack in the beginning of the vegetation. In low values of the
diseases in the planting - to 10%, the effect of fivefold spraying with bio-product was
88%. Soaking of seeds before sowing in bacterial solution of Enterobacter cloacae
with cells/ml for 8 hour increased seed germination with 13-15% and reflect to
growth of yield with 27% [104].
Application of Bacillus subtilis strain B29, against Fusarium oxysporum f.sp.
cucumerinum. After twice of 4-field-plot experiments, the control efficiencies of 100,
500 and 250 dilution times to cucumber Fusarium wilt were 70.3-88.2%, 62.3-85.9%,
and 54.7-80.6%, respectively. The average efficiency of field trials with B29 was
84.9% during 2 years and the yield of cucumber increased by 12.57%. The acute
toxicity of Bacillus subtilis strain B29 to big mouse through its mouth and skin was
examined. The application of strain B29 on cucumber, tomato, bean and seed
pumpkin was safe based on the observed seedling rate, growth and development
[105].
The cell-free culture filtrate of Bacillus subtilis B579, with a concentration of
20% (v/v), could result in the vacuolation, swelling and lysis of hyphae. Besides, it
could blacken, shrunk and hindered the germination of conidia of F. oxysporum at the
28
concentration of 80% (v/v). When applied as inoculants, B579 (108 c.f.u. ml) was
able to reduce disease incidence by 73.60%, and promote seedling growth in pot trial
studies [25].
The antagonism mechanisms between Trichoderma harzianum and cucumber
Fusarium wilt were analyzed combined with indoor selection and field efficacy.
There were distinctly inhibition effects to Fusarium spp. in all seven testing strains
through the confrontation test, the inhibition rate range from 66.7% to 85.8%.
Trichoderma harzianum effectively enhanced root resistance to Fusarium wilt. A
great deal of root cell died when infection by F. oxysporum. But the injury caused to
roots decreased if pre-inoculated with T. harzianum. Field efficacy trials showed that
the best induced effects were obtained when the concentration of spores suspension
were 108 per milliliter. T. harzianum possessed strong competition ability and
invoked a range of related genes to enhance the resistance to pathogen [106].
1.3 Inducing resistance by abiotic agents
Numerous examples of natural and synthetic chemicals have been reported to
enhance resistance systemically e.g. aluminm tris, D-phenylalanine, D-alanine, α-
aminoisobutryic acid, polyacrylic acid, salicylic acid and acetylsalicylic acid.
Although these compounds appear to have little direct effect on pathogens, when
coupled with the hosts' natural defense mechanisms, they may provide the
competitive edge required to reduce disease [107, 108].
A range of abiotic treatment reported to induce phytoalexin in various, plants
failed to induce lignification in a wounded primary wheat leaf system. The inducers
included antimetobolits, metabolic inhibitor, basic poly-peptides, oxidizing and
reducing agents, halogen anions, heavy metal inos and U.V irradiation, only mercuric
ions elicited a response [109].
Organic acids, including oxalic, tartaric, formic and acetic, were effective in
disinfecting cucumber seed against Pseudomonas syringae pv. lachrymans. When
seeds were treated by dipping in solutions of 0.1-0.4 mol. for 5-10 min and either
sown directly or dried first and then sown, germination was unimpaired. The
disinfecting effect of these acids was greatly reduced when the soil pH was increased
above 4. Similar results were obtained using both naturally and artificially
contaminated seeds, although 0.4 mol. was necessary to give complete disinfection of
the former. In trials to develop a combined seed treatment which would also control
F. wilt [F. oxysporum f.sp. cucumerinum] the best results were obtained by dipping
seeds in thiram-benomyl liquid diluted with 3% lactic acid or commercial vinegar
containing 4.2% acetic acid for 30 min, and then sowing directly without washing
[110].
Resistance is effectively induced by chemicals including benzoic acid
derivatives such as salicylic (SA) and also by ethephon when sprayed on or injected
into leaves or watered into soil. Pretreatment of cucumber plants with salicylic,
acetylsalicylic or polyacrylic acid induced local, and to a lesser extent systemic
resistance to subsequent infection with Colletrichum lagenarium [111].
Sclerotial germination for onion pathogen was less after soaking in salicylic acid
than in either phenol or gallic acid. Increasing conc. of the phenolic compounds in the
29
nutrient media led to a gradual decrease in linear growth of the fungus. Starting
formation of the sclerotia was clearly delayed at the two higher dosages of salicylic
acid and phenol (50 and 100 ppm) [112].
S-H mixture, comprising 4.4% bagasse, 8.4% rice husks, 4.25% oyster shell
powder, 8.25% urea, 1.04% KNO3, 13.16% Ca superphosphate and 60.5% mineral
ash successfully in lab., greenhouse and field tests in Taiwan against Fusarium
oxysporum f.sp. niveum on watermelon. It also reduced field incidence of F. o. f.sp.
raphani on radish and F. oxysporum f.sp. conglutinans on mustard cabbage. The
mechanisms of the disease control by the amendment are discussed. Other pathogens
controlled included Plasmodiophora brassicae on Chinese cabbage, Phytophthora
melonis on cucumber (when combined with a Ridomil (metalaxyl) spray),
Pseudomonas solanacearum on tomato, Rhizoctonia solani on rice and beans
(Phaseolus) and Sclerotium rolfsii on pepper [Capsicum] [113].
Salicylic acid, picric acid and 2,4-dinitrophenol caused significant reduction in
radial growth of S. rolfsii (by up to 90%), mycelial dry weight and viability [114].
Fractionated leaf tissue of spinach to determine if this plant contained chemicals
that were capable of inducing resistance in cucumber. After a series of fractions, they
isolated a water soluble material that was able to effectively induce resistance to
Colletotrichum lagenarium in cucumber. The active material in this fraction was
oxalic acid. These authors also found that the active resistance inducing factor in
rhubarb leaves was also oxalic acid. Modifications of the oxalate molecule (I.e.
esterification of the acid groups) eliminated the resistance inducing activity. The
induction of systemic resistance by oxalate was associated with the induction of a
local chlorotic stippling of the treated leaves [115].
Illustrated spraying cucumber leaves with salicylic acid (SA). 7-
methoxycarbonyl benzo-1, 2,3-thiadiazol and 2-chloroethyl phosphonic acid
(ethephon ) reduced the diseased area caused by Pseudoperonospora cubensis by
more than 50% in the sprayed first leaves and also in the upper second leaves
provided challenge inoculation was made 3 to 6 days but not one to 24 hr after
treatment. Electrophretic analysis of extracted proteins on polyacrylamide analysis of
analysis of extracted proteins on polyactylamide gel showed that both the SA
treatment and localized infection with P. cubensis induced several noval acid soluble
proteins in the treated and the upper untreated leaves in correlation with induced
resistance [116].
Butylated hydroxyanisol (BHA), tannic acid, ascorbic acid and dimethyl
sulfoxidants (DMSO) at a concentration of 1.0 mM controlled the disease on
cucumber fruits. Antioxidants affected Rhizopus stolonifer on grape berries but not
Botryttis cinerea or Aspergillus spp. [117].
Ethephon (2-chloroethlplosphonic acid) and cobalt sulphat as seed soaking
treatment induced resistance of cucumber to powdery mildew caused by
Sphaerotheca fuliginea. Such reaction was accompanied by increasing of free phenol
content, activation of peroxidase activity and an increase of protein with MW69 KD
in case of ethophon treatment and protein with MW33 KD in case of cobalt sulphate
treatment [118, 119].
30
Application of KMnO4 solution to the soil provided good control of Fusarium
wilt of cucumbers. Plots treated with 1:800 or 1:1000 solutions were free from the
disease, while the average rate of infected plants following treatment with a 1:1500
solution was 0.88%. Infection rates in plots treated with Shuangxiaoling,
fenaminosulf or untreated plots were 95.1, 23.31 and 40.51%, respectively. The
highest yields (112.6 kg) were obtained from plots treated with 1:1000 KMnO4
[120].
Treatment of cucumber plants with phenylthiourea and oxalic acid induced
resistance to Colletotrichum lagenarium [C. orbiculare], with reduction of 28.6-
30.5% in lesion numbers and 64.5-73.7% in lesion size. Plants with 3 expanded
leaves, sprayed with mixed chemical inducer, acquired resistance to C. orbiculare
and also to Pseudomonas [syringae pv.] lacrymans and Pseudoperonospora cubensis.
Seedlings of watermelons grown in soil treated with KT-emulsion (mixed chemical
inducer) acquired resistance to Fusarium wilt, with up to 76% control [121].
Induced resistance against Fusarium wilt of watermelon using various abiotic
inducers included different concentrations of Co as CoSo4 or ethephon (2-chloroethyl
phosphonic acid). Results indicated that the most effective treatment in reducing the
percentage of wilted plants were ethephon at 800 ppm, CO++
at 0.5 ppm. Treatment
with ethephon at 600 ppm was highly effective with cv. Giza 1 only in field
experiments [122].
Asperin (acetyl salicylic acid), Salicylic acid, cobalt sulphate and potassium
phosphate dibasic greatly reduced powdery mildew symptoms of squash causes by
Sphaerotheca fuliginea on artificially infected plants in comparison with treated
plants [123, 124].
There was a close relationship between disease severity and soil pH. Most of
the soils suppressive to cucumber Fusarium wilt had a higher pH than the non-
suppressive soils. However, suppressive soils to Phaseolus vulgaris Fusarium wilt
had lower pHs, and in these acid soils spore germination was inhibited. Cucumber
Fusarium wilt was almost completely suppressed at pH 8.0 while P. vulgaris root rot
was suppressed at pH 4.0 [125].
Salicylic acid, hydrogen peroxide, cobalt ions and Pseudomonas fluorescens
were effective for induction of resistant in watermelon against wilt pathogen in four
distinct experiments [126].
Antioxidants (ascorbic acid, propylgalate, salicylic acid and thiourea) reduced
linear growth of the tested pathogenic fungi (H. tetramera [Cochliobolus spicifer]
and F. oxysporum) on Czapek's agar medium at concentrations of 1.0mM, 5.0mM
and 10.0mM. Spore germination, sporulation and spore viability were reduced by the
antioxidants, particularly at 10mM, except thiourea which was the least effective
against spore germination of F. oxysporum. Treating seeds of cucumber with
antioxidants induced protection against C. spicifer. Antioxidants were effective in
controlling disease in soil infested with F. oxysporum [127].
Spraying the surface of the 2nd true leaf of cucumber plants with 75mMol/L
K2HPO4 enhanced the activities of chitinase, -1, 3-glucanase and peroxidase
effectively, and induced the activities of chitinase and -1, 3-glucanase to increase in
leaves 3, 4 and 5. Thus these leaves could resist anthracnose caused by
31
Colletotrichum lagenarium [C. orbiculare]. Peroxidase increased only when the leaf
was challenged directly. This research suggests that the peroxidase activity
corresponds to local induced resistance of the cucumber plant. It is concluded that
spraying the leaf with 75mMol/L K2HPO4 can induce systemic resistance to
anthracnose safely and effectively [128].
Benzoic acid, salicylic acid and ascorbic acid significantly reduced linear
growth of Fusarium oxysporum, F. solani and Rhizoctonia solani and reduced spore
germination of Fusarium spp. The 3 antioxidants significantly reduced damping- off
of tomatoes when used as a soil drench and they were more effective than tolclofos-
methyl [129].
Soaking sesame seeds cv. Giza 32 in ascorbic and salicylic acid (at 5 mM) for
12 h before sowing and then treated with ascorbic and salicylic acid 15 days after
sowing resulted in the best control against F. oxysporum f.sp. sesami compared to
Benlate, but Benlate was more effective than both acids in controlling Macrophomina
phaseolina, Mucor haemalis [68].
Oxalic acid (OA) at 2.5, 5, 10, and 20 mM was sprayed onto the green part of
the tomato plants followed by soil inoculation of by Fusarium oxysporum f.sp.
lycopersici (Fol) suspension (106 conidia/ml) at 10 ml after 2 days. OA-induced
resistance (concentration-dependent) by otherwise susceptible tomato plants was
observed [130].
Some antioxidants were tested in vitro and in vivo for their effect on the fungi
Fusarium oxysporum, F. solani and Fusarium moniliforme [Gibberella fujikuroi]. 0,
2, 4, 6, 8 and 10mM aminobutyric acid (ABA), potassium salicylate (PS), oxalic acid
(OA), salicylic acid (SA) or ascorbic acid (AA) were evaluated. Treatments were
effective in reducing mycelial growth. Spore germination was also greatly reduced by
many tested chemicals at 8mM concentration or less. The inhibitory effect of the
antioxidants increased with increasing concentrations. ABA was the only antioxidant
which induced protection in all onion cultivars against all tested Fusarium species.
PS and ABA were the most effective antioxidants. The use of antioxidants against
Fusarium species was more effective when applied as seed and transplant treatment
than when applied as soil treatment under greenhouse conditions. Results indicated
the efficacy of the antioxidants depended on the application methods, pathogen and
the cultivars [131].
The effects of four antioxidants (ascorbic acid, citric acid, mannitol and salicylic
acid) were tested against root and crown rot of strawberry. Salicylic acid, Ascorbic
acid was the most effective antioxidants on disease development, as lower percentage
of root and crown rot infection and severity. The least effective antioxidant in
controlling root and crown rot was mannitol. Whereas, the disease index for the
percentage of reduction in injury was the least. Meantime, citric acid was moderately
effective, in this respect. The yield increasing showed the same trend [132].
IBA and IAA gave the highest decrease in growth of Macrophomina phaseolina
meanwhile; KCl and H2O2 were the least effective. However, salicylic acid, Bion and
tanic acid caused intermediate decrease in growth compared with control. IAA and
IBA, caused no growth of isolates Macrophomina phaseolina M9 and M15 at 800
ppm and isolate M22 at 400 ppm. Meanwhile, salicylic acid caused no growth of the
32
3 isolates at 1600 ppm. headded also that IBA and IAA were the best chemical
inducers for decreasing sclerotial formation followed by SA, Bion, tanic acid,
whereas KCl and H2O2 were the least effective in this respect. The produced number
of sclerotia was inversely correlated with concentrations of any chemical inducer
[133].
The alleviating effects of phenolic compounds (i.e. phenolic acid, p-
hydroxybenzoic acid, p-coumaric acid and ferulic acid) on cucumber Fusarium wilt
(Fusarium oxysporum f.sp. cucumbrum were determined. The amount of phenol
compounds in the soil increased after adding organic material into the soil. The
alleviating effect of p-hydroxybenzoic acid was the best, followed by p-coumaric
acid and ferulic acid. The total amount of bacterial, actinomyces and fungus in high
phenolic compound treatments were lower than that of the control, while the amount
of microorganisms in low phenolic compound treatments increased. In addition, the
phenolic compounds inhibited the growth of the pathogen [134].
Plants are challenged by a variety of abiotic and biotic stresses. The differential
activation of distinct sets of genes or gene products in response to these challenges is
referred to as specificity. Several signaling pathways, including jasmonic acid (JA),
salicylic acid (SA), ethylene (ET), and probably hydrogen peroxide (H2O2) orchestrate
the induction of defenses. Recently, accumulating reports indicate that the SA pathway
is involved in a wide range of plant defense responses. For example, plant defense in
response to microbial attack is regulated through a complex network of signaling
pathways that involve three signaling molecules: salicylic acid, jasmonic acid and
ethylene. SA is a key regulator of pathogen-induced systemic acquired resistance (SAR),
whereas JA and ET are required for rhizobacteria-mediated induced systemic resistance
(ISR). The SA involved plant defense responses are characterized as species specific.
Even in two phylogenetic closely related plant species such as tomato and tobacco, the
SA-dependent defense pathway does not trigger the same defense responses. It also
means that the outcome of a BTH (benzothiodiazole) treatment cannot be predicted and
has to be tested for each plant-pathogen combination [135].
Salicylic acid caused the highest decrease in growth and sporulation, while,
Tannic acid caused the highest decrease in spore germination of Verticillium dahliae,
Verticillium albo-atrum and F. oxysporum in strawberry . However, Thiourea and
Catechol were the least effective. Also Antioxidants were significantly better in
improving disease control and fruit yield production than control. Salicylic acid and
Ascorbic acid were the most effective antioxidants on wilt disease and increasing the
yield [136].
Dead plants significantly decreased by pre-treating roots of strawberry (before
sowing in infected with Rhizoctonia solani) with any of the tested abiotic inducers
(salicylic acid, boric acid, ascorbic acid, CuSO4, MgSO4, KH2PO4 and Bion WF50).
In this respect, CuSO4 followed by KH2PO4, respectively were the best chemical
inducers for reducing % dead plants whether after 21 or 45 days comparing with
untreated control. The fruit yield was significantly increased also by applying tested
abiotic inducers comparing with the untreated controls. The highest yield was
produced by ascorbic acid followed by BA and CuSO4 respectively. Also dead plants
significantly decreased by pre-treating roots of strawberry (before sowing in infected
33
with Rhizoctonia solani) with any of the tested abiotic inducers (Trichoderma
harzianum, Bacillus subtilis, Pseudomonas fluorescens and Streptomyces
aureofaciens). In this respect, T. harzianum was the most effective followed by S.
aureofaciens, B. subtilis, and P. fluorescence respectively comparing with the
untreated control treatments. The fruit yield was significantly increased also by
applying tested biotic inducers comparing with the untreated controls. The highest
yield was produced by S. aureofaciens and T. harzianum followed by B. subtilis and
P. fluorescence respectively [137].
1.4 Physiological aspects of defense reaction:
For many years, the role of oxidative enzymes and their metabolic products in
defense mechanisms of infected plants has been studied. Also, peroxidase activity in
diseased plants and its effects on resistance or susceptibility in many host-pathogen
interactions have been studied. However, little attention has been given to this
enzyme in resistant plants before infection. Investigations found that peroxidase
activity is a biochemical marker, which may or may not be part of the resistance
mechanism but which can be used to predict resistance to disease.
At the early different stages of infections with various pathogens, phenolic
compounds are released to prevent the spread of the pathogenic organisms throughout
the mesophyll tissue [138].
The action of phenol system and related oxidative enzymes, besides phytoalexin
accumulation which represent the most accepted mechanism of plant resistance [139].
Exposure of root tissue from a susceptible variety of sweet potato to low
concentrations of ethylene induced a resistance to infection by Ceratotcystis
fimbriata accompaned with an increase in the activity of peroxidase and polyphenol
oxidase in the inoculated tissue [140].
Positive correlation between the degree of resistance and phenol level in healthy
plant was noticed therefore, more rapid accumulation of phenolic compounds takes
place in resistant hybrids than in the susceptible ones [141].
Increasing of peroxidase activity was found in tobacco leaves immunized by
Tobacco mosaic virus (TMV) against Pseudomonase tabacci [142].
Lower amount of carbohydrates in healthy roots of the susceptible soybean
cultivar more than the resistant cultivar was detected. Total sugar increased in both
cultivars in response to infection with M. phaseolina that causal of charcoal rot
disease. An appreciable increase was more pronounced in the resistant cultivar [143].
Nadolny and Squeira [144] determined peroxidase activities and isozyme
patterns in tobacco leaves in which disease resistance had been induced by prior
infiltration with heat-killed Pseudomonas solanacesrum B1 cells. There were no
changes in either ionically or covalently bound forms of perpxidase.
Lipopolysaccharide form K 60 cell of P. solanacearum, which induced protection,
also increased peroxidase activity and caused appearance of the isozyme band (P1).
When leaf cells were wounded by injection with asbestos fiber, peroxidase activity
increased but the (P1) band was not visible.
34
Ligification appeared to be induced almost exclusively by fungi, unlike
phytoalexin production which can also be elicited by a wide range of abiotic
treatments [109].
Peroxidase is systemically enhanced in cucumber with resistance induced
against Colletotrichum lagenarium by a previous infection with the same fungus and
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 [145]. Increased in peroxidase
activity associated with the induced resistance in cucumber [146].
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 [147].
Healthy and diseased roots of resistant soybean cultivar contained more phenolic
compounds than in susceptible cultivar. Infection with R. solani, S. rolfsii and F.
oxysporum increased the phenolic contents of the roots of both cultivars. The amount
of increase was greater in the roots of resistant cultivar than the susceptible one. On
the other hand, reducing; non-reducing and total sugars were greater in the healthy
and diseased roots of soybean susceptible cultivar than those in resistant one.
Infection with F. oxysporum, R. solani and S. rolfsii increased reducing, non-reducing
and total sugars in both cultivars. Such increase was greater in roots of susceptible
plants than in roots of resistant cultivar. He suggested that, the level of total soluble
carbohydrates may be critical factor in determine resistance [148].
Peroxidase activity of cotton infected by Fusarium oxysporum f.sp.
vasinfectum on polyacrylamide gels was studied. He concluded that the change
activity was not consistently observed and no specific trend characterized the host
reaction (i.e resistance or susceptibility). Peroxidase activity histochemically, changes
accompanying infection became apparent 7 days following inoculation and at the
same site of activity in the healthy non infected controls. The endoderm' in roots and
cambium in stems were sites of high activity of peroxidase [149].
Hammerschmidt et al. [150] have tried to elucidate the mechanism of resistance
of cucumber to non pathogens. They found that tested fungi were germinated and
formed appressoria on these plants but few penetrations were observed.
Histochemical staining revealed the deposition of lignin in upper and lateral
epidermal cell walls around the appressoria. Little or no Iignification occurred in
compatible fungus host interactions. Their results suggest that Iignification may be a
general resistance response in the cucurbitaceae.
Hammerschmidt [151] studied the relationship between lignin deposition and
disease incidence or resistance in potato tuber tissues. He found that the non-
pathogens caused a yellow discoloration in the host cell walls, first detected 8-10 hr
after inoculation. These yellow areas' of the cell walls stained positively for lignin.
Positive staining for lignin was not observed in the pathogen challenged tissues until
18-20 hr.
35
Studies with 12 tomato and four melon cultivars and breeding lines, found a high
correlation between peroxidase activity in uninfected tomato or melon and resistance
to Verticillium dahliae or Sphaerotheca fuliginea [152, 153].
Lignin biosynthesis includes the polymerization of three cinnamyl alcohols and
is mediated by the peroxidase-H202 system. Cell wall-bound peroxidases are
probably involved not only in the oxidative polymerization of hydroxylated cinnamyl
alcohols but also in the generation of hydrogen peroxide necessary for Iigniflcation
[154].
Purified acidic peroxidases from watermelon, muskmelon and cucumber that
were induced with C. lagenarium and found that the acidic peroxidases was
antigenically and electrophoretically similar among the cucurbits [155].
Enzyme activity assays and western blot analysis indicated that β-l,3-glucanases
increased in immunized by injected stem with sporangiospores of the blue mold
pathogen but not in control. Electrophoretic analysis indicated increases in amounts
of several b-proteins in immunized plants prior to challenge. A basal level of
chitinases was always detected, but increases in chitinases above this level in
immunized plants followed a profile similar to that of the β-l,3-glucanases and other
b-proteins. The increases in these proteins coincided with the onset of immunization
in plants injected with Peronospora tabcina [156].
Found that injection of stem of tobacco with Peronospora tabcina or inoculation
with tobacco mosaic virus induced systemic resistant to both pathogens. The
treatment also caused a systemic increase in peroxidase activity which was positively
correlated with induced resistance. Peroxidase activity further increased in the
induced plants and remained higher after challenge inoculation as compared to the
control plants. The isozyme patterns of peroxidases on isoelectric focusing (IEF)
showed an increase of two anionic peroxidases [157].
Healthy and diseased roots of lowest susceptible sesame genotype contained
more phenolic compounds than that in the highest susceptible one. Infection with F.
oxysporum increased the phenolic content of both genotypes. The rate of increase was
greater in roots of the lowest susceptible than the roots of the highest susceptible one.
Also, reducing, non-reducing and total sugars were higher in diseased roots of highest
and lowest susceptible cultivars than in healthy ones. Healthy roots of highest
susceptible cultivar had more reducing and total sugars than that in the lowest
susceptible. While non-reducing sugars content was more in healthy roots of the
lowest susceptible cultivar than the highest susceptible one [158].
β-1,3-glucanase, chitinase and peroxidase activities increase in tobacco with age.
These increase in activities were higher in leaf tissue from the main stalk (resistant to
blue mould) as compared to leaf tissue from suckering stems (susceptible to blue
mold) on the same plant. Isozyme patterns of β-1,3-glucanase and chitinase in all
resistant tissues are typical of those in tissues systemically protected by either stem
injection with Peronospora tabcina or foliar inoculation with TMV [159].
Many plant enzymes are involved in defense reaction against plant pathogen.
These included oxidative enzymes such as peroxidase and polyphenol oxidase which
catalase the formation of lignin and other oxidative phenols that contribute to
formation of defense barriers for reinforcing the cell structure [160].
36
A greater increase in peroxidase activity in leaves of the resistant cv. Pusa 8972
following inoculation with Macrophomina phaseolina, than those of the susceptible
Pusa 8773 was observed [161].
The clear resistance response which is known to occur in induced planes is the
ability to more rapidly lignify at the point of attempted fungal infection. The last
enzymatic step of lignification utilizes peroxidase, an enzyme that can generate lignin
polymers by catalyzing the formation of free radicals of the lignin monomer
precursors [162].
Spraying the mung bean (Vigna radiata) plants with ascorbic acid (100 ppm)
increased nodulation by Rhizobium and reduced disease intensity caused by
Macrophomina phaseolina in infested soil [163].
The induction of systemic acquired resistance (SAR) against tomato late blight
disease caused by Phytophthora infestans was accompanied by increase of
peroxidase activity in inoculated leaves as well as in upper tissue (l. 3) tomato plants
[164].
The activity of peroxidase was higher in sesame tissues infected by F.
oxysporum than in healthy tissues. The infection induced rapid increase of IAA-
oxidase activity in root and stem tissue but there were no significant changes in
activity of PAL (phenylalanin ammoialysae), however, PAL activity in leaf tissue
increased at 7 days after inoculation [165].
The chemical agent bezo (1,2,3) thiodiazole-7-carbothioic-methyle ester and the
active ingredient of plant activator Bion induced systemic acquired resistance (SAR)
in green bean against different pathogenic fungi. In chemically activated plants,
enhanced activities of the defense related enzymes chitinase, B-(1,3)-gluconase and
peroxidase were detected which are well known biochemical markers for SAR [166].
Trifluralin increased peroxidase activity and peroxidase may play an imported
role in inducing resistance of cotton seedlings to F. oxysporum f.sp. vasinfectum
[167].
Phosphate applications activate the typical defense-related enzyme like
peroxidase and polyphenol oxidase in all parts of cucumber plants [168].
The amount of total phenols, free amino acid and pectin was the maximum in
the immune cultivar and the minimum in the highly susceptible cultivar of sunflower
to charcoal rot. The activity of polyphenol oxidase was also highest in the immune
cultivar and lowest in the highly susceptible cultivar [169].
The hydrogen peroxide generated by peroxidase might act as an antifungal agent
and play a role in disease resistance [170].
Controlled of root-rot of cowpea (Vigna unguiculata) caused by R. solani and R.
bataticola (M. phaseolina) by 40.0 and 44.5% following the application of 5 and 10
ppm Cu (as copper sulphate), respectively. Reduction in disease incidence was
attributed to the increased activities of polyphenol oxidase (PPO) and peroxidase
(PO) along with higher amounts of total phenols. Peroxidase activity was several
times higher as compared to PPO specific activity and increased markedly after
infection with R. solani and M. phaseolina. Contrary to PPO and PO, the specific
activity of catalase declined sharply. Infection also caused an increase in the content
37
of total phenols, reducing sugars, Cu, Zn and Mn but a decrease in o -dihydric
phenols, flavanols, total soluble sugars, non-reducing sugars and Fe contents [171].
Free and total phenols were significantly increased in infested shoot of the tested
sunflower hybrids specially 15 days after sowing in soil infested but with non-
significant increase at 45 and 90 days. They found also, their contents were
significantly higher in the resistant hybrid than those of the moderate susceptible one
[172].
The resistant and moderately susceptible soybean cultivars contained higher
amount of phenols compared with the susceptible cultivar [173].
The biochemical defense mechanisms against wilt disease caused by F.
oxysporum f.sp. sesami following treating sesame plants with flower extract of
Helichrusum plants, B. subtilis, amino buteric acid (ABA) and KCl were investigated.
Activity of peroxidase, polyphenoloxidase and chitinase enzymes, IAA hormone and
RNA content of sesame plants may be considered as a biochemical mechanisms for
inducing systemic resistance in sesame plant against wilt disease. Free phenols of
sesame plants does not seem to be involved in induced resistance mechanisms against
wilt disease [174].
P. fluorescens isolate 4-92 induced systemic resistance against charcoal rot
disease in chickpea (C. arietinum cv. Radhey) caused by Macrophomina phaseolina
isolate CH-15. Time-course accumulation of pathogenesis-related (PR) proteins
(chitinases and glucanases) in chickpea plants inoculated with P. fluorescens was
significantly (P = 0.05) higher than in control plants [69].
After inoculation with Fusarium oxysporum f.sp. cucumerinum, the peroxidase
activity of wilt-resistant cucumber cultivars showed little change while in susceptible
cultivars activity rose sharply once the leaves became wilted. The peroxidase activity
of seedlings before inoculation showed a high correlation with resistance after
inoculation, the coefficient between activity and disease incidence being 0.949. The
peroxidase activity of cucumber seedlings can thus be used for forecasting their
resistances to Fusarium wilt since activity was stable at the seedling stage [175].
The increase in cucumber yield may be due to the role of elicitors in stimulation
of physiological processes which reflect on improving vegetative growth that
followed by active translocation of the photoassimilates from source to sink in
cucumber plant due to increasing leaf blade thickness as well as dimensions of
vascular bundles [176].
As for the effect of chemical inducers and biological control they increased
phenols content compared with control treatment. On the other hand, they decreased
the reducing, non-reducing and total sugars content in roots of strawberry plants
infected with the three wilt pathogens [136].
Biological control of cucumber Fusarium wilt with Trichoderma viride T23
was detected through bioassay The activities of defence enzymes, such as
phenylalanine ammonia-lyase (PAL), peroxidase (PO), polyphenol oxidase [catechol
oxidase] (PPO) and catalase (CAT) significantly higher levels of these enzymes were
detected in T23-treated plants than in the control. The maximum peaks of PAL, POD,
PPO and CAT activities were enhanced by 2.75, 2.49, 2.42 and 15.84 times over the
control, respectively, indicating that the production of phytoalexin or lignin might be
38
involved in the disease suppression. The enzyme activities in conidiospore-treated
cucumber reached their peaks earlier than those in chlamydospores treatment,
however, the latter showed higher enzyme activity peaks in cucumber [93].
Plant responses to pathogens are a multilayer network of defence reactions, which
try to limit and eventually stop the invading microbial pathogen. The reactions include
the rapid generation of reactive oxygen species, cross-linking of cell wall polymers, the
production of antimicrobial pathogenesis-related proteins, and low molecular weight
phytoalexins. The network of responses requires common signalling pathways and one
key compound is salicylic acid (SA).When invaded by pathogens, resistant plants induce
defense reactions both locally and in distant organs. Of interest in this study is the
regulation of gene expression by SA and its analogues which are useful tools for
elucidating SA-signalling pathways. However, SA export from plant cells has been
found in ozone-treated plants or after inoculation with pathogens. Here labeling
experiments have shown that a part of the locally synthesized SA is exported and
distributed systemically throughout the plant, which is a part of the signalling pathway to
systemic-acquired resistance in plants [177].
Phenolic acids are generally not abundant in most plants. There are a few
exceptions: gallic acid and salicylic acid (SA). Gallic acid is a precursor for the
ellagitannins and gallotannins. Salicylic acid is an important defense compound because
it mediates systemic acquired resistance (SAR), a resistance mechanism whereby SA is
used as a signaling molecule to relay information on pathogen attack to other parts of the
plant. Upon receiving the SA signal, a general defense response is activated that includes
the biosynthesis of pathogenesis-related (PR) proteins [178].
SA is also synthesized by the phenylpropanoid pathway, which also feeds into
the synthesis of phytoalexins, coumarins and lignins. The involvement of SA in
defense signaling has been extensively characterized in dicotyledonous plants. SA
and aspirin application could induce resistance against tobacco mosaic virus (TMV)
in tobacco. Since then, application of SA and its functional analogs, for example, 2,6-
dichloroisonicotinic acid (INA) and benzothiadiazole S-methyl ester (BTH) have
been found to induce expression of the PR genes and resistance against viral,
bacterial, oomycete and fungal pathogens in a variety of dicotyledonous. In case of
viruses, SA promotes the inhibition of viral replication, cell-to-cell movement and
also long-distance movement. SA has been shown to modulate HR-associated cell
death, reactive oxygen species (ROS) level, activation of lipid peroxidation and
generation of free radicals, all of which could potentially influence plant defense
against pathogens. SA at low concentrations also promotes the faster and stronger
activation of callose deposition and gene expression in response to pathogen or
microbial elicitors, a process called 'priming', which contributes to induced defense
mechanisms [179].
The lignin content in roots was increased by all tested abiotic inducers (salicylic
acid, boric acid, ascorbic acid, CuSO4, MgSO4, KH2PO4 and Bion WF50) pathogens in
comparison with the untreated controls. The twice application method and SA used
against R. fragariae and R. solani produced the highest lignin content followed by
AA. The lignin content in plant roots, regardless root rot pathogens, was increased
also by any tested biotic inducers in comparison with the untreated control. The
39
highest lignin content was induced by Bacillus subtilis followed by Pseudomonas
fluorescence, Streptomyces aureofaciens and Trichoderma harzianum respectively
comparing with the untreated control. And also all tested abiotic inducers increased
peroxidase, polyphenol oxidase (PPO and chitinase activity in shoots and roots of
strawberry plants in comparison with their untreated controls. Using ascorbic acid
against any of the tested root rot pathogens (S. rolfsii, R. fragariae and R. solani)
induced the highest increase in peroxidase activity in shoots whereas; CuSO4,
ascorbic acid and CuSO4 recorded the highest activity in roots [137].
Foliar application of elicitors showed in most cases a significant increase in
plant growth parameters. These increases may be attributed to elicitors' effect on
physiological processes in plant such as ion uptake, cell elongation, cell division,
enzymatic activation and protein synthesis. In this concern, SA enhanced growth of
plants. Jasmonic acid is a final product of the enzymatic oxidation of unsaturated
fatty acids and lipoxygenase is a pivotal enzyme in this pathway. This compound,
defined as a natural plant growth regulator, was found to be active in many
physiological systems. Plants respond to pathogen attack or elicitor treatments by
activating a wide variety of protective mechanisms designed to prevent pathogen
replication and spreading. The defense mechanisms include the fast production of
reactive oxygen species; alterations in the cell wall constitution; accumulation of
antimicrobial secondary metabolites known as phytoalexins; activation and/or
synthesis of defense peptides and proteins. In various plant species, resistance can be
induced with elicitors such as SA, MeJA and CHI against a wide range of pathogens
[180].
Root and foliar applications of 24-epibrassinolide (EBL), an immobile
phytohormone with antistress activity and significantly reduced disease severity of
Fusarium wilt of cucumber (Cucumis sativus L. cv. Jinyan No. 4) together with
improved plant growth and reduced losses in biomass regardless of application
methods. EBL treatments significantly reduced pathogen-induced accumulation of
reactive oxygen species (ROS), flavonoids, and phenolic compounds, activities of
defense-related and ROS-scavenging enzymes. The enzymes included superoxide
dismutase, ascorbate peroxidase, guaiacol peroxidase, catalase as well as
phenylalanine ammonia-lyase and polyphenoloxidase. There was no apparent
difference between two application methods used [181].
Peroxidases have been found to play a major role in the regulation of plant cell
elongation, phenolic compounds oxidation, polysaccharide cross-linking, Indole
acetic acid oxidation, cross-linking of extension monomers and mediate the final step
in the biosynthesis of lignin and other oxidative phenols. PO and PPO activities were
greater in the plants treated with mixtures of rhizobacteria and endophytic bacteria
and challenged with viruliferous aphids, compared to control plants. PPO can be
induced through octadecanoid defense signal pathway and it oxidizes phenolic
compounds to quinines, and the enzyme itself is inhibitory to viruses by inactivating
the RNA of the virus. Enhanced PPO activities against disease and insect pests have
been reported in several beneficial plant–microbe interactions [182].
The activities of plant defense-related enzyme, peroxidase (PO), polyphenol
oxidase (PPO) and phenylalanine ammonia-lyase (PAL) were significantly increased
40
in plants treated with B579. Interestingly, a higher content of IAA, an important plant
growth regulator, was detected in B579 treated plants. Furthermore, seed-soaking
with B579 exhibited a better biological control effect (Biocontrol effect 73.60%) and
plant growth promoting ability (Vigor Index 4,177.53) than root-irrigation (50.88%
and 3,575.77, respectively), suggesting the potential use of B579 as a seed-coating
agent [25].
1.5 Anatomical features of immunized plants:
Although response to vascular wilts was intensively studied and characterized in
several susceptibility pathogen interactions little histopathological and histochemical
researchers were conducted on cucumber infected with Fusarium oxysporum f.sp
cucumerinum.
Schoder and Walker [183] using paraffin – embedded pea stem noted an
increase of cambial activity in infected plants. Extensive colonization of vessels by
the fungus was observed in susceptible plants, while in resistant plants the fungus
was found only sparingly.
Tessier [184] studied the responses of peas to stem infection by race 1 and race
2 of Fusarium oxysporum f.sp. pisi by light microscopy. He found that in susceptible
interaction, wilting began 4 days after inoculation and progressed vertically through
all the leaflets until death of the plant 12-14 days after inoculation. In resistant
interactions the first leaflet above the site of inoculation exhibited yellowing between
4 and 6 days after inoculation, but no further symptom development was observed
after that time. Vascular response of the host to infection included vessel occlusion
by gels, deposition of callose in some xylem parenchyma cells, and extensive
vascular browning. The gels were composed of carbohydrates, protein, and pectin,
but tests for phenolic compounds were negative. Tyloses were not found.
Beckman et al., [185] studied callose deposition in parvascular parenchyma cell
of tomato that was inoculated after inoculation with F. oxysporum or root flora in
near isolines of tomato that was wilt-resistant or susceptible. The subsequent rate of
deposition appeared to be lower in the susceptible isoline than in the resistant isoline
inoculated with F. oxysporum. Both isolines responded strongly to root flora, but the
resistant cultivar appeared to maintain stronger level of response.
Bishop and Cooper [186] examined putative resistance mechanisms to infection
by vascular parasites in the roots of tomato and pea plants. The results indicated that
various mechanisms probably decreased the extent of initial xylem colonization,
although the potential for xylem penetration was apparently similar in resistant and
susceptible cultivars of both species. This suggests that differences in susceptibility
between cultivars may arise throughout mechanisms which operate during the
vascular phase of infection; support for this comes from preliminary studies that have
shown that resistance is still expressed in the absence roots.
Transmission electron microscopy was used to study vascular colonization in
resistant cultivar of tomato infected with Fusarium oxysporum f.sp lycopersici FOL
or V. albo-atrum and of pea infected with F. oxysporum f.sp pisi. In tomato principal
occluding reaction was the formation of tyloses and this was normally associated
with extensive accumulations of electronopaque material in the vacuoles of both the
41
xylem parenchyma cells and the tyloses themselves. Tyloses were absent in pea but
vessels were occluded by gels. The formation of both tyloses and gels were induced
by wounding [187].
