Kazakh National Agrarian University
UDС 635.64: 632.4 (574)
Copyrighted
Ibrahim Abdel-Moneim Ibahim Ismaiel
RSISTANCE INDUCTION AGAINST FUSARIUM WILT DISEASE
IN TOMATO PLANTS (Lycopersicon esculentum Mill.)
Thesis
Submitted in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy in Agricultural Botany
(6D081100 –Plant Protection and Quarantine)
SUPERVESION COMMITTE: Professor of Plant Protection; Scientific Research
Institute for Plant Protection; Academician of the
Kazakh National Academy,
Abaia Orazoli Sagitov
Professor of Plant Pathology; Botany Department;
Faculty of Agriculture; Benha University,
Gehad Mohamed El-Habbaa
Republic of Kazakhstan
Almaty 2011
This research was carried out at the Kazakh National Agricultural University
(KazNAU).
Supervision committee: Professor of Plant Protection; Scientific Research Institute
for Plant Protection; Academician of the Kazakh National
Academy,
Abaia Orazoli Sagitov
Professor of Plant Pathology; Botany Department; Faculty
of Agriculture; Benha University,
Gehad Mohamed El-Habbaa
Reviewers:
Professor of agricultural sciences,
Carapaev Amangeldi Tackalevech
Professor of agricultural sciences,
Bairakemov Cagedolla Ezbacarovech
Assertion of thesis is held in 03/08/2011 on 200 PM oclock by the governmental
certifying committee in the Kazakh National Agriculture University at the address
050010, 8 Abai St., Almaty, Kazakhstan.
This thesis will be introduced to the library of the Kazakh National Agriculture
University at the address 050010, 8 Abai St., Almaty, Kazakhstan.
Signing of Ph.D. student
_______________________
2
TABLE OF CONTENTS
Subject
Page
LIST OF ABBREVIATIONS
INTRODUCTION
2 REVIEW OF LITERATURES
3 MATERIALS AND METHODS
4 EXPERIMENTAL RESULTS
4.1.
Isolation of the causal fungi and In Vitro studies
4.1.1.
Obtained isolates
4.1.2.
Radius growth and sporulation of different isolates of FOL
4.2.
Pthogenicity test
4.2.1.
Wilt disease symptoms
4.2.2.
Percentage of wilted plants and wilt disease severity
4.2.3.
Growth parameters
4.2.3.1. Plant height (cm.)/plant (PH)
4.2.3.2. Number of leaves/plant (NL)
4.2.3.3. Fresh weight of leaves (g.)/plant (FWL)
4.2.3.4. Stem fresh weight (g.)/plant (SFW)
4.2.3.5. Root fresh weight (g.)/plant (RFW)
4.2.3.6. Root length (cm.)/plant (RL)
4.2.3.7. Weight of fruit yield (g.)/plant (WFY)
4.3.
Sensitivityof some new experimental tomato cultivars
against infection with the Fusarium wilt
4.3.1.
Percentage of wilted plants and wilt disease severity
4.3.2.
Growth parameters
4.3.2.1.
Plant height (cm.)/plant (PH)
4.3.2.2.
Number of leaves/plant (NL)
4.3.2.3.
Fresh weight of leaves/plant (FWL)
4.3.2.4.
Stem fresh weight (g.)/plant (SFW)
4.3.2.5.
Root length (cm.)/plant (RL)
4.3.2.6.
Root fresh weight (g.)/plant (RFW)
4.3.2.7.
Root volume (cm3)/plant (RV)
4.3.2.8.
Weight of Fruit yield (g.)/plant (WFY)
4.4.
In vitro Studies on some natural and chemical resistance
inducers
4.4.1.
The In vitro inhibitory effect of the tested resistance inducers at
different concentration on the radius growth
4.4.1.1.
Garlic and black pepper extracts
4.4.1.2.
Salicylic acid and riboflavin
4.4.2.
The In vitro inhibitory effect of the tested resistance inducers at
different concentration on FOL sporulation
4.4.2.1.
Garlic and black pepper extracts
4.4.2.2.
Salicylic acid and riboflavin
6 -7
8-10
11-27
28-36
37-101
37
37
37
38
38
38
40
40
43
43
43
43
43
44
3
44
44
46
46
46
46
46
52
52
52
52
53
53
53
53
55
55
55
4.5.
In Vivo studies on some natural and chemical resistance
inducers
4.5.1.
Percentage of wilted plants
4.5.1.1.
Garlic and black pepper extracts
4.5.1.2.
Salicylic acid and riboflavin
4.5.2.
Wilt disease severity
4.5.2.1.
Garlic and black pepper extracts
4.5.2.2.
Salicylic acid and riboflavin
4.5.3.
Growth characters
4.5.3.1.
Plant height
4.5.3.1.1. Garlic and black pepper extracts
4.5.3.1.2. Salicylic acid and riboflavin
4.5.3.2.
Number of leaves per plant
4.5.3.2.1. Garlic and black pepper extracts
4.5.3.2.2. Salicylic acid and riboflavin
4.5.3.3.
Fresh weight of leaves (g) per plant
4.5.3.3.1. Garlic and black pepper extracts
4.5.3.3.2. Salicylic acid and riboflavin
4.5.3.4.
Dry weight of leaves (g) per plant
4.5.3.4.1. Garlic and black pepper extracts
4.5.3.4.2. Salicylic acid and riboflavin
4.5.3.5.
Stem fresh weight (g) per plant
4.5.3.5.1. Garlic and black pepper extracts
4.5.3.5.2. Salicylic acid and riboflavin
4.5.3.6.
Root fresh weight (g) per plant
4.5.3.6.1. Garlic and black pepper extracts
4.5.3.6.2. Salicylic acid and riboflavin
4.5.3.7.
Root dry weight (g) per plant
4.5.3.7.1. Garlic and black pepper extracts
4.5.3.7.2. Salicylic acid and riboflavin
4.5.3.8.
Root length (cm) per plant
4.5.3.8.1. Garlic and black pepper extracts
4.5.3.8.2. Salicylic acid and riboflavin
4.5.3.9.
Root volume (cm3) per plant
4.5.3.9.1. Garlic and black pepper extracts
4.5.3.9.2. Salicylic acid and riboflavin
4.5.3.10. Fruit yield (g) per plant
4.5.3.10.1. Garlic and black pepper extracts
4.5.3.10.2. Salicylic acid and riboflavin
4.6.
Effect of application methods of tested inducers treatments
on leaf pigments under stress of infection with the tomato
Fusarium wilt
4.6.1.
Garlic and black pepper extracts
4.6.1.1.
Chlorophyll a
4.6.1.2.
Chlorophyll b
4
57
57
57
57
59
59
59
61
61
61
61
63
63
63
65
65
65
67
67
67
69
69
69
71
71
71
73
73
73
75
75
75
77
77
77
79
79
79
80
80
80
80
4.6.1.3.
4.6.2.
4.6.2.1.
4.6.2.2.
4.6.2.3.
4.7.
Total chlorophyll
82
Salicylic acid and riboflavin
84
Chlorophyll a
84
Chlorophyll b
85
Total chlorophyll
85
Effect of application methods of tested inducers treatments
on phenols content under stress of infection with the
tomato Fusarium wilt
87
4.7.1.
Garlic and black pepper extracts
87
4.7.1.1.
Free phenols content
87
4.7.1.2.
Conjugated phenols content
88
4.7.1.3.
Total phenols content
88
4.7.2.
Salicylic acid and riboflavin
90
4.7.2.1.
Free phenols content
90
4.7.2.2.
Conjugated phenols content
91
4.7.2.3.
Total phenols content
91
4.8.
Total soluble protein “TSP” content
93
4.8.1.
Garlic and black pepper extracts
93
4.8.2.
Salicylic acid and riboflavin
94
4.9.
Activity of the oxidative enzymes
94
4.9.1.
Polyphenoloxidase (PPO) enzyme
94
4.9.1.1.
Garlic and black pepper extracts
94
4.9.1.2.
Salicylic acid and riboflavin
95
4.9.2.
Activity of peroxidase (POD) enzyme
96
4.9.2.1.
Garlic and black pepper extracts
96
4.9.2.2.
Salicylic acid and riboflavin
97
4.10.
Anatomical studies
99
5 DISSCUSION
102-118
SUMMARY
119-126
CONCLUSION
127
REFERENCES
128-142
5
LIST OF ABBREVIATIONS
Abbreviations
%
Φ
 or m
420
430
~
±%
0
C
BP
cm.
Conc.
CP
Cv.
DS
e.x.
f. sp.
FAA
FOL
FOL
FORL
FWL
G
g or gm
IR
IR+SS
l
Increase (%)
L.S.D.
mg
ml
mm
mM
NL
nm
O.D.
PDA
pH
POD
PPO
R
RDW
Meaning
percentage
Diameter
Micron = 1/1000 millimeter
The activity at optical density 420 nm
The activity at optical density 430 nm
Nearly
Percentage of increase or decrease relative to control
Celsius degree
Black pepper extract
Centimeter = 1/100 meter
Concentration
Crude protein
Cultivar
Disease severity
For example
formae special
Formalin glacial acetic acid ethyl alcohol solution
Fusarium oxysporum f.sp. lycopersici
Fusarium oxysporum f.sp. lycopersici
F. oxysporum f.sp. radicis-lycopersici
Fresh weight of leaves/plant
Garlic extract
Gram = 1/1000 kilogram
Immersing root method
Immersing root + Spraying shoot method
Liter
= (treatment – control)/control X 100
Least significance difference
Milligram = 1/1000 gram
Milliliter = 1/1000 liter
Millimeter = 1/10 centimeter
Millimolar (10-3 molar)
Number of leaves/plant
Nanometer = 1/1000000 millimeter
Optical density
Potato dextrose agar
power of hydrogen
Peroxidase enzyme
Polyphenoloxidase enzyme
Riboflavin
Root dry weight (gm)/plant
6
Reduction (% )
RFW
RH
RL
RV
SA
SDW
SFW
SFW
SS
TSP
VB
w/v
WFY
= (control – treatment) / control X 100
Root fresh weight (gm)/plant
Relative humidity
Root length (cm)/plant
Root volume (cm3)/plant
Salicylic acid
Stem dry weight (gm)/plant
Stem fresh weight /plant
Stem fresh weight (gm)/plant
Spraying shoot method
Total soluble protein
Vascular bundle
weight to volume
Weight of fruit yield (gm)/plant
7
INTRODUCTION
Tomato (Lycopersicon esculentum Mill.) is one of the world’s most important
crops due to the high value of its fruits both for fresh market consumption and in
numerous types of processed products [75]. World volume of production has
increased approximately 10 percent since 1985, reflecting a substantial increase in
dietary use of the tomato. One of the main constraints to tomato cultivation is damage
caused by pathogens, including viruses, bacteria, nematodes and fungi, which causing
severe losses in production [26].
Fusarium oxysporum has received considerable attention from plant pathologists
because of its ability to cause vascular wilt or root rot diseases on a wide range of
plants. Despite the broad host range of the species, host specialization of individual
isolates is more circumscribed. Isolates with the same or similar host ranges are
assigned to forma specials and more than 70 formae specials have been described
[32] and [20]. The soil-borne fungus Fusarium oxysporum f. sp. radicis-lycopersici
(FORL) causes Fusarium crown and root rot of tomato, often referred to as ‘crown
rot’ [71]. Fusarium oxysporum f. sp. lycopersici (FOL) inhabits most tomato-growing
regions worldwide, causing tomato production yield losses [192].
On the other hand, F. oxysporum f. sp. lycopersici (FOL) causes Fusarium wilt
disease only of plants belonging to the genus Lycopersicon. However, some formae
specials have broader host ranges, such as F. oxysporum f. sp. radicis-lycopersici
(FORL), which cause disease on different hosts belonging to several plant families,
including tomato (L. esculentum) in the greenhouse [167] and [135]. At first, this
fungus was identified as a new race (J3) of F. oxysporum Schlecht. f. sp. lycopersici
which causes Fusarium wilt of tomato [173]. The causal agent, however, was not a
new race of FOL but a new “formae specials” of FORL based on the following
characteristics: (1) The FORL pathogen reveal distinctly different symptoms of those
caused by FOL, where, the disease symptoms in mature crops caused by FORL are
those of root and basal stalk rots rather than vascular wilt. (2) Crown and root rot
disease occurs at cool soil (18ºC) temperatures [101], [173], [102] and [189], while,
the severe FOL symptoms appears at soil temperatures of about 27ºC. (3) The host
range of FORL is larger than FOL [167] and [135]. Also, FOL is specific only to
Lycopersicon spp., when tested the pathogenicity of the fungus on 17 plant species by
inoculating different isolates of the crown rot organism, various species of the family
Leguminosae, as well as L. esculentum [167].
FOL has an extensive presence in all continents [134], [38], [68] and become
one of a limiting factor in the production of tomato and accounts for yield losses
annually [6]. It has become one of the most prevalent and damaging diseases
wherever tomatoes are grown intensively because the pathogen persists indefinitely
in infested soils [79]. Crown rot develops primarily in cool climates in both field and
greenhouse tomatoes. Substantial crop losses in infected fields have given the disease
international attention. The host range of this pathogen comprises at least 36 other
species [135]. FOL attacks only certain tomato cultivars. Plants infected by wilt
fungus show leaf yellowing and wilting that progress upward from the base of the
8
stem. The first symptom of Fusarium wilt is usually the golden yellowing of a single
leaflet or shoot, or a slight wilting and drooping of the lower leaves on a single stem.
Yellowed and wilted leaflets drop early. Initially, only one side of a leaf midrib, one
branch, or one side of a plant will be affected. The symptoms soon spread to the
remainder of the plant. Wilted leaves usually drop prematurely. Affected plants turn
to bright yellow, wilt, dry up, and usually die before maturity, producing few, if any,
fruit [68]. Fusarium diseases constitute most of the loss in tomato production
worldwide, because it spread on all geographic fields that it is so hard to find a place
without Fusarium infestation. Thus, the best way to produce tomato is developing
resistant cultivars against Fusarium species. In cultivar developing, molecular
marker assisted techniques replaced traditional breeding techniques which are high
cost and time consuming for breeders [11].
Tomato wilt, caused by FOL, has been reported in at least 32 countries
worldwide [104]. While plant disease resistance genes have been identified for the
effective control of tomato wilt, new races of the pathogen continue to develop,
overcoming deployed resistance and thwarting tomato breeding efforts [41] and [73].
Because it is a long-lived, soil-borne pathogen, infested soil remains contaminated
indefinitely, so only resistant varieties can be grown on that site. The virulence
profile of FOL isolates affecting tomatoes has been grouped into three races
according to their ability to infect a set of differential cultivars carrying distinct
resistance loci. Three Fusarium wilt resistance loci have been genetically
characterized in Lycopersicon species.
Mutants of Fusarium oxysporum f. sp. lycopersici race 1 and race 2 caused
disease symptoms on plants with resistance genes against the corresponding wild type
strains. Mutants of race 1 of the pathogen were stable, whereas, mutants of race 2 lost
the ability to cause disease symptoms in plants carrying the 1–2 resistance genes,
after prolonged maintenance on potato dextrose agar. Mutants of race 1 resembled
race 2 in pathogenicity and they were vegetatively compatible with race 2, but no
longer with race 1. These results suggest that the isolated strains with an altered
virulence pattern have mutations in loci involved in avirulence [116]. The tomato
Fusarium wilt is caused by three races of Fusarium oxysporum f. sp. lycopersici.
Races 1 and 2 are distributed worldwide whereas race 3 has a more limited
geographic distribution in California [41], Brazil [158], Florida [139]. Seven F.
oxysporum isolates were obtained from wilted tomato plants of race 1 and 2-resistant
hybrids in Brazil. Virulence assays performed using a set of the race differential
cultivars indicated that all seven isolates could be classified as F. oxysporum f. sp.
lycopersici race 3. This new Fusarium wilt might became an economically important
disease since race 3-resistant cultivars adapted to Brazil are not yet available [41].
Thirty-nine isolates of Fusarium oxysporum were collected from tomato plants
displaying wilt symptoms in a field in California 2 years after FOL race 3 was first
observed at that location. In fact, recent results indicate that new race 3 isolates could
have originated from genetic changes in the local populations of native FOL isolates
[41]. This new Fusarium wilt might became an economically important disease since
race 3-resistant cultivars adapted to Brazilian conditions are not yet available. In
addition, screening trials searching for new sources of resistance seems to be
9
necessary since the genetic plasticity associated with selective pressures due to the
use of race 3 resistant cultivars might cause the establishment of new pathogenic
races of this fungus [159].
The control of the pathogen spread mainly involves in three strategies:
husbandry practices, application of agrochemicals and use of resistant varieties [26].
The methods used to control vascular wilt are either not very efficient or are difficult
to apply. The pathogen has increased in the infested soil and become resistant to
chemical fungicides. For this reason, alternative methods with emphasis on biological
control using the resistance inducers for controlling the disease have been studied by
several researchers to reduce fungicide application and decrease cost of plant
production. Recently, there have been many reports stated that some plant extracts
and safe chemicals become a necessary to control the soil borne diseases including
tomato Fusarium wilt [201], [57], [1], [51], [6], [195], [52], [127], [218] and [219].
Also, the biological control of plant pathogens has been increasingly interested by
plant pathologists and many researchers [48], [190], [152], [181], [141] and [180].
The use of resistant varieties is the best strategy for disease control [184], [177] and
[220]. Resistant varieties are mostly produced by crossing resistant wild types and
existing cultivars developed for their properties like good taste, shape and color. A
molecular marker linked to resistance would be useful for tomato improvement
program [192]. Whatever, the best recommended way to control the disease is
selecting resistant varieties of tomato [184].
New races of Fusarium oxysporum f. sp. lycopersici could develop through
spontaneous random mutation or genetic recombination. Because F. oxysporum is an
imperfect fungus, parasexual recombination is the only mechanism by which reassortment of genetic material can occur. Heterokaryon formation, a prerequisite for
parasexual recombination, has been demonstrated in several formae speciales of F.
oxysporum, including F. o. lycopersici [182], [123] and [153].
Thus, this work was conducted to investigate the following topics:
1. Isolation of the tomato Fusarium wilt fungus, studying the in vitro growth and sporulation
of the isolated fungi and testing their pathogenicity to tomato Carolina Gold cultivar.
Also, studying the effect of infection with different fungal isolates on some plant growth
parameters and fruit yield of tested tomato cultivar.
2. Evaluation the responses of some commercial and new experimental tomato cultivars
against infection with the most virulent isolate of the Fusarium wilt in term of
percentage of wilted plants, wilt disease severity, measurements of plant growth
parameters and fruit yield.
3. Evaluation the inhibitory effect of different concentrations of some resistance inducers
(plant extracts and safe chemicals) against the most virulent isolate of the tomato
Fusarium wilt in vitro and in vivo. Different application methods were used for
evaluating the tested resistance inducers in vivo.
4. Determining the effect of tested inducers on biochemical constituents (leaf pigments,
phenols content, total soluble protein, activities of the oxidative enzymes like
polyphenoloxidase and peroxidase in plant tissues) using different application methods.
5. Studying changes in the anatomical structure of leaf petiole as affected by some tested
treatment of plant extracts.
10
2 REVIEW OF LITERATURES
Tomato (Lycopersicon esculentum Mill.) is an important vegetable crop
worldwide. Often times, its production is hindered by fungal diseases. Thew
important fungal diseases limiting tomato production are late blight, caused by
Phytophthora infestans, early blight, caused by Alternaria solani, and septoria leaf
spot, caused by Septoria lycopersici, Fusarium wilt caused by Fusarium oxysporum
f.sp. oxysporum, and Verticilium wilt caused by Verticilium dahliae. The
Phytophthora infestans is the same fungus that caused the devastating loss of potato
in Europe in 1845. A similar magnitude of crop loss in tomato has not occurred but
Phytophthora infestans has caused the complete loss of tomato crops around the
world on a small scale. Several attempts have been made through conventional
breeding and the molecular biological approaches to understand the biology of hostpathogen interaction so that the disease can be managed and crop loss prevented. In
this review, we present a comprehensive analysis of information produced by
molecular genetics and genomic experiments on host-pathogen interactions of late
blight, early blight, Septoria leaf spot, Verticilium wilt and Fusarium wilt in tomato.
Furthermore, approaches adopted to manage these diseases in tomato including
genetic transformation are presented [54].
2.1. Fusarium oxysporum
Fusarium oxysporum is a soilborne fungus that includes both nonpathogenic and
pathogenic strains [79]. Nonpathogenic strains of Fusarium oxysporum colonize the
cortex of plant roots without causing disease symptoms, whereas pathogenic strains
can move past the cortical tissue and invade the vascular tissue of susceptible hosts,
causing vascular wilt diseases. These pathogenic strains show a high level of host
specificity and are subdivided into formae speciales based on the plant species
attacked and into races based on the host cultivars attacked. F. oxysporum strains
pathogenic on tomato plants, Lycopersicon esculentum Miller) are classified into two
formae speciales, f. sp. lycopersici causing the vascular wilt disease of tomato and f.
sp. radicis-lycopersici causing Fusarium crown and root rot [20]. Fusarium wilt and
Fusarium crown rot symptoms begin as yellowing of older leaves. With Fusarium
crown rot, the leaves often turn brown or black and eventually wilt. With Fusarium
wilt, the yellow leaves turn downward and droop. Fusarium oxysporum, the cause of
both diseases, is a common tomato fungus that lives in the plant's vascular system,
which carries water from the roots to the leaves. Discolored roots indicate root rot.
Fusarium wilt causes a dark brown discoloration within the vascular tissue. Fusarium
crown rot causes a rot or canker at the base of the stem and possibly a root rot [59].
Fusarium species are among the most important phytopathogenic and toxigenic
fungi. To understand the molecular underpinnings of pathogenicity in the genus
Fusarium, we compared the genomes of three phenotypically diverse species:
Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp.
lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F.
oxysporum that include four entire chromosomes and account for more than onequarter of the genome. LS regions are rich in transposons and genes with distinct
11
evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition.