Response between susceptible and resistant peas plant against Fusarium
oxysporum f.sp pisi vascular plugs, vessel coatings, callose deposits and phenolic
compounds that accumulated as host responses were histochemically characterized.
No anatomical or ultrastructural differences in response were observed between
resistant or susceptible pathogen interactions up to 4 days after infection. After 4 days
in susceptible interaction the pathogen grew laterally from initially infected vessels
into adjacent vessels and parenchyma cells until the vascular bundle was completely
colonized, while in resistant interaction the pathogen was confined to vessels initially
infected. An increase in cytoplasmic activity of vascular parcenchyma cells was
detected in both resistant and susceptible interactions [188].
Two resistant clones of alfalfa (1079 and WL-5) were stubble inoculated with
Verticillium albo-atrum grow for two 6-wk growth cycles before being used in the
histological study. Thereafter, vascular differentiation was disrupted in clone 1079,
resulting in the absence of immature developing vessel elements and the presence of
atypically narrow metaxylem vessels in most vascular bundles in the stem.
Dissolution of vascular bundles infected with V. albo-atrum was evident by the final
week of the growth period. Confinement of V. albo-atrum to the crown untik in the
growth period appeared to account for resistance in clone 1079.
An additional resistance response was noted in stem of clone WL-S. The
response consisted of hepertrophied xylem-parenchyma cells surrounding groups of
infected vessel elements eventualy crushing and obliterating them. The hypertrophied
cell frequently tested positive for suberin. Typically narrow xylem-vessel elements
were confined to infected vascular bundles in clone WL-5 and no vascular dissolution
occurred. V. albo-atrum in the xylem vessel of both caused by Pythium
aphanidermatum. The compound triggered several host defense responses, including
the induction of structural barriers in root tissue and the stimulation of antifungal
hydrolases in both the roots and leaves.
Anatomical studies of treated watermelon plants against wilt pathogen showed
many great signs of resistance. In sections of control-infected plants, the fungus was
spread in cortex cells and in xylem vessels, a new regenerated vascular bundle was also
observed. Treated inoculated and non-inoculated plants, cell wall of epiderms was
thicker and the cortex area wider than the non-treated - non-inoculated one. Number of
xylem vessels was higher in case of treatment than non treatment. Intera-between
vascular bundles cambium (interfascicular) was regenerated under the influence of the
treatment by salicylic acid, hydrogen peroxide and cobalt ions agents. It divided to form
3-4 layers and in one case a thick walled structure appeared [126].
Histopathological changes simultaneity with elicitation of systemic acquired
resistance was tend toward growth enhancement in the sprayed plants with tested
bioinducers than those untreated ones.
Based on current knowledge of the biochemistry of resistance, it can be
concluded that SAR results from the expression of several parameters, including
changes in cell wall composition and de novo synthesis of phytoalexins and PR
42
(pathogenesis related) proteins. Changes in cell wall composition such as increased
cross-linkage among cell wall constituents and increased lignification and callose
formation are important defensive mechanisms, which frequently occur in cells
around those exhibiting programmed cell deaths. These responses inhibit penetration
by pathogens that have been able to ‘escape’ from their HR-expressing- and therefore
dying-host cell. Moreover, the local de novo synthesis of phytoalexins is often related
to the induced resistance stage. Phytoalexins are secondary plant compounds induced
by and active against microbial pathogens [189].
43
2 MATERIAL AND METHODS
2.1 Isolation and identification of cucumber Fusarium wilt:
Cucumber plants which showed wilt symptoms (yellowing, browning and
chlorosis leaves wilt, bend down, stunting, drooping and death) were collected from 3
Governorates in Egypt where cucumber are cultivated intensively in protected houses
namely Qalubiya, Ismailiya, Beheira and also from region Grac at Almaty in
Kazakhstan. The wilting plants were washed with tap water to remove any adhering
soil particles, left for drying and were separated and disinfected by sodium
hypochlorite solution 1.0% for 2 to 3 min and they were dried with sterilized filter
papers, and then cut into small pieces. Pieces were transferred into Petri-dishes which
containing water agar amended with 100 ppm streptomycin sulphate. Petri-dishes
were inoculated at 25°C for 48 hr, and then hyphal tips were transferred on potato
dextrose agar (PDA). Single spore culture technique was used for purification of
isolated Fusaria.
Identification was based mainly on cultural properties, morphology and
microscopical characteristics. Booth systems were used to identify Fusarium isolates
to the species level [190]. Identification was confirmed through the Dept. of
Taxonomy, Plant Pathology Institute, Egypt.
Formae speciales were identified according to their ability to induce wilt
symptoms on different cucurbits plants.
2.2 Pathological studies
2.2.1 Inoculum Preparation:
Inocula of Fusarium isolates for soil infestation were prepared by growing the
fungus on Petri dishes which containing potato dextrose agar (PDA). Plates were
incubated at 25°C for 8 days in the darkness. Microconidia were gently removed
from the surface of the medium with sterile water and resulting suspension was
filtered through four layers of cheesecloths to remove mycelial fragment before
adjusting the concentration to be 107 conidia/ml using haemacytomter slide (modified
from [191].
2.2.2 Pathogenicity tests and of inoculum density:
Plastic pots (20 cm in diameter) filled with autoclaved sandy loam soil, 121ºc
for 1 hr. was used. Soil was infested with five conidial suspension concentrations (i.e.
1x103, 1x10
4, 1x10
5, 1x10
6 and 1x10
7 cfu/ml) of Fusarium oxysporum.
Soil was infested with 10 m1 conidia suspension per pot from each different
concentration. Pots were irrigated to ensure the establishment of the tested isolates in
the soil. Cucumber seeds hybrid Sina 1 were surface disinfected using 1 % sodium
hypochlorite solution for one min. then thoroughly washed with sterilized water and
air dried in sterile cabinet. Five seeds were sown in each pot and after planting were
let 2 plants in each pot and the others were pulled. Pots were irrigated as needed.
Disease symptoms were observed till 30 days from planting. Plants showed wilt
symptoms were collected for re-isolation of the pathogen.
44
Disease assessment:
Cultivated plants in infested and non-infested soil were observed for wilt
incidence over 30 day's period. The symptoms of wilt was observed, plants were
counted either as healthy or disease, according to the predominant appearance of such
symptoms on plants after 30 days. The Fusarium wilt disease was recorded using a
scale containing 6 grades suggested by [52] according to formula (1, 2 and 3).
Grade: 0 : no symptoms.
1 : Plants with < 25 % of leaves with wilt symptoms.
2 : Plants with 25 to 50 % of leaves with wilt symptoms.
3 : Plants with 50 to 75 % of leaves with wilt symptoms.
4 : Plants with 76 to 100% of leaves with wilt symptoms.
5 : Plants with complete death.
Disease severity percent was determined according to equation:
Percentage of disease severity = X 100 (1)
where: a = Number of infected leaves in each category.
b = Numerical value of each category.
N = Total number of examined leaves.
K = The highest degree of infection category.
Reduction (%) = (2)
Efficacy (%) = (3)
2.2.3 Host range
The most aggressive Fusarium oxysporum (1) isolated from Qalubiya used in
this study and in all subsequent studies. Seeds of cucumber hybrid Sina1,
watermelon, Cantaloupe, luffa, melon and squash were planted in plastic pots (20 cm
in diameter) containing sandy-loam soil artificially infested with F. oxysporum as
mentioned before. Five seeds were sown in each pot and after planting were let 2
plants in each pot and the others were pulled. Three pots were used for each crop.
Control pots, for every crop, were sown by disinfested seeds in noninfested soil.
Seeds were obtained from Institute of vegetable Research, Ministry of Agriculture,
Giza.
2.2.4 Susceptibility of commercial cucumber cultivars to infection with
Fusarium wilt.
Five cucumber hybrids namely Hisham, Db 162, Db 164, Al-Zaem and Sina1
were evaluated for the resistance to Fusarium wilt under greenhouses conditions.
Plastic pots (20 cm in diameter) filled with autoclaved 121ºc for 1 hr sandy
loam, soil was used. Soil was infested with 10 m1 conidial suspensions per pot from
each concentration 1x105 cfu/ml. Pots were irrigated to ensure the establishment of
Fusarium oxysporum f.sp. cucumerinum (FOC) in the soil. Cucumber seeds were
surface disinfected using 1% sodium hypochlorite solution for one min. then
thoroughly washed with sterilized water and air dried in sterile cabinet. Five seeds
100Control
Treatment - Control
(a X b)
N X K
100Control
Control -Treatment
45
were sown in each pot and after planting 2 plants were let in each pot and the others
were pulled. Pots were irrigated as needed. Disease symptoms were observed till 30
days from planting. Plants showed wilt symptoms were collected for re-isolation of
the pathogen.
2.3 Laboratory studies
2.3.1 Studying the effect of antagonistic microorganisms in vitro against F.
Oysporum. f.sp. cucumerinum (FOC).
This study was conducted to investigate the inhibitory effect of some known
and unknown isolates of the antagonistic fungi and bacteria on the linear growth and
spore germination of FOC. The known isolates Trichoderma harzianum (3 isolates),
T. virdi (2 isolates), Cheatomium globosum and Bacillus subtilis (3 isolates) were
obtained from Biological Control Res. Dept. Agric., Res. Center Giza, Egypt while,
Chaetomium bostrycoides, Pseudomonas fluorescens (3 isolates), Pseudomonas
putida and Bacillus megtela were obtained from Onion, Garlic and Oil Crops Res.
Dept.). However, Serratia marcensens (2 isolates) and the unknown isolates
including isolates of Trichoderma spp., Chaetomium spp., Penicillium spp. and 4
isolates of Bacillus spp. were isolated from the rhizosphere of unknown species of
cucumber plants [192]. The antagonistic effects of these microorganisms were
determined as follow:
2.3.1.1 Effect of antagonistic fungi
2.3.1.1.1 Effect of antagonistic fungi on growth of F. O. f.sp. cucumerinum
(FOC) in vitro.
Two discs ( 5 mm) of 4-day-old plain agar culture of both antagonistic fungus
and FOC were inoculated simultaneously each opposite the other 1 cm apart from the
plate edge in individual plates ( 9 cm) contained 10 ml PDA medium. In control
treatment, the plates were inoculated each with 1 discs of mycelial growth of a given
isolate of FOC. Three plates were used for each particular treatment. All plates were
incubated at 25C for 5 days. Percentage of the fungal growth reduction (X) was
calculated by using the following formula (4) suggested by [193].
X = G1- G2 / G1 x 100 (4)
Where: X: fungal growth reduction.
G1: linear growth of the pathogen inoculated alone.
G2: linear growth of the pathogen inoculated against the antagonistic fungus.
2.3.1.1.2 Evaluating the effect of antagonistic fungi culture filtrates on
growth and spore germination of F. O. f.sp. cucumerinum (FOC).
The tested antagonistic fungi were inoculated separately into conical flasks 125
cc each containing 50 ml of liquid gliotoxin fermentation medium (GFM). The
inoculated flasks were incubated at 25C under complete darkness conditions to
stimulate toxin production [194]. The culture filtrates for antagonistic fungi were
collected 10 days after incubation. The obtained filtrates were centrifuged for 15
46
minutes at 4000 r.p.m. to separate the fungal growth, sterilized by filtration through
centered glass filter (G5).
2.3.1.1.3 Evaluating the effect of antagonistic fungi culture filtrates on the
linear growth of F. O. f.sp. cucumerinum (FOC).
The sterilized filtrates were added to warm sterilized Czapek's agar medium at
rate of 10, 25 and 50 % and poured before solidification into Petri dishes
(10ml/plate). Each of the treated plates was inoculated at the center with equal discs
( 9 cm) obtained from the periphery of 7 days old cultures of FOC. Plates contained
media without culture filtrates inoculated with FOC was served as control treatment.
All plates were incubated at 25C. The experiment was terminated when mycelial
mats covered medium surface in control treatment, all plates were examined and
growth reduction was calculated as mentioned before.
2.3.1.1.4 Evaluating the effect of antagonistic fungal culture filtrates on
spore germination of F. O. f.sp. cucumerinum (FOC).
The antifungal activity of fungal culture filtrates 10, 25 and 50 % was
investigated by using the method of spore counting. Spore suspension was prepared
from a 15-day-old culture of the fungus in sterile distilled water, and 100 µl fungal
suspension was added to 100 µl fungal culture filtrates, of concentration 10, 25 and
50 %, in glass vials and incubated at 25°C for 24 hours. The control vials contained
sterile distilled water instead of fungal culture filtrates. After incubation, the content
of the vials was stained with cotton blue and mounted in lactophenol. The spores
were observed under a microscope for their germination status. Percentage of spore
inhibition was calculated by using the established formula (5) according [195].
% Spore inhibition = A- B / A x 100 (5)
A: Spore germination in control
B: Spore germination in treatment
2.3.1.2. Effect of antagonistic bacteria.
2.3.1.2.1 Effect of antagonistic bacteria in vitro against F. O. f.sp.
cucumerinum (FOC).
Studying the effect of antagonistic bacteria isolates on growth of FOC were
conducted as following, individual plates contained PDA medium were streaked at
one side 1cm apart from the plate edge with a given isolate of antagonistic bacteria
with a loop full of the antagonistic bacteria (48 hrs- old) grown on liquid nutrient agar
medium (NG) and incubated for 24 hrs at 28 C. Thereafter the same plate was
inoculated at the opposite side 1cm apart from the plate edge with 9 mm disc of 4-
day-old plain agar culture of an isolate of FOC. All plates were incubated at 25 C for
5 days. The inhibition zone (in mm) between bacteria and the pathogen was measured
[196].
47
2.3.1.2.2 Evaluating the effect of antagonistic bacterial culture filtrates on
the linear growth and spore germination of F. O. f.sp. cucumerinum (FOC).
The tested antagonists bacteria were inoculated separately into conical flasks
125 cc each containing 50 ml of liquid NG media. The inoculated flasks were
incubated at 25C under complete darkness conditions to stimulate toxin production
[194]. The culture filtrates for antagonistic bacteria were collected after 3days after
incubation. The obtained filtrates were centrifuged for 15 minutes at 4000 r.p.m. to
separate the bacterial growth, sterilized by filtration through centered glass filter
(G5).
2.3.1.2.3 Evaluating the effect of antagonistic bacteria culture filtrates on
the linear growth of F. O. f.sp. cucumerinum (FOC).
The sterilized filtrates were added to warm sterilized Czapek's agar medium at
rate of 10, 25 and 50 % and poured before solidification into Petri dishes
(10ml/plate). Each of the treated plates was inoculated at the center with equal discs
( 9 cm) obtained from the periphery of 7 days old cultures of FOC. Plates contained
media without culture filtrates inoculated with FOC was served as control treatment.
All plates were incubated at 25◦C. The experiment was terminated when mycelial
mats covered medium surface in control treatment, all plates were examined and
growth reduction was calculated as mentioned before.
2.3.1.2.4 Evaluating the effect of antagonistic bacteria culture filtrates on
spore germination of F. O. f.sp. cucumerinum (FOC).
The antifungal activity of bacterial culture filtrates with concentrations 10, 25
and 50 % was investigated by using the method of spore counting. Spore suspension
of FOC was prepared from a 15-day-old culture of the fungus in sterile distilled
water, and 100 µl fungal suspension was added to 100 µl bacteria culture filtrates, of
concentration 10, 25 and 50 %, in glass vials and incubated at 25°C for 24 hours. The
control vials contained sterile distilled water in place of bacterial culture filtrates.
After incubation, the content of the vials was stained with cotton blue and mounted in
lactophenol. The spores were observed under a microscope for their germination
status. Percentage of inhibition was calculated as mentioned before.
2.3.2 Effect of different resistant inducing chemicals on the linear growth
and spore germination of F. O. f.sp. cucumerinum (FOC) in vitro:
This study was designed to investigate the inhibitory effect of some chemicals,
which used later as resistant inducing compounds, on the in vitro linear growth and
spore germination of FOC. The used chemicals were tested at 3 concentrations as
follow:
A. The antioxidants (i.e. ascorbic acid, citric acid, oxalic acid and salicylic acid)
were tested at concentration of 2.5, 5.0 and 10.0 mM.
B. Dipotassium hydrogen phosphate (K2HPO4) at concentrations 50, 100 and
200mM.
C. Cobalt sulphate (CoSO4) was tested at concentrations 1, 5 and 10 ppm.
48
D. Potassium permanganate (KMnO4) and Calcium sulphate (CaSO4) at 1000,
2500 and 5000 ppm.
2.3.2.1 Effect of different resistant inducing compounds on the linear
growth of F. O. f.sp. cucumerinum (FOC) in vitro:
The amount required for obtaining a known concentration of any chemical was
calculated and added aseptically to known amount of warm sterilized Czapek's agar
medium and poured before solidification into Petri dishes (10ml/plate) then plates
were inoculated at the center with equal discs ( 9 cm) obtained from the periphery of
7 days old cultures of FOC. Plates contained media without any chemical inoculated
with FOC was served as control treatment. Three plates were used for each particular
concentration. All plates were incubated at 25C. The experiment was terminated
when mycelial mats covered medium surface in control treatment, all plates were
examined and growth reduction was calculated as mentioned.
2.3.2.2 Effect of different resistant inducing compounds on spore
germination of F. O. f.sp. cucumerinum (FOC) in vitro:
The same chemicals at the same concentrations were tested. The amount
required for obtaining a known concentration of any chemical was calculated and
dissolved in water. Spore suspension of FOC was prepared from a 15-day-old culture
of the fungus in sterile distilled water, and 100 µl fungal suspension was added to 100
µl of each concentration, in glass vials and incubated at 25°C for 24 hours. The
control vials contained sterile distilled water in place of chemical solution. After
incubation, the content of the vials was stained with cotton blue and mounted in
lactophenol. The spores were observed under a microscope for their germination
status. Percentage inhibition was calculated as mentioned before.
2.4 Greenhouse experiments
2.4.1 Effect of treating cucumber seeds with some antagonistic fungi on
incidence with Fusarium wilt disease:
In this experiment, cucumber seeds (Sina1) were coated with suspension of any
of the following antagonistic Trichoderma harzianum (3 isolates), T. virdi (2 isolates),
Cheatomium globosum, Chaetomium bostrycoides, Trichoderma spp., Chaetomium
spp. and Penicillium spp. (prepared as described below) to evaluate their efficiency in
controlling Fusarium wilt disease incidence. The tested antagonistic Trichoderma
fungus was grown on PDA plates for 10 days at 25C then its growth was flooded
with sterile-distilled water, scraped with a camel brush then filtered thorough
sterilized filter papers. The resulted spore suspensions were found to be contained
approx. 5 X 108 conidia/ml. 10 seeds of surface sterilized cucumber seeds placed in
plastic bags was thoroughly mixed and shacked slowly for 5 minutes with mixture
consisted of 2 ml spore suspension plus 1 ml of 1% Arabic gum solution as sticker
(modified from [197].
Cucumber seeds whether treated or non-treated with antagonistic fungi were
sown in potted soils infested by FOC at five seeds in each pot and after planting 2
49
plants were let in each pot and the others were pulled. Three replicates were used for
each particular treatment. The Fusarium wilt disease was recorded as mentioned
before.
2.4.2 Effect of treating cucumber seeds with cell suspension antagonistic
bacteria isolates on incidence with Fusarium wilt disease.
Cucumber seeds (Sina1) were treated with antagonistic bacteria according to
[198]. Any of the tested antagonistic bacterial isolates (Bacillus subtilis (3 isolates),
Pseudomonas fluorescens (3 isolates), Pseudomonas putida and Bacillus megtela,
Serratia marcensens (2 isolates) and 4 isolates of Bacillus spp.) was grown for 48 hrs
at 26C on nutrient agar medium, (NA), then bacterial growth was scraped and the re-
suspended in mixture of 1.5 ml of 1.0% methyl cellulose (MC) and 1.5 ml of 0.1 M
MgSO4. Ten of surface sterilized Cucumber seeds were thoroughly mixed with 2 ml
of bacterial suspension for 5 minutes then left for 2 hrs to air dried in a laminar-flow
before planting. Bacterial population determined per seed was 1x108 c.f.u/seed
according to dilution plate assay described by [199]. Cucumber seeds whether treated
or non-treated with antagonistic bacteria were sown in potted (20 cm in diameter)
soils infested by FOC at five seeds in each pot and after planting 2 plants were let in
each pot and the others were pulled. Three replicates were used for each particular
treatment. The Fusarium wilt disease was recorded as mentioned before.
2.4.3 Effect of treating cucumber seeds or soil with some resistance
inducing chemicals on incidence with Fusarium wilt disease.
The same chemicals that were previously tested under lab conditions as
inhibitors against fungal growth and spore germination were used.
A- The antioxidants (i.e. ascorbic acid, citric acid, oxalic acid and salicylic acid)
were tested at at concentration of 2.5, 5.0 and 10.0 mM as seed soaking.
B- Dipotassium hydrogen phosphate at concentrations 50, 100 and 200mM as
seed soaking.
C- Cobalt sulphate (CoSO4) was tested at concentration 1, 5 and 10 ppm as seed
soaking.
D- Potassium permanganate (KMnO4) and calcium sulphate (CaSO4) at 1000,
2500 and 5000 pmm/ kg soil.
Ten surface sterilized cucumber seeds (Sina1) were soaked for 2.5 hours [200]
in a known concentration of any of the above mentioned chemical inducers. The
wetted seeds were spread out in a thin layer and left to 24 hours then they were sown
in pathogen-infested potted soils at five seeds in each pot and after planting 2 plants
were let in each pot and the others were pulled. Potassium permanganate (KMnO4)
and calcium sulphate (CaSO4) were used as soil drench. Seeds soaked in water were
sown in control pots. Three pots were used for each treatment as replicates. Effect of
different treatments on Fusarium wilt disease incidence was estimated as mentioned
before.
50
2.5 Experiments of commercial protected house
These experiments were conducted under commercial protected house
conditions belongs to (Санаторий Алатау) Sanatorium Alatau, Almaty, Kazakhstan.
2.5.1 Effect of treating cucumber seeds with some antagonistic Trichoderma
isolates on incidence with Fusarium wilt under protected houses:
Two experiments (during spring and autumn 2009) were conducted to evaluate
the effect of coating cucumber seeds with suspension of any of the following
antagonistic Trichoderma harzianum (3 isolates), T. virdi (2 isolates), Cheatomium
globosum, Chaetomium bostrycoides, Trichoderma spp., Chaetomium spp. and
Penicillium spp. (prepared as mentioned before) to evaluate their efficiency in
controlling Fusarium wilt disease incidence under protected houses. Ten surface
sterilized cucumber seeds (Sina1) placed in plastic bags was thoroughly mixed and
shacked slowly for 5 minutes with mixture consisted of 2 ml spore suspension plus 1
ml of 1% Arabic gum solution as sticker (modified from [197].
Cucumber seeds whether treated or non-treated with antagonistic fungi were
sown in potted (30 cm in diameter) soils infested by FOC with 50ml 105conidia/ml
spore suspension/pot at five seeds in each pot and after planting 1 plant was let in
each pot and the others were pulled. Three replicates were used for each particular
treatment. The Fusarium wilt disease was recorded as mentioned before and average
weight of fruits (Kg)/plant was measured.
2.5.2 Effect of treating cucumber seeds with cell suspension of antagonistic
bacterial isolates on incidence with Fusarium wilt under protected houses.
Two experiments (during spring and autumn 2009) were conducted to evaluate
the effect of coating cucumber seeds with suspension of any of the following
antagonistic bacterial isolates (Bacillus subtilis (3 isolates), Pseudomonas fluorescens
(3 isolates), Pseudomonas putida and Bacillus megtela, Serratia marcensens (2
isolates) and 4 isolates of Bacillus spp.) (Prepared as mentioned before) to evaluate
their efficiency in controlling Fusarium wilt disease incidence under protected
houses. Cucumber seeds (Sina1) treated with antagonistic bacteria according to [198].
Cucumber seeds were thoroughly mixed with 2 ml of bacterial suspension for 5
minutes then left for 2 hrs to air dried in a laminar-flow before planting. Cucumber
seeds whether treated or non-treated with antagonistic bacteria were sown in potted
(30 cm in diameter) soils infested by FOC at five seeds in each pot and after planting
one plant was let in each pot and the others were pulled. Three replicates were used
for each particular treatment. The Fusarium wilt disease was recorded as mentioned
before and average weight of fruits (Kg)/plant were measured.
2.5.3 Effect of treating cucumber seeds or soil with some resistance
inducing chemicals on incidence with Fusarium wilt under protected houses.
Two experiments (during spring and autumn 2009) were conducted to evaluate
the effect of the same chemicals that were previously tested under green house
conditions on incidence with Fusarium wilt disease.
51
A- The antioxidants (i.e. ascorbic acid, citric acid, oxalic acid and salicylic acid)
were tested at concentration 10.0 mM as seed soaking.
B- Dipotassium hydrogen phosphate at concentration 200mM as seed soaking.
C- Cobalt sulphate (CoSO4) was tested at concentration 10 ppm as seed soaking.
D- Potassium permanganate (KMnO4) and Calcium sulphate (CaSO4) 5000
ppm/ kg soil.
Ten surface sterilized cucumber seeds (Sina1) were soaked for 2.5 hours [200]
in known concentrations of any of the above mentioned chemical inducers. The
wetted seeds were spread out in a thin layer and left to 24 hours then they were sown
in pathogen-infested potted 30 cm in diameter soils at five seeds in each pot and after
planting one plant were let in each pot and the others were pulled. Potassium
permanganate (KMnO4) and calcium sulphate (CaSO4) were used as soil drench.
Seeds soaked in water were sown in control pots. Three pots were used for each
treatment as replicates. Effect of different treatments on Fusarium wilt disease
incidence was estimated as mentioned before and average weight of fruits (Kg)/plant
was measured.
2.6 Determination of enzymes activity, lignin content and peroxidase
isozyme:
The same antagonistic fungi, antagonistic bacteria and chemicals that were
previously tested under protected house conditions on incidence with Fusarium wilt
disease in addition to untreated control treatment on peroxidase, polyphenol-oxidase
and chitinase activity were determined. Samples were taken at 40 and 50 days after
planting.
-- Extraction of enzymes:
Samples leaves were 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
[156].
2.6.1 Peroxidase assay: Peroxidase activity was measured by incubation 0.1 ml of enzyme extract with 4
ml of guaiacol for 15 minutes at 25°C and absorbance at 470 nm was determined.
The guaiacol solution consisted of 3 ml of 0.05M potassium phosphate pH 7.0, 0.5 ml
of 2% guaiacol and 0.5 ml of 0.3% H2O2 [201]. Peroxidase activity was expressed as
the increase in absorbance at 425nm/gram fresh weigh/15 minutes.
2.6.2 Polyphenoloxidase assay: The polyphenoloxidase activity was determined according to the method
described by [202]. 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 completed
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.
52
2.6.3 Chitinase assay:
The determination was carried out according to the method of [203] one ml of
1% colloidal chitin in 0.05 M citrate 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 sugars
were 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 weight tissue / 60 minutes.
The substrate colloidal chitin was prepared from chitin powder according to the
method described by [204]. 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.
2.6.4 Determination of lignin content:
Cucumber roots were taken after 50 days from planting as samples. The
determination of lignin was carried out according to the method of [205]. Five gram
of dried cucumber roots were 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.
2.6.5 Isozyme pattern of soluble peroxidase:
Polyacrylamide gel electrophoresis (PAGE) was performed exclusively in
horizontal slab 13 mm a Mini-Gel apparatus system (3100 series), according to the
method described by [206]. 2.6.5.1 Reagents (stock solutions). A. 30% Acrylamide
29.2 g Acrylamide and 0.8 g N.N-Methylenebis-acrylamide were dissolved in
100 ml H2O.
B. 2% ammonium persulphate
0.25 g ammonium sulphate was dissolved with 10 ml H2O.
This stock must be prepared immediately before use.
C. Buffer solution
This Borate buffer pH 8.9 was used for isozyme analysis.
The stock solution was composed of 605 g tris (hydroxymethyl) aminomethane)
and 46 g boric acid dissolved in 5000 ml H2O.
D. Electrode buffer (0.125 M pH 8.9) was prepared by dilution of 300 ml of the
stock solution with 2100 ml H2O.
E. Gel preparation
35 ml of 30% Acrylamide was added with 70 ml (0.125 M pH 8.5) dilute buffer
to get 8% Acrylamide, 33 mg sodium sulphate (dissolve completely), 66 µL TEMED
53
(Teteramethy-lenediarnine) and 2.5 ml ammonium sulphate. The gel solution was
quickly poured immediately and 8 well combs were used, then gels were left for
about 30 minutes for polymerization.
2.6.5.2. Application of samples:
50 µL tissue extract of each sample was mixed with 13 µL bromophenol blue
and 13 µL glucerol. 50 µL of this mixture was applied to each groove of the prepared
gels. The run was carried out at 75 v. for about 2 hr. 2.6.5.3 Visualization of peroxidase isozyme profiles: The solution used for peroxidase visualization consisted of 0.250 mg benzidine
dihydrochloride - moistened with four drops of glacial acetic acid, 100 ml H2O and
ten drops of 1% freshly prepared hydrogen peroxide solution.
2.7 Chemical analysis of cucumber treated plants
Cucumber leaves was taken after 40 days from planting as samples. 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 [207]. 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:
2.7.1 Determination of sugar content: Total and reducing sugars were determined spectrophotometrically with picric
acid as described by [208]. 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.
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.
54
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.
2.7.2 Determination of phenolic compounds: Phenolic compounds were determined using the colorimetric method of analysis
described by [209]. Phenol reagent (Folin-Ciocalteu reagent) was prepared by boiling
a mixture of 100 g of sodium tungestate, 25 g of sodium molybdate, 700 ml of
distilled water, 50 ml of 85% phosphoric acid and 100 ml of concentrated
hydrochloric acid under reflux for 10 hours in a water bath. Then 150 g of lithium
sulphate, 50 ml of distilled water and a few drops of bromine was added to the
mixture and boiled again for 15 minutes without a reflex condenser to remove excess
bromine, then cooled, diluted to 1 liter with distilled water and filtered. The free
phenols were determined as follows, one ml of the phenol reagent and 5 ml of a 20%
solution of sodium carbonate were added to the isopropanol sample (0.2 ml) and
diluted to 10 ml with warm water, (30-35°C). The mixture was let to stand for 20
minutes and read using spectrophotometer (SPECTRONIC 20-D) at 520 nm against a
reagent blank.
For total phenols determination, 10 drops of concentrated hydrochloric acid
were added to the isopropanol sample (0.2ml) in a test tube, heated rapidly to boiling
over a free flame, with provision for condensation. Then the tubes were placed in a
boiling water bath for 10 minutes. After cooling 1ml of the reagent and 2.5 ml of
20% Na2CO3 were added to each tube. The mixture was diluted to 50 ml with
distilled water, and after 20 minutes was determined using spectrophotometer
(SPECTRONIC 20-D) at 520 nm against a reagent blank. The total and free phenol
contents were calculated for each treatment as milligrams of catechol per one-gram
fresh weight according to standard curve of catechol. The conjugated phenols were
determined by subtracting the free phenols from the total phenols.
2.7.3 Determination of total amino acid:
Total amino acid was determined using the method of analysis described by
[210]. 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
absolute methanol. The developed colour was measured at 580 nm using
spectrophotometer (SPECTRONIC 20-D) against a reagent blank. Data were
obtained referring to standard pure glycine curve.
55
2.8 Anatomical studies
It was intended to carry out a comparative anatomical study on roots of treated
plants and those of the control at 50 days after planting.
These roots specimens were then killed and fixed in F.A.A. (10 ml formalin, 5
ml glacial acetic acid and 85 ml ethyl alcohol 70%), washed in 50% ethyl alcohol,
dehydrated in a series of ethyl alcohols 70, 90, 95 and 100%, infiltrated in xylene
embedded in paraffin wax with a melting point 60-63°C, sectioned 15 microns in
thickness [211] stained with the double stain method (Fast green and safranin),
cleared in xylene and mounted in Canada balsam [212]. Four sections for each
treatment were microscopically inspected to detect histological manifestations of
noticeable responses resulted from treatments. Counts and measurements (µ) were
taken using a micrometer eye piece. Averages of readings from 4 slides / treatment
were calculated.
2.9 Carrying the best antagonistic isolates of fungi and bacteria on different
carrier materials
Preparation of antagonistic fungi and culture and inoculation of carrier
materials:
Antagonistic fungi were grown on gliotoxin fermentation medium (GFM) under
complete darkness [194] for 9 days, Meanwhile ,bacteria were grown on nutrient agar
(NG) broth for 48 hours. Culture filtrates of fungi and bacteria were collected and
centrifuged for 15 minutes at 4000 r.p.m. to separate the fungal or bacterial growth,
the concentration of fungal spore suspension was adjusted to 3x107 spore/ml,
meanwhile the concentration of bacteria cell suspension was adjusted to 3x107 cell/ml
[213]. The obtained fungal spore suspension resuspended in equal volume of each
1% talc and paraffin oil while, the obtained bacteria cell suspension resuspended in
equal volume of each 1% talc, starch and paraffin oil [214, 215, 216].
Also the obtained fungi spore suspension was carrying on sodium alginate as
the following
1. Dissolve 30g of sodium alginate in 1 liter to make a 3% solution.
2. Mix fungi spore suspension with 1% v/v of 3% (wt.) sodium alginate
solution. The concentration of sodium alginate can be varied between 6-12 %
depending on the desired hardness.
3. The beads are formed by dripping the polymer solution from a height of
approximately 20 cm into an excess (1000 ml) of stirred 0.2M CaCl2 solution with a
syringe and a needle at room temperature. The bead size can be controlled by pump
pressure and the needle gauge. Leave the beads in the calcium solution to cure for
0.5-3 hours [217].
2.10 Statistical analyses:
The similarity coefficients were then used to construct dendograms, using the
Unweighted Pair Group Method with Arithmetic Averages (UPGMA) employing the
SAHN (Sequential, Agglomerative, Hierarchical, and Nested clustering) from the
56
NTSYS–PC (Numerical Taxonomy and Multivariate Analysis System), version 1.80
(Applied Biostatistics) programs [218].
PASTA: PAleontological Statistics, runs on standard Windows computers and is
available free of charge. PAST integrates spreadsheet- type data entry with univariate
and multivariate statistics, curve fitting, time series analysis, data plotting and simple
phylogenetic analysis. Many of the functions are specific to paleontology and
ecology, and these functions are not found in standard, more extensive, statistical
packages. PAST also includes fourteen case studies (data files and exercises)
illustrating use of the program for paleontological problems, making it a complete
educational package for courses in quantitative methods (http://palaeo-
electronica.org).
Statistical analyses of all the previously designed experiments have been carried
out according to the procedures (ANOVA) reported by [219]. Treatment means were
compared by the least significant difference test “L.S.D” at 5% level of probability.
57
3 RESULTS
3.1 The casual organism
Cucumber wilted plants were collected from different locations of. Governorates
in Egypt where cucumber are cultivated intensively included, Qalubiya, Ismailiya,
Beheira and also from region Grac at Almaty in Kazakhstan and examined for the
presence of Fusarium wilt pathogen.
All cultivated stem base parts on PDA medium gave a growth of Fusarium.
Hyphal tips of such growth were transferred to slants of PDA medium. All isolates
were purified using single spore technique. The isolates obtained from one region
were assigned as isolate. Morphological features of fungal growth including, rate of
growth, mycelial color, pigmentation, macro, microconidia and chlamydospore
formation, type of conidiophore, number of septa in macro, and microconida were
observed.According to these features, the isolated fungus was identified as Fusarium
oxysporum.
3.2 Pathological studies
3.2.1 Pathogenicity tests and inoculum densities.
Pathogenicity test of the different isolates for the isolated fungus was carried out
under green house conditions. Healthy cucumber seeds cv. Sina1 were used.
In order to identify influence of inoculum densities of the tested isolates on
infection of cucumber seeds cv. Sina1, five conidial concentrations (1x103, 1x10
4,
1x105, 1x10
6 and 1x10
7 conidia/ ml) of F. oxysporum were used in this experiment.
Data presented in Table 1, show that all isolates of F. oxysporum were
pathogenic to the tested plants and caused wilt symptoms. Fusarium oxysporum (1)
was the highly virulent and caused 100% wilt while Fusarium oxysporum (4) was the
least virulence with 62.5 % wilt. The obtained results revealed that inoculum density
at 1x107 cfu of Fusarium oxysporum (1) showed the highest percentage of dead plants
100 % followed by Fusarium oxysporum (3) with conc. 1x107 cfu caused (88.00%)
dead plants. On the other hand, inoculum densities of 1x103 and 1x10
4 cfu caused the
least percentage of dead plants respectively. Generally the dead plants were
significantly increased by increasing inoculum densities.
Table 1 - Pathogenicity tests and inoculum densities of the tested isolates of F. oxysporum of
cucumber seeds cv. Sina1
Inoculum
density (cfu)
Fusarium
oxysporum (1)
Fusarium
oxysporum (2)
Fusarium
oxysporum (3)
Fusarium
oxysporum (4)
% Dead
plants
%Healthy
plants
% Dead
plants
%Healthy
plants
% Dead
plants
%Healthy
plants
% Dead
plants
%Healthy
plants
1103 12.5 87.5 23.7 76.3 20.3 79.7 12.2 87.8
1104 27.5 72.5 39.5 60.5 33.8 66.2 18.3 81.7
1105 61.7 38.3 52.2 47.8 50.3 49.8 41.1 58.9
1106 83.4 16.6 75.5 24.5 80.7 19.3 52.3 74.7
1107 100 0.0 85.6 14.4 88.00 12.0 62.5 37.5
Control 0.0 100.0 0.0 100.0 0.0 100.0 0.0 100.0
Fungi Inoculum Interaction
L.S.D. at 5% 1.16 0.87 4.50
58
3.2.2 Host range of F. oxysporum:
Six host plants (cucumber hybrid Sina1, watermelon, Cantaloupe, luffa, melon
and squash) were inoculated with Fusarium oxysporum isolate (1) with conidiophores
concentration 1105 cfu
to determine the host ranges of F. oxysporum. The obtained
results are tabulated in Table 2.
No any wilt symptoms was observed on watermelon, Cantaloupe, luffa, melon
and squash. According to these experiments, the reisolated fungus was identified as
F. oxysporum f.sp. cucumerinum (FOC).
Table 2 - Host range specify for Fusarium oxysporum f.sp. cucumerinum.
Hosts F. oxysporum f.sp. cucumerinum( FOC) (1)
% Wilt plants % Healthy plants Watermelon 0.0 100.0
Cantaloupe 0.0 100.0
Luffa 0.0 100.0
Cucumber 100.0 0.0
Melon 0.0 100.0
Squash 0.0 100.0
3.2.3 Susceptibility of commercial cucumber cultivars to infection with
Fusarium wilt. Six cucumber hybrids namely Hisham, Db 162, Db 164, Al-Zaem, China and
Sina1 were evaluated for the resistance to Fusarium wilt under greenhouses
conditions. The obtained results in Table 3, show that percentage of infection varied
among the different tested cucumber hybrids. The reaction of the tested hybrids could
be divided into four different groups (high resistant, moderately resistant, susceptible
and high susceptible). Sina1 is high susceptible because it recorded 95.83 % infected
plants. While, Al-Zaem (83.3%) and Hisham (75.0%) were susceptible. Db 162
(50.0%) and China (29.33%) was moderately resistant and Db 164 (16.67%) was
high resistant.
Table 3 - Susceptibility of commercial cucumber cultivars to infection with F. O.
f.sp. cucumerinum (FOC).
Cucumber cvs. FOC
% Infection plants % Healthy plants
Sina1 95.83 4.17
Al-Zaem 83.33 16.67
Hisham 75.00 25.00
Db 162 50.00 50.00
Db 164 16.67 83.33
China 70.67 29.33
- Infection Healthy plants
L.S.D at 5% 1.59 1.51
59
Figure 1 – Symptoms of Fusarium cucumber wilt.
3.3 Laboratory studies:
3.3.1 Effect of antagonistic fungi on the linear growth of F. O. f.sp.
cucumerinum (FOC) in vitro.
The obtained results in Table 4 and Figure 2 show that, Trichoderma harzianum
No.3, Trichoderma spp. and Trichoderma viride No.1 were the best isolates for
reducing growth of FOC and caused the highest reducing (57.14, 55,33 and 54.79%)
respectively. Trichoderma harzianum No.2 and Trichoderma harzianum No.1 (54.24
and 52.44 %) came next, whereas Cheatomium globosum and Cheatomium spp. was
the least isolates and reducing the mycelial growth by 27.67% and 29.48%
respectively.
Table 4 - Effect of Antagonistic fungi on growth of F. O. f.sp. cucumerinum (FOC).
%Efficacy Growth (mm) Antagonistic Fungi
52.44 26.3 Trichoderma harzianum 1
54.24 25.3 Trichoderma harzianum 2
57.14 23.7 Trichoderma harzianum 3
54.79 25.0 Trichoderma viride 1
49.91 27.7 Trichoderma viride 2
55.33 24.7 Trichoderma spp.
36.71 35.0 Cheatomium bostrycoides
27.67 40.0 Cheatomium globosum
29.48 39.0 Cheatomium spp.
42.68 31.7 Penicillium spp.
00.00 55.3 Control
Treatment
L.S.D. at 5% 5.99
Figure 2 - Effect of antagonistic fungi on growth of F. oxysporum f. sp. Cucumerinum (FOC) T.H.= Trichoderma harzianum, T.V = Trichoderma viride (1,2,3) number of isolate, T. sp.= Trichoderma spp., C.
b.= Cheatomium bostrycoides, C. sp. = Cheatomium spp. and C = Control
3.3.2 Evaluating the effect of antagonistic fungi culture filtrates on the
linear growth and spore germination of F. O. f.sp. cucumerinum (FOC).
The obtained results in Table 5 show that, filtrates of the all tested isolates
reduced the mycelial growth and spore germination of FOC. All Trichoderma and
Ch. bostrycoides filtrates of the tested isolates at 50% concentration completely
inhibited spore germination of FOC. Culture filtrates of Trichoderma spp., T.
harzianum No.3 and Trichoderma viride No.1 at 50% concentration were more
60
effective and reducing the mycelial growth of FOC by 91.50, 84.81 and 82.59 %
respectively. On the other hand, Ch. globosum and Trichoderma viride No.2 was the
least isolates and reducing the mycelial growth by 72.97% and 75.19% respectively.
Generally linear growth and spore germination were decreased by increasing the
concentrations of culture filtrates from 10% up to 50%.
Table 5 - Evaluation of the effect of antagonistic culture filtrates on the linear
growth and spore germination of F. O. f.sp. cucumerinum (FOC).
Antagonistic
Fungi
Dilutions
(%)
Growth
(mm)
% of spore
germination
%Efficacy
% Growth
% Spore
germination
T.harzianum1
10 37.67 23.33 58.14 76.67 25 25.33 13.33 71.86 86.67
50 19.67 00.00 78.14 100.00
T.harzianum 2
10 35.33 20.33 60.44 79.67
25 23.67 11.67 73.70 88.33 50 17.33 00.00 80.74 100.00
T.harzianum 3
10 30.67 13.67 65.92 86.33 25 17.67 7.67 80.37 92.33
50 13.67 00.00 84.81 100.00
T.viride 1
10 34.00 18.33 62.22 81.67
25 23.33 11.33 74.08 88.67
50 15.67 00.00 82.59 100.00
T.viride 2
10 39.33 25.00 56.30 75.00 25 28.00 14.33 68.89 85.67 50 22.33 00.00 75.19 100.00
Trichoderma spp.
10 27.00 8.33 70.00 91.67
25 13.33 3.33 85.19 96.67
50 7.76 00.00 91.50 100.00
Ch.bostrycoides
10 34.00 17.33 62.22 82.67 25 23.33 13.67 74.08 86.33 50 17.00 00.00 81.11 100.00
Ch. globosum
10 46.33 31.00 48.52 69.00
25 34.37 22.33 61.48 77.67
50 24.33 4.33 72.97 95.67
Cheatomium spp.
10 39.67 27.67 55.92 72.33 25 28.67 15.33 68.14 84.67 50 22.00 2.67 75.56 97.33
Penicillium spp.
10 45.33 27.00 49.63 73.00
25 33.67 14.67 62.59 85.33
50 18.00 2.33 80.00 97.67
Control 90.00 100.00 00.00 00.00
L.S.D. at 0.05 for: Growth % of spore germination
Antagonistic Trichoderma 1.48 0.95
Dilutions (%) 0.77 0.49
61
Interaction 2.56 1.65
3.3.3 Effect of antagonistic bacteria in vitro against F. O. f.sp. cucumerinum
(FOC). Data presented in Table 6 show that, Pseudomonas fluorescens No.2, Bacillus
subtilis No.2, Pseudomonas fluorescens No.3 and Bacillus spp. No.2 were the best
antagonistic bacteria for limiting growth of FOC, they caused the highest inhibition
zone (37.33, 35,67, 35,00 and 34.00 mm) respectively. Serratia marcensens No.2 and
Pseudomonas fluorescens No.1 (31.33 and 30, 67 mm) came next whereas Bacillus
spp. No3 was the lowest effective one that caused the narrowest inhibition zone
(25.33 mm).
Table 6 - Effect of antagonistic bacterial on growth of F. O. f.sp. cucumerinum (FOC).
Antagonistic bacteria Inhibition zone (mm)
Bacillus subtilis 1 30.33
Bacillus subtilis 2 35.67
Bacillus subtilis 3 26.67
Bacillus megtela 29.33
Bacillus spp. 1 30.00
Bacillus spp. 2 34.00
Bacillus spp. 3 25.33
Bacillus spp. 4 28.67
Pseudomonas fluorescens 1 30.67
Pseudomonas fluorescens 2 37.33
Pseudomonas fluorescens 3 35.00
Pseudomonas putida 27.33
Serratia marcensens 1 28.67
Serratia marcensens 2 31.33
Control 00.00
Treatment
L.S.D. at 5% 3.12
3.3.4 Evaluation of the effect of antagonistic bacteria culture filtrates on the
linear growth and spore germination of F. O. f.sp. cucumerinum (FOC).
The results in Table 7 and Figure 3&4 reveal that, all filtrates of the tested
isolates of antagonistic bacteria reduced the mycelial growth and spore germination
of FOC. All filtrates of the tested isolates at 50% concentration completely inhibited
spore germination of FOC. Culture filtrates of P.putida, S. marcensens No.2, B.
subtilis No.2 and Bacillus spp. No.2 at 50% concentration were more effective and
reducing the mycelial growth of FOC by 80.74, 80.37, 79.63 and 79.26 %
respectively. Pseudomonas fluorescens No.2 and Bacillus spp. No1 gave the same
result (77.41%) and came next whereas Bacillus spp. No4 was the lowest effective
one and reducing the growth (52.97%).
62
All Pseudomonas isolates, Serratia isolates, B. subtilis No.1 and Bacillus spp.
No.4 made lysis to mycelial of FOC. Generally linear growth and spore germination
were decreased by increasing the concentrations of culture filtrates from 10% up to
50%.
Table 7 - Evaluation of the effect of antagonistic bacterial culture filtrates on the
linear growth and spore germination of F. O. f.sp. cucumerinum (FOC).
63
Antagonistic bacterial Dilutions
(%)
Growth
(mm) % Of spore
germination
%Efficacy
%
Growth % Spore
germination
B. subtilis 1 10 38.33 17.00 57.41 83.00
25 28.00 11.00 68.89 89.00
50 20.67 00 77.03 100.00
B. subtilis 2 10 28.67 10.00 68.14 90.00
25 27.00 4.00 70.00 96.00
50 18.33 00 79.63 100.00
B. subtilis 3 10 33.33 11.00 62.97 89.00
25 27.67 8.00 69.26 82.00
50 20.67 00 71.11 100.00
B. megtela 10 56.67 17.67 37.03 82.33
25 44.67 13.00 50.37 87.00
50 22.33 00 75.18 100.00
Bacillus spp. 1 10 47.67 10.00 47.03 90.00
25 31.67 7.00 64.81 93.00
50 20.33 00 77.41 100.00
Bacillus spp. 2 10 32.00 11.00 64.44 89.00
25 24.33 4.00 72.97 96.00
50 18.67 00 79.26 100.00
Bacillus spp. 3 10 50.33 17.00 44.07 83.00
25 42.33 12.00 52.97 88.00
50 28.00 00 68.58 100.00
Bacillus spp. 4 10 57.67 20.00 35.92 80.00
25 51.67 14.00 42.59 86.00
50 42.33 00.00 52.97 100.00
P. fluorescens 1 10 58.33 17.67 35.19 82.33
25 42.67 10.33 52.59 89.67
50 27.33 00.00 69.63 100.00
P. fluorescens 2 10 55.33 11.00 38.52 89.00
25 34.33 8.00 61.86 92.00
50 20.33 00.00 77.41 100.00
P. fluorescens 3 10 57.67 12.67 35.92 87.33
25 41.67 10.00 53.70 90.00
50 25.00 00.00 72.22 100.00
P. putida 10 34.33 9.67 61.86 90.33
25 22.67 5 74.81 95.00
50 17.33 00.00 80.74 100.00
S. marcensens 1 10 38.33 11.33 57.41 88.67
25 35.33 8.67 60.74 91.33
50 21.67 00.00 75.92 100.00
S.marcensens 2 10 35.33 10.33 60.74 89.67
25 25.00 5.33 72.22 94.67
50 17.67 00.00 80.37 100.00
Control 90.00 100.00 00.00 00.00
L.S.D. at 0.05 for: Growth % of spore germination
Antagonistic bacterial 1.22 1.47
Dilutions (%) 0.55 0.66
Interaction 2.12 2.54
Figure 3 - Effect of Antagonistic Bacillus on growth of F. O. f. sp. cucumerinum (FOC). B.Sut. = Bacillus subtilis, B.M. = Bacillus megtela, B. spp. = Bacillus spp. And (1, 2, 3) number of isolate
64
Figure 4 - Effect of Antagonistic Pseudomonas and Serratia on growth of F. oxysporum f. sp.
Cucumerinum (FOC). PF.= Pseudomonas fluorescens and S. = S.erratia marcensens, PP= Pseudomonas putida and (1, 2,3) number of
isolate.
3.3.5 Effect of different resistant inducing chemicals on the linear growth
and spore germination of F. O. f.sp. cucumerinum (FOC) in vitro.
As for linear growth, the data in Table 8 and Figure 5 illustrate that, the linear
growth of FOC was significantly decreased by most tested chemical inducers.
The obtained results revealed that, all chemicals under study decreased the linear
growth and spores germination of FOC with different degrees. Oxalic acid at
concentration 10 mM completely inhibited mycelial growth of FOC followed by
oxalic acid at concentration 5 mM, salicylic acid and ascorbic acid at concentration of
10 mM reduced the linear growth of FOC by 75.92, 62.59 and 61.86% respectively.
On the other hand, Citric acid at concentration of 2.5 mM was the lowest effective one
and reduced the growth with (3.33%).
Salicylic acid, oxalic acid citric acid and Ascorbic acid at concentration 5 and 10
mM completely inhibited spore germination of FOC. Also KMnO4 at all
concentrations, (K2HPO4) at 100 and 200mM, cobalt sulphate at 10 ppm and calcium
sulphate at 5000 ppm completely inhibited spore germination of FOC.
Table 8 - Effect of some chemicals on growth and spore germination of the F. O.
f.sp. cucumerinum (FOC) in vitro.
Tested chemicals
compound Concentration
Growth (mm)
% Of spore germination
% Efficacy
% Growth % Spore
germination
Salicylic acid
2.5 mM 90.00 11.33 00.00 88.67
5.0 mM 52.33 00.00 41.86 100.00
10 mM 33.67 00.00 62.59 100.00
Oxalic acid
2.5 mM 80.67 14.33 10.37 85.67
5.0 mM 21.67 00.00 75.92 100.00
10 mM 00.00 00.00 100 100.00
Citric acid
2.5 mM 87.00 11.67 3.33 88.33
5.0 mM 49.00 00.00 45.56 100.00
10 mM 40.33 00.00 55.19 100.00
Ascorbic acid
2.5 mM 65.67 16.50 27.33 83.50
5.0 mM 45.67 00.00 49.25 100.00
10 mM 34.33 00.00 61.86 100.00
Dipotassium
hydrogen phosphate
(K2HPO4)
50 mM 79.33 13.00 11.86 87.00
100 mM 79.00 00.00 12.22 100.00
200 mM 74.33 00.00 17.41 100.00
Cobalt sulphate
(CoSO4)
1 ppm 80.33 15.30 10.74 84.70
5 ppm 76.67 10.33 14.81 89.67
10 ppm 73.33 00.00 18.52 100.00
Calcium sulphate
(CaSO4)
1000 ppm 90.00 15.43 00.00 84.57
2500 ppm 90.00 13.33 00.00 86.67
5000 ppm 74.00 00.00 17.78 100.00
Potassium 1000 ppm 86.33 00.00 4.08 100.00
65
Permanganate
(KMnO4) 2500 ppm 73.33 00.00 18.52 100.00
5000 ppm 70.00 00.00 22.22 100.00
Control 90.00 100.00 00.00 00.00
L.S.D. at 0.05 for: Growth % of spore germination
Tested compound 2.11 0.42
Dilutions (%) 1.22 0.24
Interaction 3.66 0.72
Figure 5 - Effect of some chemicals on growth of the F. O. f.sp. cucumerinum (FOC) in vitro. Cit= Citric acid, As= Ascorbic acid, Sa = Salicylic acid , Ox= Oxalic acid, C= Control, 2= 5.0 mM and 3= 10 mM.
3.4 Greenhouse experiments
3.4.1 Effect of treating cucumber seeds with some antagonistic fungi on
incidence with Fusarium wilt disease:
Ten antagonistic fungi were used in this experiment to study their effect on
disease severity of wilt pathogens on cucumber cultivars (Sina1) and the results are
tabulated in Table 9 and Figure 6.
It is clear that all tested antagonistic fungi were effective in reducing disease
severity compared to the control. T. harzianum No.3, Trichoderma spp. and
Ch.bostrycoides were the best isolates and reduced disease severity by 93.00, 92.33
and 90.00% respectively. In the other hand T. viride No.2 was the lowest effective
one and reduced disease severity by 66.67%.
Table 9 - Effect of cucumber seeds treatment with cell suspension of isolates of
antagonistic fungi on incidence with Fusarium wilt disease.
Tested antagonistic fungi Disease severity % % Efficacy T.harzianum 1 23.67 76.33
T. harzianum 2 18.33 81.67
T. harzianum 3 7.00 93.00
T. viride 1 14.33 85.67
T. viride 2 33.33 66.67
66
Trichoderma spp. 7.67 92.33
Ch. bostrycoides 10.00 90.00
Ch. globosum 30.00 70.00
Cheatomium spp. 26.67 73.33
Penicillium spp. 16.67 83.33
Control 100.00 00.00
L.S.D. at 5% 3.77
Figure 6 - Effect of cucumber seeds treatment with cell suspension of isolates of antagonistic fungi on
incidence with Fusarium wilt disease. T.V = Trichoderma viride1, T.H.= Trichoderma harzianum3, T. ssp. = Trichoderma spp., C.B.= Cheatomium
bostrycoides, C. spp. = Cheatomium spp. and P. Spp. = Penicillium spp.
3.4.2 Effect of cucumber seeds treatment with cell suspension of
antagonistic bacterial isolates on incidence with Fusarium wilt.
Fourteen antagonistic bacterial isolates were used in this experiment to study
their effect on disease severity with Fusarium wilt pathogen on cucumber cultivars
(Sina1) and the results are presented in Table 10 and Figure 7.
67
It is clear that all tested antagonistic bacterial isolates were effective in reducing
disease severity compared to the control. B.megtla, Pseudomonas fluorescens No.3
and S. marcensens No.2 were the best isolates and completely prevented the disease
incidence. Serratia marcensens No.1 B. subtilis No. 2 and Pseudomonas fluorescens
No. 2 cam next and reduced disease severity by (96.67, 93.33 and 93.13%) On the
other hand, Bacillus spp. No. 3 was the least effective isolates and reduced the
disease severity by 66.67%.
Table 10 - Effect of cucumber seeds treatment with cell suspension of isolates
of antagonistic bacteria on incidence with Fusarium wilt disease.
Antagonistic bacterial
isolates Disease severity % % Efficacy
B. subtilis 1 13.33 86.67
B. subtilis 2 6.67 93.33
B. subtilis 3 26.67 73.33
B. megtela 00.00 100
Bacillus spp. 1 11.67 88.33
Bacillus spp. 2 8.33 91.67
Bacillus spp. 3 33.33 66.67
Bacillus spp. 4 11.00 89.00
P. fluorescens 1 10.00 90.00
P. fluorescens 2 6.87 93.13
P. fluorescens 3 00.00 100
P. putida 16.67 83.33
S.marcensens 1 3.33 96.67
S. marcensens 2 00.00 100
Control 100.00 00.00
L.S.D. at 5% 3.30
3.4.3 Effect of treating cucumber seeds or treating soil with some resistance
inducing chemicals on incidence with Fusarium wilt.
In this study 8 chemical compounds (salicylic acid, oxalic acid, citric acid,
ascorbic acid, K2HPO4, CoSO4, CaSO4 and KMnO4) each with 3 concentrations were
used to test their efficacy in reducing disease incidence and disease severity of
cucumber plants caused by Fusarium wilt. The obtained results are presented in Table
11 and Figure 8. The obtained results show that, in general, both disease incidence
and disease severity of Fusarium wilt were reduced as a result of treatment by all
chemical compounds compared to the control. Percentage of disease incidence and
disease severity was decreased by increasing the concentration of tested chemicals
compound. In all cases, Salicylic acid and CaSO4 was the most effective compound
on disease development as it reduced the percentages of disease severity in addition
salicylic acid at 10 mM and CaSO4 at 2500 and 5000 ppm completely prevented the
disease followed by KMnO4 at 5000 ppm and CaSO4 at 1000 ppm which reduced the
disease severity by 97.00 and 96.67% respectively.
68
On the other hand, oxalic acid and citric acid at 2.5mM reduced the disease
severity by 80.00 and 76.33%.respectively.
Figure 7 - Effect of treating cucumber seeds with cell suspension of antagonistic bacteria
isolates on incidence with Fusarium wilt disease.
69
B.S. = B. subtilis, B. Spp. = Bacillus spp., B.M. = B. megtela, P. F. = P. fluorescens and S. M. = S.marcensens and
(1,2,3) number of isolate
Table 11 - Effect of cucumber seeds treatment with some tested chemical
compounds on incidence with Fusarium wilt.
Tested chemical
compounds Concentration Disease severity% Efficacy %
Salicylic acid
2.5 mM 16.67 83.33
5.0 mM 10.00 90.00
10 mM 00.00 100
Oxalic acid
2.5 mM 20.00 80.00
5.0 mM 11.67 88.33
10 mM 5.00 95.00
Citric acid
2.5 mM 23.33 76.67
5.0 mM 13.63 86.37
10 mM 8.67 91.32
Ascorbic acid
2.5 mM 13.33 86.67
5.0 mM 12.00 88.00
10 mM 8.83 91.17 Dipotassium
hydrogen
phosphate
(K2HPO4)
50 mM 9.00 91.00
100 mM 7.00 93.00
200 mM 4.33 95.67
Cobalt sulphate
(CoSO4)
1 ppm 10.00 90.00
5 ppm 7.00 93.00
10 ppm 4.67 95.33
Calcium sulphate
(CaSO4)
1000 ppm 3.33 96.67
2500 ppm 00.00 100
5000 ppm 00.00 100
Potassium
Permanganate
(KMnO4)
1000 ppm 5.35 84.65
2500 ppm 4.23 95.77
5000 ppm 3.00 97.00
Control 100 00.00
Chemicals Concentration Interaction
L.S.D. at 0.05 for: 1.22 1.05 3.67
70
Figure 8 - Effect of treating cucumber seeds with tested chemicals compound on incidence with
Fusarium wilt. Sa = Salicylic acid , Ox= Oxalic acid and CO = CoSO4.
3.5 Experiments of Commercial protected house:
3.5.1 Effect of cucumber seeds treatment with some antagonistic fungi on
incidence with Fusarium wilt disease under commercial3 protected house:
In these experiments ten antagonists' fungal isolates were used to study their
effect on controlling cucumber wilt disease on two successive seasons (spring 2009
and autumn 2009).
The obtained results in Table 12 and Figure 9 show that all tested antagonistic
fungi significantly reduced the disease severity of Fusarium wilt disease and
increased plants yield. In this respect, T. harzianum No.3, Trichoderma spp. and T.
viride No.1 were the best isolates and reduced disease severity by 90.27, 89.83 and
87.73% respectively. In the other hand Cheatomium spp. was the lowest effective one
71
and reduced disease severity by 81.72%. Also, all tested treatments increased the fruit
weight Kg//plant. The highest increase in fruit weight Kg//plant was induced by
T.harzianum No.3, Trichoderma spp. and T. viride No.1, which recorded 344.23,
336.54 and 320.19% respectively. Whereas Cheatomium spp. was the lowest
effective one and the increase for fruit weight Kg//plant recorded, 153.85%.
Table 12 - Effect of cucumber seeds treatment with cell suspension of
antagonistic fungal isolates on incidence with Fusarium wilt disease.
%Efficacy Mean Experiment 2
(autumn 2009)
Experiment 1
(spring 2009) Treatment
Av
era
ge
fru
its
wei
gh
t (
Kg
)/p
lan
t
%D
isea
se
sever
ity
Av
era
ge
fru
its
wei
gh
t (
Kg
)/p
lan
t
%D
isea
se
sever
ity
Av
era
ge
fru
its
wei
gh
t (
Kg
)/p
lan
t
%D
isea
se
sever
ity
Av
era
ge
fru
its
wei
gh
t (
Kg
)/p
lan
t
%D
isea
se
sever
ity
278.85 -86.45 3.94 11.12 4.25 11.00 3.63 11.24 T.harzianum 1
294.23 -87.46 4.10 10.29 4.41 10.90 3.78 10.95 T.harzianum 2
344.23 -90.27 4.62 7.98 4.94 8.10 4.30 7.86 T.harzianum 3
320.19 -87.73 4.37 10.07 4.63 9.92 4.10 10 .21 T.viride 1
277.88 -85.45 3.93 11.94 4.20 11.71 3.65 12.17 T. viride 2
336.54 -89.83 4.54 8.34 4.88 8.31 4.20 8.40 Trichoderma spp.
261.54 -84.62 3.76 12.62 4.15 12.03 3.36 13.21 Ch. bostrycoides
193.27 -82.90 3.05 14.03 3.78 13.92 2.31 14.14 Ch. globosum
153.85 -81.72 2.64 15.00 3.18 14.76 2.10 15.25 Cheatomium spp.
248.08 -83.48 3.62 13.55 4.09 13.18 3.15 13.91 Penicillium spp.
00.00 00.00 1.04 82.04 1.32 80.76 0.75 83.33 Control
L.S.D. at 0.05 for: Spring 2009 Autumn 2009
Disease severity 0.98 0.52
Average fruits weight/plant 0.83 1.14
Figure 9 - Samples of plants that treated with antagonistic fungi comparing with control.
72
3.5.2 Effect of treating cucumber seeds with cell suspension antagonistic
bacterial isolates on incidence of Fusarium wilt disease under commercial
protected house:
In these experiments fourteen antagonistic bacterial isolates were used to study
their effect on controlling wilt disease on tow successive seasons (spring 2009,
autumn 2009). The obtained results are presented in Table 13 and Figure 10.
Table 13 - Effect of cucumber seeds treatment with cell suspension of
antagonistic bacterial isolates on incidence with Fusarium wilt disease.
% Efficacy Mean Experiment 2
(autumn 2009)
Experiment 1
(spring 2009)
Treatment
Av
era
ge
fru
its
wei
gh
t
( K
g)/
pla
nt
%D
isea
se
sever
ity
Av
era
ge
fru
its
wei
gh
t
( K
g)/
pla
nt
%D
isea
se
sever
ity
Av
era
ge
fru
its
wei
gh
t
( K
g)/
pla
nt
%D
isea
se
sever
ity
Av
era
ge
fru
its
wei
gh
t
( K
g)/
pla
nt
%D
isea
se
sever
ity
241.35 -86.68 3.55 10.93 3.72 10.72 3.37 11.13 B.subtilis 1
304.81 -88.77 4.21 9.21 4.42 9.16 4.00 9.52 B. subtilis 2
207.69 -84.79 3.20 12.48 3.40 12.19 3.00 12.76 B. subtilis 3
350.00 100 4.68 00.00 4.90 00.00 4.46 00.00 B. megtela
255.77 -87.08 3.70 10.60 3.85 10.32 3.54 10.87 Bacillus spp. 1
292.31 -88.44 4.08 9.48 4.25 9.33 3.90 9.63 Bacillus spp. 2
188.46 -83.68 3.00 13.39 3.15 13.2 2.84 13.57 Bacillus spp. 3
270.19 -87.36 3.85 10.37 4.00 10.15 3.70 10.59 Bacillus spp. 4
277.89 -87.86 3.93 9.96 4.10 9.81 3.75 10.10 P. fluorescens 1
313.46 -89.44 4.30 8.66 4.50 8.41 4.1 8.90 P. fluorescens 2
333.65 90.96 4.51 7.42 4.73 7.34 4.29 7.50 P. fluorescens 3
223.08 -85.79 3.36 11.66 3.55 11.56 3.16 11.76 P. putida
321.15 -89.79 4.38 8.38 4.60 8.20 4.15 8.56 S.marcensens 1
342.31 -91.37 4.60 7.08 4.85 7.00 4.35 7.15 S. marcensens 2
00.00 00.00 1.04 82.04 1.32 80.76 0.75 83.33 Control
L.S.D. at 0.05 for: Spring 2009 Autumn 2009
Disease severity 0.65 0.58
Average fruits weight/plant 0.98 1.08
It is clear that all tested antagonistic bacterial isolates were effective in reducing
disease severity compared to the control. B. megtla was the best isolates and
completely prevented the disease incidence. S. marcensens No.2 and P. fluorescens
73
No.3 were the best isolates and reduced disease severity by 91.37 and 90.67%
respectively. S. marcensens No.1, P. fluorescens No.2 and B. subtilis No.2 came next
and reducing disease severity by 89.79, 89.44 and 88.77 % respectively. On the other
hand, Bacillus spp. No.3 was the least effective isolate and reducing the disease
severity by 83.68%.
Also, all tested treatments increased the fruit weight Kg//plant. The highest
increased in fruit weight Kg//plant was induced by B. megtla, S. marcensens No.2
and P.fluorescens No.3 by 350.00, 342.31 and 333.65 % respectively. Bacillus spp.
No. 3 was the least effective and increased fruit weight Kg//plant by 188.46%.
Figure 10 - Samples of plants that treated with antagonistic bacteria comparing with control.
3.5.3 Effect of treating cucumber seeds or treating soil with some resistance
inducing chemicals on incidence with Fusarium wilt disease.
In this study 8 chemical compounds (salicylic acid, oxalic acid, citric acid,
ascorbic acid, K2HPO4, CoSO4, CaSO4 and KMnO4) were used to test their efficacy
on controlling wilt disease on two successive seasons (spring 2009, autumn 2009).
The obtained results are presented in Table 14 and Figure 11.
The obtained results showed that, in general, both disease incidence and disease
severity of Fusarium wilt disease were reduced as a result of treatment by all
chemical compounds compared to the control. In all cases, salicylic acid and CaSO4
was the most effective compound on disease development as it reduced the
percentages of disease severity in addition salicylic acid completely prevented the
disease followed by CaSO4 and KMnO4 their reducing the disease severity by 93.24
and 92.41% respectively. On the other hand, Ascorbic acid was the least effective and
reducing the disease severity by 85.35%.
Also, all tested treatments increased the fruit weight Kg//plant. The highest
increase in fruit weight Kg//plant was induced by salicylic acid, CaSO4 and KMnO4
where they increased fruit weight Kg//plant by 343.27, 330.77 and 311.54%
respectively. Whereas ascorbic acid was the least effective and increased fruit weight
Kg//plant by 204.39%.
74
Table 14 - Effect of cucumber seeds treatment with tested chemical compounds
on incidence with Fusarium wilt disease.
% Efficacy Mean Experiment 2
(autumn 2009)
Experiment 1
(spring 2009)
Treatment
Av
era
ge
fru
its
wei
gh
t
(Kg
)/p
lan
t
%D
isea
se
sever
ity
Av
era
ge
fru
its
wei
gh
t
(Kg
)/p
lan
t
%D
isea
se
sever
ity
Av
era
ge
fru
its
wei
gh
t
(Kg
)/p
lan
t
%D
isea
se
sever
ity
Av
era
ge
fru
its
wei
gh
t
(Kg
)/p
lan
t
%D
isea
se
sever
ity
343.27 -100 4.61 00.00 4.88 0 4.34 0 Salicylic acid
269.23 - 89.53 3.84 8.59 4.15 7.80 3.52 9.37 Oxalic acid
240.39 - 88.40 3.54 9.52 3.74 8.52 3.33 10.51 Citric acid
204.81 - 85.35 3.17 12.02 3.34 10.92 3.00 13.11 Ascorbic acid
299.04 - 90.94 4.15 7.43 4.45 6.92 3.80 7.94 Dipotassium hydrogen
phosphate (K2HPO4)
282.69 - 90.35 3.98 7.92 4.30 7.30 3.65 8.53 Cobalt sulphate (CoSO4)
330.77 - 93.24 4.48 5.55 4.75 5.50 4.20 5.60 Calcium sulphate (CaSO4)
311.54 - 92.41 4.28 6.23 4.60 6.16 3.95 6.27 Potassium permanganate
(KMnO4)
00.00 00.00 1.04 82.04 1.32 80.76 0.75 83.33 Control
L.S.D. at 0.05 for: Spring 2009 Autumn 2009
Disease severity 0.94 0.67
Average fruits weight/plant 1.27 0.79
Figure 11 - Samples of plants that treated with chemical compounds comparing with control.
75
3.6 Determination of enzymes activity, lignin content and peroxidase
isozyme:
3.6.1 Effect of treating cucumber seeds with spore suspension of
antagonistic fungus isolates in peroxidase activity in cucumber plants:
The results in Table 15 and Figure 12 reveal that, all treatments significantly
increased peroxidase activity compared with control treatment in all times. The
highest activity of peroxidase was induced after 40 days by T. harzianum 3
(2628.57%) followed by Trichoderma spp. and Ch. bostrycoides their increased
peroxidase activity by (2128.57 and 2000.00%) respectively. While, T.viride1 was
the least effective and increased peroxidase activity by 114.28%. Whereas After 50
days T. harzianum 3 and Trichoderma spp. induced the highest activity of peroxidase
(554.54 and 428.40 %) respectively. followed by T. viride1 that increased peroxidase
activity by 396.02%. While, T. viride2 was the least effective and increased
peroxidase activity by 147.15%.
Table 15 - Effect of treating cucumber seeds with spore suspension of
antagonistic fungus isolates in peroxidase activity in cucumber plants as optical
density at 425nm/g fresh wight/15min.
Treatment Peroxidase activity % Efficacy
After 40 days After 50 days After 40 days After 50 days
T.harzianum 1 1.89 6.84 800.00 288.63
T.harzianum 2 3.30 7.50 1471.42 326.13
T. harzianum 3 5.73 11.52 2628.57 554.54
T. viride 1 0.45 8.73 114.28 396.02
T. viride 2 3.06 4.35 1357.14 147.15
Trichoderma spp. 4.68 9.3 2128.57 428.40
Ch. bostrycoides 4.41 7.65 2000.00 334.65
Ch. globosum 1.80 5.85 757.14 217.04
Cheatomium spp. 4.05 7.68 1828.57 336.36
Penicillium spp. 2.85 5.55 1257.14 215.34
Non-infested control with FOC 0.93 1.63 342.86 -7.39
Infested control with FOC 0.21 1.76 00.00 00.00
0
2
4
6
8
10
12
After 40 days After 50 days
T.harzianum 1
T.harzianum 2
T. harzianum 3
T. viride 1
T. viride 2
Trichoderma spp.
Ch. bostrycoides
Ch. globosum
Cheatomium spp.
Penicillium spp.
Non-infested control with FOC
Infested control with FOC
Figure 12 - Effect of treating cucumber seeds with spore suspension of antagonistic fungus isolates in
peroxidase activity in cucumber plants as optical density at 425nm/g fresh wight/15min.
76
3.6.2 Effect of treating cucumber seeds with cell suspension of antagonistic
bacterial isolates in peroxidase activity in cucumber plants:
The results in Table 16 and Figure 13 show that, all bacterial isolates
significantly increased peroxidase activity compared with control in all times. After
40 days Bacillus megtela, Pseudomonas fluorescens3 and Serratia marcensens1 and
increased peroxidase activity by 3357.14, 2885.71 and 2528.57% respectively. On the
other hand, Bacillus spp.4 was the least effective and increased peroxidase activity by
300.00%. While after 50 days Bacillus megtela and Serratia marcensens2 induced
the highest activity of peroxidase (460.79 and 348.29%) respectively, followed by
Pseudomonas fluorescens3 that increased peroxidase activity by 348.28%. While,
Bacillus spp.3 was the least effective and increased peroxidase activity by 2.27%.
Table 16 - Effect of treating cucumber seeds with cell suspension of antagonistic
bacterial isolates in peroxidase activity in cucumber plants as optical density at
425nm/g fresh wight/15min.
Treatment Peroxidase activity % Efficacy After 40 days After 50 days After 40 days After 50 days
B.subtilis 1 1.50 5.61 614.28 218.75
B.s subtilis 2 1.71 7.35 714.28 317.61
B. subtilis 3 0.95 3.99 352.38 126.70
B. megtela 7.26 9.87 3357.14 460.79
Bacillus spp. 1 1.56 5.73 642.85 225.56
Bacillus spp. 2 4.71 6.60 2142.82 275.00
Bacillus spp. 3 1.77 1.80 742.85 2.27
Bacillus spp. 4 4.08 5.04 300.00 186.36
P. fluorescens 1 4.29 5.67 1942.85 222.15
P. fluorescens 2 1.95 6.60 828.57 275.00
P.fluorescens 3 6.27 7.89 2885.71 348.28
P. putida 0.98 2.13 366.66 21.02
S. marcensens 1 5.52 7.56 2528.57 329.45
S. marcensens 2 2.28 7.89 985.71 348.29
Non-infested control with FOC 0.93 1.63 342.86 -7.39
Infested control with FOC 0.21 1.76 00.00 00.00
0
1
2
3
4
5
6
7
8
9
10
After 40 days After 50 days
B.subtilis 1
B.s subtilis 2
B. subtilis 3
B. megtela
Bacillus spp. 1
Bacillus spp. 2
Bacillus spp. 3
Bacillus spp. 4
P. fluorescens 1
P. fluorescens 2
P.fluorescens 3
P. putida
S. marcensens 1
S. marcensens 2
Non-infested control with FOC
Infested control with FOC
Figure 13 - Effect of treating cucumber seeds with cell suspension of antagonistic bacterial isolates in peroxidase
activity in cucumber plants as optical density at 425nm/g fresh wight/15min.
77
3.6.3 Effect of treating cucumber seeds with tested chemical compounds on
peroxidase activity in cucumber plants:
The results in Table 17 and Figure 14 reveal that, all chemicals compound
significantly increased peroxidase activity compared with control in all times. Cobalt
sulphate (CoSO4), salicylic acid and dipotassium hydrogen phosphate (K2HPO4) were
the best treatments and increased peroxidase activity after 40 and 50 days by
(3442.85 and 370.45), (3428.57 and 368.75) and (3042.85 and 360.22) respectively.
After 40 days potassium permanganate (KMnO4) was the least effective compound
and increased peroxidase activity by 614.28%. While After 50 days Citric acid was
the least effective compound and increased peroxidase activity by 109.65%.
Table 17 - Effect of treating cucumber seeds with tested chemical compounds in
peroxidase activity in cucumber plants as optical density at 425nm/g fresh
wight/15min.
Treatments Peroxidase activity % Efficacy
After 40
days
After
50 days
After 40
days
After
50 days
Salicylic acid 7.41 8.25 3428.57 368.75
Oxalic acid 5.16 6.00 2357.14 240.90
Citric acid 2.55 3.69 1114.28 109.65
Ascorbic acid 2.10 6.84 900.00 288.63
Dipotassium hydrogen phosphate
(K2HPO4) 6.60 8.10 3042.85 360.22
Cobalt sulphate (CoSO4) 7.44 8.28 3442.85 370.45
Calcium sulphate (CaSO4) 5.79 7.68 2657.14 336.36
Potassium Permanganate (KMnO4) 1.50 3.93 614.28 123.29
Non-infested control with FOC 0.93 1.63 342.86 -7.39
Infested control with FOC 0.21 1.76 00.00 00.00
0
1
2
3
4
5
6
7
8
9
After 40 days After 50 days
Salicylic acid
Oxalic acid
Citric acid
Ascorbic acid
Dipotassium hydrogen phosphate
(K2HPO4)Cobalt sulphate (CoSO4)
Calcium sulphate (CaSO4)
Potassium Permanganate
(KMnO4)Non-infested control with FOC
Infested control with FOC
Figure 14 - Effect of treating cucumber seeds with tested chemical compounds in peroxidase
activity in cucumber plants as optical density at 425nm/g fresh wight/15min.
78
3.6.4 Effect of treating cucumber seeds with spore suspension of
antagonistic fungus isolates in Polyphenol-oxidase activity in cucumber plants
The results in Table 18 and Figure 15 reveal that, all antagonistic fungi
significantly increased polyphenol-oxidase activity compared with control treatment
in all times. The highest activity of polyphenol-oxidase was induced after 40 days by
T. harzianum3 (90.38%) followed by T. viride1 and Trichoderma spp. where they
increased polyphenol-oxidase activity by (58.97and 47.43%) respectively. In this
respect, Ch. bostrycoides was the least effective that increased Polyphenol-oxidase
activity by 7.69%. Whereas, after 50 days T.harzianum3 and T.viride1 induced the
highest activity of polyphenol-oxidase (79.88 and 69.15%) respectively followed by
Trichoderma spp. that increased polyphenol-oxidase activity by 65.58%. while T.
viride2 was the least effective and increased polyphenol-oxidase activity by 14.36%.
Table 18 - Effect of cucumber seeds treatment with spore suspension of
antagonistic fungi isolates on Polyphenol-oxidase activity in cucumber plants as
optical density 480nm/g fresh wight/15min.