Experimentally, we demonstrate the transfer of two LS chromosomes between strains
of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS
chromosomes between otherwise genetically isolated strains explains the
polyphyletic origin of host specificity and the emergence of new pathogenic lineages
in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new
perspective [120].
2.2. Fusarium oxysporum f.sp. lycopersici
Three races i.e. 1, 2 and 3 of Fusarium oxysporum f.sp. lycopersici (FOL) are
known and could be distinguished by their pathogenicity to tomato cultivars which
possessing specific dominant resistance genes [131], [191]. Races 1 and 2 are found
in virtually all major tomato-growing regions, whereas race 3 is presently limited to
Australia [82], Florida [208], and California [49]. Very little is known about the
mechanisms involved in the development of races of FOL or imperfect fungi in
general. New races could develop through spontaneous random mutation or genetic
recombination. Because F. oxysporum is an imperfect fungus, parasexual
recombination is the only mechanism by which re-assortment of genetic material can
occur. Heterokaryon formation, a prerequisite for parasexual recombination, has been
demonstrated in several formae speciales of F. oxysporum, including FOL [182];
[123], [153].
The chronology of effects on gas exchange and chlorophyll-a fluorescence,
visible symptoms and hyphal colonization in plants of the susceptible tomato cultivar
Bonny Best inoculated with tracheomycotic fungi Fusarium oxysporum f. sp.
lycopersici or Verticillium albo-atrum were studied. The net photosynthetic rates and
related parameters of healthy [uncolonized and asymptomatic] leaves of infected
plants were affected by both the parasites. In the first uncolonized leaf, net
photosynthesis was depressed in different ways: in Fusarium-infected individuals, the
maximum detrimental effect was observed a week after inoculation, while in
Verticillium-infected plants the most severe depression was detected 21 days after
inoculation. The behaviour of the physiological parameters investigated, together
with the data relative to chlorophyll fluorescence measurements highlighted the fact
that the depression in photosynthetic activity was caused by different associated
factors in Verticillium-infected plants and was due mainly to drought stress in plants
inoculated with Fusarium [122].
Two pathogenic special forms [f. sp.] of the Fusarium oxysporum species
complex f. sp. lycopersici [FOL] and f. sp. radicis-lycopersici [FORL] are
morphologically indistinguishable. Although they are pathogenic to the same host
genus Lycopersicon [tomato], and infect the same tomato cultivar, they form distinct
diseases; FOL causes wilt while FORL causes crown rot and root rot. The isolates
were collected from geographically widespread locations [23].
Fusarium will of tomato caused by the vascular wilt pathogen Fusarium oxysporum
Schlechtend. Fr. f. sp. lycopersici (Sacc.) W. Q Snyder & H. N. Hans., is a devastating
disease that occurs in major tomato-growing regions of the world [209]; [177]. Tomato
wilt became the most serious disease of tomato throughout the Baja California Peninsula.
12
Since the winter of 2004, a disease with symptoms characteristic of those caused by a
Fusarium species has been observed in commercial fields near La Paz and Todos Santos
in the state of Baja California Sur. Symptoms include typical one-sided wilting and dark
brown vascular discoloration [95]. Recent surveys indicated that many of the commercial
cultivars with resistance to F. oxysporum f. sp. lycopersici race 1 planted in Taiwan
displayed Fusarium wilt symptoms. Yellowing on the older leaves was observed on one
side of the stems close to fruit maturity. The yellowing gradually affected most of the
foliage and was accompanied by wilting of the plants. The vascular tissue was usually
dark brown and discoloration extended to the apex. The wilting became more extensive
until plants collapsed and died [177].
Leaves on tomato plants infected with Fusarium oxysporum f. sp. lycopersici
frequently wilt unilaterally when the vascular bundles supplying the affected leaflets
are diseased. However, when the vascular bundles on one side of healthy petioles are
severed by notching the petiole base, the entire leaf remains turgid. Leaflets on the
notched side receive water by diffusion between bundles at the petiole tip. Lateral
translocation of water out of individual vessels and between bundles in diseased
xylem is impaired by the impregnation of vessel walls, intercellular spaces, and cells
adjacent to vessels with the products of vascular discoloration. Waterproofing of
vessels may play an important role in vascular dysfunction by confining water to
individual vessels and thereby increasing the importance of vessel occlusions [47].
The tomato infected transplants are stunted, the older leaves droop and curve
downward, and the plants frequently wilt and die. Symptoms on older plants generally
become apparent during the interval from blossoming to fruit maturation. Earliest symptom
is the bright yellowing of older, lower leaves, often on only one side of the plant, and the
leaflets on one side of the petiole frequently turn yellow before those on the other side. The
yellowing process gradually includes more and more of the foliage and is accompanied by
wilting of the plant during the hottest part of the day. The wilting becomes more extensive
from day to day until the plant collapses. The vascular tissue of a diseased plant is dark
brown. Browning often extends far up the stem and is especially noticeable in a petiole
scar. This browning of the vascular tissue is characteristic of the disease and can be used for
its tentative identification. Fruit infection occasionally occurs and can be detected by the
vascular tissue discoloration within the fruit. The earliest symptom of Fusarium wilt is the
bright yellowing of the lower, older leaves. These yellow leaves often develop on only one
side of the plant, and the leaflets on one side of the petiole frequently turn yellow before
those on the other side. Browning of the vascular tissue is characteristic of Fusarium wilt
and generally can be used for tentative identification. Warmer weather (82-86°F) favors
development of this pathogen. It is prevalent in acid and sandy soils. It is soilborne and
remains in soils for several years [139].
The Fusarium wilt of tomato significantly lowered the fresh weight of plant
stem, number and weight of tomato fruits in tomato plants inoculated than those noninoculated with the wilt pathogen “Fusarium oxysporum f.sp. lycopersici” [180].
2.3. Resistant cultivars
Response of near-isogenic tomato varieties to infection with Fusarium
oxysporum f.sp. lycopersici and Verticillium albo-atrum was investigated.
13
Colonization was limited in resistant but unlimited in susceptible plants. Occlusion of
vessels by tyloses was more frequent in resistant than in susceptible plants and may
have a rôle in restricting the upward spread of the pathogens in resistant plants. Six
phytoalexins were produced in the stems and roots of plants infected with
Verticillium, whereas only two were detected in these tissues following inoculation
with Fusarium. Roots and stems produced the same phytoalexins but in different
concentrations. Large amounts of phytoalexins accumulated more rapidly in resistant
than in susceptible plants [98].
Several tomato cultivars including Carolina Gold were reported to be resistant
against Fusarium wilt race 1 and race 2 whereas few were reported to be resistant
against race 3 [114]. If suitable resistant or immune varieties were not widely
available, tomato wilt caused by Fusarium oxysporum f. sp. lycopersici would
undoubtedly be the most damaging disease of tomatoes in this state [160]. While,
plant disease resistance genes have been identified for the effective control of tomato
wilt (F. oxysporum f. sp. lycopersici), new races of the pathogen continue to develop,
overcoming deployed resistance and thwarting tomato breeding efforts. Because it is
a long-lived, soil-borne pathogen, infested soil remains contaminated indefinitely, so
only resistant varieties can be grown on that site [41] and [73].
Three physiological races 1, 2 and 3 of Fusarium oxysporum f. sp. lycopersici,
based on differential cultivars, exist in Florida. Using resistant cultivars where
available for race 1 and 2 is recommended. There are some race 3 resistant cultivars
available commercially. Movement of infected plants and/or infested soil clinging to
machinery, hand tools, vehicles, trellising and staking implements, and field crates
into areas free of this pathogen should be prevented. Since flooding will spread the
fungus it is not recommended. Do not irrigate with surface water that may be
contaminated with the fungus. It is recommended that Fusarium-free transplants be
used; if transplant trays are reused these should be steam-treated between uses.
Using pre-plant soil fumigants may reduce disease incidence [139].
Infection of tomato with Fusarium (FOL) wilt significantly reduced the crop
yield and quality [1]. Tomato wilt becomes one of a limiting factor in the
production of tomato [Lycopersicon esculentum] and accounts for yield losses
annually. It has become one of the most prevalent and damaging diseases
wherever, tomatoes are grown intensively because the pathogen persists
indefinitely in infested soils. The use of resistant varieties is the best strategy for
disease control [184] and [177].
Fusarium oxysporum f. sp. lycopersici races 1 and 2 are distributed worldwide
whereas race 3 has a more limited geographic distribution with no report thus far in
Brazil. Seven F. oxysporum isolates were obtained from wilted tomato plants of race
1 and 2-resistant hybrids ‘Carmen’ and ‘Alambra’ in Brazil. Virulence assays were
performed using a set of the race differential cultivars: ‘Ponderosa’ [susceptible to all
races], ‘IPA-5’ [resistant to race 1], ‘Floradade’ [resistant to races 1 and 2] and
‘BHRS-2,3’ [resistant to race 3]. All isolates were highly virulent to ‘Ponderosa’,
‘IPA-5’ and ‘Floradade’ and were able to infect only a few plants of ‘BHRS- 2,3’. An
additional virulence test was conducted including the same set of cultivars plus
Lycopersicon pennellii ‘LA 716’. Identical results were obtained with L. pennellii
14
displaying an extreme [immune-like] resistant response. These results indicated that
all seven isolates could be classified as FOL race 3. This new Fusarium wilt might
became an economically important disease since race 3-resistant cultivars adapted to
Brazil are not yet available [158]. The best way to produce tomato is developing
resistant cultivars against Fusarium species [11].
2.4.
Plant extracts as natural resistance inducers
2.4.1. Garlic (Allium sativum) extracts
The natural plant extracts may provide an alternative to fungicides. Allium genus
revered to possess anti-bacterial and anti-fungal activities and include the powerful
antioxidants, sulfur and other numerous phenolic compounds which arouse
significant interests [30], [200], [213], [151], [91], [117], [163], [83], [28], [85]. The
inhibitory activity of garlic (Allium sativum L) against moulds has been reported by
numerous authors. It has also been observed that alliicin, thiosulfonates and other
compounds show fungistatic activities against several fungi [210], [81], [200], [8],
[18], [91]. The ajoene compound from garlic has stronger antifungal activity than
alliicin. The ajoene damages the cell walls of fungi. Activity of the garlic extract may
be due to sulfur-containing compounds such as ajoene or allicin [214]. Sprays with
the aqueous garlic extracts have antibiotic and antifungal properties and will suppress
a number of plant diseases, including powdery mildew on cucumbers and to some
extent, black spot on roses. Garlic extracts controlled diseases such as mildew, rusts,
fruit rots, blights, and black spot [154].
The antifungal activity of five plants extracts viz., Allium sativum,
Cymogopogon proxims, Carum carvi, Azadirachia indica and Eugenia caryophyllus
extracts with cold distilled water against Fusarium oxysporum f. sp. lycopersici,
Botrytis cinerea and Rhizoctonia solani was determine. The results revealed that, the
most effective plant extracts were Allium sativum, Carum carvi and Eugenia
caryophyllus. The results concluded also that plant extracts could be used as natural
fungicides to control pathogen fungi to reduce the dependence on the synthetic
fungicides [1].
The extracts of 11 species (Agave americana, Artemisia pallens, Citrus sinensis,
Dalbergia latifolia, Helianthus annus, Murraya koenigii, Ocimum basilicum,
Parthenium hysterophorus, Tagetes erecta, Thuja occidentalis and Zingiber offinale)
exhibited remarkable antisporulant effect even after 10-fold dilution of the crude
extracts while, in the case of remaining 15 plants the crude extracts loosed activity
after 10-fold dilution. The antisporulant activity of commercialised Azadirachta
preparation (Nutri-Neem) was more pronounced than that of Reynutria based one
(Milsana) and Sabadilla (veratrin), however, these botanical preparations held off the
extracts of C. gouriana and E. alsinoides and synthetic fungicides [51].
Several plant extracts were found to be highly effective on different isolates of
Fusarium wilt in the laboratory, and were tested with other control methods on two
tomato varieties artificially inoculated with the Fusarium wilt fungus. Results showed
that these extracts reduced wilt infection rate 49 days after planting on both tested
varieties. The most effective treatment after the fungicide Tachigaren was garlic
extract [6].
15
The effect of crude extracts of neem [Azadirachta indica] leaf, neem seed and
garlic [Allium sativum] at concentrations ranging from 5% to 30% of the material in
100 ml of Potato Dextrose Agar on mycelial growth of Fusarium oxysporum f. sp.
lycopersici was assessed. All the extracts inhibited mycelial growth at various levels.
Dry neem seed extract gave 100% inhibition of mycelial growth. Fresh neem leaf
extract reduced mycelial growth with increasing concentration while in garlic, no
differences in growth inhibition among the used various concentrations. However
garlic extracts decreased sporulation with increasing concentration and cultures
grown on extract amended agar plates remained viable [9].
Methanolic extracts of forty plant species commonly growing across India were
collected and have been screened for antisporulant activity against Sclerospora
graminicola (Sacc.) Schroet., the causative agent of pearl millet downy mildew. The
collection represented 38 genera of 30 families. The methanolic extracts of nine species
did not show any effect, whereas the activity of the extracts of Clematis gouriana,
Evolvulus alsinoides, Mimusops elengi, Allium sativum and Piper nigrum were
commensurable to that of the marketed botanical fungicides [51]. However, the watery
extracts of thirteen species did not show any effect, whereas the activity of watery
extracts of Allium sativum, Clematis gouriana, Evolvulus alsinoides, Mimusops elengi,
Parthenium hysterophorus, Piper nigrum and Tagetes erecta were commensurable to
that of marketed botanical fungicides and Mikal 70 wp. The crude watery extracts of 12
species [Agave americana, Aloe vera, Artemisia parviflora, Citrus limon, Citrus
sinensis, Eucalyptus globosus, Euphorbia hirta, Leucas aspera, Murraya koenigi,
Ocimum sanctum, Santalum album and Zingiber offinale] completely inhibited the
zoosprorangium formation while in the case of remaining 8 plants the crude extracts
reduced only partially the sporulation. The antisporulant activity of commercialised
Azadirachta preparation [Nutri-Neem] was more pronounced than that of Reynutria
based one [Milsana] and Sabadilla [Veratrin], however, these botanical preparations
held off synthetic fungicides and the most active watery extracts [52].
2.4.2. Black Pepper (Piper nigrum) extracts
Plant extracts of six plant species, cloves [Dianthus caryophyllus], cinnamon
[Cinnamum zeylamicum], thyme [Thymus vulgaris L.] fenugreek [Trigonella
fonicum], amme [Ammi visnagal], black pepper [Piper nigrum] and three essential
oils, geranium [Pelargonium gravedens], black cumin seeds [Nigella sativa L.] and
blue gum [Eucalyptus globulus] were evaluated for their antifungal effect on the
mycelial growth, incidence and disease severity of onion neck rot disease [Botrytis
allii]. The antifungal properties of clove extract were more effective than black
pepper on inhibiting mycelial growth and disease incidence [4]. The aqueous extracts
of 15 plant species were tested against onion white rot fungus Sclerotium cepivorum
that was grown in potato dextrose agar culture. Each extract presented a fungicidal
effect, at a concentration of 5%, when applied on allspice [Pimenta dioica] and clove
[Syzygium aromaticum]. Only clove extract retained its effect at a concentration of
1%, while allspice lost it at 3%. Cinnamon [Cinnamomum zeylanicum] and yam bean
[Pachy erosus] extracts produced total inhibition of sclerotial production besides a
poor mycelial growth. Different types of interactions were present when the extracts
16
were mixed: all combinations presented a lost of fungicidal effect [antagonistic
effect], including allspice extract; a retained fungicidal effect [single fungicidal
effect] occurred in most clove mixtures and in the combination of clove and black
pepper [Piper nigrum] the retained fungicidal effect was even below the minimal
lethal dose [synergistic effect]. The combination of extracts showed that the effect of
each plant extract could be modified by the reactions of the complex mixture of plant
compounds [140]. Several biologically important phytochemicals including alkaloids,
amides, propenyphenols, lignans, terpenes, steroid, kawapyrones, piperolides,
chalcones, dihydrochalcones, brachyamide piperine, piperolein, trichostachine,
sarmentine, sarmentosine, tricholein, retrofractamide have been extracted from P.
nigrum plants [137], [112], [25]. Concentration of alkaloids in fruits of P. nigrum
ranges from 4 to 5% [53]. Piper nigrum, commonly known as ``Black-pepper``, has
gained a global consideration because of its volume in the spice industry. This plant
has shown great potential for the discovery of novel biologically active compounds
and need for techniques to enhance the production of high quality consistent plant
material for feasible accumulation of metabolites [2].
2.5. Chemical inducers
2.5.1. Riboflavin
The nicotinic acid and to a less extent riboflavin, enhanced sugar and nitrogen
absorption and the rate of building up of cellular material in consequence was
recorded. Both riboflavin and nicotinic acid accelerated the accumulation of
carbohydrates and fat in the mycelium [145]. Treatments of sporangia and zoospores
of Phytophthora infestans race 1.2.3.4 with methionine or riboflavin for durations of
up to 8 h under fluorescent light did not affect its colonization of rye-seed agar.
Hyphal growth of races 1.2.3.4 and 0, when incubated in liquid synthetic medium,
was inhibited by free riboflavin [105].
Riboflavin (vitamin B2) is a water-soluble vitamin, which is involved in vital
metabolic processes in the cells, and is necessary for normal cell functions. Small
amounts of riboflavin are present in most animals, plants, and microbes and acts as a
coenzyme in many physiological processes of the cells. This vitamin is involved in
antioxidation and peroxidation; both processes affect the production of reactive
oxygen species (ROS). Induction of systemic resistance by foliar application of
riboflavin has been reported in some dicot plants against different pathogens e.g., in
Arabidopsis thaliana infected with Peronospora parasitica and Pesudomanas
syringae pv. tomato and tobacco infected with Tobacco mosaic virus (TMV) and
Alternaria alternata. Application of riboflavin induced systemic resistance against
different pathogens [57].
The role of riboflavin as an elicitor of systemic resistance and a plant defense
activator in rice as an important monocot plant was demonstrated. The mechanism of
riboflavin-IR and defense responses in rice against Rhizoctonia sgeath diseases was
studied. They found that riboflavin-IR can be linked to the induction of defense
pathways leading to formation of structural barriers such as lignin in rice plants.
Using riboflavin as a plant defense activator can be a new, simple, and
environmentally safe strategy to control Rhizoctonia sheath diseases of rice. The
17
lowest concentration of riboflavin tested (0.01 mM) had the best effect on induction
of resistance against R. solani and R. oryzae-sativae, the causal agents of sheath
blight and aggregate sheath spot of rice, respectively. Riboflavin did not have any
direct effect on the growth of fungi in vitro. Also, at concentrations necessary for
induction of resistance (0.01 to 2 mM), no macroscopic or microscopic cell death in
rice was observed. Therefore, riboflavin is able to activate resistance mechanisms in
rice, like dicots, in a hypersensitive response (HR)-independent manner [195].
A variety of roles have been proposed by [94] for the involvement of peroxidases
in the defense response. One possible role is the generation of reactive oxygen species
(ROS) by peroxidase–oxidative activity. The fact that the production of hydrogen
peroxide was upstream of induction of POC 1 gene expression, ruled out the possibility
of involvement of POC1 in the generation of ROS in these interactions. Another
possible function of peroxidases in the formation of structural barriers such as cell wall
enhancement and deposition of cell wall apposition, both of which can be involved in
the polymerization of lignin or suberin, the cross-linking of wall glycoproteins or
polysaccharides, and the apposition of antimicrobial phenols. Lignin formation was
investigated using phloroglucinol / HCl test [129], and lignin was detected in riboflavin
treated plants. Therefore, riboflavin-IR can be linked to the induction of defense
pathways leading to formation of structural barriers in rice plants.
Riboflavin caused induction of systemic resistance in chickpea against Fusarium
wilt and charcoal rot diseases. The dose effect of 0.01 to 20 mM riboflavin showed
that 1.0 mM concentration was sufficient for maximum induction of resistance;
higher concentration did not increase the effect. At this concentration, riboflavin
neither caused cell death of the host plant nor directly affected the pathogen’s growth.
In time course observation, it was observed that riboflavin treated chickpea plants
were inducing resistance 2 days after treatment and reached its maximum level from
5 to 7 days and then decreased. Riboflavin had no effect on salicylic acid [SA] levels
in chickpea, however, riboflavin induced plants found accumulation of phenols and a
greater activities of peroxidase than the control. Riboflavin pre-treated plants
challenged with the pathogens exhibited maximum activity of the peroxidases 4 days
after treatment [169].
Reported that the powdery mildew (Sphaerotheca fuliginea Pollacci) infection
in cucumber was significantly reduced by foliar application of a mixture of riboflavin
and methionine (RM). The effects of fungicidal activity on leaves applied with RM
were detected through restriction of progress of colonies and disease severity
compared with control plants. The initial response to foliar application of RM was
abrupt generation of hydrogen peroxide in the leaves of cucumber plants. Activities
of antioxidant enzymes such as SOD and POD were abruptly increased by foliar
application of RM. However, activities of antioxidant enzymes in control plants were
increased with disease development 9 d after pathogen inoculation. Cucumber leaves
have six major SOD isoforms. When plants were foliar-applied with RM, densities of
three SOD isozyme bands at SOD-1, SOD-2, and SOD-3 were increased 3 d after
foliar application. Leaves of cucumber plants have three major POD isozyme bands.
Densities of three POD isozyme bands were increased 3 d after foliar application with
RM. Four major PPO isozyme bands were determined in cucumber leaves. Though
18
the overall banding patterns of PPO in control and RM-applied plants were similar,
the band profiles in leaves applied with RM were characterized by high densities of
the three major isoforms. Activities of PPO in leaves applied with RM increased
rapidly during the 3 d after foliar application, and then remained relatively constant
for 15 d. Although activities of PPO in the leaves of control plants also abruptly
increased after 9 d, it was lower than those of RM-applied plants during the whole
time. The difference in lignin content between control and RM-applied plants was
detected 9 d after foliar application; it was high in leaves applied with RM [107].