Treatment
Polyphenol-oxidase
activity % Efficacy
After 40 days After 50 days After 40 days After 50 days
T.harzianum 1 16.20 19.08 15.38 26.27
T.harzianum 2 18.30 21.24 30.34 40.56
T.harzianum 3 26.73 27.18 90.38 79.88
T. viride 1 22.32 25.56 58.97 69.15
T. viride 2 16.56 17.28 17.94 14.36
Trichoderma spp. 20.70 25.02 47.43 65.58
Ch.bostrycoides 15.12 19.62 7.69 29.84
Ch. globosum 18.54 19.89 32.05 31.63
Cheatomium spp. 15.80 17.46 12.54 15.55
Penicillium spp. 17.10 18.54 21.79 22.70
Non-infested control with FOC 8.64 19.08 -38.46 26.72
Infested control with FOC 14.04 15.11 00.00 00.00
0
5
10
15
20
25
30
After 40 days After 50 days
T.harzianum 1
T.harzianum 2
T.harzianum 3
T. viride 1
T. viride 2
Trichoderma spp.
Ch.bostrycoides
Ch. globosum
Cheatomium spp.
Penicillium spp.
Non-infested control with FOC
Infested control with FOC
Figure 15 - Effect of cucumber seeds treatment with spore suspension of antagonistic fungi isolates
on Polyphenol-oxidase activity in cucumber plants as optical density 480nm/g fresh wight/15min.
79
3.6.5 Effect of treating cucumber seeds with cell suspension of antagonistic
bacteria isolates in polyphenol-oxidase activity in cucumber plants:
The results in Table 19 and Figure 16 reveal that, all antagonistic bacteria
significantly increased polyphenol-oxidase activity compared with control treatment
in all times. The highest activity of polyphenol-oxidase was induced after 40 days by
Bacillus spp.3 (43.58%) followed by B. megtela and P. fluorescens3 their increased
polyphenol-oxidase activity by (34.61 and 34.60%) respectively. While B. subtilis1
was the least effective and increased polyphenol-oxidase activity by 4.69%. Whereas
After 50 days Bacillus spp.3 and B. megtela induced the highest activity of
polyphenol-oxidase (135.27and 72.73%) respectively. followed by P. fluorescens3
that increased polyphenol-oxidase activity by 60.82%. While P. fluorescens2 was the
least effective and increased polyphenol-oxidase activity by 3.11%.
Table 19 - Effect of treatment of cucumber seeds with cell suspension of
antagonistic bacteria isolates in polyphenol-oxidase activity in cucumber plants
as optical density 480nm/g fresh wight/15min.
Treatment Polyphenol-oxidase activity % Efficacy
After 40 days After 50 days After 40 days After 50 days
B.subtilis 1 15.12 21.87 4.69 44.73
B.subtilis 2 15.66 18.36 11.53 21.51
B. subtilis 3 15.26 16.58 8.69 9.73
B. megtela 18.90 26.10 34.61 72.73
Bacillus spp. 1 15.25 22.41 8.62 48.31
Bacillus spp. 2 15.86 18.90 12.96 25.08
Bacillus spp. 3 20.16 35.55 43.58 135.27
Bacillus spp. 4 17.41 15.84 23.93 4.83
P. fluorescens 1 16.48 16.80 17.38 11.18
P. fluorescens 2 15.16 15.58 7.98 3.11
P. fluorescens 3 18.89 24.30 34.60 60.82
P. putida 16.12 21.33 14.81 41.16
S. marcensens 1 16.70 16.31 18.95 7.94
S. marcensens 2 16.30 22.50 16.10 48.90
Non-infested control with FOC 8.64 19.08 -38.46 26.72
Infested control with FOC 14.04 15.11 00.00 00.00
0
5
10
15
20
25
30
35
40
After 40 days After 50 days
B.subtilis 1
B.subtilis 2
B. subtilis 3
B. megtela
Bacillus spp. 1
Bacillus spp. 2
Bacillus spp. 3
Bacillus spp. 4
P. fluorescens 1
P. fluorescens 2
P. fluorescens 3
P. putida
S. marcensens 1
S. marcensens 2
Non-infested control with FOC
Infested control with FOC
Figure 16 - Effect of treatment of cucumber seeds with cell suspension of antagonistic bacteria isolates
in polyphenol-oxidase activity in cucumber plants as optical density 480nm/g fresh wight/15min.
80
3.6.6 Effect of treatment of cucumber seeds with tested chemicals
compound in polyphenol-oxidase activity in cucumber plants:
The results in Table 20 and Figure 17 show that, all antagonistic bacteria
significantly increased polyphenol-oxidase activity compared with control treatment
in all times. The highest activity of polyphenol-oxidase was induced after 40 days by
oxalic acid (42.74 %) followed by salicylic acid and citric acid their increased
polyphenol-oxidase activity by (36.89 and 33.05%) respectively. While dipotassium
hydrogen phosphate (K2HPO4) was the least effective and increased polyphenol-
oxidase activity by 1.28%. Whereas After 50 days oxalic acid and salicylic acid
induced the highest activity of polyphenol-oxidase (59.03 and 57.24%) respectively.
followed by potassium permanganate (KMnO4) that increased polyphenol-oxidase
activity by 55.45%. On the other hand, citric acid was the least effective and
increased polyphenol-oxidase activity by 0.46%.
Table 20 - Effect of treatment of cucumber seeds with some chemical
compounds on polyphenol-oxidase activity in cucumber plants as optical density
480nm/g fresh wight/15min.
Treatment Polyphenol-oxidase activity % Efficacy
After 40 days After 50 days After 40
days
After 50
days
Salicylic acid 19.22 23.76 36.89 57.24
Oxalic acid 20.04 24.03 42.74 59.03
Citric acid 18.68 15.18 33.05 0.46
Ascorbic acid 15.12 22.86 7.69 51.29
Dipotassium hydrogen
phosphate (K2HPO4) 14.22 18.90 1.28 25.08
Cobalt sulphate (CoSO4) 16.47 21.96 17.30 45.33
Calcium sulphate (CaSO4) 15.03 16.92 7.05 11.97
Potassium permanganate
(KMnO4) 18.28 23.49 30.20 55.45
Non-infested control with FOC 8.64 19.08 -38.46 26.72
Infested control with FOC 14.04 15.11 00.00 00.00
0
5
10
15
20
25
After 40 days After 50 days
Salicylic acid
Oxalic acid
Citric acid
Ascorbic acid
Dipotassium hydrogen phosphate
(K2HPO4)Cobalt sulphate (CoSO4)
Calcium sulphate (CaSO4)
Potassium permanganate
(KMnO4)Non-infested control with FOC
Infested control with FOC
Figure 17 - Effect of treatment of cucumber seeds with some chemical compounds on polyphenol-oxidase
activity in cucumber plants as optical density 480nm/g fresh wight/15min.
81
3.6.7 Effect of treatment of cucumber seeds with spore suspension of
antagonistic fungal isolates on chitinase activity in cucumber plants:
The results in Table 21 and Figure 18 reveal that, all antagonistic fungi
significantly increased chitinase activity compared with control treatment in all times.
The highest activity of chitinase was induced after 40 days by Penicillium spp.
(311.28%) followed by Ch. globosum and Trichoderma harzianum1 their increased
chitinase activity by (271.20 and 236.34%) respectively. On the other hand,
Cheatomium bostrycoides was the least effective that increased chitinase activity by
84.73%. Whereas After 50 days Penicillium spp. and Ch. globosum induced the
highest activity of chitinase (179.29and 165.54%) respectively. followed by
Cheatomium spp. that increased chitinase activity by 156.02%. While Ch.
bostrycoides was the least effective and increased chitinase activity by 92.54%.
Table 21 - Effect of treatment of cucumber seeds with spore suspension of
antagonistic fungal isolates on chitinase activity in cucumber plants as mM N-
acetylglucose amine equivalent released / gram fresh weigh tissue / 60 minutes.
Treatment Chitinase activity % Efficacy
After 40 days After 50 days After 40 days After 50 days
T.harzianum 1 8.11 9.41 236.34 136.97
T. harzianum 2 6.97 8.69 189.29 118.99
T. harzianum 3 7.25 8.74 200.62 120.05
T. viride 1 6.13 9.14 154.43 129.57
T. viride 2 5.75 7.67 138.75 93.07
Trichoderma spp. 5.65 10.00 134.39 151.78
Ch. bostrycoides 4.45 7.64 84.73 92.54
Ch. globosum 8.95 10.54 271.20 165.54
Cheatomium spp. 7.14 10.16 196.26 156.02
Penicillium spp. 9.91 11.09 311.28 179.29
Non-infested control with FOC 2.58 4.72 7.05 18.89
Infested control with FOC 2.41 3.97 00.00 00.00
0
2
4
6
8
10
12
After 40 days After 50 days
T.harzianum 1
T. harzianum 2
T. harzianum 3
T. viride 1
T. viride 2
Trichoderma spp.
Ch. bostrycoides
Ch. globosum
Cheatomium spp.
Penicillium spp.
Non-infested control with FOC
Infested control with FOC
Figure 18 - Effect of treatment of cucumber seeds with spore suspension of antagonistic fungal isolates on
chitinase activity in cucumber plants as mM N-acetylglucose amine equivalent released / gram fresh weigh
tissue / 60 minutes.
82
3.6.8 Effect of treatment of cucumber seeds with cell suspension of
antagonistic bacterial isolates in chitinase activity in cucumber plants:
The results in Table 22 and Figure 19 reveal that, all antagonistic bacteria
significantly increased chitinase activity compared with control treatment in all times.
The highest activity of chitinase was induced after 40 days by Bacillus megtela
(309.54%) followed by Bacillus spp.3 and Pseudomonas fluorescens3 their increased
chitinase activity by (307.80 and 271.20%) respectively. While Pseudomonas
fluorescens2 was the least effective and increased chitinase activity by 63.81%.
Whereas After 50 days Bacillus megtela and Bacillus subtilis1 induced the highest
activity of chitinase (231.13 and 215.26%) respectively. followed by Bacillus spp.3
that increased chitinase activity by 204.68%. While Pseudomonas fluorescens1 was
the least effective and increased chitinase activity by 33.29%.
Table 22 - Effect of treatment of cucumber seeds with cell suspension of
antagonistic bacterial isolates in chitinase activity in cucumber plants as mM N-
acetylglucose amine equivalent released / gram fresh weigh tissue / 60 minutes.
Treatment Chitinase activity % Efficacy
After 40 days After 50 days After 40 days After 50 days
B. subtilis 1 7.98 12.52 231.12 215.26
B. subtilis 2 5.75 8.06 138.58 103.12
B. subtilis 3 5.29 5.48 119.58 38.06
B. megtela 9.87 13.15 309.54 231.13
Bacillus spp. 1 7.56 11.42 213.69 187.75
Bacillus spp. 2 5.48 6.47 127.42 62.92
Bacillus spp. 3 9.83 12.10 307.80 204.68
Bacillus spp. 4 6.13 7.06 154.43 77.73
P. fluorescens 1 5.10 5.30 111.74 33.29
P. fluorescens 2 3.95 7.56 63.81 90.42
P. fluorescens 3 8.95 11.09 271.20 179.29
P. putida 4.47 11.30 85.60 184.58
S. marcensens 1 7.75 11.55 221.53 190.93
S. marcensens 2 5.57 8.36 130.91 110.52
Non-infested control with FOC 2.58 4.72 7.05 18.89
Infested control with FOC 2.41 3.97 00.00 00.00
0
2
4
6
8
10
12
14
After 40 days After 50 days
B. subtilis 1
B. subtilis 2
B. subtilis 3
B. megtela
Bacillus spp. 1
Bacillus spp. 2
Bacillus spp. 3
Bacillus spp. 4
P. fluorescens 1
P. fluorescens 2
P. fluorescens 3
P. putida
S. marcensens 1
S. marcensens 2
Non-infested control with FOC
Infested control with FOC
Figure 19 - Effect of treatment of cucumber seeds with cell suspension of antagonistic bacterial isolates in
chitinase activity in cucumber plants as mM N-acetylglucose amine equivalent released / gram fresh weigh
tissue / 60 minutes
83
3.6.9 Effect of treatment of cucumber seeds with tested chemical
compounds on chitinase activity in cucumber plants:
The results in Table 23 and Figure 20 show that, all antagonistic bacteria
significantly increased chitinase activity compared with control treatment in all times.
The highest activity of chitinase was induced after 40 days by cobalt sulphate
(CoSO4) (358.34%) followed by oxalic acid and dipotassium hydrogen phosphate
(K2HPO4) their increased chitinase activity by (234.60 and 146.59%) respectively.
On the other hand, citric acid was the least effective and increased chitinase activity
by 76.01%. Whereas After 50 days cobalt sulphate (CoSO4) and salicylic acid
induced the highest activity of chitinase (189.87 and 139.09%) respectively. followed
by oxalic acid that increased chitinase activity by 120.05% while citric acid was the
least effective and increased chitinase activity by 32.77%.
Table 23 - Effect of treatment of cucumber seeds with tested chemical
compounds on chitinase activity in cucumber plants as mM N-acetylglucose
amine equivalent released / gram fresh weigh tissue / 60 minutes.
Treatment Chitinase activity % Efficacy
After 40 days After 50 days After 40 days After 50 days
Salicylic acid 5.25 9.49 117.84 139.09
Oxalic acid 8.06 8.74 234.60 120.05
Citric acid 4.24 5.27 76.01 32.77
Ascorbic acid 5.63 6.99 133.52 76.14
Dipotassium hydrogen
phosphate (K2HPO4) 5.94 6.05 146.59 52.34
Cobalt sulphate (CoSO4) 11.05 11.51 358.34 189.87
Calcium sulphate (CaSO4) 5.36 5.99 122.19 50.75
Potassium Permanganate
(KMnO4) 4.75 6.68 96.92 68.21
Non-infested control with FOC 2.58 4.72 7.05 18.89
Infested control with FOC 2.41 3.97 00.00 00.00
0
2
4
6
8
10
12
After 40 days After 50 days
Salicylic acid
Oxalic acid
Citric acid
Ascorbic acid
Dipotassium hydrogen
phosphate (K2HPO4)Cobalt sulphate (CoSO4)
Calcium sulphate (CaSO4)
Potassium Permanganate
(KMnO4)Non-infested control w ith FOC
Infested control w ith FOC
Figure 20 - Effect of treatment of cucumber seeds with tested chemical compounds on chitinase activity in
cucumber plants as mM N-acetylglucose amine equivalent released / gram fresh weigh tissue / 60 minutes.
84
3.6.10 Effect of treatment of cucumber seeds with spore suspension of
antagonistic fungal isolates on lignin content in cucumber plants:
It is clear from Table 24 and Figure 21 that all tested antagonistic fungi
significantly increased lignin content compared with control treatment. The highest
increase of lignin content was induced by T. harzianum1 (290.78%) followed by Ch.
bostrycoides and T. viride1where the increase of lignin content were (279.07and
268.21%) respectively. On the other hand, Ch. globosum was the least effective and
increased lignin content by 53.12%.
Table 24 - Effect of treatment of cucumber seeds with spore suspension of
antagonistic fungal isolates on lignin content in cucumber plants.
Treatment Lignin weight (mg/1g) % Efficacy
T.harzianum 1 329.00 290.78
T. harzianum 2 148.19 76.01
T. harzianum 3 192.19 128.26
T. viride 1 310.00 268.21
T. viride 2 182.44 116.70
Trichoderma spp. 259.27 207.95
Ch. bostrycoides 319.14 279.07
Ch. globosum 128.92 53.12
Cheatomium spp. 207.44 146.39
Penicillium spp. 277.19 229.24
Non-infested control with FOC 175.77 108.78
Infested control with FOC 84.19 00.00
L.S.D. at 5% 1.81
0
50
100
150
200
250
300
350
Mg
/1g
Lignin weight
T.harzianum 1
T. harzianum 2
T. harzianum 3
T. viride 1
T. viride 2
Trichoderma spp.
Ch. bostrycoides
Ch. globosum
Cheatomium spp.
Penicillium spp.
Non-infested control with FOC
Infested control with FOC
Figure 21 - Effect of treatment of cucumber seeds with spore suspension of antagonistic fungal isolates on
lignin content in cucumber plants.
85
3.6.11 Effect of treatment of cucumber seeds with cell suspension of
antagonistic bacterial isolates on lignin content in cucumber plants:
The results in Table 25 and Figure 22 show that, all tested antagonistic bacteria
significantly increased lignin content compared with control treatment. The highest
increase of lignin content was induced with Bacillus spp.2 (286.82%) followed by B.
subtilis3 and B. subtilis2 which they increased lignin content by (211.12 and
175.56%) respectively. While P. putida was the least effective and increased lignin
content by 7.04%.
Table 25 - Effect of treatment of cucumber seeds with cell suspension of
antagonistic bacterial isolates on lignin content in cucumber plants.
Treatment Lignin weight (mg/1g) % Efficacy B. subtilis 1 164.44 95.32
B. subtilis 2 232.00 175.56
B. subtilis 3 261.94 211.12
B. megtela 173.63 106.23
Bacillus spp. 1 152.38 80.99
Bacillus spp. 2 325.67 286.82
Bacillus spp. 3 203.62 141.85
Bacillus spp. 4 207.75 146.76
P. fluorescens 1 160.07 90.13
P. fluorescens 2 176.31 109.42
P. fluorescens 3 193.31 129.61
P. putida 90.13 7.04
S. marcensens 1 195.25 131.91
S. marcensens 2 127.56 51.51
Non-infested control with FOC 175.77 108.78
Infested control with FOC 84.19 00.00
L.S.D. at 5% 1.89
0
50
100
150
200
250
300
350
Mg
/1g
Lignin weight
B. subtilis 1
B. subtilis 2
B. subtilis 3
B. megtela
Bacillus spp. 1
Bacillus spp. 2
Bacillus spp. 3
Bacillus spp. 4
P. fluorescens 1
P. fluorescens 2
P. fluorescens 3
P. putida
S. marcensens 1
S. marcensens 2
Non-infested control with FOC
Infested control with FOC
Figure 22 - Effect of treatment of cucumber seeds with cell suspension of antagonistic bacterial isolates on
lignin content in cucumber plants.
86
3.6.12 Effect of treatment of cucumber seeds with tested chemical
compounds on lignin content in cucumber plants:
It is clear from Table 26 and Figure 23 that all tested chemical compounds
significantly increased lignin content compared with control treatment. The highest
increase of lignin content was induced with citric acid (290.27%) followed by
salicylic acid and calcium sulphate (CaSO4), they increased lignin content by (288.15
and 245.53%) respectively. While dipotassium hydrogen phosphate (K2HPO4) was
the least effective and increased lignin content by 60.80%.
Table 26 - Effect of treatment of cucumber seeds with tested chemical
compounds on lignin content in cucumber plants.
Treatment Lignin weight (mg/1g) % Efficacy
Salicylic acid 326.78 288.15
Oxalic acid 234.31 178.31
Citric acid 328.57 290.27
Ascorbic acid 252.44 199.84 Dipotassium hydrogen phosphate
(K2HPO4) 135.38 60.80
Cobalt sulphate (CoSO4) 259.56 208.30
Calcium sulphate (CaSO4) 290.91 245.53
Potassium Permanganate (KMnO4) 141.81 68.44
Non-infested control with FOC 175.77 108.78
Infested control with FOC 84.19 00.00 L.S.D. at 5% 1.95
0
50
100
150
200
250
300
350
Mg
/1g
Lignin weight
Salicylic acid
Oxalic acid
Citric acid
Ascorbic acid
Dipotassium hydrogen phosphate
(K2HPO4)Cobalt sulphate (CoSO4)
Calcium sulphate (CaSO4)
Potassium Permanganate (KMnO4)
Non-infested control with FOC
Infested control with FOC
Figure 23 - Effect of treatment of cucumber seeds with tested chemical compounds on lignin content in
cucumber plants.
87
3.6.13 Effect of treatment of cucumber seeds with spore suspension of
antagonistic fungal isolates on isozyme pattern of peroxidase in cucumber
plants:
Two isoforms were detected in all treatments expect Trichoderma harzianum3
non-infested and infested control they produced one isoform. Cluster analysis and
data in Table 27, based on a Euclidian similarity matrix for antagonistic fungi traits
and two main groups, namely antagonistic fungi and the control was conducted.
Prior to this analysis, values obtained for different traits were processed for
standardization, by way of subtraction from a real value of each trait and a following
division to the standard deviation. Descriptive judging by the plot shown in Figure
24. The data confirm certain similarities of non-infested control and infested control
can be placed in the top of the first cluster followed by Ch. bostrycoides; T.
harzianum 3 and Trichoderma spp..
Finally, the last cluster occupied by T. viride1; T.harzianum1 and Penicillium
spp.. The order of clustering depicts C. bostrycoides; T. harzianum3 and Trichoderma
spp. as superior on this trait, which is followed by the T.viride1; T.harzianum1 and
Penicillium spp.. And the last one non-infested control and infested control (Figure
25).
Table 27 - Reference band, Band number, band volume, band area and
Regration fragment of peroxidase isozymes for cucumber samples that treated
with antagonistic fungi.
Rb. Bands
No.
T.harzianum1 T.harzianum3 T. viride1 Trichoderma spp.
Vo. Ar. Rf. Vo. Ar. Rf. Vo. Ar. Rf Vo. Ar. Rf
1 1 119.6 874 0.293 233.8 16721 0.301 162.7 1140 0.282 140.5 988 0.271
2 2 90.5 684 0.323 81.9 608 0.335 97.3 722 0.312
Rb. Bands
No.
Ch.bostrycoides Penicillium spp. Non-infested
Control with FOC
Infested control
with FOC
Vo. Ar. Rf Vo. Ar. Rf Vo. Ar. Rf Vo. Ar. Rf.
1 1 181.8 1254 0.237 177.4 1216 0.259 4.862 32700 0.225 3.716 26596 0.243
2 2 96.7 722 0.301 71.4 532 0.312
88
Figure 24 - Phylogenetic Tree of peroxidase isozymes of the examined cucumber
samples that treated with antagonistic fungi under the assay, non-infested control
with FOC and infested control with FOC.
0 1 2 3 4 5 6 7 8 9
-3
-2.7
-2.4
-2.1
-1.8
-1.5
-1.2
-0.9
-0.6
-0.3
0
Sim
ila
rity
y
С.
bo
stry
co
ides
bo
stry
co
ides
Т.
ha
rzia
nu
m3
Tri
cho
der
ma
sp
p.
T.v
irid
e1
T.h
arz
ian
um
1
1
Pen
icil
liu
m s
pp
.
spp
. N
on
-in
fest
ed
Co
ntr
ol
wit
h F
OC
Infe
sted
co
ntr
ol
wit
h F
OC
FO
C
89
Trichoderma harzianum1 Trichoderma harzianum3
0
Trichoderma viride1 Trichoderma spp.
Cheatomium bostrycoides Penicillium spp.
Non-infested Control with FOC Infested control with FOC
Figure 25 - Band numbers, bands highest in relation to regration fragment of peroxidase isozymes
in the examined cucumber samples that treated with antagonistic fungi under the assay and non-
infested control with FOC & Infested control with FOC.
90
3.6.14 Effect of treatment of cucumber seeds with spore suspension of
antagonistic bacterial isolates on isozyme pattern of peroxidase in cucumber
plants:
Two isoforms were detected in all treatments expect Bacillus spp.1, non-infested
and infested control they produced one isoform. Cluster analysis and data in Table
28, based on a Euclidian similarity matrix for antagonistic bacteria traits and two
main groups, namely antagonistic bacteria and the control was conducted.
Prior to this analysis, values obtained for different traits were processed for
standardization, by way of subtraction from a real value of each trait and a following
division to the standard deviation. Descriptive judging by the plot shown in Figure
26. The data confirm certain similarities of non-infested control and infested control
can be placed in the top of the first cluster followed by B. megtela; P. fluorescens3,
P.putida and S. marcensens2.
Finally, the last cluster occupied by Bacillus spp.1; B. subtilis2; Bacillus spp.2
and P. fluorescens2. The order of clustering depicts B. megtela; P. fluorescens3, P.
putida and S. marcensens2 as superior on this trait, which is followed by the Bacillus
spp.1; B. subtilis2; Bacillus spp.2 and P. fluorescens2. And the last one non-infested
control and infested control (Figure 27).
Table 28 - Reference band, Band number, band volume, band area and
Regration fragment of peroxidase isozymes for cucumber samples that treated
with antagonistic bacteria.
Rb
.
Bands
No.
Bacillus megtela Bacillus spp.1 Bacillus subtilis2 Bacillus spp.2
Vo. Ar. Rf. Vo. Ar. Rf. Vo. Ar. Rf Vo. Ar. Rf
1 1 174 1140 0.256 534 3990 0.233 135 988 0.173 142 950 0.203
2 2 117 836 0.282 518 3686 0.256 415 3078 0.248
Rb. Bands
No.
P. fluorescens3 P.fluorescens2 P. putida S.marcensens2
Vo. Ar. Rf Vo. Ar. Rf Vo. Ar. Rf Vo. Ar. Rf.
1 1 177 1178 0.203 186 1216 0.180 193 1292 0.150 180 1178 0.139
2 2 407 2964 0.248 656 4826 0.244 629 4560 0.244 612 4370 0.222
Rb. Bands
No.
Non-infested
Control with FOC
Infested control
with FOC
Vo. Ar. Rf Vo. Ar. Rf.
1 1 4,862 32700 0.225 3,716 26596 0.243
2 2
91
Figure 26 - Phylogenetic tree of peroxidase isozymes of the examined cucumber
samples that treated with antagonistic bacteria under the assay, non-infested control
with FOC and infested control with FOC.
0 1.2 2.4 3.6 4.8 6 7.2 8.4 9.6 10.8
-5
-4.5
-4
-3.5
-3
-2.5
-2
-1.5
-1
-0.5
Sim
ilari
ty
y
B.
Meg
tela
P.
pu
tid
a
S.
ma
rcen
sen
s2
Ba
cill
us
spp
.1
B.
sub
tili
s2
Ba
cill
us
spp
.2
spp
.2
P.
flu
ore
scen
s2
flu
ore
scen
s2
P.
flu
ore
scen
s3
flu
ore
scen
s3
No
n-i
nfe
sted
Co
ntr
ol
wit
h F
OC
Infe
sted
co
ntr
ol
wit
h F
OC
92
Bacillus megtela Bacillus spp.1 Bacillus subtilis2
Bacillus spp.2 Pseudomonas fluorescens3 Pseudomonas fluorescens2
Pseudomonas putida Serratia marcensens2
Non-infested Control with FOC Infested control with FOC
Figure 27 - Band numbers, bands highest in relation to regration fragment of
peroxides isozymes in the examined cucumber samples that treated with antagonistic
bacteria under the assay and non-infested control with FOC & infested control with
FOC.
93
3.6.15 Effect of treatment of cucumber seeds with tested chemical
compounds on isozyme pattern of peroxidase in cucumber plants:
Two isoforms were detected in all treatments expect oxalic acid, non-infested
and infested control they produced one isoform. Cluster analysis and data in Table
29, based on a Euclidian similarity matrix for chemical inducers traits and two main
groups, namely chemical inducers and the control was conducted.
Prior to this analysis, values obtained for different traits were processed for
standardization, by way of subtraction from a real value of each trait and a following
division to the standard deviation. Descriptive judging by the plot shown in Figure
28. The data confirm certain similarities of non-infested control and infested control
can be placed in the top of the first cluster followed by CoSO4; salicylic acid and
K2HPO4.
Finally, the last cluster occupied by oxalic acid; citric acid; ascorbic acid and
CaSO4. The order of clustering depicts CoSO4; salicylic acid and K2HPO4 as superior
on this trait, which is followed by the oxalic acid; citric acid; ascorbic acid and
CaSO4.and the last one non-infested control and infested control (Figure 29).
Table 29 - Reference band, Band number, band volume, band area and
regration fragment of peroxidase isozymes for cucumber samples that treated
with chemical inducers.
Rb
.
Bands
No.
Salicylic acid Oxalic acid Citric acid
Vo. Ar. Rf. Vo. Ar. Rf. Vo. Ar. Rf
1 1 668 5668 0.206 3,322 28558 0.271 2,671 21582 0.225
2 2 3,505 27904 0.298 1,444 12426 0.309
Rb. Bands
No.
Ascorbic acid K2HPO4 CoSO4
Vo. Ar. Rf. Vo. Ar. Rf. Vo. Ar. Rf
1 1 696 5668 0.197 2,427 18530 0.234 883 6758 0.177
2 2 2.753 21800 0.262 1.528 11990 0.280 3.349 24416 0.271
Rb. Bands
No.
CaSO4 Non-infested control
with FOC
Infested control with
FOC
Vo. Ar. Rf. Vo. Ar. Rf. Vo. Ar. Rf
1 1 3.888 27250 0.168 4.862 32700 0.225 3,716 26596 0.243
2 2 1.130 8502 0.309
94
Figure 28 - Phylogenetic tree of peroxidase isozymes of the examined cucumber samples that
treated with chemical inducers under the assay, non-infested control with FOC and infested control
with FOC.
Salicylic acid Oxalic acid Citric acid
Ascorbic acid K2HPO4 CoSO4
CaSO4 Non-infested control with FOC Infested control with FOC
Figure 29 - Band numbers, bands highest in relation to regration fragment of peroxidase isozymes in the
examined cucumber samples that treated with chemical inducers under the assay and non-infested control
with FOC & infested control with FOC.
0 1 2 3 4 5 6 7 8 9
-4
-3.6
-3.2
-2.8
-2.4
-2
-1.6
-1.2
-0.8
-0.4
0
Sim
ila
rity
y
CoS
O4
Sali
cyli
c aci
d
К2НРО
4
Oxali
c aci
d
Cit
ric
aci
d
Asc
orb
ic a
cid
CaS
O4
No
n-i
nfe
sted
Co
ntr
ol
wit
h
FO
C
Infe
sted
co
ntr
ol
wit
h F
OC
95
3.7 Chemical analysis of cucumber treated plants:
3.7.1 Effect of cucumber seeds treatment with spore suspension of
antagonistic fungal isolates on sugar content in cucumber plants:
It is clear from Table 30 that, all antagonistic fungi under study decreased the
reducing, non- reducing and total sugars content compared with the control. In this
respect, all treatments decreased the reducing sugars. The highest decrease was
induced by Trichoderma spp. (96.92%) followed by T. harzianum1 and Cheatomium
spp. which they decreased the reducing sugars by 94.48 and 93.26%, respectively less
than the control. While T. harzianum2 was the least effective and decreased the
reducing sugars by 76.12% less than the control.
All treatments decreased the non-reducing sugars. The highest decrease was
induced by T. harzianum1 and Penicillium spp. by 88.38 and 84.54% respectively
less than the control followed by T. viride1that decreased the non-reducing sugars by
73.05% less than the control. While Ch. globosum was the least effective and
decreased the non-reducing sugars by 38.44% less than the control.
As for the total sugars, T. harzianum1 and Trichoderma spp. decreased the total
sugars by 93.00 and 88.82% respectively less than the control followed by T.
viride2that decreased the total sugars by 86.04% less than the control. While T.
harzianum2 was the least effective and decreased the total sugars by 69.76% less than
the control.
Table 30 - Effect of cucumber seeds treatment with spore suspension of
antagonistic fungal isolates on sugar content in cucumber plants as mg/1g fresh
weight.
Treatment Reducing
sugar
Non
reducing
sugar
Total
sugar
% Efficacy
Reducing
sugar
Non
reducing
sugar
Total
sugar
T. harzianum 1 1.40 0.94 2.34 -94.48 -88.38 -93.00
T. harzianum 2 6.06 4.05 10.12 -76.12 -49.93 -69.76
T. harzianum 3 2.02 4.05 6.07 -92.04 -49.93 -81.86
T. viride 1 4.05 2.18 6.23 -84.04 -73.05 -81.38
T. viride 2 1.87 2.8 4.67 -92.63 -65.38 -86.04
Trichoderma spp. 0.78 2.96 3.74 -96.92 -63.41 -88.82
Ch. bostrycoides 3.43 2.95 6.38 -86.96 -63.53 -80.93
Ch. globosum 2.80 4.98 7.78 -88.96 -38.44 -76.75
Cheatomium spp. 1.71 3.43 5.14 -93.26 -57.60 -84.64
Penicillium spp. 4.67 1.25 5.92 -81.59 -84.54 -82.31
Non-infested control with
FOC 23.98 8.25 32.23 -5.87 1.94 -3.71
Infested control with FOC 25.38 8.09 33.47 00.00 00.00 00.00
Reducing sugars Non reducing sugars Total sugars
L.S.D. at 5% 0.79 0.51 0.92
96
3.7.2 Effect of treatment of cucumber seeds with cell suspension of
antagonistic bacterial isolates on sugar content in cucumber plants.
The results in Table 31 indicate that, sugars content was significantly affected
by the treatment with antagonistic bacteria. All antagonistic bacteria reduced the
reducing, non- reducing and total sugars content compared with the control except B.
megtela that increased non-reducing by 3.95%. In this respect, all treatments
decreased the reducing sugars. The highest decrease was induced by Bacillus spp.4
(98.15%) followed by B. megtela and Bacillus spp.2that decreased the reducing
sugars by 97.55 and 96.33%, respectively less than the control. While Serratia
marcensens2 was the least effective and decreased the reducing sugars by 76.15%
less than the control.
All treatments decreased the non-reducing sugars. The highest decrease was
induced by S. marcensens2 and S. marcensens1 by 94.19 and 88.50% respectively
less than the control followed by P. fluorescens3 that decreased the non-reducing
sugars by 78.86% less than the control. While Bacillus spp.4 was the least effective
and decreased the non-reducing sugars by 1.85% less than the control. As for the total
sugars, S. marcensens1 and S. marcensens2 decreased the total sugars by 87.89and
82.79% respectively less than the control followed by Bacillus spp.1 and P.
fluorescens3 that decreased the total sugars by 79.53% less than the control. While
Bacillus spp.3 was the least effective and decreased the total sugars by 68.83% less
than the control.
Table 31 - Effect of treatment of cucumber seeds with cell suspension of
antagonistic bacterial isolates on sugar content in cucumber plants as mg/1g
fresh weight.
Treatment Reducing
sugar
Non
reducing
sugar
Total
sugar
% Efficacy
Reducing
sugar
Non
reducing
sugar
Total
sugar
B. subtilis 1 4.36 4.36 8.72 -82.82 -46.10 -73.94
B.subtilis 2 2.49 5.44 7.93 -90.18 -32.75 -76.30
B. subtilis 3 3.27 6.07 9.34 -87.11 -24.96 -72.09
B. megtela 0.62 8.41 9.03 -97.55 3.95 -73.02
Bacillus spp. 1 2.18 4.67 6.85 -91.41 -42.27 -79.53
Bacillus spp. 2 0.93 6.23 7.16 -96.33 -22.99 -78.60
Bacillus spp. 3 2.96 7.47 10.43 -88.33 -7.66 -68.83
Bacillus spp. 4 0.47 7.94 8.41 -98.15 -1.85 -74.87
P. fluorescens 1 1.87 6.23 8.1 -92.63 -22.99 -75.79
P.fluorescens 2 2.49 4.98 7.47 -90.18 -38.44 -77.68
P. fluorescens 3 5.14 1.71 6.85 -79.74 -78.86 -79.53
P. putida 2.34 5.44 7.78 -90.78 -32.75 -76.75
S. marcensens 1 3.12 0.93 4.05 -87.70 -88.50 -87.89
S. marcensens 2 5.29 0.47 5.76 -79.15 -94.19 -82.79
Non-infested control with FOC 23.98 8.25 32.23 -5.87 1.94 -3.71
Infested control with FOC 25.38 8.09 33.47 00.00 00.00 00.00 Reducing sugars Non reducing sugars Total sugars
L.S.D. at 5% 0.87 0.58 0.99
97
3.7.3 Effect of treatment of cucumber seeds with tested chemicals
compound on sugar content in cucumber plants.
The results in Table 32 indicate that, sugars content was significantly affected
by the treatment with chemical compounds. All chemical compounds reduced the
reducing, non- reducing and total sugars content compared with the control.
In this respect, all treatments decreased the reducing sugars. The highest
decrease was induced by calcium sulphate (CaSO4) (95.70%) followed by Potassium
Permanganate (KMnO4) and citric acid that decreased the reducing sugars by
93.85and 90.18%, respectively less than the control. While dipotassium hydrogen
phosphate (K2HPO4) was the least effective and decreased the reducing sugars by
76.67% less than the control.
All treatments decreased the non-reducing sugars. The highest decrease was
induced by dipotassium hydrogen phosphate (K2HPO4) and oxalic acid by 94.31and
90.48% respectively less than the control followed by cobalt sulphate (CoSO4)that
decreased the non-reducing sugars by 88.50% less than the control. While Bacillus
spp.4 was the least effective and decreased the non-reducing sugars by 1.85% less
than the control.
As for the total sugars, salicylic acid and cobalt sulphate (CoSO4) decreased the
total sugars by 84.19 and 83.71% respectively less than the control followed by
oxalic acid and potassium permanganate (KMnO4)that decreased the total sugars by
83.26% less than the control. On the other hand, ascorbic acid was the least effective
and decreased the total sugars by 71.61% less than the control.
Table 32 - Effect of cucumber seeds treatment with tested chemical compounds
on sugar content in cucumber plants as mg/1g fresh weight.
Treatment Reducing
sugar
Non
reducing
sugar
Total
sugar
% Efficacy
Reducing
sugar
Non
reducing
sugar
Total
sugar
Salicylic acid 3.27 2.02 5.29 -87.11 -75.03 -84.19
Oxalic acid 4.83 0.77 5.6 -80.96 -90.48 -83.26
Citric acid 2.49 4.05 6.54 -90.18 -49.93 -80.46
Ascorbic acid 3.27 6.23 9.5 -87.11 -22.99 -71.61
Dipotassium hydrogen
phosphate (K2HPO4) 5.92 0.46 6.38 -76.67 -94.31 -80.93
Cobalt sulphate (CoSO4) 4.52 0.93 5.45 -82.19 -88.50 -83.71
Calcium sulphate (CaSO4) 1.09 6.38 7.47 -95.70 -21.13 -77.68
Potassium permanganate
(KMnO4) 1.56 4.04 5.6 -93.85 -50.06 -83.26
Non-infested control with
FOC 23.98 8.25 32.23 -5.87 1.94 -3.71
Infested control with FOC 25.38 8.09 33.47 00.00 00.00 00.00
Reducing sugars Non reducing sugars Total sugars
L.S.D. at 5% 0.81 0.67 0.69
98
3.7.4 Effect of treatment of cucumber seeds with spore suspension of
antagonistic fungal isolates on phenol content in cucumber plants.
It is clear from Table 33 that, all the free, conjugated and total phenols were
affected significantly by antagonistic fungi under study. Compared with control, all
tested antagonistic fungi, except Penicillium spp., increased the free phenols. The
highest increase in the free phenols was induced by T. viride2 (436.51%) followed by
Trichoderma spp. and T. harzianum2 by 302.76 and 271.97%, respectively. While
Ch. bostrycoides was the least effective and increased the free phenols by 71.97%.
However, Penicillium spp. did not affect the free phenols significantly although it
was decreased by 8.39%.
Also all treatments, except T. harzianum1 and Ch. bostrycoides, increased the
conjugated phenols. The highest increase was induced by T.viride2 and T. viride1 by
262.22 and 55.42% respectively followed by Penicillium spp. that increased the
conjugated phenols by 48.43%. While T. harzianum3 was the least effective and
increased the conjugated phenols by 3.78%. However, T. harzianum1 and Ch.
bostrycoides, did not affect the conjugated phenols significantly although it was
decreased by 45.38 and 11.93% respectively.
On the other hand, all treatments increased the total phenols. The highest
increase in the total phenols was induced by T. viride2 and Trichoderma spp. that
increased the total phenols by 330.67 and 132.16% respectively followed by T.
harzianum2 that increased the total phenols by 126.93%. While T. harzianum1 was
the least effective and increased the total phenols by 6.88%.