The effects of riboflavin on defense responses and secondary metabolism in
tobacco [Nicotiana tabacum cv. NC89] cell suspensions and the effects of protecting
tobacco seedlings against Phytophthora parasitica var. nicotianae and Ralstonia
solanacearum were investigated. Defense responses elicited by riboflavin in tobacco
cells included an oxidative burst, alkalinization of the extracellular medium, expression
of 4 defense-related genes with different kinetics and intensities, and accumulation of 2
total phenolic compounds, scopoletin and lignin. When applied to tobacco plants
challenged by P. parasitica and R. solanacearum, riboflavin treatment resulted in
47.9% and 48.0% protection, respectively. These results suggest that riboflavin can
both induce a series of defense responses and secondary metabolism in cell
suspensions and protect tobacco against P. parasitica and R. solanacearum [121].
2.5.2. Salicylic acid
Sclerotial germination of onion pathogen was less after soaking in salicylic acid
than in either phenol or garlic acid. Increasing concentration of the phenolic
compounds in the 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 and phemol [50 and 100 ppm] [170]. Salicylic acid, picric acid
and 2,4- dinitrophenol caused significant reduction in radial growth, mycelial dry
weight and activity of S. rolfsii [186]. Induction of resistance to Fusarium oxysporum
f.sp. lycopersici (FOL) in tomato plants [cv. Danish Export] was performed with
salicylic acid and Fusarium sp. applied to the root systems 3 weeks after sowing
under greenhouse conditions. The challenged plants were inoculated with FOL 2 days
after induction. Disease incidence was reduced in plants which had been treated with
Fusarium sp. and salicylic acid. Although, Fusarium sp. and salicylic acid initially
caused some deleterious and phytotoxic effects, respectively, the plants later
recovered and no wilt symptoms could be detected [22].
Salicylic acid is a naturally occurring phenolic in many plants and has been
shown to function as a signal compound initiating plant defense systems in response
to stress, with some reporting responses such as reduced transpiration and enhanced
adventitious root initiation [124]. SA induces manganese superoxide dismutase
(MnSOD) genes. Salicylic acid induced increases in MnSOD would serve to detoxify
superoxide radicals and protect plants from damage from oxidative stress. Foliar
application of SA before sustained UV-B stress resulted in increased antioxidant
activity and higher pigment content which were correlated with less leaf injury and
greater maintenance of canopy photochemical efficiency of Kentucky bluegrass [35];
[69]. Soil drenches and foliar applications of SA resulted in enhanced tolerance of
19
bean (Phaseolus vulgaris L.) and tomato (Lycopersicon esculentum Mill.) to heat,
chilling, and drought stresses [175].
The natural resistance to potential parasites is regulated by two fundamental
mechanisms: the “nonhost” and the “gene-for-gene” resistance, respectively. The
latter is relevant when a cultivar resistant (R) gene product recognizes an avirulence
gene product in the attacking pathogen and triggers an array of biochemical reactions
that halt the pathogen around the site of attempted invasion. To cope with virulent
pathogens, plants may benefit by some temporary immunity after a challenge
triggering such an array of defense reactions, following a localized necrotizing
infection as a possible consequence of a hypersensitive response (HR). This process,
mediated by accumulation of endogenous salicylic acid (SA), is called systemic
acquired resistance (SAR) and provides resistance, to a certain extent even against
unrelated pathogens, such as viruses, bacteria, and fungi, for a relatively long-lasting
period. SAR may be more potently activated in plants pretreated with chemical
inducers, most of which appear to act as functional analogues of SA. This review
summarizes the complex aspects of SAR as a way to prevent crop diseases by
activating the plants' own natural defenses. The following outline is taken: (1)
introduction through the historical insight of the phenomenon; (2) oxidative burst,
which produces high levels of oxygen reactive species in a way similar to the
inflammation state in animals and precedes the HR to the pathogen attack; (3) SAR
as a coordinate action of several gene products leading to the expression of defenses
well beyond the time and space limits of the HR; (4) jasmonic acid (JA) and ethylene
as other endogenous factors mediating a different pahway of induced resistance; (5)
pathogenesis related proteins (PR proteins) de novo synthesized as specific markers
of SAR; (6) exogenous inducers of SAR, which include both synthetic chemicals and
natural products; (7) the pathway of signal transduction between sensitization by
inducers and PR expression, as inferred by mutageneses, a process that is still, to a
large extent, not completely elucidated; (8) prospects and costs; (9) final remarks on
the state-of-the-art of the topic reflecting the chemical view of the author, based on
the more authoritative ones expressed by the authors of the reviewed papers [80].
Salicylic acid [SA] completely inhibited the mycelial development of Fusarium
oxysporum f.sp lycopersici [FOL] in vitro at concentrations from 0.6 mM to 1.0 mM.
[147]. Soaking sesame seeds in filtrated and autoclaved garlic extracts decreased the
charcoal rot disease severity to 3.3 and 20.0% and increased the healthy plants to 83.3
and 33.3%, respectively compared with check [soaked in water] which recorded 23.3
and 26.7% for both parameters, respectively. Soaking sesame seeds in 2, 4 and 8mM
of salicylic acid decreased charcoal rot rotted plant to 3.3, 0, 0% and increase healthy
plants to 90, 96.7 and 90%, increased peroxidase activity to 1.97, 1.44 and 1.18,
polyphenoloxidase activity to 1.51, 1.36 and 1.26 and catalase activity to 2.7, 2.6 and
2.46 comparing to check plants which recorded 0.63, 0.62 and 1.83 for the three
oxidative enzymes respectively. Also, the free phenols increased to 13.3, 10.7 and 9.6
mg/g fresh weight, conjugated phenols to 3.7, 0.4 and 0.6 mg/g fresh weight and total
phenols to 17.0, 11.1 and 10.2 mg/g fresh weight at the 3 SA concentrations,
respectively compared with 6.2, 0.6 and 6.8 in check plants [61].
20
The influence of salicylic acid (SA) doses of 50 and 250 μM, for a period of up to
7 days, on selected physiological aspects and the phenolic metabolism of Matricaria
chamomilla plants was studied. SA exhibited both growth-promoting (50 μM) and
growth-inhibiting (250 μM) properties, the latter being correlated with decrease of
chlorophylls, water content and soluble proteins. In terms of phenolic metabolism, it
seems that the higher SA dose has a toxic effect, based on the sharp increase in
phenylalanine ammonia-lyase (PAL) activity (24 h after application), which is followed
by an increase in total soluble phenolics, lignin accumulation and the majority of the 11
detected phenolic acids. Guaiacol-peroxidase activity was elevated throughout the
experiment in 250 μM SA-treated plants. In turn, some responses can be explained by
mechanisms associated with oxidative stress tolerance; these mitigate acute SA stress
(which is indicated by an increase in malondialdehyde content). However, PAL activity
decreased with prolonged exposure to SA, indicating its inhibition [115].
The intensity and timing of the reactive oxygen species (ROS) formation, lipid
peroxidation and expression of antioxidant enzymes as initial responses of tomato
(Solanum lycopersicum L.) against the invading necrotrophic pathogen Fusarium
oxysporum f. sp. lycopersici were investigated. The concentration of hydrogen
peroxide (H2O2) was 2.6 times higher at 24 h post-inoculation (hpi) and lipid
peroxidation was 4.4 times higher at 72 hpi in the extracts of inoculated roots than in
the control. An increase in total phenolic content was also detected in inoculated roots.
The activities of the antioxidative enzymes, viz., superoxide dismutase (SOD), catalase
(CAT), guaiacol peroxidase (GPX) and ascorbate peroxidase (APX), increased in
response to pathogen inoculation. SOD activity at 48 hpi in inoculated roots was 2.9
times that in the control. CAT activity showed a decrease after 24 hpi and the increase
in activities of GPX and APX was insignificant after 24 hpi in the inoculated roots. The
oxidative burst generated in the interaction between tomato and F. oxysporum f. sp.
lycopersici may be an early first line of defense by the host mounted against the
invading necrotrophic pathogen. However, seemingly less efficient antioxidative
system (particularly the decrease of CAT activity after 24 hpi) leading to sustained
accumulation of ROS and the observed higher rate of lipid peroxidation indicate that
the biochemical events are largely in favor of the pathogen, thus making this host–
pathogen interaction a compatible combination. It is discussed that the oxidative burst
served as a weapon for the necrotrophic pathogen because the antioxidative system
was not strong enough to impede the pathogen ingress in the host [126].
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 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. The salicylic acid-treated tomato plants challenged
with FOL exhibited significantly reduced vascular browning and leaf yellowing
wilting. The mycelial growth of FOL was not significantly affected by salicylic acid.
None of the three concentrations of SA tested, viz., 100 μM, 200 μM and 300 μM
were found to inhibit mycelial growth of FOL significantly as compared to control.
21
Significant increase in basal level of salicylic acid in noninoculated plants indicated
that tomato root system might have the capacity to assimilate and distribute salicylic
acid throughout the plant. The results indicated that the induced resistance observed
in tomato against FOL might be a case of salicylic acid-dependent systemic acquired
resistance. Tomato plants grown hydroponically were exogenously fed with SA
through roots and leaves, and then challenged with FOL after two days, i.e. 48 h of
last SA application. Addition of 200 μM SA, to the hydroponics medium,
significantly affected infection and development of wilt caused by FOL on tomato
plants. The percent of vascular browning and leaf yellowing wilting was markedly
reduced when plants were grown in presence of 200 μM SA. Tomato plants
inoculated with FOL conidia, but not receiving 200 μM SA treatment through roots,
exhibited typical vascular browning and leaf yellowing wilting, while the SA-treated
plants showed less than 25% vascular browning and leaf yellowing wilting after 4
weeks of the experiment. Similarly, the foliar application of 200 μM SA on the
hydroponically grown tomato plants significantly affected infection and wilt
development by FOL on tomato plants. The tomato plants inoculated with FOL
conidia, but not receiving 200 μM SA treatment as foliar spray, exhibited
characteristic vascular browning and leaf yellowing wilting, while the SA-treated
plants showed less than or equal to 25% vascular browning and leaf yellowing
wilting after 4 weeks of the experiment [127].
The effects of chemical and microbial elicitors such as β-aminobutyric acid
(BABA), salicylic acid (SA), and Pseudomonas fluorecens CHAO on hydrogen
peroxide generation and activity of the enzymes related to its metabolism, i.e.,
superoxide dismutase (SOD), guaiacol peroxidase (GPOX), and catalase (CAT) in
tomato roots infected with root-knot nematode (Meloidogyne javanica) were
investigated. Results of this study show that treating the tomato seedlings with the
above elicitors significantly reduces the nematode infection level. Among the tested
elicitors, BABA has reduced the nematode galls, number of egg masses per plant and
number of eggs per individual egg mass more than the others. Additionally, the amount
of H2O2, a product of oxidative stress, SOD and GPOX specific activities were
significantly increased in the elicitor treated plants in comparison to control. Our
observation shows that BABA also increases the H2O2 accumulation and the SOD and
GPOX activities more as compared with the other tested elicitors. Such increases have
occurred in two phases and maximum levels of them were observed at 5 days after
treatment. In contrast with the increase in SOD and GPOX activities, the CAT activity
does not show any significant increase in treated plants as compared with the control
and other tested elicitors. It can be concluded that BABA, SA, and Pseudomonas
fluorescens CHAO induce oxidative stress in tomato roots through generation of
reactive oxygen species (ROS) and the enzymes related to their metabolism [168].
2.6. Biochemical defense mechanisms
2.6.1. Oxidative enzymes and accumulation of the phenolic compoundss
Reported that, in the second stage of the Fusarium wilt disease in muskmelon,
there was still a more rapid increase of the PPO and PO activities [128]. Beside its
action on the host metabolism, the parasite also directly contributes to the increase of
22
PPO activity. Treatment of susceptible tomato plants with catechol prevented disease
symptom expression after infection by Fusarium oxysporum f. sp. lycopersici. A
marked accumulation of total phenols was observed in the catechol-treated plants.
Though, the treatment changed peroxidase and polyphenoloxidase activity, no
changes appeared in their isozyme patterns. The pathogen was recovered from both
inoculated-susceptible and catechol-treated tomato stem sections. It is suggested that
the catechol treatment renders the susceptible plants symptomless carriers. The
mechanism of this acquired resistance is discussed [161]. Treatment of susceptible
tomato plants with quinic acid increased both their phenolic content [soluble phenolslignin] and their resistance to Fusarium oxysporum. The same results were obtained
with phenylalanine, but other compounds, unrelated to phenols, were ineffective.
Quinic acid showed no fungitoxic effect by itself. The degree of resistance was
positively correlated with the induced phenolic level and plants with a stimulated
phenolic pool contained less mycelium than control plants. These data support the
views that phenolic compounds have a rôle in enhanced resistance of the tomato plant
to Fusarium; treatment with phenol precursors could provide a convenient model to
study the mechanisms of resistance involved [42].
The variations over 7–8 day of peroxidase (PO) and polyphenoloxidase (PPO)
activity have been investigated in the roots of tomato plants which exposed to stresses
(heat, chloroform and a non-pathogenic form of Fusarium oxysporum) in order to
induce resistance to Fusarium oxysporum f. sp. lycopersici. All treatments induced
increase of PO and PPO activity that reached a maximum 3 days after the treatments
in leaves, 4 days in stem and roots and were higher in leaves than in other parts.
Activity decreased to levels for the control plants after 8 days. Inoculation with
Fusarium oxysporum f. sp. lycopersici further stimulated PO and PPO activity in all
treated plants over that caused by the treatments alone. Again, activity of treated
plants was lower than in controls 7 days after inoculation. It is concluded that: 1.
increased PO and PPO activity in tomato is a systemic response to cellular injury
caused in the root by heat, chloroform and non-pathogenic Fusarium oxysporum, 2.
these treatments do not prevent the pathogen from interacting with the plants and
inducing further enzyme increase, 3. treated plants react more strongly to the
challenge inoculation than untreated plants [74].
The treatments increased photosynthetic pigments which in turn increased
carbohydrate content in plant tissues. Carbohydrates are the main repository of
photosynthetic energy, they comprise structurally polysaccharide of plant cell walls,
principally cellulose, hemicelluloses and pectin that consider a barrier against plant
pathogens invasion and phenolic compounds are associated with structural
carbohydrates, which play a major and important role in plant defense [87]. In
addition, the enhancement in chlorophyll content is resulting from stimulating
pigment formation and increasing the efficacy of photosynthetic apparatus with a
better potential for resistance as well as decreasing photophosphorylation rate, which
occurred after infection [16]. In this connection, the adaptation of plants to biotic and
abiotic stress is due to the stimulation of protective biochemical systems and
synthesis of secondary metabolites such as phenolics [162]. The increase in seed oil
content may be due to the improvement in photosynthetic pigments since there is a
23
relationship between photosynthesis processes and oil biosynthesis during seed
development in terms of inducing sucrose translocation. It was found that all tested
chemicals decreased damping-off and charcoal rot diseases and at the same time
enhanced the vegetative growth and increased the enzymatic activity, total phenols
and chlorophyll contents. Besides, these chemicals are safe for both environment and
public health [187].
Controlling of plant diseases mainly depend on fungicides treatments [156], [67].
However, fungicidal applications cause hazards to human health and increase
environmental pollution. Therefore, alternatives, eco-friendly approach treatments for
control of plant diseases are needed [1], [164], [127]. Systemic acquired resistance
(SAR) or induction of resistance to pathogen is a promising approach for controlling
plant diseases. Exogenous or endogenous factors could substantially affect host
physiology, leading to rapid and coordinated defense-gene activation in plants
normally expressing susceptibility to pathogen infection [127]. This phenomenon, that
resistance of plant to pathogens can be enhanced by the application of various biotic
and a biotic agent, called induce systemic resistance in plants [212], [39], [172], [3].
Use of chemical inducer, salicylic acid (SA) represents an interesting new
opportunity in controlling fungal and bacterial diseases within an environmental friendly
integrated crop protection system through enhancing the resistance of the plant to
pathogen [66], [24], [64], [65], [110], [14], [127]. The signal molecule SA is involved in
some signal transduction system, which induce particular enzymes catalyzing biosynthetic
reactions to form defense compounds such as polyphenols, pathogenesis- related (PR)
proteins [136], [176], [207]. There are many morphological and biochemical changes in
SAR-protected plants that then become infected. Large increase in phenolic synthesis in
plants was recorded after attack by plant pathogens [50], [64]. Phenolics that occur
constitutively and function as preformed inhibitors are generally referred to as
phytoanticipins and those that are produced in response to infection by the pathogen are
called phytoalexins and constitute an active defense response. In plants the positive
correlation between levels of polyphenol oxidase (PPO) and peroxidase (POD) and the
resistance to pathogens and herbivores is frequently observed, Evidence for the induction
of PPO in plants, particularly under conditions of stress and pathogen attack is considered.
There are some evidences indicating that the activation of peroxidase, polyphenol oxidase
plays a crucial role in the biological control and resistance of plant to pathogenic attack
[138], [198], [46], [178]. PPO also may be a pathogenic factor during the attack of fungi
on other organisms [119], [72], [130]. Enhancement of PPO and POD activity was
reported in response to pathogen inoculation in plants pretreated with SA [65], [45]. It was
reported that POD may be some of the elements of the defense systems that are stimulated
in plants in response to pathogen infection especially Fusarium oxysporum [142].
Elucidation of signaling pathways controlling the induced disease resistance is a
major objective in research on plant pathogen interactions [40], [206]. Treatments
with AM fungi, JA and SA significantly reduced % of disease incidence. Growth rate
(shoot and root) markedly inhibited in tomato plants in response to Fusarium wilt
disease as compared with healthy control. Reduction in total chlorophyll in infected
leaves significantly decreased in plants treated with SA. Also, total soluble proteins
increased in both leaves and roots of SA-treated plants as compared with infected
24
control. Results suggest that reduction in disease incidence, promotion in growth and
metabolic activities in tomato plants inoculated with bioagent (AM fungi) and
sprayed with elicitors (JA& SA) could be related to the synergistic and cooperative
effect between them; which lead to the induction and regulation of disease resistance.
Thus, two signal hormones could enhance the biological activity of AM fungi in
tomato, potentially through interaction signalling pathways. AM fungi plus JA more
effective than AM fungi plus SA [64].
Defense system of the plant against pathogen attach is the ultimate goal of any
controlling process of the pathogen. Biological control and hormonal inducers
represents an interesting strategy to stimulate the defense system of the plant
especially when applied together. Trichoderma harzianum (TH), salicylic acid (SA)
and low dose of thiophanate methyl (TM) were used as recommended fungicide as a
new strategy to enhance tomato defense response against wilt disease caused by
Fusarium oxysporum f. sp. lycopersici (FOL) under greenhouse conditions. Changes
in various physiological defenses including enzymes like polyphenol oxidase (PPO),
peroxidase (POD) and acid invertase (AI); total soluble phenols; protein and
chlorophyll content were investigated. In the present study, tomato plants infected
with FOL one week after inoculation with TH fungi (seedling root dipping and/or soil
treatment) and/or sprayed daily for one week with hormonal inducer (SA). Plants
were harvested at 35 days after pathogen infection. All applied treatments completely
protected tomato seedlings against Fusarium wilt. Disease index percentage (DI %)
was highly significantly reduced up to zeropercentage. Level of all the determined
physiological parameters greatly changed in tomato plants in response to FOL, TH
fungi and hormonal elicitor reflected many components of defense signals which
leading to the activation of power defense system in tomato against pathogen attack.
Application of TH and SA stimulated all these parameters not only to reach but also
exceed their content in healthy control [97].
Plants respond to bacterial pathogen attack by activating various defence
responses, which are associated with the accumulation of several factors like defencerelated enzymes and inhibitors which serve to prevent pathogen infection. This study
focused on the role of the defence-related enzymes phenylalanine ammonia lyase
(PAL) and polyphenol oxidase (PPO) in imparting resistance to tomato against
bacterial wilt pathogen Ralstonia solanacearum. The temporal pattern of induction of
these enzymes showed maximum activity at 12 h and 15 h for PAL and PPO,
respectively, after the pathogen inoculation (hpi) in resistant cultivars. Twenty
different tomato cultivars were analyzed for PAL, PPO and total phenol content
following pathogen inoculation. The enzyme activities and total phenol content
increased significantly (P < 0.05) in resistant cultivars upon pathogen inoculation.
The increase in enzyme activities and total phenol content were not significant in
susceptible and highly susceptible cultivars [205].
Bayoud disease is caused by Fusarium oxysporum f. sp. albedinis [FOA], is the
most damaging disease of date palm in Morocco. In the present study we have
investigated the effect of jasmonic acid [JA] on two defence-related enzymes, namely
peroxidases [POX] and polyphenoloxidases [PPO] in date palm seedlings root. Our data
show that exogenous application of JA at a concentration of 50 μM increased the activity
25
of both enzymes. The increase of POX activity in the presence of JA was much more
important than that observed following infection with the pathogen. As compared to
untreated plants, PPO activity was 2.2 and 1.3 times higher in BSTN and JHL cultivars
respectively. In addition, PAGE analysis revealed increased band intensity of the major
constitutive isoforms of POX and PPO in both JA-treated and FOA-treated seedlings.