Table 33 - Effect of treatment of cucumber seeds with spore suspension of
antagonistic fungal isolates on phenol content in cucumber plants as mg/1g fresh
weight.
Treatment Free
phenol
Conj
phenol
Total
phenol
% Efficacy Free
phenol
Conj
phenol
Total
phenol
T.harzianum 1 19.41 8.57 27.98 85.03 -45.38 6.88
T.harzianum 2 39.02 20.39 59.41 271.97 30.79 126.93
T. harzianum 3 19.5 16.18 35.68 85.89 3.78 36.29
T. viride 1 33.22 24.23 57.45 216.68 55.42 119.44
T. viride 2 56.28 56.47 112.75 436.51 262.22 330.67
T. spp. 42.25 18.53 60.78 302.76 18.86 132.16
Ch. bostrycoides 18.04 13.73 31.77 71.97 -11.93 21.35
Ch.globosum 20.3 19.9 40.2 93.52 27.65 53.55
Cheatomium spp. 32.25 23.04 55.29 207.44 47.79 111.19
Penicillium spp. 9.61 23.14 32.75 -8.39 48.43 25.10
Non-infested control with
FOC 1.08 20.98 22.06 -89.70 34.57 -15.74
Infested control with FOC 10.49 15.69 26.18 00.00 00.00 00.00
Free phenols Conjugated phenols Total phenols
L.S.D. at 5% 0.58 0.71 0.64
99
3.7.5 Effect of treatment of cucumber seeds with cell suspension of
antagonistic bacterial isolates on phenol content in cucumber plants
The results in Table 34 indicate that, the free, conjugated and total phenols were
affected significantly by the treatment with antagonistic bacteria.
All tested antagonistic bacteria increased the free phenols compared with
control. The highest increase in the free phenols was induced by Bacillus spp.2
(326.12%) followed by P. fluorescens1 and B. subtilis 3 that increased free phenols
by 293.42 and 285.99% respectively. While B. megtela was the least effective and
increased free phenols by 32.80%.
As for the total phenols all tested antagonistic bacteria increased the total
phenols. The highest increase in the total phenols was induced by Bacillus spp.2
(157.64%) followed by B.subtilis3 (137.43%) and P. fluorescens1 (103.71%). While
B. subtilis1 was the least effective and increased total phenols by 1.95%.
In aspect of the all treatments differed in their effect on the conjugated phenols,
B. subtilis3, B. megtela, Bacillus spp.2, 3, 4 and S. marcensens1 increased it by
39.00, 55.93, 45.93, 14.43, 6.29 and 83.00% respectively over control. While, B.
subtilis1,2, Bacillus spp.1, P. fluorescens1,2,3, P. putida and S. marcensens1 induced
the highest decrease by 71.71, 11.93, 44.64, 22.64, 36.50, 82.36, 62.28 and 25.97%
respectively less than control.
Table 34 - Effect of cucumber seeds treatment with cell suspension of
antagonistic bacterial isolates on phenol content in cucumber plants as mg/1g
fresh weight.
Treatment Free
phenol Conj
phenol Total
phenol
% Efficacy Free
phenol Conj
phenol Total
phenol
B.subtilis 1 22.28 4.41 26.69 112.39 -71.71 1.95
B. subtilis 2 36.47 13.73 50.2 247.66 -11.93 91.75
B. subtilis 3 40.49 21.67 62.16 285.99 39.00 137.43
Bacillus megtela 13.93 24.31 38.24 32.80 55.93 46.07
Bacillus spp. 1 18.65 8.63 27.28 77.79 -44.64 4.20
Bacillus spp. 2 44.7 22.75 67.45 326.12 45.93 157.64
Bacillus spp. 3 29.22 17.84 47.06 178.55 14.43 79.76
Bacillus spp. 4 31.27 16.57 47.84 198.10 6.29 82.73
P.fluorescens 1 41.27 12.06 53.33 293.42 -22.64 103.71
P.fluorescens 2 39.12 9.9 49.02 272.93 -36.50 87.24
P.fluorescens 3 32.74 2.75 35.49 212.11 -82.36 35.56
P. putida 37.65 5.88 43.53 258.91 -62.28 66.27
S. marcensens 1 20.59 28.53 49.12 96.28 83.00 78.62
S. marcensens 2 15.98 11.57 27.55 52.34 -25.97 5.23
Non-infested control with FOC
1.08 20.98 22.06 -89.70 34.57 -15.74
Infested control with FOC 10.49 15.69 26.18 00.00 00.00 00.00
Free phenols Conjugated phenols Total phenols
L.S.D. at 5% 0.68 0.73 0.74
100
3.7.6 Effect of treating cucumber seeds with chemical compounds on phenol
content in cucumber plants:
The results in Table 35 indicate that, phenol content was significantly affected
by the treatment with chemical compounds. Compared with control, all tested
chemical compounds increased the free phenols. The highest increase in the free
phenols was induced by citric acid (1110.3%) followed by oxalic acid (844.81%) and
ascorbic acid (579.41%). While calcium sulphate (CaSO4) was the least effective and
increased the free phenols by (78.46%).
As for the total phenol, all tested chemical compounds increased the total
phenols. The highest increase in the total phenols was induced by by citric acid
(398.05%) followed by oxalic acid (323.15%) and ascorbic acid (225.78%). While
calcium sulphate (CaSO4) was the least effective and increased the free phenols by
(33.31%).
While treatments differed in their effect on the conjugated phenols, all chemical
compounds decreased the conjugated phenols, except dipotassium hydrogen
phosphate (K2HPO4) and calcium sulphate (CaSO4) compared with the control.
In this respect, salicylic acid, oxalic acid, citric acid, ascorbic acid, cobalt
sulphate (CoSO4) and (KMnO4) decreased the conjugated phenols by 6.29, 25.14,
78.00, 10.07, 21.36 and 7.57% respectively. Whereas dipotassium hydrogen
phosphate (K2HPO4) and calcium sulphate (CaSO4) increased the conjugated phenols
by 50.29 and 3.14% respectively.
Table 35 - Effect of treating cucumber seeds with tested chemical compounds on
phenol content in cucumber plants as mg/1g fresh weight.
Treatment Free
phenol
Conju-
gated
phenol
Total
phenol
% Efficacy
Free
phenol
Conju-
gated
phenol
Total
phenol
Salicylic acid 34.02 14.61 48.63 224.31 -6.29 85.75
Oxalic acid 99.11 11.67 110.78 844.81 -25.14 323.15
Citric acid 126.96 3.43 130.39 1110.3 -78.00 398.05
Ascorbic acid 71.27 14.02 85.29 579.41 -10.07 225.78
Dipotassium hydrogen phosphate
(K2HPO4) 59.41 23.43 82.84 466.35 50.29 216.42
Cobalt sulphate (CoSO4) 20.68 12.26 32.94 97.14 -21.36 25.82
Calcium sulphate (CaSO4) 18.72 16.08 34.8 78.46 3.14 33.31
Potassium Permanganate (KMnO4) 30.88 14.41 45.29 194.38 -7.57 73.00
Non-infested control with FOC 1.08 20.98 22.06 -89.70 34.57 -15.74
Infested control with FOC 10.49 15.69 26.18 00.00 00.00 00.00
Free phenols Conjugated phenols Total phenols
L.S.D. at 5% 0.51 0.67 0.65
101
3.7.7 Effect of cucumber seeds treatment with cell suspension of
antagonistic fungal isolates on amino acids content in cucumber plants:
It is clear from Table 36 and Figure 30 that all tested antagonistic fungi
significantly decreased amino acids, except Ch. bostrycoides and C. globosum,
compared with control treatment. The highest decreased of amino acids was induced
Trichoderma spp. (55.81%) followed by T. harzianum1 and T. viride1 their decreased
amino acids by (47.57 and 31.09%) respectively. While T. viride2 was the least
effective and decreased amino acids by 5.24%.
On the other hand, Ch. bostrycoides and Ch. globosum increased amino acids by
26.23 and 6.37% respectively.
Table 36 - Effect of cucumber seeds treatment with cell suspension of
antagonistic fungal isolates on amino acids content of cucumber plants as mg/1g
fresh weight.
Treatment Amino Acids % Efficacy
T.harzianum 1 1.4 -47.57
T. harzianum 2 2.15 -19.48
T. harzianum 3 1.9 -28.84
T. viride 1 1.84 -31.09
T. viride 2 2.53 -5.24
Trichoderma spp. 1.18 -55.81
Ch.bostrycoides 3.37 26.23
Ch. globosum 2.84 6.37
Cheatomium spp. 1.94 -27.34
Penicillium spp. 1.97 -26.22
Non-infested control with FOC 1.8 -32.58
Infested control with FOC 2.67 00.00
L.S.D. at 5% 1.41
0
0.5
1
1.5
2
2.5
3
3.5
Mg
/1g
Amino Acid
T.harzianum 1
T. harzianum 2
T. harzianum 3
T. viride 1
T. viride 2
Trichoderma spp.
Ch.bostrycoides
Ch. globosum
Cheatomium spp.
Penicillium spp.
Non-infested control with FOC
Infested control with FOC
Figure 30 - Effect of cucumber seeds treatment with cell suspension of antagonistic fungal isolates on
amino acids content of cucumber plants as mg/1g fresh weight.
102
3.7.8 Effect of cucumber seeds treatment with cell suspension of
antagonistic bacterial isolates in amino acids content in cucumber plants:
The results in Table 37 and Figure 31 show that, all tested antagonistic bacteria
significantly increased amino acids, except P. fluorescens2, compared with control
treatment. The highest decreased of amino acids was induced S. marcensens2
(78.65%) followed by B. megtela and S. marcensens1 their decreased amino acids by
(76.78 and 72.66%) respectively. While Bacillus spp.3 was the least effective and
decreased amino acids by 6.00%.Whereas, P. fluorescens2 increased amino acids by
5.25%.
Table 37 - Effect of cucumber seeds treatment with cell suspension of
antagonistic bacterial isolates on amino acids content in cucumber plants as
mg/1g fresh weight.
Treatment Amino acids % Efficacy B. subtilis 1 1.15 -56.93
B. subtilis 2 0.98 -63.30
B. subtilis 3 1.96 -26.59
B. megtela 0.62 -76.78
Bacillus spp. 1 1.29 -51.69
Bacillus spp. 2 1.17 -56.18
Bacillus spp. 3 2.51 -6.00
Bacillus spp. 4 1.38 -48.31
P. fluorescens 1 0.93 -65.17
P. fluorescens 2 2.81 5.25
P. fluorescens 3 1.27 -52.43
P. putida 1.24 -53.56
S. marcensens 1 0.73 -72.66
S. marcensens 2 0.57 -78.65
Non-infested control with FOC 1.8 -32.58
Infested control with FOC 2.67 00.00
L.S.D. at 5% 1.32
0
0.5
1
1.5
2
2.5
3
Mg
/1g
Amino acids
B. subtilis 1
B. subtilis 2
B. subtilis 3
B. megtela
Bacillus spp. 1
Bacillus spp. 2
Bacillus spp. 3
Bacillus spp. 4
P. fluorescens 1
P. fluorescens 2
P. fluorescens 3
P. putida
S. marcensens 1
S. marcensens 2
Non-infested control with FOC
Infested control with FOC
Figure 31 - Effect of cucumber seeds treatment with cell suspension of antagonistic bacterial isolates on
amino acids content in cucumber plants as mg/1g fresh weight.
103
3.7.9 Effect of treating cucumber seeds with tested chemicals compound in
amino acids content in cucumber plants:
It is clear from Table 38 and Figure 32 that all tested chemical compounds
significantly increased amino acids compared with control treatment. The highest
increase of amino acids was induced by calcium sulphate (CaSO4) (68.54%) followed
by (KMnO4) and ascorbic acid, they increased amino acids by (65.54 and 46.07%)
respectively while dipotassium hydrogen phosphate (K2HPO4) was the least effective
and decreased amino acids by 13.11%. On the other hand, citric acid increased amino
acids by 23.97%.
Table 38 - Effect of cucumber seeds treatment with tested chemical compounds
on amino acids content in cucumber plants as mg/1g fresh weight.
Treatment Amino acids % Efficacy
Salicylic acid 1.86 -30.34
Oxalic acid 1.75 -34.46
Citric acid 3.31 23.97
Ascorbic acid 1.44 -46.07
Dipotassium hydrogen phosphate
(K2HPO4) 2.32 -13.11
Cobalt sulphate (CoSO4) 1.86 -30.34
Calcium sulphate (CaSO4) 0.84 -68.54
Potassium Permanganate (KMnO4) 0.92 -65.54
Non-infested control with FOC 1.8 -32.58
Infested control with FOC 2.67 00.00
L.S.D. at 5% 1.14
0
0.5
1
1.5
2
2.5
3
3.5
Mg
/1g
Amino acids
Salicylic acid
Oxalic acid
Citric acid
Ascorbic acid
Dipotassium hydrogen
phosphate (K2HPO4)Cobalt sulphate (CoSO4)
Calcium sulphate (CaSO4)
Potassium Permanganate
(KMnO4)Non-infested control with
FOCInfested control with FOC
Figure 32 - Effect of cucumber seeds treatment with tested chemical compounds on amino acids content in
cucumber plants as mg/1g fresh weight.
104
3.8 Anatomical studies:
3.8.1 Effect of cucumber seeds treatment with tested antagonistic fungal
isolates on the mean counts and measurements of certain histological features of
main cucumber root at 50 days after planting as affected by antagonistic fungal
isolates treatments.
The results in Table 39 and Figure 33 reveal that, out of 22 anatomical
characters investigated in cucumber root, 1, 7, 8, 10, 12, 14, 16, 17, 18, 19, 20 and 21
were positively changed in the treated plants with all fungal isolates comparing to the
infested control with FOC. The main changes were occurred in number of xylem
vessels (NXV) in the vascular bundle that seemed to be correlated with the resistance
against the Fusarium wilt disease. NXV in large vascular bundle recorded 42, 44.5,
72, 48, 45.5 and 31.5 by T. harzianum1, T. harzianum3, T.viride1, Trichoderma spp.,
Ch. bostrycoides and Penicillium spp. respectively comparing with NXV 19.5 in the
infested control with FOC. Number of fiber layer, thickness of fiber layer, wall
thickness of the fiber cell and cambium region thickness seemed strong barrier to
infection with FOC and positively changed in the treated plants with all fungal
isolates comparing to the infested control with FOC.
The obtained results under protected house showed that, T. harzianum No.3,
Trichoderma spp. and T. viride No.1 were the best isolates and reduced disease
severity by 90.27, 89.83 and 87.73% respectively. And induced the highest increase
in fruit weight Kg/plant by T. harzianum No.3, Trichoderma spp. and T. viride No.1,
which recorded 344.23, 336.54 and 320.19% respectively.
And according to the data in Table 39 the same isolates gave the positively
changed in anatomical characters that were investigated in cucumber root.
Figure 33 - Effect of cucumber seeds treatment with tested antagonistic fungal isolates on the mean counts
and measurements of certain histological features of main cucumber root.
105
Table 39 - Effect of treating cucumber seeds with tested antagonistic fungal
isolates on the mean counts and measurements of certain histological features of
main cucumber root at 50 days after planting as affected by antagonistic fungal
isolates treatment.
Treatments
T.h
arz
ian
um
1
T.h
arz
ian
um
3
T.
viri
de
1
Tri
chod
erm
a
spp
.
Ch
eato
miu
m
bo
stry
coid
es
Pen
icil
liu
m
spp
.
No
n-i
nfe
sted
con
tro
l w
ith
FO
C
Infe
sted
con
tro
l w
ith
FO
C
Histological
characteristics
(micron)
1 Root diameter 7005.90* 6790.30* 9989.84* 6681.12* 9146.20* 7925.50* 7089.16* 6394.3
0
2 Epidermal thickness 33.20 32.60 34.65 36.40 35.20 52.80* 33.00 37.40
3 No. of cortex layer 6 5 6 6 8 5.50 7 10.50
4 Thickness of cortex
layers 364.10 319.00 415.8 346.50 330.0 272.80 426.80 663.85
5 Mean thickness. of
cortex layers 60.68 63.80* 69.30* 57.75 41.25 49.60 60.97 63.22
6 No. of vascular
bundles V.B. 6 6 6.5* 6 5 5.67 6 6
7 No. of fiber layer 11.50* 13* 16.75* 10.34* 17* 13* 12.67* 7
8 Thickness of fiber
layer 398.75* 298.10* 550.00* 410.30* 487.85* 402.05* 401.50* 224.35
9 Mean thickness of
fiber layers 34.67 22.93 32.83 39.68 28.70 30.93 31.69 40.62
10 Wall thickness of the
fiber cell 25.85* 20.53* 26.40* 21.45* 19.80* 20.35* 21.27* 17.60
11 outer phloem
thickness 248.60 157.30 286.37 214.50 107.80 162.80 231.55 308.00
12 Cambium region
thickness 185.90* 143.00* 179.30* 123.20* 84.70* 152.90* 80.30* 71.50
13 Xylem thickness 1137.95 1382.15* 1989.90* 1225.96 1189.10 1080.75 1358.13* 1244.6
5
14 No. of xylem vessels
in large V.B. 42* 44.50* 72.00* 48.00* 45.50* 31.50* 44* 19.50
15
Diameter of the
widest xylem vessels
in large V.B.
187.55 191.95 246.95* 195.07 160.05 160.05 198.28 243.10
16
Wall thickness of the
widest xylem vessels
in large V.B.
28.60* 23.54 28.05* 31.53* 22.55 29.70* 23.10 24.75
17 Inner phloem
thickness 352.00* 231.00* 220.00* 254.10* 254.10* 286.00* 360.80* 158.04
18 Length of vascular
bundles 2323.20* 2211.55* 3225.57* 2224.06* 2123.55* 2084.50* 1209.28
2066.9
0
19 No. of pith layers 12.00* 13.00* 8 20.00* 18* 20.00* 16* 9
20 Thickness of pith
layers 1631.30* 1729.20* 1064.30* 1540.00* 2552.00* 2225.30* 3751.00* 858.00
21 Mean thickness of pith
layers 135.94* 133.02* 133.10* 77.00 141.78* 111.27* 234.44* 95.34
22 Hollow pith diameter 00 00 1573.00* 00 1617.00* 880.00* 00 00
V.B. = Vascular bundle
* Positive changed character comparing to the control
106
3.8.2 Effect of cucumber seeds treatment with tested antagonistic bacterial
isolates on the mean counts and measurements of certain histological features of
main cucumber root at 50 days after planting as affected by antagonistic
bacterial isolates treatment.
The results in Table 40 and Figure 34 reveal that, out of 22 anatomical
characters investigated in cucumber root, 1, 2, 7, 8, 10, 12, 14, 16, 17, 18, 19, 20 and
21 were positively changed in the treated plants with all bacterial isolates comparing
to the untreated control. The main changes were occurred in number of xylem vessels
(NXV) in the vascular bundle that seemed to be correlated with the resistance against
the Fusarium wilt disease. NXV in large vascular bundle recorded 42, 43, 61.5, 41,
34.67, 51.5, 42 and 42.5 by B. subtilis2, B. megtela, Bacillus spp.1, Bacillus spp.2, P.
fluorescens2, P. fluorescens3, P. putida and S. marcensens2 comparing with NXV
19.5 in the infested control with FOC. And also number of fiber layer, thickness of
fiber layer, wall thickness of the fiber cell and cambium region thickness seemed
strong barrier to infection with FOC and positively changed in the treated plants with
all bacterial isolates comparing to the infested control with FOC.
The obtained results under protected house showed that, B. megtla was the best
isolates and completely prevented the disease incidence followed by S. marcensens
No.2 and P. fluorescens No.3 and reduced disease severity by 91.37 and 90.67%
respectively. And also, the highest increased in fruit weight Kg//plant was induced by
B. megtla, S. marcensens No.2 and P.fluorescens No.3 by 350.00, 342.31 and 333.65
% respectively.
And according to the data in Table 40 the same isolates gave the positively
changed in anatomical characters that were investigated in cucumber root.
Figure 34 - Effect of cucumber seeds treatment with tested antagonistic bacterial isolates on the
mean counts and measurements of certain histological features of main cucumber root.
107
Table 40 - Effect of cucumber seeds treatment with tested antagonistic bacterial isolates on
the mean counts and measurements of certain histological features of main cucumber root at
50 days after planting as affected by antagonistic bacterial isolates treatments.
Treatments
B.s
ub
tili
s 2
B.m
egte
la
Ba
cill
us
spp
. 1
Ba
cill
us
spp
. 2
P.f
luo
resc
ens
2
P.
flu
ore
scen
s 3
P.
pu
tid
a
Ser
rati
a
ma
rcen
sen
s 2
No
n-i
nfe
sted
con
tro
l w
ith
FO
C
Infe
sted
con
tro
l w
ith
FO
C
Histological
characteristics
(micron)
1 Root diameter 8199.40* 6367.30* 6694.60* 7436.00* 6193.36 8205.52* 7482.20* 8643.74* 7089.16* 6394.3
2 Epidermal
thickness 40.70* 41.25* 30.80 35.20 36.30 41.80* 52.80* 35.50 33.00 37.40
3 No. of cortex
layer 6 9.0 7 5 9.00 8 7.50 8 7 10.50
4 Thickness of
cortex layers 339.90 418.00 341.00 300.30 546.70 485.96 416.90 368.50 426.80 663.85
5
Mean
thickness. of
Cortex layers 56.65 46.44 48.71 60.06 60.74 60.75 55.59 46.06 60.97 63.22
6 No. of vascular
bundles V.B. 4 6 6 5.50 6 6 5.50 6 6 6
7 No. of fiber
layer 16.50* 17* 11.50* 14.50* 12.34* 17* 14.50* 9* 12.67* 7
8 Thickness of
fiber layer 610.50* 701.80* 390.50* 557.70* 366.85* 393.8* 408.65* 323.37* 401.50* 224.35
9 Mean thickness
of fiber layers 37.00 41.28 33.96 38.46 29.73 23.16 28.18 35.93 31.69 40.62
10 Wall thickness
of the fiber cell 19.80* 23.10* 25.30* 28.05* 25.67* 23.10* 18.70* 27.50* 21.27* 17.60
11 outer phloem
thickness 220.00 212.30 201.30 217.80 97.90 145.20 227.70 239.80 231.55 308.00
12
Cambium
region
thickness 132.00* 176.00* 99.00* 112.20* 118.80* 143.00* 135.30* 151.80* 80.30* 71.50
13 Xylem
thickness 1131.90 1165.45 1315.50* 1184.70 1148.03 1603.80 1062.05 1630.20* 1358.13* 1244.65
14
No. of xylem
vessels in large
V.B. 42* 43* 61.50* 41* 34.67* 51.50* 42* 42.50* 44* 19.50
15
Diameter of
the widest
xylem vessels
in large V.B.
189.75 200.20 209.00 226.60 236.50 248.05* 191.40 247.50* 198.28 243.10
16
Wall thickness
of the widest
xylem vessels
in large V.B.
22.37 26.40* 16.50 48.40* 27.87* 22.37 21.45 31.35* 23.10 24.75
17 Inner phloem
thickness 392.70* 324.50* 300.30* 309.10* 162.80* 189.20* 267.30* 242.00* 360.80* 158.04
18
Length of
vascular
bundles 2487.10* 2580.05* 2305.60* 2381.50* 1894.38 2475.00* 2101.00* 2587.17* 1209.28 2066.9
19 No. of pith
layers 15* 15.00* 15* 18* 10* 8 11* 14* 16* 9
20 Thickness of
pith layers 2310.00* 2255* 1339.80* 2002.00* 1238.60* 1039.50* 1350.80* 1236.40* 3751.0* 858.00
21 Mean thickness
of pith layers 990.00* 150.33* 89.32 111.22* 123.86 129.94* 122.80* 88.31 234.44* 95.34
22 Hollow pith
diameter 154.00* 1073.60* 00 00 00 1160.50* 990.00* 1496.00* 00 00
V.B. = Vascular bundle * Positive changed character comparing to the control
108
3.8.3 Effect of cucumber seeds treatment with tested chemical compounds
on the mean counts and measurements of certain histological features of main
cucumber root at 50 days after planting as affected by chemical compounds
treatments.
The results in Table 41 and Figure 35 reveal that, out of 22 anatomical
characters investigated in cucumber root, 1, 7, 8, 12, 14, 17, 20 and 21 were
positively changed in the treated plants with all chemical compounds comparing to
the untreated control. The main changes were occurred in number of xylem vessels
(NXV) in the vascular bundle that seemed to be correlated with the resistance against
the Fusarium wilt disease. NXV in large vascular bundle recorded 28, 26.5, 47.5, 40
and 47 by salicylic acid, oxalic acid, K2HPO4, CoSO4 and CaSO4 comparing with
NXV 19.5 in the infested control with FOC. And also number of fiber layer,
thickness of fiber layer and cambium region thickness seemed strong barrier to
infection with FOC and positively changed in the treated plants with all chemical
compounds comparing to the infested control with FOC.
The obtained results under protected house showed that, salicylic acid
completely prevented the disease followed by CaSO4 and dipotassium hydrogen
phosphate (K2HPO4) they reducing the disease severity by 93.24 and 90.94%
respectively. And also induced the highest increased in fruit weight Kg/plant 343.27,
330.77 and 299.04 % respectively.
And according to the data in Table 41 the same treatments gave the positively
changed in anatomical characters that were investigated in cucumber root.
Figure 35 - Effect of cucumber seeds treatment with tested chemical compounds on the mean counts
and measurements of certain histological features of main cucumber root.
109
Table 41 - Effect of treating cucumber seeds with tested chemicals compound on the
mean counts and measurements of certain histological features of main cucumber root
at 50 days after planting as affected by chemicals compound treatments.
Treatments
Sali
cyli
c aci
d
Oxali
c aci
d
K2H
PO
4
CoS
O4
CaS
o4
No
n-i
nfe
sted
con
trol
wit
h
FO
C
Infe
sted
con
trol
wit
h
FO
C
Histological
characteristics
(micron)
1 Root diameter 7624.04* 5769.9 7026.8* 8146.60* 7983.80* 7089.16* 6394.30
2 Epidermal thickness 30.80 37.40 33.00 35.20 31.90 33.00 37.40
3 No. of cortex layer 6 7 6 7 7 7 10.50
4 Thickness of cortex
layers 297.00 418.00 243.10 396.00 273.90 426.80 663.85
5 Mean thickness. of
cortex layers 49.5 59.71 40.52 56.57 39.13 60.97 63.22
6 No. of vascular
bundles V.B. 6 4 6 6 6 6 6
7 No. of fiber layer 14* 13* 15* 14.50* 14* 12.67* 7
8 Thickness of fiber
layer 369.60* 413.05* 446.6* 357.50* 293.15* 401.50* 224.35
9 Mean thickness of
fiber layers 26.40 31.77 29.77 24.66 20.94 31.69 40.62
10 Wall thickness of the
fiber cell 19.25* 17.05 11.00 17.05 17.05 21.27* 17.60
11 outer phloem
thickness 163.90 258.50 225.50 179.30 231.00 231.55 308.00
12 Cambiumal region
thickness 151.07* 129.80* 138.60* 83.60* 155.10* 80.30* 71.50
13 Xylem thickness 888.80 975.70 1248.5* 1452.00* 1255.65* 1358.13* 1244.65
14 No. of xylem vessels
in large V.B. 28.00* 26.50* 47.5* 40.00* 47.00* 44* 19.50
15
Diameter of the
widest xylem vessels
in large V.B.
168.30 206.53 211.93 228.80 135.30 198.28 243.10
16
Wall thickness of the
widest xylem vessels
in large V.B.
22.73 17.60 33.55* 20.35 15.95 23.10 24.75
17 Inner phloem
thickness 282.15* 250.80* 297.00* 359.70* 343.20* 360.80* 158.04
18 Length of vascular
bundles 1855.52 2027.85 2341.35* 2432.10* 2366.10* 1209.28 2066.90
19 No. of pith layers 17* 8 10* 9 13* 16* 9
20 Thickness of pith
layers 3260.40* 803.00* 1791.90* 1540.00* 1885.40* 3751.00* 858.00
21 Mean thickness of
pith layers 191.79* 100.38* 179.19* 171.11* 145.03* 234.44* 95.34
22 Hollow pith diameter 00 00 00 880.00* 754.60* 00 00
V.B. = Vascular bundle
* Positive changed character comparing to the control
110
3.9 Effect of carrying the best antagonistic isolates of fungi and bacteria on
different carrier materials on infection with Fusarium wilt.
3.9.1 Comparison between some different carrier materials of antagonistic
fungal isolates on cucumber seeds on infection with Fusarium wilt.
The percentage of wilted cucumber plants were significantly reduced by using
different carrier materials inoculated by antagonistic fungal isolates as compared with
control. The results in Table 42 show that, treatment with paraffin oil as a carrier
material was the best effective for decreasing incidence of disease. Wilted plants
were ranged between 3.00-35.00%. T. viride on all carriers was the best effective
isolates and reduced wilted plants from 89.00% in control to 3.00% in treated plants
followed by Trichoderma spp. that reduced wilted plants to 5.00% in case of
treatment with paraffin oil. While in case of talc T. viride followed by T. harzianum
were the best effective isolates and reduced wilted plants from 89.00% in control to
8.00 and 14.00% respectively.
Table 42 - Comparison between some different carrier materials of antagonistic
fungal isolates on cucumber seeds on infection with Fusarium wilt.
Carrier materials Sodium alginate Paraffin oil Talc
Isolates % Dead
plants
%Healthy
plants
% Dead
plants
%Healthy
plants
% Dead
plants
%Healthy
plants
T.harzianum (1, 2,3) 15.00 75.00 12.00 88.00 14.00 86.00
T. viride (1,2) 5.00 95.00 3.00 97.00 8.00 92.00
Trichoderma spp. 30.00 70.00 5.00 95.00 27.00 73.00
Ch. bostrycoides 32.00 68.00 19.00 81.00 16.00 84.00
Penicillium spp. 35.00 65.00 17.00 83.00 19.00 81.00
Infested control with FOC 89.00 11.00 89.00 11.00 89.00 11.00
Figure 36 - Antagonistic fungal isolates on different carrier materials.
3.9.2 Comparison between some different carrier materials of antagonistic
bacterial isolates on cucumber seeds on infection with Fusarium wilt.
The percentage of wilted cucumber plants were significantly reduced by using
different carrier materials inoculated by antagonistic fungal isolates as compared with
control. The results in Table 43 show that, paraffin oil followed by talc were the most
effective for decreasing incidence of disease. Wilted plants were ranged 3.00-
31.00%. B. megtela was the best effective isolates and reducing wilted plants from
89.00% in control to (3.00 and 8.00%) in treated plants followed by P. fluorescens
that reduced wilted plants to (5.00 and 12%) on the paraffin oil and talc respectively.
Table 43 - Comparison between some different carrier materials of antagonistic
bacterial isolates on cucumber seeds on infection with Fusarium wilt.
111
Carrier materials Starch Paraffin oil Talc
Isolates % Dead plants %Healthy
plants
% Dead
plants
%Healthy
plants
% Dead
plants
%Healthy
plants
B. subtilis (1,2,3) 19.00 81.00 11.00 89.00 17.00 83.00
B. megtela 31.00 69.00 3.00 97.00 8.00 92.00
P. fluorescens (1,2,3) 14.00 86.00 5.00 95.00 12.00 88.00
P. putida 24.00 76.00 7.00 93.00 14.00 86.00
S. marcensens (1,2) 16.00 84.00 8.00 92.00 15.00 85.00
Infested control 89.00 11.00 89.00 11.00 89.00 11.00
Figure 37 - Antagonistic bactrial isolates on different carrier materials.
4 DISCUSSION
Cucumber (Cucumis sativus L.) is one of the most important economically
vegetable crops. It 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 and in protected houses. Cucumber is one of the
important vegetable crops in Egypt and also Kazakhstan. Egypt is the eighth in world
production, 595732 metric tons of cucumber in the year 2008 while Kazakhstan takes
the seventeenth grade in the world, and their production 268010 metric tons of
cucumber in the year 2008 and the total world production reached 40 million tons
[220].
Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum (FO), is one
of the major diseases in cucumber (Cucumis sativus) production [181]. Greenhouse
cucumber plants infected with Fusarium oxysporum showed the following symptoms;
root and stem rot w increasing in frequency and severity. Affected plants wilt at the
fruit-bearing stage, especially at temperatures over 27 C, and mycelial growth and
orange spore masses develop on the crown and stem [18].
Chemical fungicides have been used for a long time as the main strategy for
control in order manage fungal diseases and subsequently increase yield production
[221, 222, 223].On the other hand, the fungicides resistant races of the pathogen have
been reported [224, 225]. Also, these are reports on the side effects of fungicides on
human health [226, 227] and the environment [228, 229]. Therefore development of
nontoxic alternative to chemical fungicides would be useful in reducing the
undesirable effects of their uses.
112
In general, methods for controlling of Fusarium wilt, root, and stem rots on
greenhouse crops have emphasized development of resistant cultivars and avoidance
of primary inoculum. Breeding for resistance, however, can be difficult if resistance
genes have not been identified, as is the case for Fusarium root and stem rot in
cucumber [17]. Recently disease-resistant cucurbit rootstocks were identified onto
which susceptible cucumber varieties could be grafted. Reduction of primary
inoculum (e.g., on seed, planting stock, and in growing media) is an alternative
option, through the use of fungicides, heat or chemical treatments, disease-free
planting stock, sanitation, and fumigation [230]. Several reports have also
demonstrated the successful use of biological control agents (mostly bacteria and
fungi) against diseases caused by various formae speciales of F. oxysporum on a
range of hosts [231, 232, 62, 233, 52, 234].
4.1 Isolation of the causal of cucumber fusarium wilt:
In the present work, four isolates of Fusarium oxysporum were isolated from
cucumber wilted plants showing different degrees of vascular discoloration. All
isolates could grow on PDA medium forming delicate white to pink mycelia, often
with a purple tinge; and are sparse to abundant. Microconidia are abundant, oval-
ellipsoid, straight to curved and nonseptate. Macroconidia are sparse to abundant,
borne on branched conidiophores and are thin walled, three- to five-septate, fusoid-
subulate and pointed at both ends, have a pedicellate base, three to five-septate. The
three-septate spores are more common. Chlamydospores, both smooth and rough
walled, are abundant and form terminally or on an intercalary basis [190, 235].
4.2 Pathological studies
Pathogenicity test of the different isolates of the isolated fungus was carried out
under green house conditions. Healthy cucumber seeds cv. Sina1 were used. In order
to identify influence of inoculum densities of the tested isolates on infection of
cucumber seeds cv. Sina1, five conidial concentrations (1x103, 1x10
4, 1x10
5, 1x10
6
and 1x107
cfu) of F. oxysporum were used Inoculum density of 1x 107 of each of F.
oxysporum showed the highest percentage of dead plants, while inoculum densities of
103 and 10
4 conidia/ml caused the least percentage of dead plants [236, 237, 238,
239], with disease incidence and severity have increasing by increasing inoculum
density.
Six host plants (Cucumber hybrid Sina1, Watermelon, Cantaloupe, luffa, Melon
and squash) were inoculated with Fusarium oxysporum isolate (1) with conidiophores
concentration 1105
to determine the host ranges of F. oxysporum. No any wilt
symptoms was observed on watermelon, Cantaloupe, luffa, melon and squash.
According to these experiments, the isolated fungus was identified as F. oxysporum
f.sp. cucumerinum (FOC). These results are in agreement with those of Armstrong
and Armstrong [240] who reported that, plant pathogenic forms of F. oxysporum
were divided into formae speciales based on the hosts they attack.
Six cucumber hybrids namely Hisham, Db 162, Db 164, Al-Zaem, China and
Sina1 were evaluated for the resistance to Fusarium wilt under greenhouses
conditions. The obtained results showed that percentage of infection varied among
113
the different tested cucumber hybrids. The reaction of the tested hybrids could be
divided into four different groups (highly resistant, moderately resistant, susceptible
and highly susceptible).Sina1was considered highly susceptible because it recorded
95.83 % infected plants. Woever Al-Zaem (83.3%) and Hisham (75.0%) were
susceptible. Db 162 (50.0%) and China (29.33%) was moderately resistant and Db
164 (16.67%) was highly resistant. The obtained results are in harmony with those
obtained by Dong and Chen [20] who found that, 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. 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 fungi is Fusarium oxysporum forma specialis radicis-cucumerinum)
[18].
4.3 Effect of some resistance-inducers on growth and spore
germination of cucumber Fusarium wilt fungus in vitro:
Studying the effect of antagonistic fungi on the linear growth of F. oxysporum
f.sp. cucumerinum (FOC) in vitro as dual culture showed that, Trichoderma
harzianum No.3, Trichoderma spp. and Trichoderma viride No.1 were the most
effective isolates for reducing growth of FOC and caused the highest
reduction(57.22, 55,41 and 54.82%) respectively. Growth of T. viride was various in
the dual culture [37]. Trichoderma spp. was an effective hyperparasite, penetration
and coiling Fusarium oxysporum hyphae. Trichoderma glaucom produced effective
metabolites, while, T. album caused lysis and inhibited the pathogen [49].
Investigation of the effect of antagonistic fungal culture filtrates at three
concentrations on the linear growth and spore germination of F. oxysporum f.sp.
cucumerinum (FOC) revealed that, filtrates of all Trichoderma isolates and
Cheatomium bostrycoides at 50% concentration completely inhibited spore
germination of FOC. Culture filtrates of Trichoderma spp., Trichoderma harzianum
No.3 and Trichoderma viride No.1 at 50% were more effective and reduced the
mycelial growth of FOC by 91.50, 84.81 and 82.59 %, respectively. This is in
agreement with the observance ofother wokers [43, 48, 80]. In this respect, Dennis
and Webster [241] found that, Trichoderma spp. produced the antibiotic
"Trichodermol". This antibiotic can inhibit the growth of several fungi. Other
previous work showed that, Chaetomium globosum A, completely inhibited spore
germination of V. dahliae at 32µg/ml. and was also active against V. albo-atrum and
Rhizoctonia solani, Trichoderma viride, T. koningii, T. harzianum, Gliocladium
virens and that G. catenulatum showed the greatest potential in controlling the
growth of Fusarium oxysporum and Rhizoctonia solani on grasses [59]. Trichoderma
spp. has been widely used as antagonistic fungal agents against several pests as well
as plant growth enhancers. Faster metabolic rates, anti-microbial metabolites, and
114
physiological conformation are key factors which chiefly contribute to antagonism of
these fungi. Mycoparasitism, spatial and nutrient competition, antibiosis by enzymes
and secondary metabolites, and induction of plant defense system are typical
biocontrol actions of these fungi. On the other hand, Trichoderma spp. have also been
used in a wide range of commercial enzyme productions, namely, cellulases,
hemicellulases, proteases, and 1,3-glucanase [242].
Evaluation the effect of antagonistic bacteria on the linear growth and spore
germination of F. oxysporum f.sp. cucumerinum (FOC) revealed that, Pseudomonas
fluorescens No.2, Bacillus Subtilis No.2, Pseudomonas fluorescens No.3 and
Bacillus spp. No.2 were the most effective antagonistic bacteria for limiting growth
of FOC where they caused the highest inhibition zone (37.33, 35,67, 35,00 and 34.00
mm) respectively. Serratia marcensens No.2 and Pseudomonas fluorescens No.1
(31.33 and 30, 67 mm). D’Ercole et al. [243] noted variable types of antagonism of
which, the formation of free zone between the fungi; lytic phenomena or complete
covering of the pathogen by the antagonist was found.
Evaluation the effect of antagonistic bacterial culture filtrates at three
concentrations on the linear growth and spore germination of F. oxysporum f.sp.
cucumerinum (FOC) revealed that, all filtrates of the tested bacterial isolates at 50%
concentration completely inhibited spore germination of FOC. Culture filtrates of
Pseudomonas putida, Serratia marcensens No.2, Bacillus Subtilis No.2 and Bacillus
spp. No.2 at 50% concentration were the highest effective and reduced the mycelial
growth of FOC by 80.74, 80.37, 79.63 and 79.26 % respectively. Pseudomonas
fluorescens No.2 and Bacillus spp. No1 (77.41%) came next whereas Bacillus spp.
No4 was the least effective one and reduced the growth (52.97%). All Pseudomonas
isolates, Serratia isolates, Bacillus subtilis No.1 and Bacillus spp. No.4 made lysis to
mycelial of FOC. Generally linear growth and spore germination were decreased by
increasing the concentrations of culture filtrates from 10% up to 50%. The previous
results are in harmony with those obtained by others workers [244, 245, 246]. In this
respect, Pusey and Wilson [247] reported that B. subtilis exerted a heat stable
antibiotic interfering with spore germination. Other previous work showed that,
Pseudomonas fluorescens inhibited the mycelial growth of Fusarium oxysporum f.
sp. lycopersici and suppressed the Fusarium wilt of tomato [99]. Pseudomonas putida
and Serratia marcescens significantly reduced Fusarium wilt of cucumber when
applied as root treatments [52].
Eight chemical compounds (salicylic acid, oxalic acid, citric acid, ascorbic acid,
K2HPO4, CoSO4, CaSO4 and KMnO4) each with 3 concentrations were tested for their
effects on the linear growth and spore germination of FOC in vitro. The obtained
results reveal that all chemicals under study decreased the linear growth and spores
germination of FOC with different degrees. Oxalic acid at concentration 10 mM
completely inhibited mycelial growth of FOC followed by oxalic acid at
concentration 5 mM, salicylic acid and ascorbic acid at concentration of 10 mM
reducing the linear growth of FOC by 75.92, 62.59 and 61.86% respectively.
However, citric acid at concentration of 2.5 mM was the least effective one and
reducing the growth (3.33%). Salicylic acid, oxalic acid citric acid and ascorbic acid at
concentration 5 and 10 mM completely inhibited spore germination of FOC. Also
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KMnO4 at all concentrations, K2HPO4 at 100 and 200mM, Cobalt sulphate at 10 ppm
and calcium sulphate at 5000 ppm completely inhibited spore germination of FOC.
These results agree with those of El-Kolaly [132]. Who found that salicylic acid and
ascorbic acid were the most effective antioxidants against root and crown rot of
strawberry. Working with other hosts of other workers [112, 114, 163, 68, 133] found
that salicylic acid and ascorbic acid caused significant reduction in radial growth.
4.4 Efficiency of some resistance-inducers on induced cucumber resistance
against Fusarium wilt fungus under greenhouse: -
Under greenhouse conditions ten antagonistic fungi were tested for their efficacy
in induced cucumber resistance against Fusarium wilt, (FOC) on cucumber. The
results indicated that, all tested antagonistic fungi were effective in reducing disease
severity. Trichoderma harzianum No.3, Trichoderma spp. and Chaetomium
bostrycoides were the most effective isolates and reduced disease severity by 93.00,
92.33 and 90.00% respectively. In the other hand, Trichoderma viride No.2 was the
least effective one and reduced disease severity by 66.67%. These results are in
agreement with those obtained by other workers [61, 59, and 58]. In this respect,
Moon et al., [54] found that, Trichoderma harzianum suppressed Fusarium wilt
caused by Fusarium oxysporum f.sp. fragariae in strawberries. The wheat bran or
rice straw culture of T. harzianum suppressed disease incidence more effectively than
the other culture substrates. T. harzianum cultured on wheat bran or rice decreased
disease incidence to 68% of the control. A conidial suspension of T. harzianum alone
or a suspension mixed with crab shell also reduced disease incidence. T. harzianum
was highly effective in controlling disease in acidic soil (pH 3.5 – 5.5). Disease
incidence and population density of F.o. f.sp. fragariae decreased in a sandy loam
soil inoculated with T. harzianum. There were no similar effects on inoculated loam
soil. Trichoderma viride, T. koningii, T. harzianum, Gliocladium virens and G.
catenulatum showed the greatest potential in controlling the growth of Fusarium
oxysporum and Rhizoctonia solani on grasses [59].Trichoderma acting with different
mechanisms including mycoparasitism [194, 248, 249] act as acontrolling agent
through production of antifungal substances [250, 251]. Trichoderma spp. also act
through production of destructive enzymes i.e. chitinase [252, 253, 254].
Under greenhouse conditions, fourteen antagonistic bacterial isolates were tested
for their efficacy in inducing cucumber resistance against Fusarium wilt, (FOC) on
cucumber. The results showed that, all tested antagonistic bacterial isolates were
effective in reducing disease severity. Bacillus megtla, Pseudomonas fluorescens
No.3 and Serratia marcensens No.2 were the most effective isolates and completely
prevented the disease incidence. Serratia marcensens No.1, B. subtilis No. 2 and
Pseudomonas fluorescens No.2 were came next and reduced disease severity by
(96.67, 93.33 and 93.13%)respectively. On the other hand, Bacillus spp. No. 3 was
the least effective isolates and reduced the disease severity by 66.67%. These results
are in agreement with those obtained by others [255, 256, 257, 258], who found that
the bacterium protected different crop seedlings against infection by F. oxysperum
and V. daliae. Bacillus subtilis also showed considerable effects in controlling of
infection. This might be due to the bacterium producing more antibiotics (Bacteriocin
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and subtilisin) which act as inhibitors to pathogenic fungi [259, 260, 261].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
[262].
Under greenhouse conditions, 8 chemical compounds (salicylic acid, oxalic acid,
citric acid, ascorbic acid, K2HPO4, CoSO4, CaSO4 and KMnO4) each with 3
concentrations were tested for their efficacy in inducing cucumber resistance against
Fusarium wilt, FOC on cucumber. The obtained results indicate that, both disease
incidence and disease severity of Fusarium wilt disease decreased as a result of
treatment by all chemical compounds. Percentage of disease incidence and disease
severity decreased by increasing the concentration of tested chemical compounds. In
all cases, salicylic acid and CaSO4 was the most effective compound on disease
development as it reduced the percentages of disease severity in addition salicylic
acid at 10 mM and CaSO4 at 2500 and 5000 ppm completely prevented the disease
followed by KMnO4 at 5000 ppm and CaSO4 at 1000 ppm reducing the disease
severity by 97.00 and 96.67% respectively. The current results are in harmony with
the results obtained by [112, 114, 68, 132, 137], who found that salicylic acid gave
the most effective control against many pathogens. Benzoic acid, salicylic acid and
ascorbic acid significantly reduced linear growth of Fusarium oxysporum, F. solani
and Rhizoctonia solani and reduced spore germination of Fusarium spp. The 3
antioxidants significantly reduced damping-off of tomatoes when used as a soil
drench and they were more effective than tolclofos-methyl [129]. Salicylic acid,
hydrogen peroxide and cobalt ions were effective for induction of resistance in
watermelon against wilt pathogen in four distinct experiments [126].
4.5 Efficiency of some resistance-inducers on induced cucumber resistance
against Fusarium wilt fungus under commercial protected house:
Under commercial protected agriculture ten antagonistic fungi were tested on
two successive seasons (spring 2009, autumn 2009) for their effect on disease
severity of wilt pathogen (FOC) on cucumber plants. The results showed that, all
tested antagonistic fungal isolates significantly reduced the disease severity of wilt
disease and increased plants yield. In this respect, Trichoderma harzianum No.3,
Trichoderma spp. and Trichoderma viride No.2 were the most effective isolates and
reduced disease severity by 90.27, 89.83 and 87.73%, respectively. In the other hand,
Chaetomium spp. was the least effective one and reduced disease severity by 81.72%.
Also, all tested treatments increased the fruit weight/plant. The highest increase in
fruit weight/plant was induced by Trichoderma harzianum No.3, Trichoderma spp.
and Trichoderma viride No.2, 342.31, 336.54 and 320.19% respectively. However,
Chaetomium spp. was the least effective one and increased fruits weight/plant by
153.85%. The current results are in harmony with those obtained by [83, 90, 104].
Ahmed [263] reported that spraying cucumber plants with biological control agent to
control powdery mildew disease 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
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extract + Trichoderma filtrate (38.41and 40.06%) and Trichoderma filtrate + Bacillus
filtrate (37.50 and 39.17%). This high potentiality in antagonism might be due to that
Trichoderma spp. act through different mechanisms including mycoparasitism [194,
248, 249] also, through production of antifungal substances [250, 251]. Trichoderma
spp. also acts through production of destructive enzymes i.e. chitinase or antifungal
substances [252, 253, 254], or also stimulate resistant in the host [264, 265].
Under commercial protected agriculture fourteen antagonistic bacterial isolates
were tested on two successive seasons (spring and autumn 2009) for their effect on
disease severity of wilt pathogens FOC on cucumber plants. The obtained results
show that, all tested antagonistic bacterial isolates were effective in reducing disease
severity. Bacillus megtla was the most effective isolates and completely prevented
the disease incidence followed by Serratia marcensens No.2 and Pseudomonas
fluorescens No.3 where reduced disease severity by 91.37 and 90.67% respectively
Whereas, Serratia marcensens No.1, Pseudomonas fluorescens No.2 and B. subtilis
No.2 reduced disease severity by 89.79, 89.44 and 88.77 % respectively. On the other
hand, Bacillus spp. No.3 was the least effective isolate and reduced the disease
severity by 83.68%. All tested treatments increased the fruits weight/plant. The
highest increase in fruit weight/plant was recorded by Bacillus megtla, Serratia
marcensens No.2 and Pseudomonas fluorescens No.3 being 350.00, 342.31 and
333.65 % respectively. Bacillus spp. No. 3 was the least effective one and increased
fruits weight/plant by 188.46%. The current results are in harmony with those
obtained by other workers [69, 85, 84, 96]. Pseudomonas fluorescens strain WCS
417, known for its ability to suppress Fusarium wilt diseases (WCS 417), reduced
incidenceof banana with Fusarium wilt by 87.4%. These isolates should be further
evaluated for potential application in the field, independently and in combination
[97]. The cell-free culture filtrate of Bacillus subtilis, with a concentration of 20%
(v/v), could result in the vacuolation, swelling and lysis of hyphae. Besides these may
have been shrunk and hindered germination of conidia of F. oxysporum at the
concentration of 80% (v/v). When applied as inoculants, Bacillus subtilis (108 cfu.
ml) was able to reduce disease incidence by 73.60%, and promote seedling growth in
pot trial studies [25]. Bacillus subtilis also showed considerable effect in controlling
Fusarium wilt. This might be due to the bacterium producing more antibiotics
(Bacteriocin and subtilisin) which act as inhibitors to pathogenic fungi [259, 260,
261]; 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 have been due to
competition for spaces or nutrients [262].
Under commercial protected agriculture 8 chemical compounds were tested on
two successive seasons (spring and autumn 2009) for their effects on disease severity
of wilt pathogens (FOC) of cucumber. The obtained results indicate that, both disease
incidence and disease severity of Fusarium wilt disease were reduced as a result of
treatment by all chemical compounds. In all cases, salicylic acid and CaSO4 were the
most effective compounds on disease development as it reduced the percentages of
disease severity in addition salicylic acid completely prevented the disease incidence
followed by CaSO4 and KMnO4 being reduced disease severity by 93.24 and 92.41%
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respectively. On the other hand, ascorbic acid was the least effective and reduced the
disease severity by 85.35%. Also, all tested treatments increased fruits weight/plant.
The highest increase in fruit weight/plant was recorded by salicylic acid, CaSO4 and
KMnO4 being increased fruits weight/plant by 342.31, 330.77 and 311.54%
respectively. However, ascorbic acid was the least effective and increased fruit
weight/plant by 204.39%. The presented results are in agreement with Mansour [136]
who reported that salicylic acid caused the highest decrease in growth and sporulation
of Verticillium dahliae, Verticillium albo-atrum and F. oxysporum. Tannic acid
caused the highest decrease in spore germination of Verticillium dahliae, Verticillium
albo-atrum and F. oxysporum in strawberry. Antioxidants were significantly more
effective in improving disease control and fruit yield production. Salicylic acid and
ascorbic acid were the most effective antioxidants on wilt disease and increasing the
yield. Application of KMnO4 solution to the soil provided effective control of
Fusarium wilt of cucumbers. Plots treated with 1:800 or 1:1000 solutions were free
from the disease, while the average rate of infected plants following treatment with a
1:1500 solution was 0.88%. Infection rates in untreated plots was 40.51%. The
highest yields (112.6 kg) were obtained from plots treated with 1:1000 KMnO4 [120].
Working with other hosts other workers [112, 127, 126,130, 131] found that chemical
inducers and antioxidants caused significant reduction of Fusarium wilt and increased
fruits yield.
4.6 Efficacy of treating cucumber seeds with some resistance-inducers on
enzymes activity, lignin content and peroxidase isozyme:
Ten antagonistic fungi (Trichoderma harzianum (3 isolates), T. virdi (2 isolates),
Chaetomium globosum, Chaetomium bostrycoides, Trichoderma spp., Chaetomium
spp. and Penicillium spp.), fourteen antagonistic bacterial isolates (Bacillus subtilis (3
isolates), Pseudomonas fluorescens (3 isolates), Pseudomonas putida, Bacillus
megtela, Serratia marcensens (2 isolates) and 4 isolates of Bacillus spp.) and eight
chemical compounds (salicylic acid, oxalic acid, citric acid, ascorbic acid, K2HPO4,
CoSO4, CaSO4 and KMnO4) were evaluated for their activity on peroxidase,
polyphenol-oxidase and chitinase enzymes at 40 and 50 days after planting. Results
indicate that all tested treatments significantly increased the activity of all enzymes
tested. Trichoderma harzianum 3 was the most effective isolate of fungi and induced
the highest activity of peroxidase and polyphenol-oxidase in all times, whereas,
Penicillium spp. recorded the most effective isolate of fungi and reported the highest
activity of chitinase in all times. On the other hand, Bacillus megtela was the most
effective effective isolate of bacterial isolates and induced the highest activity of
peroxidase and chitinase in all times, while, Bacillus spp.3 was the most effective
isolate of bacterial isolates and induced the highest activity of polyphenol-oxidase in
all times. Cobalt sulphate (CoSO4) was the most effective effective chemical
compounds and recorded the highest activity of peroxidase and chitinase in all times,
while, Oxalic acid was the most effective chemical compound, and showed the highest
activity of polyphenol-oxidase in all times. The present results concerning the
increase in peroxidase, polyphenol-oxidase and chitinase enzymes activity are in
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agreement with results reported by others [266, 267, 268, 269, 270, 271]. Smith and
Hammerschmidt [155] found that induced resistance in cucurbit plants accompanied
by a marked increase in intercellular peroxidase isozymes. Induced resistance in
cucumber plans with K2HPO4 increased the activity of peroxidase and chitinase
enzymes [272] and -1.3-glucanase [160]. Induced resistance in cucumber plants
with acetylsalicylic acid (aspirin) was reported to increase the activity of chitinase, -
l,3-glucanase, peroxidase polyphenol-oxidase and phenylalanine ammonia [273].
Many plant enzymes are involved in defense reactions against plant pathogens.
Oxidative enzymes such as peroxidase and polyphenol oxidase enhance formation of
lignin, while other oxidative phenols contribute in formation of defense barriers for
reinforcing the cell structure [160]. Enzyme activity plays an important role in plant
disease resistance through increasing plant defense mechanisms that are considered
the main tool of varietals resistance [274]. In the current study following inoculation
with Fusarium oxysporum f.sp. cucumerinum, peroxidase activity of wilt-resistant
cucumber cultivars showed little change while in susceptible cultivars the activity
rose sharply once the leaves became wilted. The peroxidase activity of seedlings
before inoculation showed highly significant correlation with resistance after
inoculation, peroxidase activity of cucumber seedlings can be used for forecasting
their resistances to Fusarium wilt since activity is stable at the seedling stage [175].
Abiotic inducers increased peroxidase, polyphenol oxidase (PPO) and chitinase
activity in shoots and roots of strawberry plants. Using ascorbic acid against root rot
pathogens S. rolfsii, R. fragariae and R. solani induced the highest increase in
peroxidase activity in shoots whereas; CuSO4, ascorbic acid and CuSO4 recorded the
highest activity in roots [137]. Experiments on root and foliar applications of 24-
epibrassinolide (EBL), which is an immobile phytohormone with antistress activity
[181] showed a decrease in disease severity of Fusarium wilt of cucumber (Cucumis
sativus L. cv. Jinyan No. 4) and increased plant growth with reduced losses in
biomass.Also, EBL reduced pathogen-induced accumulation of reactive oxygen
species (ROS), flavonoids, and phenolic compounds, activities of defense-related and
ROS-scavenging enzymes. The enzymes included superoxide dismutase, ascorbate
peroxidase, guaiacol peroxidase, catalase as well as phenylalanine ammonia-lyase
and polyphenoloxidase. The activities of plant defense-related enzyme, peroxidase
(POX), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) were
increased in plants treated with Bacillus subtilis. A higher content of indoleacetic acid
(an important plant growth regulator) was detected in Bacillus subtilis treated plants.
Furthermore, seed-soaking with Bacillus subtilis exhibited a more efficient biological
control (Biocontrol effect 73.60%) and promoted plant growth (Vigor Index 4,
177.53) than root-irrigation (50.88% and Vigor Index 3, 575.77, respectively),
suggesting the potential use of Bacillus subtilis as a seed-coating agent [25].
The present work evaluated the effect of ten antagonistic fungi (3 isolates of
Trichoderma harzianum, 2 isolates of T. viride, Chaetomium globosum, Chaetomium
bostrycoides, Trichoderma spp., Chaetomium spp. and Penicillium spp.). Also
fourteen antagonistic bacterial isolates were evaluated (3 isolates of Bacillus subtilis ,
3 isolates of Pseudomonas fluorescens, Pseudomonas putida, Bacillus megtela, 2
isolates of Serratia marcensens and 4 isolates of Bacillus spp.) Besides 8 chemical
120
compounds (salicylic acid, oxalic acid, citric acid, ascorbic acid, K2HPO4, CoSO4,
CaSO4 and KMnO4) were assed singly on lignin content in cucumber plants. Results
indicate that all treatments increased the lignin content.
Trichoderma harzianum1 was the most effective isolate of fungi and increased
lignin content by290.78%. However, two Bacillus spp. were the most effective isolate
among bacterial isolates and increased lignin content by 286.82%. On the other hand,
citric acid was the most effective chemical and increased lignin content by 290.27%.
The present results concerning the increase in lignin content are in agreement with
those reported by others [109, 150, 151, 154]. Lignification plays its role as defense
mechanisms, increasing the mechanical resistance of the host cell wall, restricting
diffusion of pathotoxins and nutrients and inhibiting growth of pathogens due to the
toxicity of lignin precursors and lignifications of the pathogen [147]. Dean and Kuc
[107] reported more rapid lignifications following challenge in protected leaves of
immunized plants. The rate of lignifications increased more rapidly in immunized
plants after wounding by pricking leaves with a pin. Thus immunization may induce
cucumber to respond rapidly upon injury or infection. Rapid lignification in resistant
or immunized cucumber after penetration by Cladosporium cucumenmim or
Colletotrichum lagenarium and their fungal mycelia was observed in the presence of
confiferyl, hydrogen peroxide and peroxidase prepared from immunized cucumber
leaves [145]. Spraying cucumber plants with K2HPO4 at 100 mM before ten days
from inoculation with powdery mildew was reported to have increased lignin content
by 65% [270]. Induced resistance in various plants showed positive significant
correlation with enhancement of chitinase activity and -1,3-glucanase enzymes
which hydrolyses the hyphal cell wall of pathogenic fungi [275, 276]. The lignin
content in roots was increased by the abiotic inducers (salicylic acid, boric acid,
ascorbic acid, CuSO4, MgSO4, KH2PO4 and Bion WF50) . The twice application
methods and SA used against R. fragariae and R. solani produced the highest lignin
content followed by ascorbic acid. The lignin content in plant roots, regardless of
root rot pathogens, also increased by the biotic inducers. Highest lignin content was
reported to have induced by Bacillus subtilis followed by Pseudomonas fluorescence,
Streptomyces aureofaciens and Trichoderma harzianum respectively [137].
Isozyme pattern of peroxidase in cucumber extract contained two bands expect
Trichoderma harzianum3, Bacillus spp.1, oxalic acid, non-infested and infested
control. One additional band was found in the case of immunized plants. Bands
volume of immunized plants was denser than non-infested and infested control. The
role of oxidative enzymes in relation to disease resistance was subject to research in
many laboratories all over the world [277, 278, 145]. Peroxidases have not been
shown to have direct antifungal properties, unlike other systemic inducible enzymes
such as chitinases [279, 280]. Peroxidase may function in disease resistance
indirectly by affecting biochemical processes which in turn influence disease
resistance. Increased activities of peroxidase were found in all cellular fractions in
induced plants. High specific activities of peroxidase in extract from intercellular
spaces and from cell walls suggest that peroxidase is secreted outside of tobacco leaf
cells, either bound to cell walls or present in the intercellular spaces. Although
biological functions of all the peroxidases are not completely understood, they
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reported to have a role in secondary wall biosynthesis [281, 282, 283]. Peroxidases
also participate in superoxide generation and lignification [281, 282, 283, 284, 285,
286, 287]. A systemic increase in peroxidase activity is associated with induced
systemic resistance in cucumber [146].
4.7 Efficacy of treating cucumber seeds with some resistance-inducers on
sugars, phenols and amino acids contens:
The present work evaluated the effect of ten antagonistic fungi (3 isolates of
Trichoderma harzianum, 2 isolates of T. viride, Chaetomium globosum, Chaetomium
bostrycoides, Trichoderma spp., Chaetomium spp. and Penicillium spp.). Also
fourteen antagonistic bacterial isolates were evaluated (3 isolates of Bacillus subtilis ,
3 isolates of Pseudomonas fluorescens, Pseudomonas putida, Bacillus megtela, 2
isolates of Serratia marcensens and 4 isolates of Bacillus spp.) Besides 8 chemical
compounds (salicylic acid, oxalic acid, citric acid, ascorbic acid, K2HPO4, CoSO4,
CaSO4 and KMnO4) were assed, each on sugar content. All treatments significantly
decreased sugar content.
All antagonistic fungi under study reduced the reducing, non- reducing and total
sugars content. In this respect, all treatments decreased the reducing sugars. The
highest decrease was induced by Trichoderma spp. followed by Trichoderma
harzianum1. However, the highest decrease in non-reducing sugars was induced by
Trichoderma harzianum1 and Penicillium spp.. As for the total sugars, Trichoderma
harzianum1 and Trichoderma spp. induced the highest decrease in the total sugars.
All antagonistic bacteria decreased the reducing, non- reducing and total sugars
content except Bacillus megtela which increased non-reducing sugars. In this respect,
all treatments decreased the reducing sugars. The highest decrease was induced by
Bacillus spp.4 followed by Bacillus megtela. Serratia marcensens2 and Serratia
marcensens1 recorded the highest decrease of non-reducing sugars. As for total
sugars, Serratia marcensens1 and Serratia marcensens2 induced the highest decrease
of the total sugars.
All chemical compounds decreased the reducing, non- reducing and total sugars
content. In this respect, all treatments decreased the reducing sugars. The highest
decrease occurred with calcium sulphate (CaSO4) followed by potassium
permanganate. The highest decrease of the non-reducing sugars was recorded by
dipotassium hydrogen phosphate (K2HPO4) and oxalic acid. As for the total sugars,
salicylic acid and cobalt sulphate (CoSO4) recorded the highest decrease of the total
sugars. These results are in agreement with those of Gangopadhyay et al. [143] who
found that, lower amount of carbohydrates in healthy roots of the susceptible soybean
cultivar was noticed compared with the resistant one. Total sugar increased in both
cultivars in response to infection with M. phaseolina that caused charcoal rot disease.
An appreciable increase was more pronounced in the resistant cultivar. Resistance
was positively correlated with sugar content in the leaves of resistant cultivars [288,
289, 290]. On the other hand, high sugar content in the susceptible cultivars was
reported by other workers [291, 292]. Awad [293] reported that sugars tended to
increase the susceptibility of detached leaves to fungal parasites by providing an extra
source of energy for the invader. Chemical inducers and biological control were
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reported to decrease reducing, non-reducing and total sugars in roots of strawberry
plants infected with the three wilt pathogens [136].
The present work evaluated the effect of ten antagonistic fungi (3 isolates of
Trichoderma harzianum, 2 isolates of T. viride, Chaetomium globosum, Chaetomium
bostrycoides, Trichoderma spp., Chaetomium spp. and Penicillium spp.). Also
fourteen antagonistic bacterial isolates were evaluated (3 isolates of Bacillus subtilis ,
3 isolates of Pseudomonas fluorescens, Pseudomonas putida, Bacillus megtela, 2
isolates of Serratia marcensens and 4 isolates of Bacillus spp.) Besides 8 chemical
compounds (salicylic acid, oxalic acid, citric acid, ascorbic acid, K2HPO4, CoSO4,
CaSO4 and KMnO4) were assed ,each on phenol content. The obtained results show
that, all treatments significantly increased phenol content. All tested antagonistic
fungi, except Penicillium spp., increased the free phenols. The highest increase in the
free phenols was induced by Trichoderma viride2 followed by Trichoderma spp.
Also all treatments, except Trichoderma harzianum1 and Chaetomium bostrycoides,
increased the conjugated phenols. The highest increase was recorded by Trichoderma
viride2 and Trichoderma viride1. The highest increase in the total phenols was
recorded by Trichoderma viride2 and Trichoderma spp. All tested antagonistic
bacteria increased the free phenols. The highest increase in the free phenols was
induced by Bacillus spp.2 followed by Pseudomonas fluorescens1 and Bacillus
subtilis3. As for the total phenols all tested antagonistic bacteria increased the total
phenols. The highest increase in the total phenols was induced by Bacillus spp.2
followed by Bacillus subtilis3.Treatments differed in the effect on the conjugated
phenols, Bacillus subtilis3, Bacillus megtela, Bacillus spp.2, 3, 4 and Serratia
marcensens1 increased it. While, Bacillus subtilis12, Bacillus spp.1, Pseudomonas
fluorescens1, 2, 3, Pseudomonas putida and Serratia marcensens1 induced the
highest decrease. All tested chemical compounds increased the free phenols. The
highest increase was recorded by citric acid followed by oxalic acid. As for the total
phenol, all tested chemical compounds increased the total phenols. The highest
increase in the total phenols was induced by citric acid followed by oxalic acid.
Treatments were different in their effects on the conjugated phenols.All chemical
compounds decreased the conjugated phenols, except (K2HPO4) and Calcium
sulphate (CaSO4). This increase in the total phenol levels surely gave an increase in
the capability of plants to defend against disease infection process and disease
development. The present results concerning the increase in total phenol contents,
indicate the role of secondary metabolic substances (such as phenolic compounds in
disease resistance mechanisms [294]. Moreover, toxic phenolic compounds in plant
cells act through: (1) the structure of bond form with cell wall components of plant
tissues [295] (2) enhance host resistant by stimulating host defense mechanisms [296]
(3) prevent the extent of fungal growth in plant tissues [297] and (4) penetrate the
microorganisms and cause considerable damage to the cell metabolisms [298]. In this
respect, Mandal et al., [299] demonstrated that exogenous application of 200 μM
salicylic acid through root feeding and foliar spray could induce resistance against
Fusarium oxysporum f. sp. lycopersici (Fol) in tomato. The activities of
phenylalanine ammonia lyase (PAL) and peroxidase (POD) were 5.9 and 4.7 times
higher, respectively than the control plants at 168 h of salicylic acid feeding through
123
the roots. The increase in PAL and POD activities was 3.7 and 3.3 times higher,
respectively at 168 h of salicylic acid treatments through foliar spray than control
plants. Cucumber plants treated by combination of 7 mM SA (foliar spray) and
Bacillus subtilis (soil drench), prior to fungal infection by Fusarium oxysporum f.sp.
radicis cucumerinum, exhibited reduction of fungal infection and increased plant
growth . Evaluation of total phenol content and polyphenol oxidase activities show
that the combined application of SA and Bacillus subtilis significantly increased the
above plant defense compounds compared to SA and Bacillus subtilis alone and
control. The peak levels of them were observed in 7 and 5 days after elicitors'
application, respectively. According to these results, SA as a chemical elicitor and
Bacillus subtilis as biocontrol agent and plant growth promoter can be integrated for
effective protection of cucumber plants against FORC infection [300].
The amount of total phenols, free amino acid and pectin was reported by Pathak
et al,[169] in maximum levels in the immune cultivar to charcoal rot and minimum
in the highly susceptible cultivar of sunflower. They added that free and total phenols
were increased in infested shoot of the tested sunflower hybrids specially 15 days
after sowing in soil infested but with non-significant increase at 45 and 90 days.
However others found that the contents were significantly higher in the resistant
hybrid than in the moderately susceptible one [175]. Chemical inducers and
biological control increased phenols contents in strawberry plants infected with wilt
pathogens, [136]. Vermerris and Nicholson [178] reported that phenolic acids are
generally not abundant in most plants. There were a few exceptions: gallic acid and
salicylic acid (SA). Gallic acid was a precursor for the ellagitannins and gallotannins.
Salicylic acid was an important defense compound because it mediates systemic
acquired resistance (SAR), a resistance mechanism whereby SA was used as a signaling
molecule to relay information on pathogen attack to other parts of the plant. Upon
receiving the SA signal, a general defense response was activated which included
biosynthesis of pathogenesis-related (PR) proteins.
The present work evaluated the effect of ten antagonistic fungi (3 isolates of
Trichoderma harzianum, 2 isolates of T. viride, Chaetomium globosum, Chaetomium
bostrycoides, Trichoderma spp., Chaetomium spp. and Penicillium spp.). Also
fourteen antagonistic bacterial isolates were evaluated (3 isolates of Bacillus subtilis ,
3 isolates of Pseudomonas fluorescens, Pseudomonas putida, Bacillus megtela, 2
isolates of Serratia marcensens and 4 isolates of Bacillus spp.) Besides 8 chemical
compounds (salicylic acid, oxalic acid, citric acid, ascorbic acid, K2HPO4, CoSO4,
CaSO4 and KMnO4) were assed, each on amino acids content. The obtained results
indicated that, all treatments significantly decreased amino acids content. All tested
antagonistic fungi significantly decreased amino acids, except Chaetomium
bostrycoides and Chaetomium globosum. The highest decrease of amino acids was
induced by Trichoderma spp. followed by Trichoderma harzianum1 and
Trichoderma viride1. Also all tested antagonistic bacteria significantly decreased
amino acids, except Pseudomonas fluorescens2. The highest decrease of amino acids
was induced Serratia marcensens2 followed by Bacillus megtela and Serratia
marcensens1. All tested chemical compounds significantly decreased amino acids.
The highest decrease of amino acids was induced by calcium sulphate (CaSO4)
124
followed by (KMnO4) and ascorbic acid. The treatments decreased total amino acid
as a result of decreasing the Fusarium wilt disease severity. Resistant variety
compared with susceptible variety plants against many diseases was reported by El-
Shanawani et al. [289] who indicated that the highly susceptible variety of cucumber
contained higher amounts of total free amino acids in healthy leaves than the highly
resistant one. The increase in total free amino acids was more pronounced in the
highly susceptible variety than in the highly resistant one.
4.8 Anatomical studies:
Six antagonistic fungi (2 isolates Trichoderma harzianum, T. viride,
Chaetomium bostrycoides, Trichoderma spp. and Penicillium spp.).Also,eight
antagonistic bacterial isolates (Bacillus subtilis, 2 isolates of Pseudomonas
fluorescens, Pseudomonas putida, Bacillus megtela, Serratia marcensens and 2
isolates of Bacillus spp.) as well as five chemical compounds (salicylic acid, oxalic
acid, K2HPO4, CoSO4 and CaSO4 ) were evaluated on certain histological features of
main cucumber root at 50 days after planting. The obtained results show many signs
of resistance. All treatments increased the number of xylem vessels (NXV) in the
vascular bundle that seemed to be correlated with the resistance against the Fusarium
wilt disease. Also the number of fiber layer, thickness of fiber layer, wall thickness of
the fiber cell and cambium region thickness seemed strong barrier to infection with
FOC and was positively changed in the treated plants. A new regenerated vascular
bundle was also observed in the treated plants. Thus, treating cucumber seeds before
planting induced positive changes in their water conductive elements.They resist the
wilt disease development by facilitating absorbing more water as the plants need. In
fact, the functional water-conducting system, the tracheary elements of the xylem, is
required to sustain plant growth and development [301]. The enlarged number of
xylem vessels and width of the vascular bundles caused by treatments might be a
probable induced defense mechanism against the cucumber Fusarium wilt. Neither
conidia nor mycelia of the tomato Fusarium wilt pathogen were detected in leaf
petioles of treated and untreated tomato plants [302]. Pennypacker [303] stated that,
no conidia were observed in advance of the mycelium in xylem vessel elements of
carnation infected with Fusarium oxysporum f.sp. dianthi. They added that, absence
of conidia in advance of mycelium in the xylem vessel elements is probably the
primary reason for the success of culture indexing as a controlling measure for
Fusarium wilt of carnation. In fact, xylem plays an important role in strengthening
plant bodies as well as in transporting water and minerals. It is a complex tissue
composed of vessels, tracheids, fibres and parenchyma. In arabidopsis, secondary
xylem does not develop in immature fluorescence stems shorter than 10 cm, although
primary xylem does exist in them [304]. Anatomical studies of treated watermelon
plants against wilt pathogen showed many marked signs of resistance. In sections of
control-infected plants, the fungus was spread in cortex cells and in xylem vessels.
Treated inoculated and non-inoculated plants, cell wall of epiderms was thicker and the
cortex area wider than the non-treated - non-inoculated one. Number of xylem vessels
was higher in case of treatment than non-treatment. Intera-between vascular bundles
cambium (interfascicular) was regenerated under the influence of the treatment by
125
salicylic acid, hydrogen peroxide and cobalt ions agents. It was divided to form 3 to 4
layers and in one case a thick walled structure appeared [126].
4.9 Carrying the most effective antagonistic isolates of fungi and bacteria on
different carrier material The percentage of wilted cucumber plants were significantly reduced by using
different carrier materials inoculated by antagonistic fungal isolates. Paraffin oil was
the most effective for decreasing incidence of disease. Wilted plants were ranged
between 3.00 to 35.00%. Trichoderma viride on all carriers was the most effective
fungal isolates and reducing wilted plants from 89.00% in control to 3.00% in treated
plants. Bacillus megtela was the most effective effective bacterial isolates and
reducing wilted plants from 89.00% in control to (3.00%) in treated plants followed
by Pseudomonas fluorescens reducing wilted plants to 5.00%. These results are in
agreement with the finding of Abd El-Ghafar et al. [55] who reported that the
percentage of pre-emergence and wilted cucumber and watermelon plants was
significantly reduced by using different carrier materials inoculated by Pseudomonas
fluorescens. Antagonistic bacteria are usually applied with a carrier or adhesive
materials are peat, methyl cellulose, xanthan, gum, talc or gum Arabic [305]. Van
Peer et al. [244] found that Pseudomonas sp. strain suppressed Fusarium wilt in
carnation. Kloepper and Tuzun [306] reported that strains of plant growth-
promoting rhizobacteria (PGPR) induced systemic resistance against F. oxysporum f.
sp. cucumerinum. Mechanisms of biological control of Fusarium wilt by beneficial
microorganisms induced through competition for nutrients as iron, composition for
infection sites on roots and production of antibiotics [307, 308, 233]. They mentioned
that treatment with PGPR reduced spread of F. oxysporum f. sp. cucumerinum in
internal stems and petioles cucumber plants.
126
Conclusion
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 agriculture 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. Cucumber plants
are affected by several fungal pathogens, and Fusarium oxysporum Schlechtend.:Fr.
is among the most important pathogens. The causal agent of wilt disease in cucumber
Fusarium oxysporum f. sp. cucumerinum is economically important wilting pathogen
and causing significant yield losses in greenhouse of cucumber. The new trends now
in the entire world in the field plant pathology aimed at developing alternative
approaches for managing crop diseases to reducing use of fungicides in the control of
diseases.
Basic scientific results research.
1- F. oxysporum f.sp. cucumerinum (FOC) was the causal agent of cucumber
Fusarium wilt.
2- Inoculum density of 1x 107 of FOC showed the highest percentage of dead
plants, while, inoculum densities of 1 x 103 and 1 x 10
4 were the least effective.
3- All Trichoderma and Chaetomium bostrycoides filtrates and all bacterial
isolates at 50% concentration completely inhibited spore germination of FOC and
inhibiting the linear growth of FOC.
4- Oxalic acid at 10 mM completely inhibited mycelial growth of FOC, while,
salicylic acid, oxalic acid, citric acid and ascorbic acid at concentration 5 and 10 mM
completely inhibited spore germination of FOC.
5- Trichoderma harzianum3, Trichoderma spp. and Chaetomium bostrycoides
were the most effective in reducing disease incidence and disease severity of
cucumber wilt.
5- Bacillus megtla, Pseudomonas fluorescens3 and Serratia marcensens2 were
the most effective isolates and completely prevented the disease incidence.
6- Salicylic acid and CaSO4 were the most effective chemicals in reducing
disease incidence and disease severity of cucumber wilt under greenhouse conditions.
7- Under protected agriculture houses Trichoderma harzianum No.3,
Trichoderma spp. and Trichoderma viride No.1 were the most effective isolates and
reduced disease severity by 90.27, 89.83 and 87.73% respectively and increased the
fruits weight (Kg)/plant by 344.23, 336.54 and 320.19% respectively.
8- Under protected agriculture houses Bacillus megtla was the most effective
isolates and completely prevented the disease incidence followed by Serratia
marcensens No.2 and Pseudomonas fluorescens No.3 and reduced disease severity by
91.37 and 90.67% respectively, also increased fruits weight (Kg)/plant which
increased by 350.00, 342.31 and 333.65 % respectively.
9- Under protected agriculture houses salicylic acid completely prevented the
disease followed by CaSO4 and KMnO4.They reduced the disease severity by 93.24
127
and 92.41% respectively whereas, increased fruits weight/plant by 343.27, 330.77
and 311.54% respectively.
10- As for the effect of biotic agents and chemical inducers they increased
activity of peroxidase, polyphenol-oxidase, chitinase enzymes and also lignin,
phenols content. On the other hand, they decreased the reducing, non-reducing and
total sugars and amino acids in cucumber plants.
11- Isozyme pattern of peroxidase in cucumber extract contained two bands
expect in case of Trichoderma harzianum3, Bacillus spp.1, oxalic acid, non-infested
and infested control. One additional band was found in case of immunized plants.
Bands volume of immunized plants was denser than non-infested and infested
control.
12- Treating cucumber seeds with fungal and bacterial antagonistic isolates or
soaking seeds in chemical inducers solutions, before planting induced positive
changes in their water conductive elements, reasonably they resist the wilt disease
development by facilitating absorbing more water as the plants are need. Also
number of fiber layer, thickness of fiber layer and cambium region thickness that
seemed strong barrier to infection with FOC positively changed in the treated plants
with all fungal and bacterial antagonistic isolates or soaking seeds in chemical
inducers solutions, before planting comparing to the infested control with FOC.