Close examination of symptomatic and asymptomatic plants showed that root tissues of
symptomatic plants were massively colonized by FOA. Also, disease development in
these plants appeared to involve a marked degradation of the host cell walls early during
the process of pathogen invasion. In contrast, the presence of FOA in asymptomatic
plants induced limited necrotic lesions [hypersensitive-reaction like lesions] that were
probably involved in reducing the progression of the pathogen. Together, our findings
indicate that JA is capable of enhancing date palm root resistance to infection by FOA
via the activation of defence-related enzymes such as PPO and POX. A close
relationship was found between resistance in date palm against FOA and the activation
of POX and PPO enzymes. Furthermore, the resistance induced by JA on date palm
seedlings is associated with increased POX and PPO activities. [100]
2.6.2. Accumulation of specific proteins
The accumulation of hydroxyproline-rich glycoproteins (HRGPs) was
investigated after induction of resistance in pearl millet against downy mildew caused
by Sclerospora graminicola. Treatment of susceptible pearl millet seeds with various
biotic and abiotic elicitors resulted in increased HRGP content in the cell walls of
coleoptiles at 9 h after inoculation. Similar results with increased accumulation at 4–
6 h after inoculation were obtained in suspension cells of pearl millet. Maximum
HRGP accumulation was observed in seedlings raised from susceptible seeds treated
with chitosan and Pseudomonas fluorescens. Peroxidase and hydrogen peroxide,
essential components for HRGP cross-linking, were also increased in samples treated
with these elicitors. A tissue specific increase in HRGP at the regions around vascular
bundles was observed upon chitosan treatment. The results presented will have a
presumed importance in identifying the susceptible pearl millet varieties and
improving those using elicitors of defense for field applications [193].
The interaction between tomato and Fusarium oxysporum f. sp. lycopersici
(FOL) has become a model system for the study of the molecular basis of disease
resistance and susceptibility. Gene-for-gene interactions in this system have provided
the basis for the development of tomato cultivars resistant to Fusarium wilt disease.
Over the last 6 years, new insights into the molecular basis of these gene-for-gene
interactions have been obtained. Highlights are the identification of three avirulence
genes in FOL and the development of a molecular switch model for I-2, a nucleotidebinding and leucine-rich repeat-type resistance protein which mediates the
recognition of the Avr2 protein. We summarize these findings here and present
possible scenarios for the ongoing molecular arms race between tomato and FOL in
both nature and agriculture [196].
2.7. Anatomy
When vascular elements of tomato plants become infected with Fusarium
oxysporum f. sp. lycopersici or root microflora, contact parenchyma cells that
26
ensheath the vessels commonly respond by depositing a callose-containing substance
along the intervening wall and especially at pit sites. These callose-containing
deposits were first identified in contact parenchyma cells by light microscopy using
thick sections and alkaline aniline blue staining and then characterized
ultrastructurally using TEM examination of sequential thin sections from the same
cells. They could be differentiated ultrastructurally from pit membranes in both
protoxylem and metaxylem tissues, and from the protective layer which has been
demonstrated to occur in metaxylem contact cells as part of normal developmental
and maturation processes. The callose-containing deposits often appear as electrontranslucent globules with electron-opaque centres. The globules become fused into
variously osmiophilic and electron-opaque aggregates, conferring a layered or
marbled appearance that is distinctive to these deposits [143].
The disease-resistance response correlates with changes in cell biochemistry and
physiology that are accompanied by structural modifications including the formation
of callose-enriched wall appositions and the infiltration of phenolic compounds at
sites of potential pathogen penetration. Ultrastructural and cytochemical approaches
have the potential to significantly improve our knowledge of how plants defend
themselves and how plant disease resistance is expressed at the cell level [27].
The effects of some chemical resistance-inducers on some anatomical features
of squash leaves were studied. His results revealed that, applying boric acid (BA) at
10mM, KH2PO4 at 50mM, MnSO4 at 10 & 20mM did not caused significant effect on
the length of the main vascular bundle (LVB) while salicylic acid (SA) at 10mM
decreased it significantly (-20.2%) in comparison with control. Oxalic acid at 5mM
induced the highest increase (91.2%) followed by KH2PO4 at 100mM (78.2%),
KH2PO4 at 200mM (76.7%) and BA at 20mM (62.7%). The width of the main
vascular bundle was significantly increased by all tested treatments in comparison
with control. Among tested interactions, KH2PO4 at 100mM was the most effective
for increasing the width of the main vascular bundle “WVB” (284.3%) followed by
BA at 5 mM (208.8%) and COCl2 at 20 mM (204.9%) while, SA at 10mM induced
the lowest significant increase (14.7%) in comparison with control. The number of
xylem vessels in the main vascular bundle was significantly increased by all tested
treatments in comparison with control. Applying COCL2 at 20mM was the best for
increasing the number of xylem vessels in the main vascular bundle “NXVB” (29)
followed by KH2PO4 at 100mM (28). While, SA at 10mM and BA at 10mM induced
the lowest significant increase in number of xylem vessels (11) compared with (7) in
control treatment. Diameter of xylem vessels (DXV) in the main vascular bundle was
responded positively and significantly by the tested treatments. The tested chemical
compounds OA, KH2PO4, COCl2, AA, BA, CuSO4, SA, MnSO4, Penconazole
increased average DXV by 97.3, 64.0, 57.3, 53.3, 46.7, 42.7, 41.3, 24.0 and 24.0%,
respectively over control. However, OA at 5mM induced the highest increase in the
DXV (128.0%) followed by OA at 10mM and KH2PO4 at 50mM (92.0%) and OA at
20mM, AA at 20mM and COCl2 at 20mM (72.0%). While, CuSO4 at 10 mM induced
the lowest increase (12.0%) compared with control [63].
27
3 MATERIALS AND METHODS
3.1. Isolation of the Fusarium wilt pathogen
Samples of tomato (Lycopersicon esculentum Mill.) plants showing typical
symptoms of Fusarium wilt disease were selected from different tomato cultivars
grown under glasshouses conditions in Almaty province of Kazakhstan during May
2008 season. Lower stem portions (3 cm long) of wilted plant viewed different
degrees of vascular discoloration were cut and placed in polyethylene bags then
brought immediately to the laboratory. The taken plant portions were rinsed
thoroughly in tap water, surface sterilized for 2 min in 1% sodium hypochlorite
solution, removed, rinsed three times in sterile distilled water then dried between
sterile filter paper. The peripheral ends (about ½ cm length) of each plant portion
were cut and discarded while the 2 cm-long middle part was cut longitudinally into 4
pieces under aseptic conditions. The 4 cut pieces of each fragment were plated onto
Potato Dextrose Agar (PDA) medium amended with streptomycin sulphate (300
mg/l) according to [17]. The PDA plates were incubated at 25ºC for 3-5 days [108].
3.2. Purification and identification of the isolated fungi
Hyphal tips taken randomly from the peripheral ends of the growing colonies
were transferred to platted potato dextrose agar (PDA). The resultant growths of the
isolated fungi were further purified using hyphal tip and/or single spore techniques.
The morphological characteristics of the macroconidia, phialids, microconidia,
chlamydospors, and colony growth traits with aid of the light microscope were used
for identification of the obtained isolates of Fusarium oxysporium [146]; [118].
3.3. In vitro growth and sporulation of different isolates of Fusarium
oxysporum
Nine isolates of Fusarium oxysporum were isolated from stems and roots of
tomato wilted plants showing different degrees of vascular discoloration, grown
under glasshouse conditions at different locations of Almaty, Kazakhstan. Each of
these isolates was allowed to grow for 5 days on PDA medium at 28ºC., then their
mycelial growth diameter (mm) and sporulation capacity (1.0x10 6 spores/ml) using
the haemosytometer slide were determined. All the obtained Fusarium-isolates
formed colonies, conidia and mycelia with morphological characteristics typical of F.
oxysporum according to [32] and [194].
3.4. Pathogenicity test of the Fusarium oxysporum isolates
Pathogenicity tests of the obtained isolates of Fusarium oxysporum were carried
out during May 2008 under glasshouse conditions. The tomato (Lycopersicon
esculentum Mill.) cv. Carolina Gold was used in this study. Transplanting and
inoculation was carried out as following:
3.4.1. Transplanting tomato cultivars and pathogen inoculation
Four weeks old tomato transplants (cv. Carolina Gold) were planted into plastic
pots (30 cm. in diameter) each containing 11 Kg of natural soil mixture consisted of clay
and sand at rate of 2:1 (by weight) at rate of 5 seedlings per pot. Twenty days after
transplanting, thinning took-place as only 3 seedlings were left per each pot then spore
28
suspension of a particular Fusarium isolate which prepared as mentioned at 106
spores/ml, was poured over stem base at rate of 20 ml/seedling. In control (noninoculated), plain water was used instead of spore suspension. Pots were irrigated and
maintained in a glasshouse at 25-30ºC and 70% relative humidity (RH). Three pots
(replicates) were used for each particular isolate. The inoculated treatments were
arranged in a completely randomized block design in the glasshouse. Plants were
observed for symptoms of wilt for 2 months after inoculation. Irrigation was carried out
two times weekly. The plants were sprayed with the pesticide malathion three times
every 3-4 weeks to protect tomato against the spreaded pests during the growing season.
3.4.2. Preparing of spore suspension
Fresh, mature (14-days old) PDA plated cultures of each particular Fusarium
isolate grown at 28ºC were used for preparing their inocula (spore suspension). Each
Fusarium-culture was flooded with 5 ml of sterile distilled water and exhaustive
scraping of the surface by hair brush. Spore suspension of 5 to 6 cultured PDA plates
for each Fusarium isolate were collected together and filtered through cheesecloth to
remove the majority of the hyphal fragments. Number of spores in the resultant spore
suspension was adjusted to be contained about 1.0x106 spores/ml [29] and [17] by
microscopic enumeration with the aid of a cell-counting haemocytometer.
3.4.3. Disease assessment
Two months after inoculation, tomato plants were investigated for wilt disease
incidence, plant growth characters and fruit yield. The wilt disease symptoms were
observed and photographed. Percentages of wilted and/or dead plants as well as, the
wilt disease severity (DS) was carried out according to the following visual scale and
description as suggested by [202]:
Infection grade
Description
No wilting symptoms (healthy plant).
0
Plant slightly wilted, vascular discoloration found in main root
1
region.
Plant moderately wilted, yellowing of old leaves, spreading
2
vascular browning.
Plant severely wilted, dying of all leaves except end leaves.
3
Dead plant, seedling entirely wilted.
4
Plants were uprooted and the lower stem and tap root were longitudinally
dissected for examination of internal tissues discoloration. The wilt disease severity
(DS) was determined and calculated using the following formula [188]:
Disease severity (DS) % = (1A+2B+3C+4D)/4T×100, Where, A, B, C and D are the
number of plants corresponding to the infection numerical grade, 1, 2,3 and 4
respectively and 4T is the total number of plants (T) multiplied by the maximum
discoloration grade 4, where T=A+B+C+D.
3.4.4. Effect of inoculation with different Fusarium isolates on tomato
plant growth and yield production
After 2 months post inoculation with the different isolates of Fusarium
oxysporum, the following growth parameters were also investigated:
29
1) Plant height (cm.);
2) Number of leaves/plant;
3) Fresh weight of leaves (g.)/plant;
4) Stem fresh weight (g.)/plant;
5) Root fresh weight (g.)/plant;
6) Root length (cm.);
7) Root volume (cm3)/plant; and
8) Weight of fruit yield (g.)/plant.
3.5. Evaluation of some commercial and new experimental tomato cultivars
against infection with the tomato wilt pathogen (Fusarium oxysorum f. sp.
lycopersici) under glasshouse conditions
In this study, some tomato cultivars [Table, 1] were evaluated against infection
with the tomato Fusarium wilt infection. Fusarium oxysporum f. sp. lycopersici
isolate A which proved the most virulent during the pathogenicity test was used in
this work. Tomato transplants of different cultivars (4 weeks old) were planted in
pots (30 cm Φ/ 11 kg soil/ 3 seedlings per pot) and placed under glasshouse
conditions at 25-30C with 70% RH and watered as required.
Table 1 - The tested tomato cultivars (provided by the seed company (Rijk Zwaan
Ltd. - Uzbekistan
Used name
Experimental code
Lot number
Exp 1
EXP 8340 Tomato Seeds
088340
Exp 2
EXP 8355 Tomato Seeds
088355
Exp 3
EXP 8416 Tomato Seeds
088416
Exp 4
EXP 8420 Tomato Seeds
088420
Exp 5
EXP 8576 Tomato Seeds
088576
Carolina Gold
Dona
Two weeks after transplanting, spore suspension of the tested Fusarium isolate
(A) was added at stem base of plants (20 ml for each). Three pots (replicates) were
used for each cultivar. The inoculated pots were arranged in a completely
randomized block design in the glasshouse. For preparing spore suspension, the
FOL-A isolate was plated onto Potato Dextrose Agar (PDA) medium amended with
streptomycin sulfate (300 mg/l) [17] at 28ºC for 14 days. Cultures of FOL-A isolate
were used for preparing fungal spore suspension. The resultant spore suspension was
adjusted to be about “1.0x106 spores/ml” [29] and [17] by microscopic enumeration
with the aid of a cell-counting haemocytometer.Percentages of diseased and/or dead
plants as well as wilt disease severity (DS) for each particular cultivar were
determined 2 months after inoculation as above mentioned according to [188]. The
plant height (cm), root length (cm), root fresh weight and total fruit yield/plant (g)
were also determined for all tested tomato cultivars. For each treatment 9 plants were
used (3 plants per pot). Plants were uprooted and the lower stem and tap root were
longitudinally dissected in order to examine the discoloration of the internal tissues.
30
Percentage of reduction in a given growth variable was calculated using the
following formula [62]:
Reduction (%) = value of control – value of treatment/ value of control × 100%.
3.6. Effect of garlic and black pepper extracts at different concentration on
growth and sporulation of different isolates of the tomato wilt pathogen
(Fusarium oxysorum f. sp. lycopersici) in vitro
3.6.1. Preparation of plant exracts
Fresh bulbs of garlic (Allium sativum) and dried seeds of black pepper (Pepper
nigrum) were obtained from the local market of Almaty province, Kazakhstan. The
garlic fresh bulbs were peeled while, the dried seeds of black pepper were grounded to a
fine powder, then a proper amount (200 g) of each of peeled garlic bulbs of powder of
black pepper seeds was blended separately with 200 ml sterile distilled water in an
electric blender for 10 min. The resultant suspension of each plant material was filtered
through two layers of muslin cloth followed by filtration through Whatman No. 1 filters
paper. The resultant crude extract of each plant material (garlic and black pepper) was
considered as 100% stock solutions which were stored at -4ºC until used.
3.6.2. Antifungal activity assay of plant extracts
The antifungal activities of the prepared extracts of garlic and black pepper
against growth and sporulation of different isolates of the tomato wilt pathogen
(Fusarium oxysporum f. sp. lycopersici) were investigated in vitro. The potato
dextrose agar (PDA) medium was used in this study. Set of conical flasks containing
PDA medium was prepared and autoclaved at 120ºC for 30 min as usual.
Aqueous extract of garlic or black pepper was sterilized by Millipore filter then
added immediately to the warmed (40-45ºC) PDA medium at 0.5, 1.0, 2.0, 3.0 and 4.0%
concentrations. In all cases, the normal concentration of agar (2%), dextrose (2%) and
peeled potato (200g/l) was taken into consideration. The warmed treated or untreated
(control) PDA medium in each conical flasks was gently shacked then poured with
constant volume (15 ml) into sterilized Petri plates (9 cm diameter) and left to solidify.
The medium without extracts served as control. After the solidification of the medium,
three plates (replicates) of known treatment was inoculated aseptically at the center with
0.5 cm diameter disk taken from 7-days-old cultures of a tested Fusarium isolate. All
inoculated plates were incubated at 27˚C. The plates were daily observed until fungal
growth covered the surface of the medium in any treatment. After 5 days of incubation,
colony diameter (mm) of each plate was measured by averaging the two diameters taken
at right angles for each colony. Also, the spore account per milliliter for each treatment
was calculated by microscopic enumeration with a cell-counting haemocytometer.
3.7. Effect of riboflavin and salicylic acid at different concentration on
growth and sporulation of different isolates of the tomato wilt pathogen
(Fusarium oxysorum f. sp. lycopersici) in vitro
3.7.1. Preparation of salicylic acid and riboflavin
Stock solution each of SA (HO.C6H4.COOH, MW 138.12 g/mol) and riboflavin
(C17H20N4O6, MW 376.36 g/mol) at 50 mM was prepared by dissolving a weight of
6.91 and 18.82g of SA and riboflavin, respectively with distilled water, then
completed to 1000ml.
31
3.7.2. Antifungal activity assay of chemical inducers
The antifungal activities of the prepared chemical inducers against growth and
sporulation of different isolates of the tomato wilt pathogen (Fusarium oxysporum f.
sp. lycopercici) were investigated in vitro. The potato dextrose agar (PDA) medium
was used in this study. Set of conical flasks containing PDA medium was prepared
and autoclaved at 120˚C for 30 min as usual.
Aqueous chemical inducers of salicylic acid or riboflavin was sterilized by
Millipore filter then added immediately to the warmed (40-45ºC) PDA medium at 0.1,
0.5, 1.5, 5.0 and 10.0 mM concentrations. In all cases, the normal concentration of agar
(2.0%), dextrose (2.0%) and peeled potato (200g/l) was taken into consideration. The
warmed treated or untreated (control) PDA medium in each conical flasks was gently
shacked, then appropriate and constant volume of the medium was poured into sterilized
Petri-dishes (9 cm diameter) and allowed to solidify. The medium without extracts
served as control. After the solidification of the medium, three plates (replicates) of
known treatment was inoculated aseptically at the center with 0.5 cm diameter disk
taken from 7-days-old cultures of a tested Fusarium isolate. All inoculated plates were
incubated at 27˚C. The plates were daily observed until fungal growth covered the
surface of the medium in any treatment. After 5 days of incubation, colony diameter
(mm) of each plate was measured by averaging the two diameters taken at right angles
for each colony. Also, the spore account per milliliter for each treatment was calculated
by microscopic enumeration with a cell-counting haemocytometer.
3.8. Effect of treatment with plant extracts and safe chemicals on
controlling tomato Fusarium wilt pathogen, plant growth and fruit yield in
vivo
In this study, three application methods namely immersing roots (IR), spraying
shoots (SS) and combined method (IR+SS) were used for treating tomato seedlings (4weeks old) of the tomato cv. Carolina gold with different inducer treatments i.e., garlic
(G) at 0.5 and 4.0%, black pepper (BP) extracts at 0.5 and 4.0, salicylic acid (SA) at 0.1
and 10.0mM and riboflavin (R) at 0.1 and 10mM. Tap water was used instead of the
control treatment (untreated). All treatments were conducted simultaneously and
immediately before transplanting. The treated and untreated seedlings were transplanted
in 30 cm diameter plastic pots (at rate of 3 seedlings per pot) under glasshouse
conditions. Three pots were used for each particular treatment. Each seedling was
inoculated, one week after planting, with 20 ml of spore suspension (1.0x106 spores/ml)
of the tomato wilt pathogen (F. oxysporum f.sp. lycopersici).
Effects of tested treatments on the induction of resistance against infection with
the tomato Fusarium wilt in terms of percentage of wilted and/or dead plants and
disease severity (DS) were determined as above mentioned after two months from
treatment. In addition, the following parameters:
1) Plant height (cm.);
2) Number of leaves/plant;
3) Fresh weight of leaves (g.)/plant;
4) Dry weight of leaves (g.)/plant;
5) Stem fresh weight (g.)/plant;
32
6) Root fresh weight (g.)/plant;
7) Root dry weight (g.)/plant;
8) Root length (cm.);
9) Root volume (cm3)/plant; and
10) Weight of fruit yield (g.)/plant.
As well as, percentage of increase in a given growth variable was calculated
using the following formula [62]:
Increase (%) = value of treatment - value of control / value of control × 100%.
3.9. Biochemical changes associated with different treatments of plant
extracts and safe chemical inducers
After two months from treatment, samples of the fifth plant leaf were taken from
each one of tomato (Carolina Gold cv.) plants under investigation wether treated with
the tomato Fusarium wilt (FOL) pathogen or untreated (control). The collected plant
leaves for each particular treatment were used to investigate the effects of tested
treatments on the following topics:
3.9.1. Chlorophyll's content
Extraction of chlorophyll's from the collected fifth leaf samples was determined
according to [21]. All steps of extraction were performed in dim light and as rapidly as
possible. In this respect, 0.2 g of a known fresh leaf sample was weighed, taken in a
mortar with small amounts of CaCO3 and acid washed sand in addition to 10 ml of 85%
aqueous acetone solution. All mixture components in the mortar were ground till the
slurry is completely homogenized. The homogenate was filtered through filter paper
(Whattman No.1) in a 25 cc measuring flask. The residue of homogenate in the mortar
was rewashed several times by using small volumes of 85% aqueous acetone solution
until the mortar is devoid of green colour. The washing filtrate was added to the previous
extract in the measuring flask and completed to the mark with 85% acetone solution.
Chlorophyll contents were adjusted to mg/g fresh weight. The absorbance (A) of
Chlorophyll’s concentration was calculated according to the following equations, [19]:
Chlorophyll a (mg/l) = (A663 x 12.7) – (A645 x 2.69);
Chlorophyll b (mg/l) = (A645 x 22.9) – (A663 x 4.68); and
Total chlorophyll (mg/l) = (A663 x 8.02) – (A645 x 20.2)
AS: A663 = optical density (O.D.) at 663 nm and A645 = O.D. at 645 nm.
3.9.2. Phenols and total soluble protein contents
In this study, samples representing the fifth plant leaves were taken as above
mentioned. The leaf samples taken from each particular treatment were extracted
separately by using the method suggested by [185]. A known weight (1 g) of the fifth
leaves was cut into small portions and immediately dispensed in a brown glass bottle
containing 95% ethyl alcohol and kept in the dark at room temperature for two
weeks. Then, tissues were grounded with ethyl alcohol and centrifuged for 10 min at
3000 rpm. The ethanolic extracts were prepared by subjecting these extracts to hot air
current at 40-45ºC till near dryness. They were then quantitatively transferred to
small bottles, compeleted to 10 ml with 50% isopropyl alcohol and frozen until using.
Extracts from each particular treatment were used for different chemical analysis as
follows:
33
3.9.2.4. Preparation of phenol reagent
Phenolic compounds were determined using the colourimetric method of
analysis described by [37]. 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 reflex 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.
3.9.2.5. Determination of free phenols compounds
One ml of the phenol reagent and 5 ml of a 20% solution of sodium carbonate to
the isopropanol sample (0.2 ml) diluted to 10 ml with warm water, 30-35°C. The
mixture was left to stand for 20 minutes and read using spectrophotometer
(SPECTRONIC 20-D) at 520 nm against a reagent blank.
3.9.2.6. Determination of total phenols compounds
For determination of total (free and conjugated) phenols, 10 drops of
concentrated hydrochloric acid were added to the isopropanol sample extract (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 then readings were
determined after 20 minutes using spectrophotometer (SPECTRONIC 20-D) at 520
nm against a reagent blank. The total phenols as well as the free phenol contents were
calculated for each treatment as milligrams of catechol per one gram fresh weight
according to standard curve of catechol.
3.9.2.7. Determination of conjugated phenols compounds
The conjugated phenols were determined by subtracting the free phenols from
the total phenols. The results were expressed as mg catechol 100 g -1 fresh weight.
3.10. Determination of oxidative enzymatic activities and total soluble
protein assay
3.10.1. Preparation of crud extract
The fifth leaf of each treated and non-treated plants was carefully cut at the leaf
base level after 60 days from treatment. The collected leaves for each particular
treatment were placed in polyethylene bags which were tightly closed and frozen
immediately. Each leaf sample was homogenized individually with 0.1 M of
phosphate buffer (pH 6.8) at rate of 3.0 ml/g fresh weight and centrifuged under
cooling (2ºC) at 17.000g for 15 min. The clear supernatant was taken as crude extract
for assaying the peroxidase, polyphenoloxidase activities and the total soluble
proteins [149]. The supernatant was collected and stored at -20ºC until use.
Peroxidase, Polyphenoloxidase enzymes and total soluble protein assays were
carried out using a (SPECTRONIC 20-D) spectrophotometer at 272C. Readings of
the spectrophotometer were recorded every 30 Sec, for 5 min for the two enzymes.
The reference cuvette for the spectrophotometer always contained the same
34
concentrations of components as the sample cuvette, except that the substrate solution
was replaced by extraction buffer.
3.10.2. Peroxidase assay
The activity of peroxidase enzyme was measured as described by [43]. The
obtained enzyme extract (0.3 ml) was added to 0.1 ml of 100 mM potassium
phosphate buffer (pH 7.0), (prepared by mixing 38.5ml of 100mM potassium
phosphate monobasic (KH2PO4) and 61.5ml of 100mM potassium phosphate dibasic
(K2HPO4)); 0.32 ml of 5% pyrogallol; 0.16 ml of 0.5% hydrogen peroxide in sample
cuvette (final volume of 3.0 ml) and the rest of distilled water. The initial rate
increase in absorbance at 420 nm was regarded as an arbitrary unit of enzyme
activity. Enzyme activity was expressed as 420/min/g fresh weight.
3.10.3. Polyphenoloxidase assay
Polyphenoloxidase was assayed following the method of [197]. The reaction
mixture contained 2 ml of 1% catechol solution as substrate, 0.2 ml of enzyme extract
and the rest of 0.05 M sodium phosphate buffer pH 6.8 in a final volume of 4 ml.
Enzyme activity was expressed as 430/min/g fresh weight.
3.10.4. Soluble protein assay
Protein content was determined using crystalline bovine serum albumin (BSA)
as a standard. Five ml of the Bradford dye (reagent) were added to 100 L of protein
extract, vortexed and absorbance was measured at 595 nm after 2 min and before one
hour. Protein concentration was calculated as mg/g-1 fresh weight from a standard
curve of bovine serum albumin. Reagent was prepared as follow: 100 mg of
Coomassie Brilliant Blue G-250 was dissolved in 50 ml of 95 % ethanol then 100 ml
of 85 % phosphoric acid was added to the dye solution. The resulting solution was
diluted to a Haul volume of one liter with distilled water [36].
3.11. Leaf anatomical structure
Two months after treatments, samples represented petioles of fifth leaves were
taken from the main stem basically of each treatment. Specimens were killed and
fixed for 48 hr. in FAA solution composed of formalin, glacial acetic acid and ethyl
alcohol 70 % at rate of 10:5:85 (by volume), respectively. The selected materials
were removed from the FAA solution, washed in 50% ethyl alcohol, dehydrated in a
normal ethyl alcohol series, embedded in paraffin wax (melting point 56ºC.),
sectioned to a thickness of 15-25 microns, double stained with safranine-fast green,
cleared in xylene and mounted in canada balsam [211]. Sections were examined
microscopically and read to detect anatomical manifestations of noticeable responses
resulted from investigated treatments.
Light photomicrographs were taken with a digital camera (Panasonic, DMCFX100, Osaka, Japan) fitted to the microscope. The following anatomical characters
were determined for each particular treatment:
{01}- Thickness of cuticle (µm),
{02}- Thickness of epidermal layer (µm),
{03}- Number of collenchyma layers,
{04}- Thickness of collenchyma layers (µm),
35
{05}- Number of parenchyma layers,
{06}- Thickness of parenchyma layers (µm),
{07}- Thickness of cortex (µm),
{08}- Thickness of outer phloem in the bi-collateral vascular bundle [VB] (µm),
{09}- Thickness of cambium in VB (µm),
{10}- Thickness of xylem in VB (µm),
{11}- Number of xylem vessels in VB,
{12}- Thickness of largest vessels in VB (µm),
{13}- Thickness of inner phloem in VB (µm),
{14}- Length of Vascular Bundle (µm),
{15}- Widest of VB (µm),
{16}- Number of pith layers,
{17}- Pith layers thickness (µm), and
{18}- Whole section thickness (µm).
3.12. Statistical analysis
All Data were analyzed statistically for the least significant difference (L.S.D.)
according to [78].
36
4 EXPERIMENTAL RESULTS
4.1. Isolation of the causal fungi and In Vitro studies
4.1.1. Obtained isolates
Nine isolates of Fusarium oxysporum were isolated from stem and roots of
tomato wilted plants showing different degrees of vascular discoloration, grown
under glasshouse conditions at different locations of Almaty, Kazakhstan. All isolates
formed colonies, conidia and mycelia with morphological characteristics typical of F.
oxysporum. These isolates were used for inoculation seedlings of the Carolina Gold
cultivar grown in plastic pots under glasshouse conditions. Wilt symptoms were
observed after two months from inoculation particularly brown vascular discoloration
in stem. This was the first record about presence of tomato wilt caused by Fusarium
oxysporum f. sp. lycopersici (FOL) in Kazakhstan.
4.1.2. Radius growth and sporulation of different isolates of FOL
Fusarium isolates were grown on potato dextrose agar (PDA) medium. The in
vitro growth (mm) and sporulation (106/ml) were determined after 5 days from
incubation at 28C. The results in Table (2) and Fig. 1a indicated that the colony
diameter and sporulation capacity of the tested Fusarium isolates were significantly
varied. In descending order, colony diameter for isolates F, D, E, I, A, C, B, H and G
recorded 77.7, 75.0, 70.3, 67.3, 65.7, 65.3, 63.0, 60.7 and 55 mm., respectively. In
this respect, colony diameters of isolates C and A were significantly equal meanwhile
it was significantly varied between each pair of the other isolates.As for sporulation
capacity (106 spores/ml), the results in the same table and Fig. 1b indicated that,
Fusarium isolate H recorded the highest sporulation (1.46), followed by isolates G
(1.14) with significant difference between them. Isolate A came next (0.63) followed
by isolates F (0.58), D (0.57), B (0.32), C (0.28), E (0.24) and I (0.20), respectively.
No significant differences were found between isolates A, F and D or between
isolates B, C, E and I.
Table 2 - Radius growth (mm.) and sporulation (106 spores/ml) of different
Fusarium oxysporum f. sp. lycopersici (FOL) isolates grown on PDA medium and
incubated at 28C for 5 days in vitro
Tested Fusarium isolates
Growth (mm.)
Spores/ml (106)
65.7
0.631
Isolate A
63.0
0.320
Isolate B
65.3
0.284
Isolate C
75.0
0.569
Isolate D
70.3
0.240
Isolate E
77.7
0.578
Isolate F
55.0
1.138
Isolate G
60.7
1.458
Isolate H
67.3
0.196
Isolate I
L.S.D. at 5%
0.758
0.136
37
Figure 1b - Sporulation (106 spores/ml) of
different Fusarium oxysporum f. sp.
lycopersici (FOL) isolates grown on PDA
medium and incubated at 28C for 5 days in
vitro
Figure 1a - Radius growth (mm.) of
different Fusarium oxysporum f. sp.
lycopersici (FOL) isolates grown on PDA
medium and incubated at 28C for 5 days
in vitro
4.2.
Pathogenicity test
Pathogenicity test was carried out under glasshouse condition at Almaty
province, Kazakhstan using the tomato Carolina Gold cultivar. The obtained results
were as following:
4.2.1. Wilt disease symptoms
The earliest symptom of wilt disease on tomato plants is the yellowing of the
older leaves. This often develops on only one side of the plant, and the leaflets on one
side of a petiole frequently turn yellow before those on the other side. The yellowing
gradually affects most of the foliage and is accompanied by wilting of the plant
during the hottest part of the day. The wilting becomes more extensive from day to
day until the plant collapses and dies (Photo 1a). The vascular tissue of stem and
roots of a diseased plant are usually dark brown. This browning extends far up the
stem. The browning of the vascular system is characteristic of the disease and
generally can be used for its identification. The pith remains healthy (Photo 1b).
4.2.2. Percentage of wilted plants and wilt disease severity
The percentage wilted plants and disease severity (DS) was significantly affected
by tested FOL isolates. Isolates A and G recorded the highest % wilted plants (77.8%)
followed by isolate F (66.7%) and B (55.6%) without significant difference between
them. Isolate D came next (44.4%) and C and E (33.3%). However, isolates H and I
which recorded 11.1% wilted plants seemed be non-pathogenic to tomato cultivar
Carolina Gold particularly when compared with the non-inoculated control (Table, 3).
The same data proved that both isolates A and G were the most virulent as they
recorded the highest significant increase in DS (52.8%) followed by isolate F (41.7%)
with clear significant differences in between. Isolate B came the next (33.3%), isolate
D (25.0%, isolates C and E (22.2%) without significant differences between the first
and the latter two isolates. However, isolate H was the least virulent in term of wilt
disease severity as it recorded the lowest significant increase (11.1%) compared with
the non-inoculated control (Table, 3 & Fig., 2).
38
Photo 1a - Healthy (H) and wilted (1-5) tomato plants (Carolina Gold cv.) inoculated
with Fusarium oxysporum f. sp. lycopercici (FOL) isolate
(two-months after inoculation)
Photo 1b - Longitudinal sections in stem (above) and roots (below) of healthy
(control and severely wilted tomato plants (Carolina Gold cv.) inoculated with
different FOL isolates (two-months after inoculation). Notice vascular discoloration
on stem and roots of diseased plants
39
Table 3 - Effect of inoculation with nine isolates of Fusarium oxysporum f. sp.
lycopersici (FOL) on wilted plants % and disease severity % (DS) of tomato cultivar
Carolina Gold (two-months after inoculation)
Tested Fusarium isolates
Wilted plants %
Disease severity %
77.8
52.8
Isolate A
55.6
33.3
Isolate B
33.3
22.2
Isolate C
44.4
25.0
Isolate D
33.3
22.2
Isolate E
66.7
41.7
Isolate F
77.8
52.8
Isolate G
11.1
11.1
Isolate H
11.1
8.33
Isolate I
Control
0.00
0.00
L.S.D. at 0.05
31.714
9.173
Fig. 2 - Effect of inoculation with nine isolates of Fusarium oxysporum f. sp.
lycopersici (FOL) on disease severity % (DS) of tomato cultivar Carolina Gold
(two-months after inoculation)
4.2.3. Growth parameters
4.2.3.1. Plant height (cm.)/plant (PH)
The tomato plant’s height was negatively and significantly affected by all tested
FOL isolates comparing to the un-inoculated (control) treatment. The highest
significant reduction in plant height (32.26%) was caused by isolate A followed by
isolates G, C and H which reduced plant height by 26.45, 24.51 and 22.58%,
respectively without significant variations between the later three isolates. In the same
point, the lowest significant reduction in plant height was induced by isolate I (9.35%)
whereas, isolates D, E, F and H showed moderate significant reduction in plant height
i.e. 18.71, 16.77, 19.67 and 14.84%, respectively (Table, 4a; 4b & Fig., 3).
40
Table 4a - Effect of inoculation with nine isolates of Fusarium oxysporum f. sp.
lycopersici (FOL) on growth parameters of tomato cultivar Carolina Gold (twomonths after inoculation)
Tested
Fusarium
*PH
NL
FWL SFW RFW
RL
WFY
isolates
70.00
9.00 10.18 16.79 10.00 27.33 24.55
Isolate A
80.00 10.33 11.09 22.10 11.90 33.33 30.67
Isolate B
78.00
9.67 12.71 20.08 13.13 34.00 23.42
Isolate C
84.00 10.00 11.43 19.09 11.17 30.00 40.65
Isolate D
86.00 11.33 14.88 20.77 13.53 35.33 37.32
Isolate E
83.00 11.33 13.80 19.11 14.21 35.33 42.06
Isolate F
76.00
9.00 10.45 17.17 9.88 28.67 26.72
Isolate G
88.00 12.33 17.14 22.80 12.56 37.67 39.25
Isolate H
93.67 13.33 17.18 21.47 16.17 39.33 55.18
Isolate I
Control
(non103.33 15.00 47.72 32.78 22.89 40.67 71.92
inoculated)
L.S.D. at 0.05
4.371 0.941 5.098 4.257 3.007 3.767 9.834
*PH= plant height (cm.), NL= Number of leaves, FWL= Fresh weight of leaves
(g.)/plant, SFW= Stem fresh weight (g.)/plant, RFW= Root fresh weight (g.)/plant,
RL= Root length (cm.)/plant, WFY= Weight of fruit yield (g.)/plant.
Table 4b - Effect of inoculation with nine isolates of Fusarium oxysporum f. sp.
lycopersici (FOL) on growth parameters reduction % of tomato cultivar Carolina
Gold (two-months after inoculation)
*Reduction % for
Tested Fusarium isolates
**PH NL FWL SFW RFW RL WFY
32.26 40.00 78.67 48.78 56.31 32.80 65.86
Isolate A
22.58 31.13 76.76 32.58 48.01 18.05 57.36
Isolate B
24.51 35.53 73.37 38.74 42.64 16.40 67.44
Isolate C
18.71 33.33 76.05 41.76 51.20 26.24 43.48
Isolate D
16.77 24.47 68.82 36.64 40.89 13.13 48.11
Isolate E
19.67 24.47 71.08 41.70 37.92 13.13 41.52
Isolate F
26.45 40.00 78.10 47.62 56.84 29.51 62.85
Isolate G
14.84 17.80 64.08 30.45 45.13 7.38 45.43
Isolate H
9.35 11.13 64.00 34.50 29.36 3.29 23.28
Isolate I
Control
0.00
0.00
0.00
0.00
0.00
0.00
0.00
* Reduction (%) = (control – treatment) / control X 100
**PH= plant height (cm.), NL= Number of leaves, FWL= Fresh weight of leaves
(g.)/plant, SFW= Stem fresh weight (g.)/plant, RFW= Root fresh weight (g.)/plant,
41
RL= Root length (cm.)/plant, WFY= Weight of fruit yield (g.)/plant.
Fig. 3 - Effect of inoculation with nine
isolates of Fusarium oxysporum f. sp.
lycopersici (FOL) on plant height
reduction % of tomato cultivar Carolina
Gold (two-months after inoculation)
Fig. 4 - Effect of inoculation with nine
isolates of Fusarium oxysporum f. sp.
lycopersici (FOL) on number of leaves
reduction % of tomato cultivar Carolina
Gold (two-months after inoculation)
Fig. 5 - Effect of inoculation with nine
isolates of Fusarium oxysporum f. sp.
lycopersici (FOL) on fresh weight of
leaves reduction % of tomato cultivar
Carolina Gold (two-months after
inoculation)
Fig. 6 - Effect of inoculation with nine
isolates of Fusarium oxysporum f. sp.
lycopersici (FOL) on stem fresh weight
reduction % of tomato cultivar Carolina
Gold (two-months after inoculation)
42
4.2.3.2. Number of leaves/plant (NL)
Number of leaves per tomato plant (NL) was significantly and negatively
decreased by all tested FOL isolates (9.0-13.33) comparing with the non-inoculated
control plants (15.0). It is clear that, the highest significant reduction in the NL was
recorded by isolates A and G (40.0%) and isolate C (35.53%) without significant
differences in between followed by isolate D (33.33%), isolates E and F (24.47%)
whereas the lowest significant reduction was recorded by isolates H (17.8%) and I
(11.13%) comparing with the non-inoculated control plants (Table, 4a; 4b & Fig., 4).
4.2.3.3. Fresh weight of leaves (g.)/plant (FWL)
All tested FOL isolates caused high and dangerous losses in fresh weight of
leaves g/plant (FWL). The reduction in FWL ranged between 64.0% and 78.67% in
inoculated plants comparing with the un-inoculated check plants. Isolates A and G
caused the highest reduction in the FWL i.e. 78.67 and 68.82%, respectively whereas,
isolates H and I recorded lower reduction (64.0%) comparing with the former two
isolates. The remained isolates showed intermediate effect in this respect. These
results might be due to the high defoliation and dryness in leaves of the wilted plants
(Table, 4a; 4b and Fig., 5).
4.2.3.4. Stem fresh weight (g.)/plant (SFW)
The fresh weight of stem (g)/plant (SFW) in tomato plants inoculated with any
of tested FOL isolate was significantly lower than the un-inoculated (control) plants.
The SFW reduced by 30.45-48.78% comparing to the check treatment. In this regard,
the heights significant reduction was recorded by FOL isolates A, G, D and F, which
reduced SFW by 48.78, 47.62, 41.76 and 41.7%, respectively without significant
differences between them. While, isolates C, E, I, B and H reduced SFW by 38.74,
36.64, 34.5, 32.58 and 30.45%, respectively comparing to the non-inoculated control
plants (Table, 4a; 4b and Fig., 6).
4.2.3.5. Root fresh weight (g.)/plant (RFW)
Inoculation of tomato plants with different FOL isolates significantly decreased
the fresh weight of roots (g)/plant (RFW) comparing to the check (un-inoculated)
treatment. In this regard, the heights significant reduction in RFW was recorded by
isolate G (56.84%), isolate A (56.31%), isolate D (51.2%), isolate B (48.01%) and
isolate H (45.13%) without significant differences in between. Whereas, the lowest
significant reduction in the RFW was recorded by isolate I (29.36%), isolate F
(37.92%) and isolate E (40.89%) without significant differences between them.
However, isolate C reduced RFW by 42.64% and significantly varied compared with
isolate G or isolate I (Table, 4a; 4b and Fig., 7).
4.2.3.6. Root length (cm.)/plant (RL)
It is clear that, inoculation of tomato plants with most FOL isolates caused
significant reduction in their root length (RL). The three isolates A, G and D caused
43
the heights significant reduction in RL as they recorded 32.8, 29.51 and 26.4%
reduction, respectively without significant differences between them. However,
isolates B, C and E produced the lowest significant reduction in RL meanwhile;
isolates H and I had no significant effect in this regard comparing to the noninoculated control plants (Table, 4a; 4b and Fig., 8).
Fig. 7 - Effect of inoculation with nine
isolates of Fusarium oxysporum f. sp.
lycopersici (FOL) on root fresh weight
reduction % of tomato cultivar Carolina
Gold (two-months after inoculation)
Fig. 8 - Effect of inoculation with nine
isolates of Fusarium oxysporum f. sp.
lycopersici (FOL) on root length
reduction % of tomato cultivar Carolina
Gold (two-months after inoculation)
Fig. 9 - Effect of inoculation with nine isolates of Fusarium oxysporum f. sp.
lycopersici (FOL) on weight of fruit yield reduction % of tomato cultivar Carolina
Gold (two-months after inoculation)
4.2.3.7. Weight of fruit yield (g.)/plant (WFY)
Tomato plants inoculated with different tested isolates of FOL pathogen
produced significantly lower weights of tomato fruit yield (FWY) compared with the
non-inoculated plants (control). In this regard, isolates C, A, G and B were
significantly equal and caused the highest significant reduction in the WFY i.e. 67.44,
65.86, 62.85 and 57.36%, respectively whereas; isolate I caused the lowest significant
reduction in the FWY (23.28%). On the other hand, isolates E, H, D and F were
moderately effective and recorded 48.11, 45.43, 43.48 and 41.52% reduction in the
WFY, respectively (Table, 4a; 4b and Fig., 9).
44
4.3. Sensitivity of some new experimental tomato cultivars against
infection with the Fusarium wilt
4.3.1. Percentage of wilted plants and wilt disease severity
The tested tomato cultivars responded differently against artificial inoculation
with the tomato wilt pathogen, Fusarium oxysporum f. sp. lycopersici (FOL) (Table,
5 and Fig., 10). Regardless inoculation, the tomato cultivars Carolina Gold and Dona
recorded similar highest increase in the percentage of wilted plants (38.9%) but they
significantly varied in disease severity as they recorded 26.4 and 22.2%, respectively.
The tomato cultivar EXP 4 came the next (27.8% wilted plants and 12.5%
disease severity), EXP 5 (16.7% wilted plants and 8.3% disease severity), and EXP 3
(11.1% wilted plants and 2.8% disease severity) with significant differences between
them particularly in % wilted plants. Similar trend was noticed concerning the wilt
disease severity. Similar trend was noticed concerning inoculation treatments.
However, EXP 1 and EXP 2 cultivars were the most resistant against FOL infection;
they remained wilt disease-free looks like non-inoculated plants.