13- The percentage of wilted cucumber plants were significantly reduced by
using different carrier materials inoculated by antagonistic fungal isolates as
compared with control. Paraffin oil was the most effective for decreasing incidence of
disease. Wilted plants were ranged 3.00-35.00%. Trichoderma viride on all carriers
was the most effective fungal isolates and reducing wilted plants from 89.00% in
control to 3.00% in treated plants.
14- The percentage of wilted cucumber plants were significantly reduced by
using different carrier materials inoculated by antagonistic bacterial isolates as
compared with control. Paraffin oil was the most effective for decreasing incidence of
disease. Bacillus megtela was the best effective bacterial isolate and reduced wilted
plants from 89.00% in control to 3.00% in treated plants.
Assessment of the completeness of the work tasks: the tasks set in the
dissertation have been completed successfully:
1- Biotic and abiotic agents were used successfully to induce resistance of
cucumber against attack with fusarium wilt disease under protected houses.
2- The most effective biotic agents were produced in commercial products as
alternatives to fungicides, to reducing use of fungicides in the control of cucumber
fusarium wilt disease under protected houses.
Recommendations on the present research. The results of present
dissertational work may be applied in the use of materials for programs aimed to
production of bio-control agents in commercial products.
The results of present dissertational work may be implied a lecture material for
general and special courses in "Plant pathology", "Biological control of plant
diseases", "New trends in controlling of plant pathology" and "Dynamics of plant
resistance to diseases" in higher educational institutions for plant pathology.
128
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
pathology
Implementation level and application field: For the first time in Kazakhstan to
study induction cucumber resistance against Fusarium wilt in Kazakhstan. Studying
the possibility to use biotic and a biotic agents to induce resistance of cucumber
against Fusarium revealed that, many of biotic isolates and abiotic agents can be used
to induce resistance of cucumber against Fusarium wilt and also abiotic agent as
methods to control. The most effective antagonistic fungal and bacterial isolates and
also biotic agents that reduced the diseases severity of cucumber fusarium wilt also
positively changed in anatomical characters that were investigated in cucumber root.
Activity of peroxidase, polyphenol-oxidase, chitinase enzymes and also lignin
content also positively affected in treated plants with biotic isolates and abiotic
agents. This investigation explains mechanism of induced cucumber resistance
against Fusarium wilt.
Economical effectiveness or work significance: This study is the first study of
induction cucumber resistance against Fusarium wilt in Kazakhstan. The results of
the research that, production the biotic agents in commercial products that 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 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
vegetables plant that produced under protected houses.
129
References
1 Hanam J.J., Holley W.D., Goldsberry K.L. Greenhouse management //
Springer-Verlag, Berlin. - 1978.
2 Vakalounakis D.J. Diseases and pests of vegetable crops and their control //
Technological Education Institute, Heraklio, Greece. - 1988.
3 Lumsden R.D., Rldout C.J., Vendemia M.E, Harrlson D.J., Waters R.M.,
Walter J.F. Characterization of major secondary metabolites produced in soilless mix
by a formulated strain of the biocontrol fungus Gliocladium virens // Can. J.
Microbiol. - 1992. - Vol. 38. - P. 1274-1280.
4 Zhang B.X., Ge O.X., Chen D.H., Wang Z.Y., He S.S. Biological and
chemical control of root diseases on vegetable seedlings in Zhejiang province, China
// 1990. - P. 181-91. In: Biological Control of Soil-Borne Plant Pathogens. Hornby,
D.E.; Cook, R.J.; Henis, Y.; KO, W.H.; Rovira, A.D.; Schippers, B. and Scott, P.R.
(eds.). Cab International, Walling Ford.
5 Kuc J. Plant immunization and its applicability for disease control // 1987.
- P. 255-274 in: Innovative Approaches to Plant Disease Control. I. Chet, ed. John
Wiley and Sons, New York.
6 Misaghi I.J. Physiology and Biochemistry of Plant Pathogens Interactions.
Plenum Press. New York and London. - 1982. 287pp.
7 Lyon G.D., Newton A.C. Implementation of elicitor mediated induced
resistance in agriculture // 1999. - P. 299-318. In: Induced Plant Defences Against
Pathogens and Herbivores. Agrawal, A.A.; Tuzan, S. and Ent, E.B. (eds.). Aps Press.
St. Paul, USA.
8 Oostendrop M., Kumz W., Dietricch B., Staub T. Induced disease
resistance in plants by chemicals // Euro. J. Plant Pathol. - 2001. - Vol. 107. - P. 19-
28.
9 Larkin R.P., Hopkins D.L., Martin F.N. Suppression of Fusarium wilt of
watermelon by nonpathogenic Fusarium oxysporum and other microorganisms
recovered. Phytopathology. - 1996. - Vol. 86. - P. 812-819.
10 Leeman M., Den Ouden F.M., Van Pelt J.A., Cornelissen C., Matamala
Garros A., Bakker P.A.H.M., Schippers B. Suppression of Fusarium wilt of radish by
co-inoculation of fluorescent Pseudomonas spp. and root-colonizing fungi. Eur. J.
Plant Pathol. - 1996. - Vol. 102. - P. 21-31.
11 Lemanceau P., Bakker P.A.H.M., Dekogel W.J., Alabouvette C., Schippers
B. Effect of pseudobactin 358 production by Pseudomonas putida WCS358 on
suppression of Fusarium wilt of carnations by nonpathogenic Fusarium oxysporum
Fo47. Appl. Environ. Microbiol. - 1992. - Vol. 58. - P. 2978-2982.
12 Raaijmakers J.M., Leeman M., Van Oorschot M.M.P., van der Sluis
Schippers B., Bakker P.A.H.M. Dose-response relationships in biological control of
fusarium wilt of radish by Pseudomonas spp. Phytopathology. - 1995. - Vol. 85. - P.
1075-1081.
13 Vakalounakis D.J., Fragkiadakis G.A. Plant pathobreeding with emphasis
in tomato and cucurbits (in Greek) // Vakalounakis, Heraklio, Greece. - 2003.
130
14 Vakalounakis D.J., Wang Z., Fragkiadakis G.A., Skaracis G.N., Li D.B.
Characterization of Fusarium oxysporum isolates obtained from cucumber (Cucumis
sativus) in China bypathogeni-city, VCGs and RAPD // Plant Dis. - 2004. - Vol. 88. -
P. 645-649.
15 Vakalounakis D.J. Root and stem rot of cucumber caused by Fusarium
oxysporum f.sp. radicis-cucumerinum // Plant Disease. - 1996. - Vol. 80. - P. 313-
316.
16 Vakalounakis D.J., Fragkiadakis G.A. Genetic Diversity of Fusarium
oxysporum Isolates from Cucumber: Differentiation by Pathogenicity, Vegetative
Compatibility, and RAPD Fingerprinting // Phytopathology. - 1999. - Vol. 89. - P.
161-168.
17 Pavlou G.C., Vakalounakis D.J., Ligoxigakis E.K. Control of root and stem
rot of cucumber, caused by Fusarium oxysporum f. sp. radicis-cucumerinum, by
grafting onto resistant rootstocks // Plant Dis. - 2002. - Vol. 86. - P. 379-382.
18 Punja Z. K., Parker M. Development of Fusarium root and stem rot, a new
disease on greenhouse cucumbers in British Columbia caused by Fusarium
oxysporum f. sp. radicis-cucumerinum // Can. J. Plant Pathol. - 2000. - Vol. 22. - P.
349-363.
19 Adachi K., Kobayashi M., Takahashi E. Development of wilt symptom
caused by fusaric acid on intact and semi-intact cucumber plants // Japanese-Journal-
of-Soil-Science-and-Plant-Nutrition. - 1991. - Vol. 62, № 2. - P. 101-106.
20 Dong W. Chen L. Identification of resistance to Fusarium wilt in Sichuan
cucumber cultivars // Crop Genetic Resources. - 1993. - Vol. 3. - P. 16.
21 Martinez R., Aguilar M.I., Guirado M.L., Alvarez A., Gomez J. First report
of fusarium wilt of cucumber caused by Fusarium oxysporum in Spain // Plant
Pathology. - 2003. - Vol. 52, № 3. - P.410.
22 Balaz F., Stojsin V., Jasnic S., Inic D., Bagi F., Budakov D. The most
important fungal diseases in greenhouse production // Biljni Lekar (Plant Doctor). -
2009. - Vol. 37, № 5. - P. 468-493.
23 Ding J., Shi K., Zhou Y.H., Yu J.Q. (a). Microbial community responses
associated with the development of Fusarium oxysporum f. sp. cucumerinum after
24-epibrassinolide applications to shoots and roots in cucumber // European Journal
of Plant Pathology. - 2009. - Vol. 124, № 1. - P. 141-150.
24 Zhou X.G., Wu F.Z., Wang X.Z., Yuan Y. Progresses in the mechanism of
resistance to Fusarium wilt in cucumber (Cucumis sativus L.) // Journal of Northeast
Agricultural University (English Edition). - 2008. - Vol. 15, № 3. - P. 1-6.
25 Chen F., Wang M., Zheng Y., Luo J.M., Yang X.R., Wang X.L.
Quantitative changes of plant defense enzymes and phytohormone in biocontrol of
cucumber Fusarium wilt by Bacillus subtilis B579 // World Journal of Microbiology
& Biotechnology. - 2010. - Vol. 26, № 40. - P. 675-684.
26 Dormanns-Simon E. Biological agents for the control of soil-borne pests //
Technical Workshop on non-chemical alternatives to replace methyl bromide as a soil
fumigant, Budapest, Hungary, 26-28 June - 2007. - P. 99-104.
131
27 Shimotsuma M., Kuc J., Jones C.M. The effects of prior inoculations with
non-pathogenic fungi on Fusarium wilt of watermelon // HortScience. - 1972. - Vol.
7. - P. 72-73.
28 Kuc J., Schockley G., Kearney K. Protection of cucumber against
Colletotrichum lagenarium by Colletotrichum lagenarium // Physiol. Plant
Pathol. - 1975. - Vol. 7. - P. 195-199.
29 Tigchelaar E.C., Dick J.B. Induced resistance from simultaneous
inoculation of tomato with Fusarium oxysporum Sacc. and Verticillium albo-
atrum Reinke & Berth // HortScience. - 1975. - Vol. 10. - P. 623-624.
30 Caruso F.L., Kuc J. Field protection of cucumber, watermelon and
muskmelon against Colletotrichum lagenarium by Colletotrichum lagenarium //
Phytopathology. - 1977. - Vol. 67. - P. 1290-1292.
31 Tjamas E.C. Induction of resistance to verticillum wilt in cucumber
(Cucumis sativus) // Physiol. Plant Pathol. - 1979. - Vol. 15. - P. 223-227.
32 Ishiba C., Toni T., Murata M. Protection of cucumber against anthracnose
by a hypovirulant strain of Fusarium oxysporum f sp. cucumerineum // Ann.
Phytopathol. Soc. Jpn. - 1981. - Vol. 47. - P. 352-359.
33 Sirry A.R., Salem S.H., Zayed M.A., Anwar Dawlat A. Rhizpsphere
microflora of sesame plants infected with root-rot disease and their activities in
antagonizing the main pathogens // Egypt. J. Microbiol. - 1981. - Vol. 18, № 1-2. - P.
65-78.
34 Gessler C., Kuc J. Appearance of a host protein in cucumber plants
Infected with viruses, bacteria and fungi // Journal of Experimental Botany. - 1982. -
Vol. 33, № 132. - P. 58-66.
35 Iida W., Nakano T., Amemiya Y., Hirano K. Effect of soil amendment with
ground crab shell on Fusarium wilt of cucumber caused by Fusarium oxysporum f.sp.
cucumerinum // Technical-Bulletin,-Faculty-of-Horticulture-Chiba-University. -
1985. - Vol. 36. - P. 127-134.
36 Martyn R.D. Differential cross protection of watermelon to Fusarium will
by related formae speciales // (Abstr.) Phytopathology. - 1985. - Vol. 75. - P. 1304.
37 Morshed M. S. In vitro antagonism of different species of some seed-borne
fungi of bean (Phaseolus vulgaris L.) // Banglades J.of Botany. - 1985. - Vol. 14, №
2. - P. 119-126.
38 D’Ercole N., Nipoti P. Biological control of Fusarium and Verticillium
infections in tomatoes under protected cultivation // Colture–Protette. - 1986. - Vol.
15, № 3. - P. 55-59.
39 Seo I.S. Effect of organic matter on the occurrence of Fusarium wilt in
cucumber // Korean-Journal-of-Plant-Pathology. - 1986. - Vol. 2, №1. - P. 43-47.
40 D’Ercole N., Nipoti P., Finessi L.E., Manzali D. Review of several years
of research in Italy on the biological control of soil fungi with Trichoderma spp //
Bulletin. OEPP. - 1988. - Vol. 18, № 1. - P. 95-102.
41 Park C.S., Paulitz T.C., Baker R. Biocontrol of Fusarium wilt of cucumber
resulting from interactions between Pseudomonas putida and nonpathogenic isolates
of Fusarium oxysporum // Phytopathology. - 1988. - Vol. 78, № 2. - P. 190-194.
132
42 Tari P.H., Anderson A.J. Fusarium Wilt Suppression and Agglutinability
of Pseudomonas putida // Appl Environ Microbiol. - 1988. - Vol. 54, № 8. - P. 2037-
2041.
43 Moon B.J., Chung H.S., Cho C.T. Studies on antagonism of Trichoderma
species to Fusarium oxysporum f.sp. fragariae. I- Isolation, identification and
antagonistic properties of Trichoderma species // Korean Journal of Plant Pathology.
- 1988. -Vol. 4, № 2. - P. 111-123.
44 Cho C.T., Moon B.J. Ha S.Y. Biological control of Fusarium oxysporum
f.sp. cucumerinum causing cucumber wilt by Gliocladium virens and Trichoderma
harzianum // Korean Journal of Plant Pathology. - 1989. - Vol. 5, № 3. - P. 239-249.
45 Abd-El-Moity T.H., Eisa H.A. Amer (Afaf) H. Evaluation of some
biocontrol agents in controlling cotton seedling disease // Zagazig J. Agric. Res. -
1990. - Vol. 17. - P. 1187-1194.
46 Calvet C. Pera J., Barea J.M. Interactions of Trichoderma spp. with Glomus
mosseae and two wilt pathogenic Fungi // Agriculture, Ecosystems and Environment.
- 1990. - Vol. 29, № 1-4. - P. 59 – 65.
47 Lemanceau P., Alabouvette C. Biological control of Fusarium diseases by
fluorescent Pseudomonas and non-pathogenic Fusarium // Crop Prot. - 1991. -
Vol. 10. - P. 279-286.
48 Ziedan E.H.E. Studies on Fusarium wilt disease of sesame in ARE // M. Sc.
Thesis, Fac. of Agric. Ain-Shams Univ. - 1993.
49 Amemiya Y., Kondo A., Hirano K., Hirukawa T., Kato T. Antifungal
substances produced by Cheatomium globosum // Technical Bulletin of Faculty of
Horticulture, Chiba University. - 1994. - Vol. 48. - P. 13-18.
50 Castrejon Sanguino A. Detection, in vitro, of fungi antagonistic to
Verticillium dahliae Kleb. race T.9 // ITEA Produccion Vegetal. - 1994. - Vol. 90, №
2. - P. 129-131.
51 Bae Y.S.; Shim C.K.; Park C.S., Kim H.K. Synergistic effects of
Gliocladium virens and Pseudomonas putida in the cucumber rhizosphere on the
suppression of cucumber Fusarium wilt // Korean-Journal-of-Plant-Pathology. -
1995. - Vol.11, № 4. - P. 287-291.
52 Liu L., Kloepper J.W., Tuzun S. Induction of systemic resistance in
cucumber against Fusarium wilt by plant growth-promoting rhizobacteria //
Phytopathology. - 1995. - Vol. 85, № 6. - P. 695-698.
53 Minuto A., Migheli Q., Garibaldi A., Vanachter A. Integrated control of
soil-borne plant pathogens by solar heating and antagonistic microorganisms //
Fourth international symposium on soil and substrate infestation and disinfestation,
Leuven, Belgium, 6-12 September 1993. Acta-Horticulturae. - 1995. - Vol. 382. - P.
138-143.
54 Moon B.J., Chung H.S., Park H.C. Studies on antagonism of Trichoderma
species to Fusarium oxysporum f.sp. fragariae. Biological control of Fusarium wilt
of strawberry by a mycoparasity Trichoderma harzianum // Korean Journal of Plant
Pathology. - 1995. - Vol. 11, № 4. - P. 298 – 303.
133
55 Abd El-Ghafar N.Y., Mervat Amara A.T., Nagwa Gamil A.M. Biocontrol
of some fusarium wilt diseases using different carrier materials for Pseudomonas
fluorescein // Al-Azhar J. Agric. Res. - 1996. - Vol. 23. - P. 53-67.
56 Dinakaran D., Marimuthu T. Inhibition of Macrophomina phaseolina
(Tassi.) Goid by mutants of Trichoderma viride Pers // ex Fr. Journal of Biological
Control. - 1997. - Vol. 11, № 1/2. - P. 43-47.
57 Jeong M.J., Jang S.S., Park C.S. Influence of soil pH and salinity on
antagonistic activity and rhizosphere competence of biocontrol agents // Korean-
Journal-of-Plant-Pathology. - 1997. - Vol. 13, № 6. - P. 416-420.
58 Khalifa M.M.A. Studies of root-rot and wilt diseases of sesame plants // M.
Sc. Thesis, Fac. Agric., Moshtohor, Zagazig Univ. Benha branch. - 1997. 158 pp.
59 Kowalik M. Trichoderma spp. and Gliocladium spp. As factors controlling
the occurrence of pathogenic fungi in stands of alfalfa and grasses // Progress in Plant
Protection. - 1997. - Vol. 37, № 2. - P. 390-393.
60 Xu J.H., Wang J.B., Li R.Q., Miao C. Histopathological study on
cucumber infected by Fusarium wilt // Acta Phytopathologica Sinica. - 1997. - Vol.
27, № 4. - P. 349-352.
61 Larkin R.P., Fravel D.R., Dugger P., Richter D. Biological control of wilt
pathogens with fungal antagonists // Proceedings Beltwide Cotton Conferences, San
Diego, California, USA. - 1998. 5-9 January. - Vol. 1. - P. 125-127.
62 Larkin R.P., Fravel D.R. Efficacy of various fungal and bacterial biocontrol
organisms for control of Fusarium wilt of tomato // Plant-Disease. - 1998. - Vol. 82,
№ 9. - P. 1022-1028.
63 Hamed H.A. Biological control of basal stem rot and wilt of cucumber
caused by Pythium ultimum and Fusarium oxysporum f.sp. cucumerinum // African-
Journal-of-Mycology-and-Biotechnology. - 1999. - Vol. 7, № 1. - P. 81-91.
64 Hammad A.M.M., El-Mohandes M.A.O. Controlling Fusarium wilt disease
of cucumber plants via antagonistic microorganisms in free and immobilized states //
Microbiological-Research. - 1999. - Vol. 154, № 2. - P. 113-117.
65 Singh P.P., Shin Y.C., Park C.S., Chung Y.R., Shin Y.C., Park C.S., Chung
Y.R. Biological control of Fusarium wilt of cucumber by chitinolytic bacteria //
Phytopathology. -1999. -Vol. 89, № 1. -P. 92-99.
66 Mathivanan N., Srinivasan K., Chelliah S. Biological control of soil-borne
diseases of cotton, eggplant, okra and sunflower by Trichoderm aviride // Zeitschrift-
fur-Pflanzenkrankheiten-und-Pflanzenschutz. - 2000. - Vol. 107, № 3. - P. 235-244.
67 Paulitz T., Nowak T.B., Gamard P., Tsang E., Loper J. A novel antifungal
furanone from Pseudomonas aureofaciens, a biocontrol agent of fungal plant
pathogens // Journal-of-Chemical-Ecology. - 2000. - Vol. 26, № 6. - P. 1515-1524.
68 Abdou E., Abd-Alla H.M., Galal A.A. Survey of sesame root- rot and wilt
disease in Minia and their possible control by ascorbic and salicylic acids // Assiut
Journal of Agricultural Sciences. - 2001. - Vol. 32, № 3. - P. 135-152.
69 Srivastava A.K., Tanuja S., Tana T.K., Arora D.K. Induced resistance and
control of charcoal rot in Cicer arietinum (chickpea) by Pseudomonas fluorscens //
Canadian Journal of Botany. - 2001. - Vol. 79, № 7. - P. 787-795.
134
70 Cotes A.M., Elad Y. (ed.), Freeman S. (ed.), Monte E. Biocontrol of
fungal plant pathogens - from the discovery of potential biocontrol agents to the
implementation of formulated products // IOBC-WPRS Working Group "Biological
Control of Fungal and Bacterial Plant Pathogens". Proceedings of the sixth meeting,
Biocontrol Agents: Mode of Action and Interaction with Other Means of Control,
Sevilla, Spain, November 30 - December 3, 2000. Bulletin-OILB-SROP. - 2001. -
Vol. 24, № 3. - P. 43-47.
71 Hao B.Q., Ma L.P., Qiao X.W., Gao F., Hao B.Q., Ma L.P., Qiao X.W.,
Gao F. Bioassay of antagonistic bacteria against cucumber fusarium wilt // Chinese-
Journal-of-Applied-and-Environmental-Biology. - 2001. - Vol. 7, № 2. - P. 155-157.
72 Koike N., Hyakumachi M., Kageyama K., Tsuyumu S., Doke N. Induction
of systemic resistance in cucumber against several diseases by plant growth-
promoting fungi: lignification and superoxide generation // European-Journal-of-
Plant-Pathology. - 2001. - Vol. 107, № 5. - P. 523-533.
73 Gottlieb M. Factors affecting the ability to colonisation of root system by
bacteria from the genus Pseudomonas // Postepy-Mikrobiologii. - 2002. - Vol. 41, №
3. - P. 277-297.
74 Lutz M.P., Wenger S., Maurhofer M., Defago G., Elad Y. (ed.), Kohl J.
(ed.), Shtienberg D. Mixture of two antagonists: influence on expression of their key
biocontrol factors // IOBC-WPRS Working Group 'Biological Control of Fungal and
Bacterial Plant Pathogens'. Proceedings of the 7th working group meeting, Influence
of abiotic and biotic factors on biocontrol agents at Pine Bay, Kusadasi, Turkey, 22-
25 May 2002. Bulletin-OILB-SROP. - 2002. - Vol. 25, № 10. - P. 237-239.
75 Mazzola M., Veen J.H. (ed.), Laanbroek H.J. (ed.), Vos W.M.
Mechanisms of natural soil suppressiveness to soilborne diseases // Proceedings of
the 9th International Symposium on Microbial Ecology, Amsterdam, Netherlands,
August 2001. Antonie-van-Leeuwenhoek. - 2002. - Vol. 81, № 1-4. - P. 557-564.
76 Rocha R.V., Omero C., Chet I., Horwitz B.A., Herrera E.A. Trichoderma
atroviride G-protein alpha-subunit gene tga1 is involved in mycoparasitic coiling and
conidiation // Eukaryotic-Cell. - 2002. - Vol. 1, № 4. - P. 594-605.
77 Srikanta D., Biswapati M., Maity D., Roy S.K., Dey S., Mondal B.
Different techniques of seed treatment in the management of seedling disease of
sugarbeet // Journal-of-Mycopathological-Research. - 2002. - Vol. 40, № 2. - P. 175-
179.
78 Larena P., Sabuquillo P., Melgarejo P., Cal A. Biocontrol of fusarium and
verticillium wilt of tomato by Penicillium oxalicum under greenhouse and field
conditions // Journal-of-Phytopathology. -2003. -Vol. 151, № 9. -P. 507-512.
79 Mascher F., Schnider K.U., Haas D., Defago G., Moenne L.Y. Persistence
and cell culturability of biocontrol strain Pseudomonas fluorescens CHA0 under
plough pan conditions in soil and influence of the anaerobic regulator gene anr //
Environmental-Microbiology. - 2003. - Vol. 5, № 2. - P. 103-115.
80 Mayur D., Deshmukh V.V. Effect of bio-agents and soil amendments on
chickpea wilt caused by Fusarium oxysporium f. sp. Ciceri // Research-on-Crops. -
2003. - Vol. 4, № 1. - P. 141-143.
135
81 Ratul S., Tanuja S., Rakesh K., Juhi S., Alok K.S., Kiran S. Dilip K.A.
Role of salicylic acid in systemic resistance induced by Pseudomonas fluorescens
against Fusarium oxysporum f. sp. ciceri in chickpea // Microbiological Research. -
2003. - Vol. 158 № 3. - P. 203-213.
82 Cal A., Larena I., Sabuquillo P., Melgarejo P. Biological control of tomato
wilts // Recent-Research-Developments-in-Crop-Science. - 2004. - Vol. 1, № 1. - P.
97-115.
83 Khan M. R., Khan S.M., Mohiddin F.A. Biological control of Fusarium
wilt of chickpea through seed treatment with the commercial formulation of
Trichoderma harzianum and/or Pseudomonas fluorescens // Phytopathologia-
Mediterranea. - 2004. - Vol. 43, № 1. - P. 20-25.
84 Landa B.B., Navas C.J.A., Jimenez D.R.M. Integrated management of
Fusarium wilt of chickpea with sowing date, host resistance, and biological control //
Phytopathology. - 2004. - Vol. 94, № 9. - P. 946-960.
85 85 Jeun Y.C., Park K.S., Kim C.H., Fowler W.D., Kloepper J.W.
Cytological observations of cucumber plants during induced resistance elicited by
rhizobacteria // Biological Control. - 2004. - Vol. 29. - P. 34–42
86 Sangle U.R., Bambawale O.M. New strains of Trichoderma spp. strongly
antagonistic against Fusarium oxysporum f. sp. Sesame // Journal-of-Mycology-and-
Plant-Pathology. - 2004. - Vol. 34, № 1. - P. 107-109.
87 Vagelas I.K., Gravanis F.T., Gowen S.R. Soilborne fungi and bacteria
symbiotically associated with Steinernema spp. acting as biological agents against
Fusarium wilt of tomato // Bulletin-OILB/SROP. - 2004. - Vol. 27, № 1. - P. 279-
284.
88 Zhu T.H., Xing X.P., Sun S.D. The antagonism mechanisms and diseases
control trials of Trichoderma strain T97 against several plant fungal pathogens in
greenhouse // Acta-Phytophylacica-Sinica. - 2004. - Vol. 31, № 2. - P. 139-144.
89 Hao Z.P., Christie P., Qin L., Wang C.X., Li X.L. Control of fusarium wilt
of cucumber seedlings by inoculation with an arbuscular mycorrhizal fungus //
Journal-of-Plant-Nutrition. - 2005. - Vol. 28, № 11. - P. 1961-1974.
90 Hari C., Surender S. Control of chickpea wilt (Fusarium oxysporum f sp.
ciceri) using bioagents and plant extracts // Indian-Journal-of-Agricultural-Sciences. -
2005. - Vol. 75, № 2. - P. 115-116.
91 Rudresh D.L., Shivaprakash M.K., Prasad R.D. Potential of Trichoderma
spp. as biocontrol agents of pathogens involved in wilt complex of chickpea (Cicer
arietinum L.) // Journal-of-Biological-Control. - 2005. - Vol. 19, № 2. - P. 157-166.
92 Shalini V., Dohroo N.P. Comparative efficacy of biocontrol agents against
Fusarium wilt of pea // Integrated-plant-disease-management-Challenging-problems-
in-horticultural-and-forest-pathology,-Solan,-India,-14-to-15-November-2003. -
2005. - P. 93-99.
93 Zhuang J.H., Gao Z.G., Yang C.C., Chen J., Xue C.Y., Mu L.X. Biocontrol
of Fusarium wilt and induction of defense enzyme activities on cucumber by
Trichoderma viride strain T23 // Acta Phytopathologica Sinica. - 2005. - Vol. 35, №
2. - P. 179-183.
136
94 Ayed F., Daami R.M., Jabnoun K.H., El-Mahjoub M. Potato vascular
Fusarium wilt in Tunisia: incidence and biocontrol by Trichoderma spp // Plant-
Pathology-Journal-Faisalabad. - 2006. - Vol. 5, № 1. - P. 92-98.
95 Bonjar G.H.S., Farrokhi P.R., Shafii B., Aghighi S., Mahdavi M.J.,
Aghelizadeh A. Laboratory preparation of a new antifungal agent from Streptomyces
olivaceus in control of Fusarium oxysporum f.sp. melonis of cucurbits in greenhouse
// Journal-of-Applied-Sciences. - 2006. - Vol. 6, № 3. - P. 607-610.
96 Domenech J., Reddy M.S., Kloepper J.W., Ramos B., Gutierrez M,J.
Combined application of the biological product LS213 with Bacillus, Pseudomonas
or Chryseobacterium for growth promotion and biological control of soil-borne
diseases in pepper and tomato // BioControl. - 2006. - Vol. 51, № 2. - P. 245-258.
97 Nel B., Steinberg C., Labuschagne N., Viljoen A. The potential of
nonpathogenic Fusarium oxysporum and other biological control organisms for
suppressing fusarium wilt of banana // Plant-Pathology. - 2006. - Vol. 55, № 2. - P.
217-223.
98 Sharma S.N., Chandel S.S. Biological control of gladiolus wilt caused by
Fusarium oxysporum f.sp. gladioli // Indian-Journal-of-Plant-Protection. - 2006. -
Vol. 34, № 1. - P. 97-100.
99 Someya N., Tsuchiya K., Yoshida T., Noguchi M.T., Sawada H. Combined
use of the biocontrol bacterium Pseudomonas fluorescens strain LRB3W1 with
reduced fungicide application for the control of tomato Fusarium wilt // Biocontrol-
Science. - 2006. - Vol. 11, № 2. - P. 75-80.
100 Chen X.L., Wang G.H., Jin J., Lu B.L. Biocontrol effect of Paenibacillus
polymyxa BRF-1 and Bacillus subtilis BRF-2 on fusarium wilt disease of cucumber
and tomato // Chinese Journal of Eco-Agriculture. - 2008. - Vol. 16, № 2. - P. 446-
450.
101 Li W., Hu J.C. Wang S.J. Growth-promotion and biocontrol of Cucumber
fusarium wilt by marine Bacillus subtilis 3512A // Journal of Shenyang
Agricultural University. - 2008. - Vol. 39, № 2. - P. 182-185.
102 Yang C.L., Xi Y.D., Liu B.W., Zhang M., Peng H.X. Primary study on
growth-promoting and biological control effects of Trichoderma harzianum T-h-30
on vegetables// Southwest China Journal of Agricultural Sciences. - 2008. - Vol. 21,
№ 6. - P. 1603-1607.
103 Zhang S.S., Raza W., Yang X.M., Hu J.A., Huang Q.W., Xu Y.C., Liu
X.H., Ran W., Shen Q.R. Control of Fusarium wilt disease of cucumber plants with
the application of a bioorganic fertilizer // Biology and Fertility of Soils. - 2008. -
Vol. 44, № 8. - P. 1073-1080.
104 Georgieva O.A., Georgiev G.A. Biological control of diseases on main
vegetables-researches and practice in Maritsa vegetable crops institute // Acta
Horticulturae. -2009. -Vol. 830. -P. 511-518.
105 Li J., Yang Q., Zhang S.M., Wang Y.X., Zhao X.Y. Evaluation of
biocontrol efficiency and security of A Bacillus subtilis strain B29 against cucumber
Fusarium wilt in field // China Vegetables. - 2009. - Vol. 2. - P. 30-33.
106 Liu A.R., Chen S.C., Chen K., Lin X.M., Wang F.H. Antagonism effect of
Trichoderma harzianum against Fusarium oxysporum on cucumber and related genes
137
expression analysis // Acta Phytophylacica Sinica. - 2010. - Vol. 37, № 3. - P. 249-
254.
107 Dean R.A., Kuc J. Rapid-lignification in response to wounding and
infection as a mechanism for induced system protection in cucumber // Physiology
and Molecular Plant Pathology. - 1987. - Vol. 31. - P. 69-81.
108 Kessmann H., Staub T., Hofmann C., Maetzke T., Herzog J., Ward E.,
Uknes S., Ryals J. Induction of Systemic Acquired Disease Resistance in Plants by
Chemicals // Annual Review of Phytopathology. - 1994. - Vol. 32, № 9. - P. 439-459.
109 Pearce R.B., Ride J.P. Specificity of induction of the lignification response
in wounded wheat leaves // Physiological Plant Pathology. - 1980. - Vol. 16, № 2. -
P. 197-198.
110 Komoto Y., Kimura T. Seed disinfection against angular leaf spot of
cucumber by organic acids and simultaneous seed disinfection against the disease and
Fusarium wilt // Bulletin-of-the-Chugoku-National-Agricultural-Experiment-
Station,-E. - 1983. - Vol. 21. - P. 21-36.
111 Mills P.R., Wood R.K.S. The effects of polyacrylic acid, acetylsalicylic
acid and salicylic acid on resistance of cucumber to Colletotrichum lagenarium //
Phytopathologische Zeitschrift. - 1984. - Vol. 111. - P. 209-216.
112 Salama A.A.M., Ismail I.M.K., Ouf S.A. Soaking Sclerotium cepivorum in
phenolic compounds and their effect on germination, growth and sclerotial formation
// Bulletin of the Faculty of Sci., Cairo Univ. - 1985. - Vol. 53, № 1. - P. 309-319.
113 Sun S.K., Huang J.W. Formulated soil amendment for controlling
Fusarium wilt and other soilborne diseases // Plant-Disease. - 1985. - Vol. 69(11). - P.
917-920.
114 Singh P.K., Dwivedi R.S. Chemical control of Sclerotium rolfsii Sacc., a
foot-rot pathogen of barley // National Academy Science Letters, India. - 1987. - Vol.
10, № 12. - P. 409-411.
115 Dubrava N.S., Dean R.A., Kuc J. Induction of systemic resistance to
anthracnose caused by Colletotrichum Lagenarium in cucumber by oxalates and extracts
from spinach and rhubarb leaves // Physiol. Mol. Plant Pathol. - 1988. - Vol. 33. - P. 69-
79.
116 Okuno T., Nakayama M., Okajima N., Furasawa I. Systemic resistance to
downy mildew and appearance of acid soluble proteins in cucumber leaves treated
with biotic and abiotic inducers // Annals of the Phytopathological Society of Japan. -
1991. - Vol. 57. - P. 203-211.
117 117 Elad Y. The use of antioxidants (free radical scavengers) to control
grey mould (Botrytis cinerea) and white mould (Sclerotinia sclerotiomm) in various crops
// Plant Pathology. - 1992. - Vol. 41, № 4. - P. 417-426.
118 Harfoush D.I., Salama D.S. (a). Pathological and biochemical response of
cucumber against powdery mildew associated with induced resistance by ethephon //
Annals Agric. Sci. Mansoura Univ. - 1992. - Vol. 17. - P. 3555-3565.
119 Harfoush D.I., Salama, D.S. (b). Induction of systemic resistance to
powdery mildew in cucumber leaves by seed soaking application with cobalt //
Annals Agric. Sci. Mansoura Univ. - 1995. - Vol. 33, № 17. - P. 3226-3237
138
120 Li E.B., Ma Y.Q., Ceng F.B. Preliminary study on the control of Fusarium
wilt in cucumber using KMnO4 solution // Chinese-Vegetables. - 1992. - Vol. 2. - P.
20-21.
121 Zhang Y.N., Liu Y.H. Studies on cucurbit diseases control by non-
fungicidal compounds // Acta-Phytopathologica-Sinica. - 1992. - Vol. 22, № 3. - P.
241-244.
122 Abd-El-Kareem F., Ashour W.E., Diab M.M., Aly M.M. Induction of
resistance in watermelon plants against Fusarium wilt using biotic and chemical
inducers // 5th
Nat. Conf. of Pests and Dis. of Veg. and Fruits, Ismailia, Egypt. -
1993. - P. 447-455.
123 Gamil N.A.M. (a). Induced resistance in squash plants against powdery
mildew by cobalt and phosphate sprays // Annals of Agricultural Science, Moshtohor.
- 1995. - Vol. 33. - P. 183-194.
124 Gamil N.A.M. (b). Aspirin induces resistance to powdery mildew in squash
plants // Annals of Agricultural Science, Moshtohor. - 1995. - Vol. 33. - P. 681-691.
125 Kobayashi N., Komada H. Screening of suppressive soils to Fusarium wilt
from Kanto, Tozan and Tokai areas in Japan, and analysis of their suppressiveness //
Soil Microorganisms. - 1995. - Vol. 45. - P. 21-32.
126 Gado E.A.M. Studies on the mechanism of induced resistance to
fusarium wilt of watermelon // MsC. Thesis, Fac. of Agric., Ain Shams University,
Egypt. - 1997. 153pp. 127 Shaat M.M.N. Virulence of Helminthosporium tetramera and Fusarium
oxysporum on cucumber plants // Assiut Journal of Agric. Sci. - 1998. - Vol. 29, №
5. - P. 85-101.
128 Zhu Y.Y., Shen Q.R., Xie X.D., Wang Y., Liang Y.C. Enzymatic activities
during the induced systemic resistance of cucumber by K2HPO4 // Journal of Nanjing
Agricultural University. - 1999. - Vol. 22, № 50. -P. 54 (Abstract).
129 Shahda W.T. Biological control of tomato damping-off of seedlings //
Alexandria-Journal of Agricultural Research. - 2000. - Vol. 45, № 1. - P. 317-329.
130 Attitalla I.H., Brishammar S. Oxalic-acid elicited resistance to fusarium
wilt in Lycopersicon esculentum Mill // Plant-Protection-Science. - 2002. - Vol. 38, № 1. - P. 128-131.
131 El-Ganaieny R.M.A., El-Sayed A.M. Gebrial M. Induced resistance to
fusarial disease in onion plants by treatment with antioxidants // Assiut J. of Agric.
Sci. - 2002. - Vol. 33. - P. 133-147.
132 El-Kolaly Ghada A.A. Pathological studies on root and crown rots of
strawberry in Egypt // Ph.D. Thesis, Fac. of Agric., Cairo University. - 2003. 151 p.
133 Khalifa M.M.A. Pathological studies on charcoal rot disease of sesame //
Ph, D. Thesis, Fac. Agric., Moshtohor, Zagazig Univ. Benha branch. - 2003. 236 pp.
134 Yuan F., Zhang C.L., Shen Q.R., Yuan F., Zhang C.L., Shen Q.R.
Alleviating effect of phenol compounds on cucumber fusarium wilt and mechanism //
Agricultural-Sciences-in-China. - 2003. - Vol. 2, № 6. - P. 647-652.
135 Peng J.Y., Deng X.J., Huang J.H., Jia S.H., Miao X.X., Huang Y.P. Role of
salicylic acid in tomato defense against cotton bollworm Helicoverpa armigera //
139
Hubner. Zeitschrift Fur Naturforschung C-A Journal of Biosciences. - 2004. - Vol.
59. - P. 856-862
136 Mansour A.S. Pathological studies on wilt disease of strawberry in Egypt //
Ph.D. Thesis Fac of Agric. Moshtohor, Benha Univ. - 2005. 137pp.
137 Abdel-Ghany R.E.A. Induced resistance for controlling root-rot disease of
strawberry and their side effects on biological activities in soil // Ph.D. Thesis, Fac. of
Agric., Mosh. Univ., Benha. - 2008. 179pp.
138 Vonfleet D.S. Histochemistry and junction of the endodermis // Bot.Rev. -
1962. - Vol. 27. - P. 65-220
139 Hare R .C. Physiology of resistant to fungal disease in plants // Bot. Rev. -
1966. - Vol. 32. - P. 95-137.
140 Stahmann M. A., Clare B.G, Woodbury W. Increased disease resistance
and enzyme activities induced by ethylene and ethylene production by black rot
infected sweet potato tissue // Plant Physiol. - 1966. - Vol. 41. - P. 1505-1512.
141 Goodman R.N., Kiraly Z., Zaitlin M. The biochemistry and physiology of
infectious plant disease // D.Van Nostrand Co.Inc. London. - 1967. 354 pp.
142 Loverkovich L., Loverkovich H., Stahmann M.A. Tobacco mosaic virus-
induced resistance to Pseudomonas tabaci in tobacco // Phytopathology. - 1968. -
Vol. 58. - P. 1034-1035
143 Gangopadhyay S., Wyllie T.D., Oswald T.H. Utilization of soybean galactose
by Macrophomina phaseolina in charcoal rot disease // Phytopathology. Z. - 1974. -
Vol. 80. - P. 60-66.
144 Nadolny L., Sequeira L. Increase in peroxidase activity is not directly
involved in induced resistance in tobacco // Physiological Plant Pathology. - 1980. -
Vol. 16, № 1. - P. 8.
145 Hammerschmidt R., Kuc J. Lignification as mechanism for induced
systemic resistance in cucumber // Physiological Plant Pathology. - 1982. - Vol. 20. -
P. 61-71.
146 Hammerschmidt R., Nuckles E.M., Kuc J. Association of enhanced
peroxidase activity with induced systemic resistance of cucumber to
Colletotrichum lagenarium // Physiol. Plant Pathol. - 1982. - Vol. 20. - P. 73-
82.
147 Kuc J. Induced immunity to plant disease. Bioscience // Bioscience. - 1982.
- Vol. 32. - P. 854-860.
148 Abd-El-Kader M.A.M. Studies on certain diseases of soybean // Ph.D.
Thesis, Fac., Agric. Assiut Univ. -1983.
149 El- Akkad E.A.F. Comparative studies on some Fusarium and verticillium
wilt disease in Egypt // Ph.D. Thesis, Fac. of Agric., Cairo University, Egypt. - 1983.
163pp.
150 Hammerschmidt R., Bonnen A.M., Bergstrom G.C. Association of
lignification with non-host resistance of cucurbits // Phytopathology. - 1983. - Vol.
73. - P. 829 (Abstract)
151 Hammerschmidt R. Rapid deposition of lignin in potato tuber tissue as a
response to fungi non-pathogenic on potato // Physiological Plant Pathology. - 1984. -
Vol. 24. - P. 33-42.
140
152 Reuveni R., Ferreira J.F. The relationship between peroxidase activity and
the resistance of tomatoes (Lycopersicon esculentum) to Verticillium dahliae
// Phytopathol. Z. - 1985. - Vol. 112. - P. 193-197.
153 Reuveni R., Bothnia G.C. The relationship between peroxidase activity and
the resistance to Sphaerotheca fuliginea in melons // Phytopathol. Z. - 1985. -
Vol. 114. - P. 260-267.
154 Goldberg R. Liberman M. Mathieu C. Pierron M., Catesson A. M.
Development of epidermal cell wall peroxidases along the mung bean hypocotyl:
possible involvement in the cell wall stiffening process // J. Exp. Bot. - 1987. - Vol.
38, № 8. - P. 1378-1390.
155 Smith J., Hammerschmidt R. Comparative study of acidic peroxidase
associated with induced resistance in cucumber, muskmelon and watermelon //
Physiol. Mol. Plant Pathol. - 1988. - Vol. 33. - P. 255-261.
156 156 Tuzun S., Rao M.N., Vogeli U., Schardl C.L., Ku J.A. Induced
systemic resistance to blue mold: early induction and accumulation of ,1,3-
gluconases, chitinases, and other pathogenisis-related proteins (b-proteins) in
immunized tobacco // Phytopathology. - 1989. - Vol. 79. - P. 979-983.
157 Ye X.S., Pan S.Q., Kuc J. Activity, isoenzyme pattem, and cellular
localization of peroxidase as related to systemic resistance of (Perosporu tabaciiza)
and to tobacco mosaic tobacco to blue mold virus // Phytopathology. - 1990. - Vol.
80. - P. 1295-1298
158 Zahra A. M. Studies on wilt disease of sesame (Sesamum indicum L.) in upper
Egypt // Ph. D. Thesis, Fac. Agric., Assuit Univ. -1990.
159 Wyatt E., Sarah S., Pan Q., Kuć J. β-1,3-Glucanase, chitinase, and
peroxidase activities in tobacco tissues resistant and susceptible to blue mould as
related to flowering, age and sucker development // Physiological and Molecular
Plant Pathology. - 1991. - Vol. 39, № 6. - P. 433-440.
160 Avdiushko S.A., Ye X.S., Kuc J. Detection of several enzmetic activities in
leaf prints of cucumber plants // Physiol.and Mol.Plant Pathol. - 1993. - Vol. 42. - P.
441-454.
161 Jagdish C., Tyagi R.N.S. Peroxidase activity associated with leaf blight of
mung bean (Vigna radiata (Linn.) Wilczek // Indian Journal of Mycology and Plant
Pathology. - 1993. - Vol. 23, № 2. - P. 184-186.
162 Hammcrschmidt R., Yang-Cashman P. Induced resistance in cucurbits // -
1995. - P. 63-85 in: Induced Resistance to Disease in Plants. Developments in Plant
Pathology 4. R. Hammcrschmidt and J. Kuc, cds. Kluwcr Academic Publishers,
Dordrecht, the Netherlands.
163 Bhattacharyya P. Effect of ascorbic acid on nodulation and disease
intensity of mung bean // Journal of Mycopathological Research. - 1996. - Vol. 34, №
1. - P. 59-62.
164 Anfoka G., Buchenauer H. Induced systemic resistance in tomato and
tobacco plants against cucumber mosaic virus // J. of Plant Disease and Protection. -
1997. - Vol. 104, № 5. - P. 506-516.
141
165 Chiang M.H., Hyun, J.W., Park W.M. Enzyme activities of defense-related
proteins in sesame tissues infected by Fusarium oxysporium // J. of the Korean
Society for Horticultural Sci. - 1997. - Vol. 38, № 5. - P. 502-505.
166 Siegrist J., Glenewinkel (Dagmar), Kolle (Karmen), Schmedtke (Margetet).
Chemically inducer resistance in green bean against bacterial and fungal pathogens //
J. of Plant Disease and Protection. - 1997. - Vol. 104, № 6. - P. 599-610.
167 Song F.M., Zheng Z., Ge X.C., Song F.M., Zheng Z., Ge X.C. Role of
peroxidase in the resistance of cotton seedling to Fusarium oxysporum f.sp.
vasinfectum // J. of Zhejiang Agric. Univ. - 1997. - Vol. 23, № 2. - P. 143-148.
168 Orober M., Siegrist J., Buchenauer H. Induction of systemic aquired
resistance in cucumber by foliar phosphate application // In H., Russel PE, Dehne H.
W., Sisler, H. D., eds. Modern Fungicides and Antifungal compounds II. Germany.
Andover. - 1998. - P.339-348.
169 Pathak D. Srivastava M.P., Deka S.C. Biochemical basis of charcoal rot
[Rhizoctonia bactericola (Taub.) Buter] resistant and susceptible cultivars of
sunflower in relation to total phenols, free amino acids, insoluble pectin and
polyphenol oxidase activity // Annals of Biology (Ludhiana). - 1998. - Vol. 14, № 2. -
P. 189-193.
170 Podile A.R., Laxmi V.D.V. Seed bacterization with B. subtilis AF1
increased phenylalanine ammonia-lyase and reduced the incidence of Fusarium wilt
in pigeonpea // J. Phytopath. - 1998. - Vol. 146. - P. 225-259.
171 Shahina K., Luthra Y.P., Gandhi S.K. Copper induced effect on
biochemical constituents in cowpea susceptible to Rhizoctonia species // Acta
Phytopathologica et Entomologica Hungarica. -1999. -Vol. 34, № 3. -P. 199-210.
172 Tohamy (Eman), Y., El-Mougith A.A., El-Deeb A.A., Fleifel (Elham),
H.H. Nitrogen metabolism, phenolic compounds and some enzymatic activities in
some sunflower hybrids grown in infested and non-infested soil by Macrophomina
phaseolina // Al-Azhar J. of Microbiol. - 1999. - Vol. 44. - P. 43-54.
173 Ahmed Hoda, A.M., El-Moneem K.H.A., Allam A.D., Fahymy F.G.M.
Biological control of root rots and wilt diseases of cotton // Assuit J. of Agric. Sci. -
2000. - Vol. 31, № 2. - P. 269-285.
174 Shalaby S.I.M., Saeed M.N.A. Biochemical defense mechanisms
associated with the systemic induced resistance in sesame plants against Fusarium
wilt disease // Zagazig J. Agric. Res. - 2000. - Vol. 27, № 1. - P. 105-113.
175 Xu Q.X., Yu J.Z., Lu S.J. Variation rhythm of peroxidase activity in
cucumber during seedling stage and its relation to Fusarium wilt resistance // Acta
Agriculturae Shanghai. - 1994. - Vol. 10, № 3. - P. 58-62.
176 Sharma S., Sharma S.S., Rau V.K. Reversal by phenolic compounds of
abscussic acid-induced inhibition of in vitro activity of amylase from seeds of
Triticum aestivum L // New Phytol. - 1986. - Vol. 103, № 2. - P. 293-297.
177 Eichhorn H. Klinghammer M. Becht P., Tenhake R. Isolation of a novel
ABC-transporter gene from soybean induced by salicylic acid // J. Exp. Bot. - 2006. -
Vol. 57, № 10. - P. 2193-2201.
178 Vermerris W., Nicholson R.L. Phenolic Compound Biochemistry. 2006. 284
pp. Published by Springer, P.O. Box 17, 3300 AA Dordrecht, the Netherlands.
142
179 Hayat S., Ahmad A. Salicylic Acid: A Plant Hormone // 1st Ed. Published
by Springer, Dordrecht, The Netherlands. - 2007. 401pp.
180 Farouk S., Ghoneem K.M., Ali Abeer A. Induction and expression of
systemic resistance to downy mildew disease in cucumber by elicitors // Egypt. J.
Phytopathol. - 2008. - Vol. 36, № 1-2. - P. 95-111.
181 181 Ding J., Shi K., Zhou Y.H., Yu J.Q. (b). Effects of root and foliar
applications of 24-epibrassinolide on fusarium wilt and antioxidant metabolism in
cucumber roots // HortScience. - 2009. - Vol. 44, № 5. - P. 1340-1345.
182 Harish S., Kavino M., Kumar N., Balasubramanian P., Samiyappan R.
Induction of defense-related proteins by mixtures of plant growth promoting
endophytic bacteria against Banana bunchy top virus // Biological Control. - 2009. -
Vol. 51, № 1. - P. 16-25.
183 Schroeder W.T., Walker J.C. Influence of controlled environment and
nutrition of garden pea to Fusarium wilt // J.Agric.Res. - 1942. - Vol. 65. - P. 221-
248.
184 Tessier B.J. Propagule build-up and distribution of Fusarium oxysporum
f.sp. pisi race 1 and 2 in wilt resistant and susceptible pea (Pisum sativum L.)
cultivars // M.Sc thesis, Rod Island. - 1980. 95 pp. (c.f. Tessier et al., (1990).
185 Beckman C.H., Mueller W.C., Tessier B.J., Harrison N.A. Recognition and
callose deposition in response to vascular infection in fusarium wilt resistant or
susceptible tomato plants // Physiological Plant Pathology. - 1982. - Vol. 20. - P. 1-
10.
186 Bishop C.D., Cooper R.M. An ultrastructural study of root invasion in three
vascular wilt diseases // Physiological Plant Pathology. - 1983. - Vol. 22. - P. 15-27
187 1867 Bishop C.D., Cooper R.M. Ultrastructure of vascular colonization by
fungal wilt pathogens. II. Invasion of resistant cultivars // Physiological Plant
Pathology. - 1984. - Vol. 22. - P. 277-289.
188 Tessier B.J., Mueller W.C., Morgham A.T. Histopathology and
ultrastructure of vascular responses in peas resistant and susceptible to Fusarium
oxysporum f.sp. pisi // Phytopathology. - 1990. - Vol. 80. - P. 756-764.
189 Walter D., Newton A., Lyon G.D. Induced Resistance for Plant Defence: A
Sustainable Approach to Crop Protection // 1st. ed., Blackwell Publishing Ltd,
Oxford, UK. - 2007. 272pp.
190 Booth C. The genus Fusarium. Commonwealth mycological institute, Key,
surrey, England. - 1971.
191 Martyn R.D., Netzer D. Resistance to races 0, 1, and 2 of Fusarium wilt of
watermelon in Citrullis sp. PI 296341-FR // HortScience. - 1991. - Vol. 26. - P. 429-
432.
192 Comm O.A.J. Endospore – forming rods and cocci, Genus Bacillus. 1955. -
P. 201-217 (in the shorter Bergey,s manual of Determinative Bacteriology . Holt, J.
G. (Ed), 1981, 8 Th. Ed the Williams and Wilkins Company, Baltimore).
193 Abd-El-Moity T.H. Effect of single and mixture of Trichoderma harzianum
isolates on controlling three different soil-borne pathogens // Egypt. J, Microbiol. -
1985. - P.111-120.
143
194 Abd-El-Moity T.H., Shatla M.N. Biological control of white rot disease of
onion Sclerotium cepivorum by Trichoderma harzianum // Phytopathology Z. - 1981.
- Vol. 100. - P. 29-35.
195 Bindu S., Padma K. In vitro antifungal potency of some plant extracts against
Fusarium oxysporum // International Journal of Green Pharmacy. January. - 2009.
196 Maurhofer M., Keel C., Hass, D., Defago G. Influence of plant species on
disease suppression by Pseudomonas fluorescens strain CHAO with enhanced
antibiotic production // Plant Pathol. - 1995. - Vol. 44. - P. 40-50.
197 Harman G.E. Chet I., Baker R. Trichoderma hamatum effects on seed and
seedling disease induced in radish and pea by Pythium spp. or Rhizotonia solani //
Phytopathology. - 1980. - Vol. 70, № 12. - P. 1167-1172.
198 Park J.L., Rand R.E., Joy A.E., King E.B., Biological control of Pythium
damping off and Aphanomyces root-rot of peas by application of Pseudomonas
cepacia or P. fluorescent to seed // Plant Dis. - 1991. - Vol. 75. - P. 987-992.
199 Callan N.W., Mather D.E., Miller J.B. Biopriming seed treatment for
biological control of Pythium ultimum pre-emergence damping off in sh 2 sweet corn.
Plant Dis. - 1990. - Vol. 74. - P. 368-372.
200 Shalaby S.I.M. Effect of fungicidal treatment of sesame seeds on root rot
infection, plant growth and chemical components // Bulletin of Faculty of
Agriculture, University of Cairo. - 1997. - Vol. 48, № 2. - P. 397-411.
201 Abeles F.B., Bosshart R.P., Forrence L.E., Habig W.H. Preparation and
purification of glucanase and chitinase from bean leaves // Plant Physiol. - 1971. -
Vol. 47. - P. 129-134.
202 Matta A., Dimond A.E. Symptoms of Fusarium wilt in relation to quantity
of fungus and enzyme activity in tomato stems // Phytopathology. - 1963. - Vol. 53. -
P. 574-587.
203 Monreal J., Reese E.T. The chitinase of Serratia marcescens // Canadian J.
of Microbiology. - I969. - Vol. 15. - P. 689-696.
204 Ried J.D., Ogryd-Ziak D.M. Chitinase over producing mutant of Serratia
marcescens // Appl. and Environ. Microbiol. - 1981. - Vol. 41. - P. 664-669.
205 Bjorkman A. Studies on finely divided wood // Part 1. Extraction of lignin
with neutral solvents. Svensk Papperstidn. - 1956. - Vol. 59. - P. 447-485.
206 Stegemann H., Afify A.M.R., Hussein K.R.F. Cultivar identification of
dates (Phoenix dactylifera) by protein patterns // Second International Symposium of
Biochemical Approaches to Identification of Cultivars. Braunschweing, West
Germany. - 1985. - P. 44.
207 Bozarth R.F., Diener T.O. Changes in concentration of free amino acids
and –12-amids in tobacco plants by potato virus X and potato virus Y // Virology. -
1963. - Vol. 21. - P. 188-193.
208 Thomas W., Dutcher R.A. The colorimetric determination of carbohydrates
in plants by the picric acid reduction method. 1. The estimation of reducing sugars
and sucrose // Journal of American Chemical Society. - 1924. - Vol. 46. - P. 1662-
1669.
209 Bary H.G., Thorpe W.V. Analysis of phenolic compounds of interest in
metabolism // Methods of chemical analysis. - 1954. - Vol. 1. - P. 27-51.
144
210 Muting D., Kaiser E. Hoppe-Sayler,s Zschr // Physiol. Chem. - 1963. - P.
332-376.
211 Sass J.E. Botanical microtechnique // Iowa state college press, Ames, Iowa.
- 1951. - P. 228.
212 Johanson D.V. Plant microtechnique // New York, London, McGrow-Hill
Book Co. Inc. - 1940. - P. 27-154.
213 Abd-El-Moneim (Maisa) L. Evaluation of some non-chemical methods to
control some soilborne fungi and foliage diseases of cucumber // Ph.D. Thesis, Agric.
Zagazig Univ. - 2001.
214 Fukui R., Schroth M.N. Hendson M., Hancock J.G. (a). Interaction between
strains of Pseudomonads in sugar beet spermospheres and their relationships to
pericarp colonization by Pythium ultimum in soil // Phytopathology. - 1994. - Vol. 84.
- P. 1322-1330.
215 Fukui R., Schroth M.N. Hendson M., Hancock J.G. (b). Growth patterns
and metabolic activity of Pseudomonads in sugar beet spermospheres: Relationship to
pericarp colonization by pythium ultimum // Phytopathology. - 1994. - Vol. 84. - P.
1331-1338.
216 Pierson E.A., Weller D.M. Use of mixtures of fluorescent pseudomonads to
suppress take-all and improve the growth of wheat // Phytopathology. - 1994. - Vol.
84. - P. 940-947.
217 Ohlson S., Larsson P.O., Mosbach K. Steroid transformation by living
cells immobilized in calcium alginate // European J. Appl. Microbiol. Biotechnol. -
1979. - Vol. 7. - P. 103.
218 Rohlf F. J. Feature extraction applied to systematics // 1993. Chapter 25, -
P. 375-392, in Fortuner, R.(ed.) Advances in computer methods for systematic
biology. Johns Hopkins Univ. Press: Baltimore. 560 pp.
219 Snedecor G.W., Cochran W.G. Statistical methods // Oxford and J. PH.
Publishing Com. 8th
edition. - 1989.
220 FAOStat Database. Food and Agriculture Organization, United Nations.
C.f. Economic Research Service, USDA. - 2008.
221 Ghewande M.P. Management of foliar diseases of groundnut (Arachis
hypogaea) using plant extracts. Indian J. Agric. Sci. - 1989. - Vol. 59, № 2. - P. 133-
134 (c.f. Rev. Pl. Pathol. - 1990. - Vol. 69, № 5. - P. 242).
222 El-Naggar M.A.A. Effect of some extracts as germicides on controlling of
powdery mildew disease of pepper under plastic house // 8th
Cong. Egypt
Phytopathol. Soc., Cairo. - 1997. - P. 173-185.
223 Nada M. G. A. Studies on antifungal activities of some Egyptian medicinal
and aromatic plants // Ph.D. Thesis, Agric., Zagazig Univ. - 2002.
224 O,Brien R.g. fungicide resistances in population of cucurbit powdery
mildew (Sphaerotheca fuliginea) // Crop Hort. Sci. - 1994. - Vol. 22. - P. 145-149.
225 McGrath M.T., Staniszewska H. Management of powdery mildew in
summer squash with host resistance, disease threshold based fungicide programs or
an integrated program // Plant Dis. - 1996. - Vol. 80, № 9. - P. 1044-1052.
226 Eckert J.W., Ogawa J.M. The chemical control of postharvest diseases.
Deciduous fruits, berries, vegetable and root/tuber crops // Annu. Rev. Phytopathol. -
145
1988. - Vol. 26. - P. 433-469.
227 Durmusoglu E., Massfeld W., Sengonca C. Determination of the exposure
of workers to two different pesticides in a greenhouse with roses // Mitteilungen der
Deutscheo Gesellschaft far Allgemeine und Angewandte Entomolgie (German). -
1997. - Vol. 2. - P. 319-322 (c.f. Rev. Pl. Pathol. - 1998. Vol. 77, № 7. - P. 716).
228 Horst R.K., Kawamoto S.O., Porter L.L. Effect of sodium bicarbonate and
oils on the control of powdery mildew and black spot of roses // Plant Disease. -
1992. - Vol. 76, № 3. - P. 247-251.
229 Garcia J.E. Pesticides as contaminants // Turrialba (Costa Rica). - 1993. -
Vol. 43, № 3. - P. 221-229 (c.f. Rev. Pl. Pathol. - 1995. - Vol. 74, № 6. - P. 409).
230 Agrios G.N. Plant Pathology. 4th ed.Academic Press Ltd., San Diego, CA.
- 1997.
231 Datnoff L.E., Nemec S., Pernezny K. Biological control of Fusarium crown
and root rot of tomato in Florida using Trichoderma harzianum and Glomus
intraradices // Biol. Contr. - 1995. - Vol. 5. - P. 427-431.
232 Kumar B.S.D. Fusarial wilt suppression and crop improvement through
two rhizobacterial strains in chickpea growing in soils infested with Fusarium
oxysporum f. sp. Ciceris // Biol. Fertil. Soils. - 1999. - Vol. 29. - P. 87-91.
233 Mandeel Q., Baker R. Mechanisms involved in biological control of
fusarium wilt of cucumber with strains of nonpathogenic Fusarium oxysporum //
Phytopathology. - 1991. - Vol. 81. - P. 462-469.
234 Mishra P.K., Mukhopadhyay A.N., Fox R.T.V. Integrated and biological
control of gladiolus corm rot and wilt caused by Fusarium oxysporum f. sp.
Gladioli // Ann. Appl. Biol. - 2000. - Vol. 137. - P. 361-364.
235 Gordon T.R., Martyn R.D. The evolutionary biology of Fusarium
oxysporum. Annu. Rev. Phytopathol. - 1997. - Vol. 35.- P. 111-128.
236 Mace M.E., Veech J.A. Fusarium wilt of susceptible and resistant tomato iso-
lines, host colonization`// Phytopathology. - 1971. - Vol. 61, № 7. - P. 834-840.
237 Shaw D.V., Gubler W.D., Hansen J. Field resistance of California
strawberries to Verticillium dahliae at three conidial inoculums concentrations //
HortScience. - 1997. - Vol. 32, № 4. - P. 711-713.
238 Xiao C.L., Subbarao K.V. Relationships between Verticillium dahliae
inoculum density and wilt incidence, severity, and growth of cauliflower //
Phytopathology. - 1998. - Vol. 88, № 10. - P. 1108-1115.
239 Khan A., Atibalentja N., Eastburn D.M. Influence of inoculum density of
Verticillium dahliae on root discoloration of horseradish // Plant Disease. - 2000. -
Vol. 84, № 3. - P. 309-315.
240 Armstrong M.J., Armstrong J.K. Formae speciales and races of Fusarium
oxysporum causing wilt diseases // In: Nelson PE, Toussoun TA, Cook RJ, eds.
Fusarium: diseases, biology, and taxonomy. London, UK: Pennsylvania State
University Press. - 1981. - P. 391-399.
241 Dennis C., Webster J. Antagonistic properties of species-groups of
Trichoderma, I. Production of non-volatile antibiotics. II. Production of volatile
antibiotics // Trans. Br. Mycol. Soc. - 1971. - Vol. 57, № 1. - P. 25-39. (cv. Rev. PI.
Pathol. - 1972. - Vol. 51, № 3. - P. 202).
146
242 Verma M., Brar S.K., Tyagi R.D., Surampalli R.Y., Valero J.R.
Antagonistic fungi, Trichoderma spp. Panoply of biological control // Biochemical
Engineering Journal. - 2007. - Vol. 37. - P. 1-20.
243 D’Ercole N., Sportelli M., Nipti P. Different types of antagonism of
Trichoderma sp. towards plant pathogenic soil fungi. Informatore Filopathologico. -
1984. - Vol. 34, № 11. - P. 43-47. (c.f. El-Garhy, 1994).
244 Van Peer R., Niemann G.J., Schippers B. Induced resistance and
phytoalexin in accumulation in biological control of Fusarium wilt of carnation by
Pseudomonas spp. strain WCS 417r // Phytopathology. - 1991. - Vol. 81.- P. 728-
733.
245 Wei G., Kloepper J. W., Tuzun S. Induction of systemic resistance of
cucumber to Colletotrichum orbiculare by select strains of plant growth-promoting
rhizobacteria // Phytopathology. - 1992. - Vol. 81. - P. 1508-1512.
246 Zhou T. Paulitz T.C. Induced resistance in the biocontrol of Pythium
aphanidermatum by Pseudomonas spp. on cucumber // J. Phytopathol. - 1994. -
Vol. 142. - P. 51-63.
247 Pusey P.L., Wilson C.L. Post-harvest biological control of stone fruit
brown rot by Bacillus subitlis // Plant Dis. - 1984. - Vol. 68. - P. 753-756.
248 Martin F.N., Hancock J.C. The use of Pythium oligandrum for biological
control of pre-emergence damping-off caused by P. ultimum // Phytopathology. -
1987. - Vol. 77. - P. 1013-1020.
249 Benhamou W., Chet I. Hyphal interaction between Trichoderma harzianum
and Rhizoctonia solani ultrastructure and gold cytochemistry of the mycoparasitic
process // Phytopathology. - 1993. - Vol. 83. - P. 1062-1071.
250 Tuner W.B. Fungal metabolites // Academis Press, London, New York. -
1971. - P. 446
251 Hayes C.K. Improvement of Trichodema and Gliocladium by genetic
manipulation // In Biological control of Plant disease progress and challenges for the
future. - 1992. - P. 227-286. (Tjamos, E.C.; G.C; Papavizas and R.J. Cook, (ed.))
Plenum Press, New York and London, Published in cooperation with NATO
Scientific Affairs Division. P. 462.
252 Abd-El-Moity T.H. Further studies on the biological control of white rot
disease of onion // Ph.D. Thesis, Fac, Agric., Minufiya Univ. Egypt. - 1981.
253 Paderes D.F., Hockenhull J., Jensen D.F., Mathur S.B. In vivo screening of
Trichoderma isolates for antagonism against Sclerotium rolfsii using rice seedlings //
Bulletin Oil B/SROP. - 1992. - Vol. 15, № 1. - P. 33-35. (c.f Rev. PI. Pathol. - 1993.
- Vol. 72, № 1. - P. 208).
254 Bolar J.P., Norelli J.L., Wong K.W., Hayes C.K., Harman Q.E.,
Aldwinckle H.S. Expression of Endochitinase from Trichoderma harzianum in
transgenic apple increases resistance to apple scab and reduces vigour //
Phytopathology. - 2000. - Vol. 90. - P. 72-77.
255 Berg G., Knaape G., Seidel D. Biological control of Verticillium dahliae
Kleb. By natural occurring rhizosphere bacteria // Archives of Phytopathology and
Plant Protection. - 1994. - Vol. 29, № 3. - P. 249-262.
147
256 Saddlers H.M. Use of bacteria in controlling fungal diseases // Gemuse
(Munchen). - 1996. - Vol. 32, № 3. - P. 180-189.
257 Sankar P., Jeyarajan R. Biological control of Sesamum root rot by seed
treatment with Trichoderma spp. and Bacillus subtilis // Indian Journal of Mycology
and Plant Pathology. - 1996. - Vol. 26, № 2. - P. 217-220.
258 Wan, Z.W., Li X.Z., Liu Y.L., Wang J.J. Biological control of strawberry
wilt antagonistic microbes // Chinese Journal of Biological Control. -1999. - Vol. 15,
№ 4. - P. 187.
259 Ferreira J.H.S., Matthee F.N., Thomas A.C. Biological control of Eutypa
lota on grapevine by an antagonistic strain Bacillus subtilis // Phytopathology. - 1991.
- Vol. 81. - P. 283-287.
260 Asaka O., Shoda M. Biocontrol of Rhizoctonia solani damping off of
tomato with Bacillus subtilis RB14 // Applied and Environmental Microbiology. -
1996. - Vol. 62, № 11. - P. 4081-4085.
261 Farahat A.A. Biological control of some potato bacterial diseases // Ph.D
Thesis Fac. Agric. Minufiya Univ. - 1998.
262 Wolk M., Sorkar S. Antagonism in vivo of Bacillus spp. Against
Rhizoctonia solani and Pythium spp // Azeiger fur schadling skundey pflanzenschutz,
Umweltschutz. - 1994. - Vol. 67, № 1. - P. 1-5. (cv. Rev. Pl. Path. - 1994. - Vol. 73,
№ 6. - P. 4601).
263 Ahmad G.A. Using plant extracts to control powdery mildew disease that
attack cucumber plants under protected houses // M. Sc. Fac. of Agric., Moshtohor.
Zagazig Univ., Benha Branch. - 2005. 170 pp.
264 Elad Y., Kirshner B., Yehuda N., Sztejnberg A. Management of powdery
mildew and gray mould of cucumber by Trichoderma harzianum T39 and
Ampelomyces quisqualis AQ10 // BioControl. - 1998. - Vol. 43, № 2. - P. 241-251.
265 Howell C.R., Hanson L.E., Stipenovic R.D., Puckhaber L.S. Induction of
terpenoid synthesis in cotton roots and control of Rhizoctonia solani by seed
treatment with Trichoderma virens // Phytopathology. - 2000. - Vol. 90, № 3. - P. 24-
52.
266 Matta A., Abattista Gentile I., Ferraris L. Stimulation of β,1,3-glucanase
and chitinase by stresses that induce resistance to fusarium wilt in tomato //
Phytopath. Medit. - 1988. - Vol. 27. - P. 45-50.
267 Yurina O.V., Yurina T.P., Anikina L. Peroxidase activity of the leaves in
cucumber as a test for resistance to mildew // SeI'Skokhozyaistvennaya-Biology. -
1993. - Vol. 1. - P. 113-117 (c.f. Data Base of CAB International).
268 Mosa A. A. Effect of foliar application of phosphates on cucumber
powdery mildew // Annals of Agricultural Science (Cairo). - 1997. - Vol. 42, № 1. -
P. 241-255.
269 Reuveni M., Agapov V., Reuveni R. A foliar spray of micronutrient
solutions induces local and systemic protection against powdery mildew
(Sphaerotheca fuliginea) in cucumber plants // European Journal of Plant Pathology.
- 1997. - Vol. 103, № 7. - P. 581-588.
148
270 Abd-El-Kareem F.M.A. Induction of resistance to some diseases of
cucumber plants grown under greenhouse conditions // Ph.D. Thesis, Agric. Ain
Shams Univ. - 1998.
271 El-Habbak M.H. Induction of resistance to powdery mildew disease of
Squash plants // M.Sc. thesis, Fac. of Agric., Mosh. Zagazig Univ., Benha Branch. -
2003.
272 Irving H.R., Kuc J. Local and systemic induction of peroxidase, chitinase
and resistance in cucumber plants by potassium phosphate monobasic //
Physiological and Molecular Plant Pathology. - 1990. - Vol. 37. - P. 355-366.
273 Schneider S., Ullrich W.R. Differential induction of resistance and
enhanced enzyme activities in cucumber and tobacco caused by treatment with
various abiotic and biotic inducers // Physiological and Molecular Plant Pathology. -
1994. - Vol. 45. - P. 291-304.
274 Takuo S., Tatsuji S., Johan H., Erick V. Pectin, Pectinase and
Protopectinase: protection, properties and applications // Adv. Appl. Microbiol. -
1993. - Vol. 39. - P. 213-294.
275 Abd-El-Kareem F.M.A., Abd-Alla M.A., El-Mohamedy R.S.R. Induced
resistance in potato plants for controlling early blight disease under field conditions //
Egypt. J. Phytopath. - 2002. - Vol. 29, № 2. - P. 27-41.
276 El-Gamal (Nadia), G. Usage of some biotic and abiotic agents for Induction
of resistance to cucumber powdery mildew under plastic house conditions // Egypt. J.
Phytopath. - 2003. - Vol. 31, № 1-2. - P. 129-140.
277 Biles C.L., Martyn R.D. Peroxidase, polyphenoloxidase and shikimate
dehydrogenase isozymes in relation to the tissue type, maturity and pathogen
induction of watermelon seedlings // Plant Physiol. Bioch. - 1993. - Vol. 31. - P. 499-
506.
278 Akhtar M., Garraway M.O. Changes in maize peroxidase associated with
variation in susceptibility to Bipolaris maydis race T // Phytopathology. - 1987. -
Vol. 77. - P. 1739 (Abstract).
279 Boiler T. Hydrolytic enzymes in plant disease resistance // Plant-Microbe
Interactions Molecular and Genetic Perspectives. - 1987. - Vol. 2. - P. 385-413.
280 Schlumbaum A., Mauch F., Vogeli U., Boiler T. Plant chitinases are potent
inhibiters of fungal growth // Nature. - 1986. - Vol. 324. - P. 365-367.
281 Grisebach H. Lignins // Academic Press, New York. The Biochemistry of
Plants. - 1981. - Vol. 7. - P. 451-478.
282 Gaspar T., Penel C., Thorpe T., Greppin H. Peroxidases, a survey of their
biochemical and physiological roles in higher plants // University of Geneva Press,
Geneva, Switzerland. - 1982.
283 Lamport D.T.A. Roles for peroxidases in cell wall genesis //
Molecular and Physiological Aspects of Plant Peroxidases. - 1986. - P. 199-207.
284 Mader M., Ungemach J., Schloss P. The role of peroxidase isozyme
groups of Nicotiana tabacum in hydrogen peroxide formation // Planta. - 1980. - Vol.
147. - P. 467-470.
149
285 Vance C.P., Anderson J.O., Sherwood R.T. Soluble and cell wall
peroxidases in reed canarygrass in relation to disease resistance and localized lignin
formation // Plant Physiol. - 1976. - Vol. 57. - P. 920-922.
286 Vance C.P., Sherwood R.T. Regulation of lignin formation in reed
canarygrass in relation to disease resistance // Plant Physiol. - 1976. - Vol. 57. - P.
915-919.
287 Vance P.C, Sherwood R.T., Kirk T.K. Lignification as amechanism of
disease resistance // Annu. Rev. Phytopathol. - 1980. - Vol. 18. - P. 259-288.
288 Helal R.M., Zaki M.S., Fadl F.A. Physiological studies on the nature of
resistance to powdery mildew in cucumber // Res. Bull. Ain Shams Univ., Cairo. -
1978. - Vol. 923. - P. 12.
289 El-Shanawani M., Mohamed S.A., Awad M. El-Desouky S.h.
Morphological and physiological resistance to powdery mildew in cucumber // 6th
Con. of Phytopathol. Cairo, March. 1990.
290 Mohamed S.A. Virulence of L. taurica (Lèv) Arn. on some pepper cultivars
and its control // Minufiya J. Agric. Res. - 1994. - Vol. 19, № 6. - P. 2883-2902.
291 Omar S.S. Studies on powdery mildew of wheat in Egypt // M.Sc. Thesis,
Agric., Cairo Univ. - 1977. 125 pp.
292 Farahat A.A. Studies on powdery mildew of some leguminous plants //
Ph.D. Thesis, Agric. Ain Shams Univ. - 1980.
293 Awad N.G.H. Reaction of some cucurbits against S. fuliginea in relation to
their physiological and histopathological changes // Arab Univ. J. Agric. Sci. Ain
Shams Univ. Cairo. - 2000. - Vol. 8, № 3. - P. 829-851.
294 Kalaichelvan P.T., Nagarajan G. A fungitoxic alkaloid from Crotalaria
paleda. Indian Phytopathol. - 1992. - Vol.45, № 2. - P. 252-253.
295 Mahadevan A., Sridhar K. Methods of Physiological Plant 3rd
Edition //
Sivakami Pub. Madras. - 1986.
296 Subba Rao P.V., Geigen J.P., Einhorn J., Rio B., Malosse C., Nicole M.,
Savary S., Ravise A. Host defence mechanisms against groundnut rust // Internal.
Arahis Newslett. - 1988. - Vol. 4. - P. 16-18.
297 Soni G.L., Sedha R.K. Khanna P.K., Garcha H.S. Growth inhibition of
Fusarium oxysporum by phenolic compounds // Indian J. Microbiol. - 1992. - Vol.
32. - P. 45-49 (c.f. Data Base of CAB International).
298 Kalaichelvan P.T., Elangovan N. Effect of phenolics on Drechslera oryzae
// Indian Phytopathol. - 1995. - Vol. 48, № 3. - P. 271-274.
299 Mandal S., Mallick N., Mitra A. Salicylic acid-induced resistance to
Fusarium oxysporum f. sp. lycopersici in tomato. Plant Physiol.Biochem. - 2009. -
Vol. 47. - P. 642-649.
300 Yousefi H., Sahebani N., Mirabolfathy M., Faravardeh L., Mahdavi V. The
effect of salicylic acid and Bacillus subtilis on cucumber root and stem rot, caused by
Fusarium oxysporum f.sp. radicis cucumerinum // Iran. J. Plant Path. - 2010. - Vol.
46, № 4. - P. 85-87.
301 Ismail I.O. Function and regulation of xylem cysteine protease 1 and xylem
cysteine protease 2 in Arabidopsis // P.HD. Dissertation, Faculty of the Virginia
Polytechnic Institute and State University. - 2004. - 116 p.
150
302 Sagitov A.O., El-Habbaa G.M., Ismaiel F.H., El-Fiki I.A. Inducing
anatomical resistance against infection with tomato fusarium wilt by using garlic and
black pepper extracts // исследование результаты. - 2010. - Vol. 4. - P. 171-177.
303 Pennypacker B.W., Nelson P.E. Histopathology of carnation infected with
Fusarium oxysporum f.sp. dianthi. Phytopathology. - 1972. - Vol. 62. - P. 1318-1326.
304 Ko J.H., Han K.H., Park S., Yang J. Plant body weight-induced secondary
growth in Arabidopsis and its transcription phenotype revealed by whole-
transcriptome profiling // Plant Physiology. - 2004. - Vol. 135. - P. 1069-1083.
305 Cook R.J., Baker K.F. The nature and practice of biological control of plant
pathogens // APS Press, American Phytopathological Society, Hand book. - 1989.
539 pp.
306 Kloepper J.W., Tuzun S. Induction of systemic resistance in cucumber
against Fusarium wilt by plant growth-promoting rhizobacteria // Phytopathology. -
1995. - Vol. 85. - P. 595-698.
307 Elad Y., Baker R. The role of competition for iron in suppression of
chalmydospore germination of Fusarium spp. by Pseudomonas spp. //
Phytopathology. - 1985. - Vol. 75. - P. 1053-1059.
308 Wetter B.M. Biological control of soilbome plant pathogens in the
rhizosphere with bacteria // Annu. Rev. Phytopathol. - 1988. - Vol.26. - P. 379-407.
151
Appendix A
The difference between SAR and ISR