Table 5 - Effect of inoculation with Fusarium oxysporum f. sp. lycopersici isolate A
on wilted plants % of different tomato cultivars (two-months after inoculation)
wilted plants %
Disease severity %
Tested
Healthy
Disease
Healthy
Mea Disease
Mea
cultivars
(control
d
(control)
n
d
n
)
77.8
0.0
52.8
0.0
Carolina Gold
38.9
26.4
77.8
0.0
44.4
0.0
Dona
38.9
22.2
0.0
0.0
0.0
0.0
EXP 1
0.0
0.0
0.0
0.0
0.0
0.0
EXP 2
0.0
0.0
22.2
0.0
5.6
0.0
EXP 3
11.1
2.8
55.6
0.0
25.0
0.0
EXP 4
27.8
12.5
33.3
0.0
16.7
0.0
EXP 5
16.7
8.3
Mean
38.1
0.0
20.6
0.0
L.S.D. at 5%
Inoculation
1.44
1.10
Cultivars
5.03
3.85
Interaction
10.07
7.71
45
.
Fig. 10 - Effect of inoculation with Fusarium oxysporum f. sp. lycopersici isolate A
on wilted plants % of different tomato cultivars (two-months after inoculation)
4.3.2. Growth parameters
4.3.2.1. Plant height (cm.)/plant (PH)
The data in Table (6a) and Fig., (11) stated that, the plant height of tomato
plants, regardless cultivar, was significantly affected by inoculation with FOL. Plant
height was significantly decreased from 105.9 cm in the control (non-inoculated)
plants to 96.6 cm in inoculated plants. The plant height of the three tomato cultivars
Carolina Gold, Dona and EXP 4 only was significantly reduced by 32.3, 13.6 and
7.8% comparing to their non-inoculated plants, respectively. However, plant height in
the remained tested cultivars viz., EXP 1, EXP 2, EXP 3 and EXP 5 was not
significantly affected by inoculation with FOL when compared with their respective
non-inoculated control.
4.3.2.2. Number of leaves/plant (NL)
The data in Table (6a) indicated that, the number of leaves per tomato plant
(NL) was significantly varied between tested tomato cultivar and inoculation
treatments but not affected by the interaction between the two factors (cultivar and
inoculation). The NL was significantly lower in plants inoculated with the Fusarium
wilt (12.86) than the non-inoculated ones (14.76). Regardless inoculation treatment,
the tomato cultivar EXP 1 produced the highest significant NL (17.5) followed by
EXP 3 and EXP 2 (14.83 & 14.5), Carolina Gold and EXP (13.0), Dona (12.5) and
EXP 4 (11.33), respectively. In general, inoculation with the Fusarium wilt reduces
NL by 26.7, 21.4, 14.3, 11.1, 10.6, 6.7 and 1.9% in the tomato culativars Carolina
Gold, Dona, EXP 5, EXP4, EXP 3, EXP 2 and EXP 1, respectively (Fig., 12).
4.3.2.3. Fresh weight of leaves (g.)/plant (FWL)
The data in Table (6a) stated that, the fresh weight of leaves/plant (FWL) was
significantly lower in tomato plants inoculated with FOL (33.3 g) than the non-inoculated
ones (41.4 g). The tested tomato cultivar was significantly varied in this respect.
Regardless inoculation, the tomato cultivar EXP 1 recorded the highest significant FWL
(55.7 g) followed by EXP 5 (51.0 g), EXP 3 (42.3 g), Carolina Gold (35.1 g), EXP 4 (31.2
g), Dona (23.4 g) and EXP 2 (22.9 g). Comparing with healthy (non- inoculated) plants,
46
the FWL of inoculated plants of these tomato cultivars was reduced by 5.9, 15.7, 5.0, 52.9,
15.3, 35.6, and 10.0%, respectively (Fig., 13). These results proved that the highest
defoliation, due to FOL inoculation, was recorded by Carolina Gold and Dona, the most
susceptible tomato cultivars meanwhile the least defoliation was recorded by EXP 3, EXP
1 and EXP 2, the most resistant tomato cultivars.
4.3.2.4. Stem fresh weight (g.)/plant (SFW)
The data in Table (6b) and Fig., (14) stated that, the stem fresh weight /plant
(SFW) was significantly lower in tomato plants inoculated with FOL (13.7 g) than
the non-inoculated ones (16.5 g). The tested tomato cultivar was significantly varied
in this respect. Regardless inoculation, the tomato cultivar EXP 4 recorded the
highest significant SFW (18.7 g) followed by EXP 1 (16.5 g), EXP 2 (15.8 g), EXP 5
(14.5 g), EXP 3 (13.8 g), Carolina Gold (13.7 g), and Dona (12.6 g), respectively.
The difference between SFW of the latter three cultivars was not significantly varied.
Comparing with healthy (non-inoculated) plants, the SFW of inoculated plants of
these tomato cultivar was reduced by 7.2, 2.0, 13.2, 14.5, 7.0, 45.1, and 30%,
respectively. These results stated that the highest reduction in the SFW, due to FOL
inoculation, was recorded by Carolina Gold and Dona (30.0-45.1%), the most
susceptible tomato cultivars meanwhile the least reduction was recorded by the new
experimental tomato cultivars (2.0-14.5%) (Fig.,14).
Table 6a - Effect of inoculation with Fusarium oxysporum f. sp. lycopersici isolate A
on growth parameters of different tomato cultivars (two-months after inoculation)
Plant height (cm.)
* Reduction
Tested cultivars
%
Diseased Healthy (control)
Mean
70.0
103.3
32.3
Carolina Gold
86.7
87.0
100.7
13.6
Dona
93.8
107.0
109.7
2.4
EXP 1
108.3
95.7
97.3
1.7
EXP 2
96.5
114.3
115.3
0.9
EXP 3
114.8
102.3
111.0
7.8
EXP 4
106.7
100.0
103.7
3.5
EXP 5
101.8
Mean
96.6
105.9
L.S.D. at 5%
Inoculation
0.7
Cultivars
2.5
Interaction
5.0
Number of leaves/plant
* Reduction
Tested cultivars
%
Diseased Healthy (control)
Mean
11.00
15.00
26.70
Carolina Gold
13.00
11.00
14.00
21.40
Dona
12.50
17.33
17.67
1.90
EXP 1
17.50
14.00
15.00
6.70
EXP 2
14.50
14.00
15.67
10.60
EXP 3
14.83
47
EXP 4
EXP 5
Mean
L.S.D. at 5%
Inoculation
Cultivars
Interaction
Tested
cultivars
Carolina Gold
Dona
EXP 1
EXP 2
EXP 3
EXP 4
EXP 5
Mean
L.S.D. at 5%
Inoculation
Cultivars
Interaction
10.67
12.00
96.6
12.00
14.00
105.9
11.33
13.00
0.16
0.57
NS
Fresh weight of leaves (g.)/plant
Diseased
Healthy (control)
Mean
22.5
47.7
35.1
18.3
28.5
23.4
54.0
57.4
55.7
21.7
24.1
22.9
41.2
43.3
42.3
28.6
33.8
31.2
46.7
55.3
51.0
33.3
41.4
11.10
14.30
* Reduction
%
52.8
35.6
5.9
10.0
5.0
15.3
15.7
0.55
1.92
3.84
Table 6b - Effect of inoculation with Fusarium oxysporum f. sp. lycopersici isolate A
on growth parameters of different tomato cultivars (two-months after inoculation)
Stem fresh weight (g.)/plant
Tested cultivars
* Reduction %
Diseased
Healthy (control)
Mean
9.7
17.7
45.1
Carolina Gold
13.7
10.4
14.9
30.0
Dona
12.6
16.3
16.7
2.0
EXP 1
16.5
14.7
16.9
13.2
EXP 2
15.8
13.3
14.3
7.0
EXP 3
13.8
18.0
19.4
7.2
EXP 4
18.7
13.3
15.6
14.5
EXP 5
14.5
Mean
13.7
16.5
L.S.D. at 5%
Inoculation
0.29
Cultivars
1.02
Interaction
2.03
Root length (cm.)/plant
Tested cultivars
* Reduction %
Diseased
Healthy (control)
Mean
30.3
40.7
25.4
Carolina Gold
35.5
48
Dona
EXP 1
EXP 2
EXP 3
EXP 4
EXP 5
Mean
L.S.D. at 5%
Inoculation
Cultivars
Interaction
Tested cultivars
Carolina Gold
Dona
EXP 1
EXP 2
EXP 3
EXP 4
EXP 5
Mean
L.S.D. at 5%
Inoculation
Cultivars
Interaction
30.0
41.7
30.3
37.3
30.0
27.0
32.4
37.7
44.7
32.0
40.7
35.7
32.3
37.7
33.8
43.2
31.2
39.0
32.8
29.7
20.4
6.7
5.2
8.2
15.9
16.5
0.2
0.8
1.7
Root fresh weight (g.)/plant
Diseased
Healthy (control)
Mean
13.3
22.9
18.1
12.1
20.8
16.4
19.3
20.3
19.8
14.3
14.8
14.6
18.3
20.2
19.3
18.3
19.9
19.1
11.2
13.6
12.4
15.3
18.9
* Reduction %
41.8
42.0
4.9
3.0
9.3
7.8
17.1
0.23
0.79
1.58
Table 6c - Effect of inoculation with Fusarium oxysporum f. sp. lycopersici isolate A
on growth parameters of different tomato cultivars (two-months after inoculation)
Root volume (cm.3)/plant
Tested cultivars
* Reduction %
Diseased Healthy (control)
Mean
9.8
15.9
38.1
Carolina Gold
12.9
13.9
18.0
22.8
Dona
15.9
18.7
20.7
9.7
EXP 1
19.7
49
EXP 2
EXP 3
EXP 4
EXP 5
Mean
L.S.D. at 5%
Inoculation
Cultivars
Interaction
Tested cultivars
Carolina Gold
Dona
EXP 1
EXP 2
EXP 3
EXP 4
EXP 5
Mean
L.S.D. at 5%
Inoculation
Cultivars
Interaction
13.7
22.0
12.0
13.0
14.7
14.7
23.7
14.2
14.1
17.3
14.2
22.8
13.1
13.6
6.8
7.0
15.6
7.9
0.31
1.08
NS
Fruit yield (g.)/plant
Diseased Healthy (control)
24.6
71.9
30.4
69.2
149.0
153.8
75.7
78.2
133.3
143.3
205.7
236.3
90.3
116.7
101.3
124.2
0.8
2.9
5.9
* Reduction (%) = (control – treatment) / control X 100
50
Mean
48.2
49.8
151.4
76.9
138.3
221.0
103.5
* Reduction %
65.9
56.1
3.1
3.2
7.0
13.0
22.6
Fig. 11 - Effect of inoculation with
Fusarium oxysporum f. sp. lycopersici
isolate A on plant height reduction % of
different tomato cultivars
(two-months after inoculation)
Fig. 12 - Effect of inoculation with
Fusarium oxysporum f. sp. lycopersici
isolate A number of leaves reduction %
of different tomato cultivars
(two-months after inoculation)
Fig. 13 - Effect of inoculation with
Fusarium oxysporum f. sp. lycopersici
isolate A fresh weight of leaves reduction
% of different tomato cultivars
(two-months after inoculation)
Fig. 14 - Effect of inoculation with
Fusarium oxysporum f. sp. lycopersici
isolate A stem fresh weight reduction %
of different tomato cultivars
(two-months after inoculation)
51
Fig. 15 - Effect of inoculation with
Fusarium oxysporum f. sp. lycopersici
isolate A root length reduction % of
different tomato cultivars
(two-months after inoculation)
Fig. 16 - Effect of inoculation with
Fusarium oxysporum f. sp. lycopersici
isolate A on root fresh weight reduction
% of different tomato cultivars
(two-months after inoculation)
Fig. 17 - Effect of inoculation with
Fusarium oxysporum f. sp. lycopersici
isolate A on root volume reduction % of
different tomato cultivars
(two-months after inoculation)
Fig. 18 - Effect of inoculation with
Fusarium oxysporum f. sp. lycopersici
isolate A on weight of fruit yield
reduction % of different tomato cultivars
(two-months after inoculation)
4.3.2.5. Root length (cm.)/plant (RL)
The data in Table (6b) declared that, the root length (RL) was significantly
affected by inoculation with FOL. It was significantly decreased from 37.7 in the
52
non-inoculated plants to 32.4 cm in the inoculated ones. Comparing to the RL in the
non-inoculate tomato plants, the RL was decreased by 25.4, 20.4, 16.5, 15.9, 8,2, 6.7,
and 5.2% in Carolina Gold, Dona cultivars and the new experimental tomato cultivars
EXP 5, EXP 4, EXP 3, EXP 1 and EXP 2, respectively. The difference in root length,
regardless inoculation, reached the significant level (at 5%) between any pair of these
tomato cultivars. However, the RL in the inoculated plants of the two new
experimental cultivars EXP 1 and EXP 2 were not varied significantly when
compared with their respective controls. The healthy plant of the new experimental
cultivar EXP 1 recorded the highest RL (44.7 cm) followed by Carolina Gold (40.7
cm), EXP 3 (40.7 cm), Dona (37.7 cm), EXP 4 (35.7 cm), EXP 5 (29.7 cm), and EXP
2 (32.0 cm), respectively. In the inoculated plants, the RL recorded 41.7, 37.3, 30.3,
30.3, 30.0, and 30 cm in the tomato cultivars EXP 1, EXP 3, Carolina gold, EXP 2,
Dona, and EXP 4, respectively without significant differences between the later four
cultivars. The lowest RL (27.0 cm) was recorded in the inoculated plants of tomato
cultivar EXP 5 (Fig., 15).
4.3.2.6. Root fresh weight (g.)/plant (RFW)
Inoculation of tomato plants with the tomato wilt pathogen (FOL) significantly
decreased the RFW (Table, 6b) of most tested tomato cultivars comparing to their
respective un-inoculated control plants. The RFW in the most resistant cultivars, EXP
1 and EXP 2, was significantly equal in both inoculated and un-inoculated plants
meanwhile were significantly decreased to different extents in the remained tested
tomato cultivars. The most susceptible cultivars Carolina Gold and Dona recorded the
highest reduction in RFW (41.8-42.0%). In the moderately resistant cultivars (EXP 3,
EXP 4 and EXP 5), their RFW was decreased, due to inoculation with FOL by 7.817.1% (Fig., 16).
4.3.2.7. Root volume (cm3)/plant (RV)
The data in Table (6c) indicated that the average of the root volume (RV) was
significantly lower in tomato plants inoculated with FOL, (14.7 cm3 /plant) than the
non-inoculated control plants (17.3 cm3 /plant). Regardless inoculation, the new
experimental tomato cultivar EXP 3 recorded the highest RV (22.8 cm3) followed by
EXP 1 (19.7 cm3) Dona (15.9 cm3), EXP 2 (14.2 cm3), EXP 5 (13.6 cm3), EXP 4
(13.1 cm3) and Carolina Gold (12.9 cm3) without significant differences between the
latter three tomato cultivars. The RV was not significantly affected by the interaction
between inoculation treatments (inoculated or not) and tomato cultivars. However,
the most susceptible cultivar Carolina Gold, due to inoculation) shows the highest
reduction in the RV (38.1%) followed by Dona (22.8%), EXP 4 (15.6%), EXP 1
(9.7%), and EXP 5, EXP 3 and EXP 2 (6.8-7.9%), respectively (Fig., 17).
4.3.2.8. Weight of Fruit yield (g.)/plant (WFY)
The data in Table (6c) proved that the weight of fruit yield/plant (WFY) was
significantly lower in FOL inoculated (101.3 g) than the non-inoculated control (124.2
g). Regardless inoculation treatments, the highest WFY was produced by the new
experimental tomato cultivar EXP 4 (221.0 g) followed by EXP 1 (151.4 g), EXP 3
(138.3 g), EXP 5 (103.5 g), EXP 2 (76.9 g), Dona (49.8 g) and Carolina Gold (48.2 g)
without significant differences between the latter two susceptible cultivars. The
53
interaction between inoculation and tomato cultivars indicated that the fruit yield of the
new experimental cultivar EXP 1 only was not significantly affected by FOL
inoculation comparing with its respective non-inoculated control. However, the WFY
of the remained tested cultivars was significantly lower in inoculated than the noninoculated plants. In this regard, the most susceptible cultivars Carolina Gold and Dona
recorded highest significant reduction in their WFY (56.1-65.9%) followed by EXP 5
(22.6%), EXP 4 (13.0%), EXP 3 7.0%) and EXP 2 (3.2%), respectively (Fig., 18).
4.4.
In vitro Studies on some natural and chemical resistance inducers
4.4.1. The In vitro inhibitory effect of the tested resistance inducers at
different concentration on the radius growth
4.4.1.1. Garlic and black pepper extracts
The radius growth of the tomato wilt fungus, Fusarium oxysporum f. sp.
lycopercici (FOL), was significantly affected by tested inducer treatments [garlic (G),
black pepper (BP) extracts], their concentrations as well as by the interaction between
them (Table, 7a anf Fig., 19a). Regardless concentrations, the garlic extract showed
the highest inhibitory effect, recorded lower radius growth (29.3 mm) than black
pepper extract (42.7 mm) with significant differences between them. The garlic and
black pepper extracts reduced the FOL radius growth by 55.41 and 35.03%,
respectively, reduction in the FOL radius growth was increased successively by
increasing their concentrations. The radius growth was reduced by 11.2, 21.8, 72.3,
82.7 and 83.3% at 0.5, 1.0, 2.0, 3.0 and 4.0% concentrations, respectively comparing
to the untreated control. As for interactions, the radius growth was completely
inhibited (100.0% inhibition) by garlic extract at concentration of ≥2.0% whereas,
black pepper extract allowed FOL to still grew even at 4.0% concentration (22.0mM),
reducing it by 66.5% comparing to the untreated control (Fig., 19a).
4.4.1.2. Salicylic acid and riboflavin
The radius growth of FOL was significantly affected by tested inducer treatments
[salicylic acid (SA) and riboflavin (R)], their concentrations as well as by the interaction
between them (Table, 7b and Fig., 19b). Regardless concentrations, riboflavin showed
higher inhibitory effect than salicylic acid. They recording radius growth of 44.0 and
48.3 mm which reduced by 32.99 and 26.4%, respectively with significant differences
between them comparing to the untreated control. Reduction in the FOL radius growth
was increased successively as tested concentrations increased. Thus, the radius growth
was reduced to 62.2, 57.7, 33.8, 14.3, and 6.6 mm at concentrations of 0.4, 0.5, 1.5, 5.0
and 10.0 mM, respectively comparing to the untreated control, which recorded 65.7 mm.
As for interactions, the radius growth was completely inhibited (100.0% inhibition) at
concentration of ≥5.0 and 10.0mM for riboflavin and salicylic acid, respectively. It is
interest to state that, the FOL radius growth was not significantly varied in PDA medium
treated by salicylic acid at 0.1 and 0.5mM while it was significantly enhanced in
medium treated by riboflavin at 0.1mM (70.3mM) or 0.5mM (69.0mM) comparing to
the untreated control medium (65.7mM).
54
Table 7a - Effect of garlic (G) and black pepper (BP) extracts at different
concentrations on the in vitro radius growth (mm.) of Fusarium oxysporum f.sp.
lycopersici isolate A
Radius growth (mm.)
* Reduction %
Tested concentrations
Mean
Mean
G
BP
G
BP
59.0
57.7
10.2
12.2
at 0.5%
58.3
11.2
51.0
51.7
22.3
21.3
at 1.0%
51.3
21.8
0.0
36.3
100.0
44.7
at 2.0%
18.2
72.3
0.0
22.7
100.0
65.5
at 3.0%
11.3
82.7
0.0
22.0
100.0
66.5
at 4.0%
11.0
83.3
65.7
65.7
0.0
0.0
Control
65.7
0.0
Mean
29.3
42.7
55.4
35.0
L.S.D. at 5% for:
Inducers
0.26
Concentrations
0.17
Interaction
1.57
* Reduction (%) = (control – treatment) / control X 100
Table 7b - Effect of salicylic acid (SA) and riboflavin (R) at different concentrations
on the in vitro radius growth (mm.) of Fusarium oxysporum f.sp. lycopersici isolate
A
Radius growth
* Reduction %
Tested
Mea
(mm.)
Mean
concentrations
n
SA
R
SA
R
65.7
70.3
0.0
-7.1
0.1mM
68.0
-3.6
65.3
69.0
0.5
-5.1
0.5mM
67.2
-2.3
55.7
59.0
15.2
10.2
1.5mM
57.3
12.7
37.7
0.0
42.6
100.0
5.0mM
18.8
71.3
0.0
0.0
100.0
100.0
10.0mM
0.0
100.0
65.7
65.7
0.0
0.0
Control
65.7
0.0
Mean
48.3
44.0
26.4
33.0
L.S.D. at 5% for:
Inducers
0.15
Concentrations
0.10
Interaction
0.92
* Reduction (%) = (control – treatment) / control X 100
55
Fig. 19b - Effect of salicylic acid (SA)
and riboflavin (R) at different
concentrations on the in vitro radius
growth reduction % of Fusarium
oxysporum f.sp. lycopersici
isolate A
Fig. 19a - Effect of garlic (G) and black
pepper (BP) extracts at different
concentrations on the in vitro radius
growth reduction % of Fusarium
oxysporum f.sp. lycopersici isolate A
4.4.2. The In vitro inhibitory effect of the tested resistance inducers at
different concentration on FOL sporulation
4.4.2.1. Garlic and black pepper extracts
The data in Table (8a) proved that, the spore count (106 spores/ml) produced by
the tomato wilt fungus, Fusarium oxysporum f. sp. lycopercici (FOL), was responded
against tested inducers [garlic (G), black pepper (BP) extracts] in similar way as
described for it radius growth. The G extract inhibited the FOL sporulation more than
BP, decreasing it by 59.8 and 39.9% comparing to the untreated control. The FOL
sporulation was progressively and significantly decreased as inducer’s concentration
increased. It was decreased by 29.5, 39.3, 72.7, 74.0 and 83.8% at 0.5, 1.0, 2.0, 3.0
and 4.0% concentrations, respectively comparing to the untreated control (Fig., 20a).
As for interactions, spore production by the FOL was completely inhibited (100.0%
inhibition) by using G at concentration ≥ 2.0% whereas, black pepper at
concentration 4.0% decreased it by 66.5% only comparing to the untreated control.
4.4.2.2. Salicylic acid and riboflavin
The data in Table (8b) proved that, the spore count (106 spores/ml) produced by
FOL was affected significantly by tested inducers [salicylic acid (SA) and riboflavin
(R)] in similar way as described for it radius growth. Regardless concentrations, R
was more effective for reducing the FOL sporulation than SA. R and SA reduced
sporulation by 58.5 and 59.0%, respectively comparing to the untreated control. The
reduction in FOL sporulation was progressively and significantly increased as
inducer’s concentration increased. It was decreased by 44.5, 47.9, 69.2, 90.8 and
100.0% at concentrations of 0.4, 0.5, 1.5, 5.0 and 10.0mM, respectively comparing to
the untreated control (Fig., 20b). As for interactions, spore production by the FOL
was completely inhibited (100.0% inhibition) by using riboflavin at concentration ≥
5.0mM and salicylic acid at concentration of 10.0mM.
56
Table 8a - Effect of garlic (G) and black pepper (BP) extracts at different
concentrations on the in vitro sporulation (106 spores/ml) of Fusarium oxysporum
f.sp. lycopersici isolate A
Sporulation (106
* Reduction % Mea
Tested
spores/ml.)
Mean
concentrations
n
G
BP
G
BP
1.111
0.934
23.4
35.6
0.5%
1.022
29.5
0.933
0.827
35.7
43.0
1.0%
0.880
39.3
0.000
0.791
45.5
2.0%
0.396 100.0
72.7
0.000
0.756
47.9
3.0%
0.378 100.0
74.0
0.000
0.471
67.5
4.0%
0.236 100.0
83.8
1.451
1.451
0.0
0.0
Control
1.451
0.0
Mean
0.583
0.871
59.8
39.9
L.S.D. at 5% for:
Inducers
0.04
Concentrations
0.11
Interaction
0.22
* Reduction (%) = (control – treatment) / control X 100
Table 8b - Effect of salicylic acid (SA) and riboflavin (R) at different concentrations
on the in vitro sporulation (106 spores/ml) of Fusarium oxysporum f.sp. lycopersici
isolate A
Sporulation (106
* Reduction
Tested
Mea
Mea
spores/ml)
%
concentrations
n
n
SA
R
SA
R
0.800
0.810
44.2
0.5%
0.805 44.9
44.5
0.782
0.729
49.8
1.0%
0.756 46.1
47.9
0.316
0.578
60.2
2.0%
0.447 78.2
69.2
0.267
0.000
3.0%
0.133 81.6 100.0
90.8
0.000
0.000
4.0%
0.000 100.0 100.0 100.0
1.451
1.451
0.0
Control
1.451 0.0
0.0
Mean
0.603
0.595
58.5
59.0
L.S.D. at 5% for:
Inducers
0.04
Concentrations
0.11
Interaction
0.23
* Reduction (%) = (control – treatment) / control X 100
57
Fig. 20b - Effect of salicylic acid (SA)
Fig. 20a - Effect of garlic (G) and black
and riboflavin (R) at different
pepper (BP) extracts at different
concentrations on the in vitro sporulation
concentrations on the in vitro sporulation
reduction % of Fusarium oxysporum f.sp.
reduction % of Fusarium oxysporum f.sp.
lycopersici
lycopersici isolate A
isolate A
4.5. In Vivo studies on some natural and chemical resistance inducers
In this study, three application methods namely immersing roots (IR), spraying
shoots (SS) and combined method (IR+SS) were used for treating tomato seedlings
(4-weeks old) of the tomato cultivar Carolina gold with different inducer treatments
i.e. garlic (G) at 0.5 and 4.0%, black pepper (BP) extracts at 0.5 and 4.0, salicylic
acid (SA) at 0.1 and 10.0mM and riboflavin (R) at 0.1 and 10mM. The treated and
untreated seedlings were transplanted in 30 cm diameter plastic pots (at rate of 3
seedlings per pot) under glasshouse conditions. Three pots were used for each
particular treatment. Each seedling was inoculated, one week after planting, with 20
ml of spore suspension (106spores/ml) of the tomato wilt pathogen (F. oxysporum
f.sp. lycopersici). After two months, the following parameters were measured.
4.5.1. Percentage of wilted plants
4.5.1.1. Garlic and black pepper extracts
The data in Table (9a) stated that the percentage of wilted tomato plants was
significantly affected only by tested treatments but not by methods or
method/treatment interactions. The SS method recorded the lowest % wilted plants
(33.3%) followed by IR (40.0%) and IR+SS (43.3%) without significant difference
between them. As for treatments, BP at 4.0% and G at 4.0% were the most effective,
decreasing % wilted plants by 86.7 and 80.0% whereas, G at 0.5% was the least
effective, decreased % wilted plants by 46.7% comparing to the untreated control. The
investigated interactions proved that using IR/G and SS/G at 4.0% in addition to SS/BP
at 4.0% were the most effective which completely suppressed disease infection
(100.0% reduction) followed by IR/BP at 4.0%, IR+SS/BP at 4.0% and IR+SS/BP at
0.5% (80.0% reduction) while, IR/BP at 0.5% was the least effective as it decreased
wilt infection only by 20.0% comparing to the untreated control (Fig., 21a).
4.5.1.2. Salicylic acid and riboflavin
58
The data in Table (9b) showed that the percentage of wilted tomato plants was
significantly affected only by tested treatments but not by methods or
method/treatment interactions. However, the IR+SS method recorded the lowest %
wilted plants (36.7%) followed by SS (40.0%) and IR (43.3%) without significant
difference between them. As for treatments, SA at 10.0mM and R at 10.0mM were
the most effective, reduced % wilted plants by 73.3% followed by SA at 0.1mM
(60.0%) and R at 0.1mM (53.3%) compared with the control treatment. Regarding
interactions, SS and IR+SS with SA or R at 10.0mM were the most effective
interactions as they decreased % wilted plants by 80.0% whereas, SS/SA at 0.1mM
and IR/R at 0.1mM were the least effective as they decreased it by 40.0% comparing
to the untreated control (Fig., 21b).
Table 9a - Effect of garlic (G) and black pepper (BP) extracts at 0.5 % 4.0% using
different application methods on wilted plants % of tomato Carolina Gold cv. under
stress of infection with FOL isolate A (two-months after inoculation)
Wilted plants %
** Reduction %
Treatments
Mean
Mean
* IR
SS IR+SS
IR
SS IR+SS
33.3 50.0
50.0
40.0
G extract at 0.5%
44.4 60.0 40.0
46.7
0.0
0.0
50.0
G extract at 4.0%
16.7 100.0 100.0 40.0
80.0
16.7
80.0
BP extract at 0.5% 66.7 33.3
38.9 20.0 60.0
53.3
16.7
0.0
16.7
BP extract at 4%
11.1 80.0 100.0 80.0
86.7
83.3
0.0
0.0
0.0
Control (untreated) 83.3 83.3
83.3
0.0
Mean
40.0 33.3
43.3
52.0 60.0
48.0
L.S.D. at 5% for:
Methods
NS
Treatments
11.69
Interaction
NS
* IR = immersing roots, SS = spraying shoots.
** Reduction (%) = (control – treatment) / control X 100
Table 9b - Effect of salicylic acid (SA) and riboflavin (R) at 0.1 and 10.0mM using
different application methods on wilted plants % of tomato Carolina Gold cv. under
stress of infection with FOL isolate A (two-months after inoculation)
Wilted plants %
** Reduction %
Mea
Mea
Treatments
* IR
SS
IR+SS
n
IR
SS IR+SS
n
16.7 50.0
33.3
SA at 0.1mM
33.3 80.0 40.0 60.0
60.0
33.3 16.7
16.7
SA at 10.0mM
22.2 60.0 80.0 80.0
73.3
50.0 33.3
33.3
R at 0.1mM
38.9 40.0 60.0 60.0
53.3
33.3 16.7
16.7
R at 10.0mM
22.2 60.0 80.0 80.0
73.3
83.3 83.3
83.3
0.0
Control (untreated)
83.3 0.0 0.0
0.0
Mean
43.3 40.0
36.7
48.0 52.0 56.0
L.S.D. at 5% for:
Methods
NS
Treatments
14.93
59
Interaction
NS
Fig. 21a - Effect of garlic (G) and black
pepper (BP) extracts at 0.5 % 4.0% using
different application methods on wilted
plants reduction % of tomato Carolina
Gold cv. under stress of infection with
FOL isolate A (two-months after
inoculation).
Fig. 21b - Effect of salicylic acid (SA)
and riboflavin (R) at 0.1 and 10.0mM
using different application methods on
wilted plants reduction % of tomato
Carolina Gold cv. under stress of
infection with FOL
isolate A (two-months after inoculation).
4.5.2. Wilt disease severity
4.5.2.1. Garlic and black pepper extracts
The data in Table (10a) showed that the tested application methods were not
significantly varied concerning % wilt disease severity (DS). The recorded DS for IR,
SS and IR+SS methods were 11.1, 8.3 and 8.6%, respectively. As for treatments, the
BP extract at 4.0% was the most effective treatments as it decreased the DS by 94.1%
followed by G at 4.0% (84.3%), G at 0.5% (62.7%), and BB at 0.5% (60.8%),
respectively comparing to the untreated control (0.0% reduction). Concerning
interactions, IR/G at 4.0%, SS/G at 4.0% and SS/BP at 4.0% completely suppressed
disease development (100.0% reduction in DS) followed by IR/BP at 4.0% and
IR+SS/BP at 0.5% (94.1% reduction), SS/BP at 0.5% (70.6% reduction) whereas
IR/BP at 0.5% was the least effective in this respect, as it decreased the DS by 17.6%
comparing with the control treatment (Fig., 22a).
4.5.2.2. Salicylic acid and riboflavin
As described above, the wilt disease severity (DS) was not significantly affected
by application method (Table (10b)). However, the DS recorded by IR, SS and
IR+SS application methods was 7.8, 11.7 and 8.3%, respectively. As for treatments,
SA at 10.0mM was the most effective as it decreased the DS by 86.3% followed by R
at 10.0mM (74.5%), SA at 0.1mM (72.5%) and R at 0.1mM (70.6%), respectively
comparing to the untreated control (0.0% reduction). Concerning interactions, the
highest significant reduction in DS was recorded by IR/SA at 0.1mM (94.1%)
followed by IR/SA at 10.0mM, SS/SA at 10.0mM and IR+SS/R at 10.0mM (88.2%
reduction), IR/R at 10.0mM, IR+SS/R at 0.1mM and IR+SS/SA at 10.0mM (82.4%
60
reduction), IR/R at 0.1mM and IR+SS/SA at 0.1mM (70.6% reduction), respectively
comparing with the control treatment (Fig., 22b).
Table 10a - Effect of garlic (G) and black pepper (BP) extracts at 0.5 % 4.0% using
different application methods on wilt disease severity % of tomato Carolina Gold cv.
under stress of infection with FOL isolate A (two-months after inoculation)
Disease severity %
** Reduction %
Treatments
Mean
Mean
* IR
SS IR+SS
IR
SS IR+SS
11.1 11.1
4.2
52.9 52.9
82.3
G extract at 0.5%
8.8
62.7
0.0
0.0
11.1
G extract at 4.0%
3.7 100.0 100.0 52.9
84.3
6.9
1.4
17.6 70.6
94.1
BP extract at 0.5% 19.4
9.3
60.8
1.4
0.0
2.8
94.1 100.0 88.2
BP extract at 4%
1.4
94.1
23.6
0.0
0.0
0.0
Control (untreated) 23.6 23.6
23.6
0.0
Mean
11.1
8.3
8.6
52.9 64.7
63.5
L.S.D. at 5% for:
Methods
NS
Treatments
2.96
Interaction
8.89
* IR = immersing roots, SS = spraying shoots
** Reduction (%) = (control – treatment) / control X 100
Table 10b - Effect of salicylic acid (SA) and riboflavin (R) at 0.1 and 10.0mM using
different application methods on wilt disease severity % of tomato Carolina Gold cv.
under stress of infection with FOL isolate A (two-months after inoculation)
Disease severity %
** Reduction %
Treatments
Mean
Mean
* IR
SS IR+SS
IR
SS IR+SS
1.4
11.1
6.9
SA at 0.1mM
6.5 94.1 52.9 70.6
72.5
2.8
2.8
4.2
SA at 10.0mM
3.2 88.2 88.2 82.4
86.3
6.9
9.7
4.2
R at 0.1mM
6.9 70.7 58.8 82.4
70.6
4.2
11.1
2.8
R at 10.0mM
6.0 82.4 52.9 88.2
74.5
23.6 23.6
23.6
0.0
Control (untreated)
23.6 0.0 0.0
0.0
Mean
7.8
11.7
8.3
67.1 50.6 64.7
L.S.D. at 5% for:
Methods
NS
Treatments
4.12
Interaction
12.36
* IR = immersing roots, SS = spraying shoots
** Reduction (% ) = (control – treatment) / control X 100
61
Fig. 22a - Effect of garlic (G) and black
pepper (BP) extracts at 0.5 % 4.0% using
different application methods on wilt
disease severity reduction % of tomato
Carolina Gold cv. under stress of
infection with FOL isolate A (twomonths after inoculation)
Fig. 22b - Effect of salicylic acid (SA)
and riboflavin (R) at 0.1 and 10.0mM
using different application methods on
wilt disease severity reduction % of
tomato Carolina Gold cv. under stress of
infection with FOL isolate A (twomonths after inoculation)
4.5.3.
Growth characters
4.5.3.1. Plant height
4.5.3.1.1. Garlic and black pepper extracts
The data in Tables (11a) stated that, the plant height was significantly affected
by tested application methods, inducer treatments as well as by method/treatment
interactions. As for application methods, the IR+SS recorded the tallest plant height
(153.7cm) followed by IR (138.8 cm) and SS (135.1 cm), respectively. All treatments
significantly increased plant height comparing to the untreated control. In this
respect, G at 0.5% was the best treatment for recording the highest average of plant
height (163.9 cm) followed by BP at 4.0% (150.5 cm), G at 4.0% (137.6 cm) and BP
at 0.5% (136.6 cm) without significant difference between the latter two treatments.
These four treatments increased plant height by 32.0, 21.2, 10.8 and 10.0%,
respectively comparing to the untreated control. Regarding interactions, IR+SS/BP at
4.0 was best of all and increasing plant height by 40.8% (174.8 cm) followed by IR/G
at 0.5% which increased plant height by 38.7% (172.2 cm) while, the lowest
significant increase was recorded by IR/BP at 0.5% which increased the plant height
by 7.4% (133.3 cm). However, IR/BP at 0.5%, SS/G at 4.0% and SS/BP at 0.5%
showed no significant effects on the plant height when compared with the untreated
control (Fig., 23a).
4.5.3.1.2. Salicylic acid and riboflavin
The data in Table (11b) stated that, the plant height was significantly affected
by application methods, inducer treatments as well as by method/treatment
interactions. As for application methods, the IR recorded the tallest plant height
(150.2 cm) followed by SS (141.6 cm) and IR+SS (136.2 cm), respectively. All
treatments significantly increased plant height comparing to the untreated control. In
62
this respect, SA at 0.1mM was the best treatment for recording the highest plant
height (162.2 cm) followed by R at 10.0mM (149.1cm) and SA at 10.0mM (148.6
cm) without significant difference between the latter two treatments. Thus, these
three treatments increased plant height by 30.6, 20.0 and 19.69%, respectively
comparing to the untreated control. The lowest significant increase (4.1%) was
recorded by using R at 0.1mM (129.3 cm) comparing to the untreated control (124.2
cm). Among tested interactions, IR/SA at 0.1mM was best of all, increasing plant
height by 53.4% (190.5cm) comparing to the control. However, IR/R at 0.1mM, SS/R
at 10.0mM and IR+SS/SA at 0.1mM showed no significant effects on the plant height
when compared with the untreated control (Fig., 23b).
Table 11a - Effect of garlic (G) and black pepper (BP) extracts at 0.5 % 4.0% using
different application methods on plant height (cm.) of tomato Carolina Gold cv.
under stress of infection with FOL isolate A (two-months after inoculation)
Plant height (cm.)/plant
** Increase (%)
Treatments
Mean
Mean
* IR
SS
IR+SS
*IR SS IR+SS
172.2 154.2 165.3 163.9 38.7 24.2 33.2
G extract at 0.5%
32.0
137.5 125.0 150.3 137.6 10.7 0.7 21.1
G extract at 4.0%
10.8
BP extract at 0.5% 127.0 128.8 154.0 136.6 2.3 3.8 24.0
10.0
133.3 143.3 174.8 150.5 7.4 15.4 40.8
BP extract at 4%
21.2
0.0
Control (untreated) 124.2 124.2 124.2 124.2 0.0 0.0
0.0
Mean
138.8 135.1 153.7
11.8 8.8 23.8
L.S.D. at 5% for:
Methods
1.31
Treatments
2.18
Interaction
6.54
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
Table 11b - Effect of salicylic acid (SA) and riboflavin (R) at 0.1 and 10.0mM using
different application methods on plant height (cm.) of tomato Carolina Gold cv.
under stress of infection with FOL isolate A (two-months after inoculation)
Plant height
** Increase %
(cm.)/plant
Treatments
Mean
Mean
IR+S
* IR
SS
IR+SS
IR SS
S
190.5 171.5 124.7 162.2 53.4 38.1 0.4
SA at 0.1mM
30.6
148.2 153.5 144.2 148.6 19.3 23.6 16.1
SA at 10.0mM
19.7
127.8 133.5 126.5 129.3 3.0 7.5
1.9
R at 0.1mM
4.1
160.3 125.2 161.7 149.1 29.1 0.8 30.2
R at 10.0mM
20.0
0.0
Control (untreated) 124.2 124.2 124.2 124.2 0.0 0.0
0.0
Mean
150.2 141.6 136.2
21.0 14.0 9.7
L.S.D. at 5% for:
63
Methods
0.74
Treatments
1.23
Interaction
3.69
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
Fig. 23a - Effect of garlic (G) and black
pepper (BP) extracts at 0.5 % 4.0% using
different application methods on plant
height increase % of tomato Carolina
Gold cv. under stress of infection with
FOL isolate A (two-months after
inoculation)
Fig. 23b - Effect of salicylic acid (SA)
and riboflavin (R) at 0.1 and 10.0mM
using different application methods on
plant height increase % of tomato
Carolina Gold cv. under stress of
infection with FOL isolate A (twomonths after inoculation)
4.5.3.2. Number of leaves per plant
4.5.3.2.1. Garlic and black pepper extracts
The data in Table (12a) stated that, the number of leaves per plant (NL) was
significantly affected by application methods, inducer treatments as well as by
method/treatment interactions. As for application methods, the IR+SS as well as SS
methods were significantly equal recording the highest significant NL (13.7-13.8)
followed by the IR method (12.4). All treatments significantly increased the NL
comparing to the untreated control. In this respect, G at 0.5% recorded the highest NL
(15.4) followed by BP at 4.0% (15.2), G at 4.0% (14.1) and BP at 0.5% (12.8)
comparing to the untreated control (9.0). These inducer treatments, however,
increased NL by 71.6, 68.5, 56.8 and 42.6%, respectively comparing to the control.
Regarding method/treatment interactions, IR+SS/BP at 4.0% was the best for
increasing NL (77.8% increase) following by IR/G at 0.5% and SS/G at 0.5% (74.1%
increase) comparing to the control. However, the NL produced by IR/G at 0.5%
interaction (9.5) was not significantly varied when compared with the untreated
control (Fig., 24a).
4.5.3.2.2. Salicylic acid and riboflavin
The data in Table (12b) stated that, the number of leaves per plant (NL) was
significantly affected by inducer treatments and method/treatment interactions. The
tested application methods were not significantly varied in this respect. All inducer
treatments significantly increased the NL comparing to the untreated control. The
highest NL was recorded by R at 10.0mM (15.3) while, R at 0.1mM recorded the
64
lowest NL (15.2), both treatments increased NL by 70.4 and 58.0%, respectively
comparing to the untreated control. Among tested method/treatment interactions,
IR+SS/R at 10.0mM recorded the highest increase in the NL (90.7%) followed by
SS/SA at 0.1mM (74.1%) while, IR/R at 0.1mM recorded the lowest significant
increase (42.6%) comparing to the control (Fig., 24b).
Table 12a - Effect of garlic (G) and black pepper (BP) extracts at 0.5 % 4.0% using
different application methods on number of leaves/plant of tomato Carolina Gold cv.
under stress of infection with FOL isolate A (two-months after inoculation)
Number of leaves/plant
** Increase %
Treatments
Mean
Mean
* IR
SS
IR+SS
IR SS IR+SS
15.7
15.7
15.0
G extract at 0.5%
15.4 74.1 74.1 66.7
71.6
13.7
14.3
14.3
G extract at 4.0%
14.1 51.9 59.3 59.3
56.8
9.5
14.7
14.3
BP extract at 0.5%
12.8 5.6 63.0 59.3
42.6
14.2
15.3
16.0
BP extract at 4%
15.2 57.4 70.4 77.8
68.5
9.0
9.0
9.0
0.0 0.0
0.0
Control (untreated)
9.0
0.0
Mean
12.4
13.8
13.7
37.8 53.3 52.6
L.S.D. at 5% for:
Methods
0.17
Treatments
0.29
Interaction
0.87
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
Table 12b - Effect of salicylic acid (SA) and riboflavin (R) at 0.1 and 10.0mM using
different application methods on number of leaves/plant of tomato Carolina Gold cv.
under stress of infection with FOL isolate A (two-months after inoculation)
Number of leaves/plant
** Increase %
Treatments
Mean
Mean
* IR
SS
IR+SS
IR SS IR+SS
15.2
15.7
13.2
SA at 0.1mM
14.7 68.5 74.1 46.3
63.0
15.0
15.2
14.3
SA at 10.0mM
14.8 66.7 68.5 59.3
64.8
12.8
14.8
15.0
R at 0.1mM
14.2 42.6 64.8 66.7
58.0
15.2
13.7
17.2
R at 10.0mM
15.3 68.5 51.9 90.7
70.4
9.0
9.0
9.0
0.0 0.0
0.0
Control (untreated)
9.0
0.0
Mean
13.4
13.7
13.7
49.3 51.9 52.6
L.S.D. at 5% for:
Methods
NS
Treatments
0.32
Interaction
0.95
65
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
Fig. 24a - Effect of garlic (G) and black
pepper (BP) extracts at 0.5 % 4.0% using
different application methods on number
of leaves increase % of tomato Carolina
Gold cv. under stress of infection with
FOL isolate A (two-months after
inoculation)
Fig. 24b - Effect of salicylic acid (SA)
and riboflavin (R) at 0.1 and 10.0mM
using different application methods on
number of leaves increase % of tomato
Carolina Gold cv. under stress of
infection with FOL isolate A (twomonths after inoculation)
4.5.3.3. Fresh weight of leaves (g) per plant
4.5.3.3.1. Garlic and black pepper extracts
The data in Table (13a) indicated that, the fresh weight of leaves (g) per plant
(FWL) was significantly affected by tested application methods, inducer treatments
as well as by the interaction between application methods and inducer treatments.The
IR method recorded the highest FWL (91.0 g) followed by IR+SS (87.8 g) and SS
method (83.1 g), respectively. All tested inducer treatments showed significant
positive effect on the FWL comparing to the untreated control. In this respect, BP at
4.0% and G at 0.5% were the best treatments which increased FWL by 49.2 and
47.9%, respectively whereas, G at 4.0% recorded the lowest significant increase in
FWL (33.1%) comparing to the control which recorded (65.4g/plant). In general, the
highest increase in the FWL was recorded by IR+SS/BP at 0.5% (87.1%) and IR/BP
at 4.0% (85.1%) followed by SS/G at 0.5% (65.6%) and IR/G at 4.0% (59.6%)
whereas, the lowest significant increase was recorded by SS/BP at 0.5% (14.4%)
comparing to the untreated control. The increases in FWL caused by IR+SS/G at
4.0% (12.0%) and IR/BP at 0.5% (8.5%) were not significantly varied when
compared to the untreated control (Fig., 25a).
4.5.3.3.2. Salicylic acid and riboflavin
The data in Table (13b) indicated that, the SS method produces higher FWL
(93.5 g) than IR method (88.3 g) or IR+SS method (88.2 g). However, all tested
inducer treatments showed significant positive effect on the FWL comparing to the
66
untreated control. In this respect, SA at 0.1mM was the best treatment which
increased FWL by 65.3% followed by R at 10.0mM (51.6%), SA at 10.0mM (39.6%)
and R at 0.1mM (30.8%), respectively comparing to the untreated control. Among
tested method/treatment interactions, IR/R at 0.1mM, IR/SA 0.1mM, SS/SA at
0.1mM and SS/R at 0.1mM were the best of all which increased the FWL by 84.1,
83.0, 77.6 and 75.6% comparing to the control. The lowest significant increase in the
FWL was induced by IR+SS/SA at 0.1mM (35.3%) whereas, many interactions i.e.
IR/SA at 10.0mM, IR/R at 0.1mM, SS/R at 10.0mM and IE+SS/R at 0.1mM showed
no significant effect in this respect comparing to the untreated control (Fig., 25b).
Table 13a - Effect of garlic (G) and black pepper (BP) extracts at 0.5 % 4.0% using
different application methods on the fresh weight of leaves (g.)/plant of tomato
Carolina Gold cv. under stress of infection with FOL isolate A (two-months after
inoculation)
Fresh weight of leaves
** Increase %
(g.)
Mea
Mea
Treatments
n
IR+S
n
* IR
SS
IR+SS
IR SS
S
41. 65.
92.8
108.4
89.2
36.3
G extract at 0.5%
96.8
47.9
8
6
59. 27.
104.5
83.6
73.3
12.0
G extract at 4.0%
87.1
33.1
6
7
14.
74.9
122.4
87.1
BP extract at 0.5% 71.0
89.4 8.5
36.7
4
85. 27.
121.1
83.1
88.8
35.7
BP extract at 4%
97.7
49.2
1
0
Control
65.4
65.4
65.4
0.0
65.4 0.0 0.0
0.0
(untreated)
39. 26.
Mean
91.0
83.1
87.8
34.2
0
9
L.S.D. at 5% for:
Methods
1.73
Treatments
2.89
Interaction
8.66
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
Table 13b - Effect of salicylic acid (SA) and riboflavin (R) at 0.1 and 10.0mM using
different application methods on the fresh weight of leaves (g.)/plant of tomato
Carolina Gold cv. under stress of infection with FOL isolate A (two-months after
inoculation)
Fresh weight of leaves
Mea
Mea
Treatments
** Increase %
(g.)
n
n
67
* IR
SS
IR+SS
IR
SS
SA at 0.1mM
119.8
116.3
88.6
108.2
83.
0
106.7
91.4
1.3
114.9
72.6
85.6
5.9
77.
6
54.
5
75.
6
SA at 10.0mM
66.3
101.1
R at 0.1mM
69.3
R at 10.0mM
120.5
69.5
107.7
99.2
84.
1
Control
(untreated)
65.4
65.4
65.4
65.4
Mean
88.3
93.5
88.2
IR+S
S
35.3
65.3
63.1
39.6
10.9
30.8
6.2
64.6
51.6
0.0
0.0
0.0
0.0
34.
8
42.
8
34.8
L.S.D. at 5% for:
Methods
1.68
Treatments
2.80
Interaction
8.39
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
Fig. 25b - Effect of salicylic acid (SA)
and riboflavin (R) at 0.1 and 10.0mM
using different application methods on
the fresh weight of leaves increase % of
tomato Carolina Gold cv. under stress of
infection with FOL isolate A
(two-months after inoculation)
Fig. 25a - Effect of garlic (G) and black
pepper (BP) extracts at 0.5 % 4.0% using
different application methods on the fresh
weight of leaves increase % of tomato
Carolina Gold cv. under stress of
infection with FOL isolate A
(two-months after inoculation)
4.5.3.4. Dry weight of leaves (g) per plant
4.5.3.4.1. Garlic and black pepper extracts
68
The data in Tables (14a) indicated that, the highest significant dry weight of
leaves per plant (DWL) was recorded by IR method (26.0 g) followed by IR+SS
method (24.8 g) and SS method (24.3 g), respectively. All tested inducer treatments
significantly increased DWL comparing to the untreated control. BP at 0.5% (27.54
g/plant) and G at 0.5% (27.44 g/plant) were the best in this respect which increased
DWL by 35.5 and 35.0%, respectively without significant differences between them.
However, the lowest significant increase was induced by G at 4.0% (19.6%)
comparing to the untreated control. Regarding method/treatment interactions, SS/BP
at 0.5% induces the highest increase in the DWL (93.5%) followed by SS/G at 0.5%
(62.0%), IR/BP at 4.0% (53.2%) while, the lowest significant increases i.e. 13.2 and
14.2% were induced by G and BP at 4.0%, respectively comparing to the untreated
control. On the other hand, the increases in the DWL/plant caused by IR/BP at 0.5%,
SS/BP at 0.5%, IR+SS/G at 0.5 and 4.0% IR+SS/BP at 4.0% were not significantly
varied comparing to the untreated control (Fig., 26a).
4.5.3.4.2. Salicylic acid and riboflavin
The data in Tables (14b) indicated that, the highest significant dry weight of
leaves per plant (DWL) was recorded by SS method (27.5 g) followed by IR method
(26.6 g) and IR+SS method (24.4 g), respectively. All tested inducer treatments
significantly increased DWL comparing to the untreated control. SA at 0.1mM
induces the highest significant increase (57.4%) followed by R at 10.0mM (42.2%),
SA at 10.0mM (24.6%) and R at 0.1mM (19.7%), respectively comparing to the
untreated control. Regarding method/treatment interactions, SS/SA at 0.1mM induces
the highest increase in the DWL (99.5%) followed by IR/R at 10.0mM (72.2%) and
IR/SA at 0.1mM (71.1%) while, the lowest significant increase was induced by
SS/SA at 10.0mM (18.2%) comparing to the untreated control. Some interactions i.e.
IR/SA at 10.0mM, IR/R at 0.1mM, SS/R at 10.0mM, IR+SS/SA at 0.1mM and
IR+SS/R at 0.1mM showed no significant effect on the DWL if compared with the
untreated control (Fig., 26b).
Table 14a - Effect of garlic (G) and black pepper (BP) extracts at 0.5 % 4.0% using
different application methods on the dry weight of leaves (g.)/plant of tomato
Carolina Gold cv. under stress of infection with FOL isolate A (two-months after
inoculation)
Dry weight of leaves
** Increase %
(g.)/plant
Treatments
Mean
Mean
*
IR+S
SS
IR+SS
IR
SS
IR
S
27.
62.
32.9
21.5
37.5
5.6
G extract at 0.5%
27.4
35.0
9
0
29.
13.
23.0
20.6
44.1
1.4
G extract at 4.0%
24.3
19.6
3
2
21.
22.0
39.3
5.0 8.1 93.5
BP extract at 0.5%
27.5
35.5
3
31. 23.2
22.2
53.2 14.
9.0
BP extract at 4%
25.5
25.5
69
Control
(untreated)
Mean
1
20.
3
26.
0
2
20.3
20.3
24.3
24.8
20.3
0.0
0.0
0.0
28.0
19.
5
21.9
0.0
L.S.D. at 5% for:
Methods
0.38
Treatments
0.64
Interaction
1.91
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
Table 14b - Effect of salicylic acid (SA) and riboflavin (R) at 0.1 and 10.0mM using
different application methods on the dry weight of leaves (g.)/plant of tomato
Carolina Gold cv. under stress of infection with FOL isolate A (two-months after
inoculation)
Dry weight of leaves
** Increase %
(g.)/plant
Treatments
Mean
Mean
IR+S
* IR
SS
IR+SS
IR
SS
S
99.
34.8 40.5
20.6
1.5
SA at 0.1mM
32.0 71.1
57.4
5
18.
22.1 24.0
29.8
8.8
46.9
SA at 10.0mM
25.3
24.6
2
49.
20.7 30.3
21.9
2.1
7.9
R at 0.1mM
24.3
19.7
3
10.
35.0 22.4
29.3
44.0
R at 10.0mM
28.9 72.2
42.2
2
Control
20.3 20.3
20.3
0.0 0.0
0.0
20.3
0.0
(untreated)
35.
Mean
26.6 27.5
24.4
30.8
20.1
4
L.S.D. at 5% for:
Methods
0.46
Treatments
0.77
Interaction
2.32
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
70
Fig. 26a - Effect of garlic (G) and black
pepper (BP) extracts at 0.5 % 4.0% using
different application methods on the dry
weight of leaves increase % of tomato
Carolina Gold cv. under stress of
infection with FOL isolate A
(two-months after inoculation)
Fig. 26b - Effect of salicylic acid (SA)
and riboflavin (R) at 0.1 and 10.0mM
using different application methods on
the dry weight of leaves increase % of
tomato Carolina Gold cv. under stress of
infection with FOL isolate A
(two-months after inoculation)
4.5.3.5. Stem fresh weight (g) per plant
4.5.3.5.1. Garlic and black pepper extracts
The data in Tables (15a) indicated that, the fresh weight of stem (g)/plant
(SFW) was significantly affected by tested application methods, inducer treatments as
well as by the interaction in between. The IR+SS method recorded the highest
significant SFW (78.8 g) followed by IR (73.7 g) and SS (71.3 g), respectively with
significant differences in between. All tested inducer treatments significantly
increased SFW comparing to the untreated control. In this respect, G at 0.5% induces
the highest increase in the SFW (26.5%) followed by BP at 0.5% (21.5%), BP at
4.0% (10.7%) and G at 0.5% (8.1%), respectively without significant differences
between the latter two treatments, comparing to the control. Concerning
method/treatment interactions, IR+SS/BP at 0.5% was the best which increased SFW
by 57.7% followed by IR+SS/G at 0.5%, IR/G at 0.5%, SS/G at 0.5%, IR/G at 4.0%,
IR/BP at 4.0%, IR+SS/BP at 4.0% and SS/BP at 0.5% which increased it by 29.3,
25.7, 24.4, 19.9, 12.0, 11.1 and 9.0%, respectively comparing to the control. The
remained interactions showed no significant differences on the SFW when compared
with the untreated control (Fig., 27a).
4.5.3.5.2. Salicylic acid and riboflavin
The data in Tables (15b) indicated that, the fresh weight of stem (g)/plant
(SFW) was significantly affected by tested application methods, inducer treatments as
well as by the interaction in between. As for application methods, SS recorded the
highest significant increase in the SFW (4.2 g) followed by IR (82.9 g) and IR+SS
(69.5 g), respectively. All tested inducer treatments significantly increased SFW
comparing to the untreated control. In this respect, SA at 0.1mM induces the highest
significant increase in the SFW (46.2%) followed by SA at 10.0mM (24.6%), R at
10.0mM (22.2%), and R at 0.1mM (6.1%), respectively. Concerning
method/treatment interactions, SS/SA at 0.1MM causes the highest increase (72.3%)
71
followed by IR/SA at 0.1mM (62.6%) and IR/R at 10.0mM (61.1%) whereas the
lowest significant increase was induced by SS/R at 0.1mM (13.1%). On the other
hand, IR/SA at 10.0mM, IR/R at 0.1mM, SS/R at 10.0mM, SS/R at 10.0mM and
IR+SS/R at 10.0mM showed no significant differences in the SFW when compared
with the untreated control (Fig., 27b).
Table 15a - Effect of garlic (G) and black pepper (BP) extracts at 0.5 % 4.0% using
different application methods on stem fresh weight (g.)/plant of tomato Carolina Gold
cv. under stress of infection with FOL isolate A (two-months after inoculation)
Fresh weight of stem
** Increase %
(g.)/plant
Treatments
Mean
Mean
IR+S
* IR SS
IR+SS
IR
SS
S
24.
82.7 81.9
85.1
25.7
29.3
G extract at 0.5%
83.2
26.5
4
78.9 67.9
66.6
19.9 3.2
1.3
G extract at 4.0%
71.1
8.1
2.3 5.0 57.1
BP extract at 0.5% 67.3 69.1 103.4
79.9
21.5
73.7 71.7
73.1
12.0 9.0 11.1
BP extract at 4%
72.8
10.7
Control
65.8 65.8
65.8
0.0 0.0
0.0
65.8
0.0
(untreated)
Mean
73.7 71.3
78.8
12.0 8.3 19.8
L.S.D. at 5% for:
Methods
1.01
Treatments
1.69
Interaction
5.07
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
Table 15b - Effect of salicylic acid (SA) and riboflavin (R) at 0.1 and 10.0mM using
different application methods on stem fresh weight (g.)/plant of tomato Carolina Gold
cv. under stress of infection with FOL isolate A (two-months after inoculation)
Fresh weight of stem
** Increase %
(g.)/plant
Treatments
Mean
Mean
IR+S
* IR
SS
IR+SS
IR SS
S
62. 72.
107.0 113.4
68.2
3.7
SA at 0.1mM
96.2
46.2
6
3
49.
68.0
98.2
79.8
21.3
SA at 10.0mM
82.0 3.4
24.6
2
13.
67.8
74.4
67.3
2.3
R at 0.1mM
69.8 3.0
6.1
1
61.
105.9 69.1
66.2
5.1
0.6
R at 10.0mM
80.4
22.2
0
65.8
65.8
65.8
0.0
Control
65.8 0.0 0.0
0.0
72
(untreated)
Mean
82.9
84.2
69.5
26.
0
28.
0
5.6
L.S.D. at 5% for:
Methods
1.04
Treatments
1.73
Interaction
5.19
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
Fig. 27a - Effect of garlic (G) and black
pepper (BP) extracts at 0.5 % 4.0% using
different application methods on stem
fresh weight increase % of tomato
Carolina Gold cv. under stress of
infection with FOL isolate A (twomonths after inoculation)
Fig. 27b - Effect of salicylic acid (SA)
and riboflavin (R) at 0.1 and 10.0mM
using different application methods on
stem fresh weight increase % of tomato
Carolina Gold cv. under stress of
infection with FOL isolate A (twomonths after inoculation)
4.5.3.6. Root fresh weight (g) per plant
4.5.3.6.1. Garlic and black pepper extracts
The data in Tables (16a) indicated that, the root fresh weight (g)/plant (RFW)
was significantly affected by tested application methods, inducer treatments as well
as by the interaction in between. Regarding application methods, IR+SS recorded the
highest RFW (19.4 g) followed by IR method (18.9 g) and SS method (18.6 g),
respectively without significant differences between the latter two methods. All tested
inducer treatments significantly increased RFW comparing to the untreated control.
In this respect, G at 0.5% induces the highest increase in RFW (26.8%) followed by
BP at 4.0% (24.5%), G at 4.0% (20.5%) and BP at 0.5% (19.2%), respectively
comparing to the untreated control. All interactions between methods and inducer
treatments showed significant increase in the RFW comparing to the untreated
control. In this regard, IR+SS/G at 0.5 and 4.0% caused the highest (50.2%) and
lowest significant increase (11.4%), respectively (Fig., 28a).
4.5.3.6.2. Salicylic acid and riboflavin
The data in Tables (16b) and Fig., (28b) indicated that, the fresh weight of roots
(g)/plant (RFW) was significantly affected by tested application methods, inducer
73
treatments as well as by the interaction in between. IR method recorded the highest
significant increase in the RFW (22.2 g) followed by SS method (17.7 g) and IR+SS
method (17.1 g), respectively. All tested inducer treatments significantly increased
RFW comparing to the untreated control. In this respect, SA at 0.1mM recorded the
highest increase (35.1%) followed by R at 10.0mM (26.7%), SA at 10.0mM (17.2%)
and R at 0.1mM (12.2%), respectively comparing to the untreated control. As for
interactions, the highest significant increase in the RFW was recorded by IR/R at
10.0mM (74.3%) followed by IR/SA at 0.1mM (69.9%) while, the lowest significant
increase (15.1%) was recorded by IR+SS/SA at 0.1mM compared with the untreated
control. On the other hand, IR/R at 10.0mM, SS/SA at 10.0mM, SS/R at 10.0mM,
IR+SS/SA at 10.0mM, IR+SS/R at 10.0mM and IR+SS/R at 10.0mM showed no
significant differences in the RFW when compared with the control treatment.
Table 16a - Effect of garlic (G) and black pepper (BP) extracts at 0.5 % 4.0% using
different application methods on root fresh weight (g.)/plant of tomato Carolina Gold
cv. under stress of infection with FOL isolate A (two-months after inoculation)
Fresh weight of roots
** Increase %
(g.)/plant
Treatments
Mean
Mean
IR+S
IR+S
* IR
SS
IR
SS
S
S
13.
18.8
18.2
24.1
50.2
G extract at 0.5%
20.4 17.0
26.8
1
22.
20.5
19.7
17.9
11.4
G extract at 4.0%
19.4 27.6
20.6
8
14.
18.4
20.5
27.4
BP extract at 0.5% 18.5
19.1 15.4
19.2
7
29.
20.6
20.8
18.6
16.0
BP extract at 4%
20.0 28.2
24.5
3
Control
16.1
16.1
16.1
0.0 0.0
0.0
16.1
0.0
(untreated)
16.
Mean
18.9
18.6
19.4
17.6
21.0
0
L.S.D. at 5% for:
Methods
0.31
Treatments
0.51
Interaction
1.53
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
Table 16b - Effect of salicylic acid (SA) and riboflavin (R) at 0.1 and 10.0mM using
different application methods on root fresh weight (g.)/plant of tomato Carolina Gold
cv. under stress of infection with FOL isolate A (two-months after inoculation)
74
Treatments
Fresh weight of roots
(g.)/plant
Mean
IR+S
* IR
SS
S
27.3
19.3
18.5
21.7
21.4
17.2
17.8
18.8
18.1
19.1
16.9
18.0
28.0
16.9
16.2
20.4
** Increase %
IR
69.9
33.4
12.9
74.3
SA at 0.1mM
SA at 10.0mM
R at 0.1mM
R at 10.0mM
Control
16.1
16.1
16.1
0.0
(untreated)
16.1
Mean
22.2
17.7
17.1
38.1
L.S.D. at 5% for:
Methods
0.47
Treatments
0.79
Interaction
2.36
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
Fig. 28a - Effect of garlic (G) and black
pepper (BP) extracts at 0.5 % 4.0% using
different application methods on root
fresh weight increase % of tomato
Carolina Gold cv. under stress of
infection with FOL isolate A (twomonths after inoculation)
SS
20.3
7.3
18.7
5.2
IR+S
S
15.1
11.0
5.0
0.6
0.0
10.3
0.0
6.3
Mean
35.1
17.2
12.2
26.7
0.0
Fig. 28b - Effect of salicylic acid (SA)
and riboflavin (R) at 0.1 and 10.0mM
using different application methods on
root fresh weight increase % of tomato
Carolina Gold cv. under stress of
infection with FOL isolate A (twomonths after inoculation)
4.5.3.7. Root dry weight (g) per plant
4.5.3.7.1. Garlic and black pepper extracts
The data in Table (17a) indicated that, the dry weight of roots (g)/plant (RDW)
was not significantly affected by tested application methods. However, all tested
inducer treatments significantly increased RDW (6.6-7.9 g/plant) comparing to the
untreated control (5.0 g/plant. In this respect, BP at 4.0% induces the highest increase
(41.1%) followed by BP at 0.5% (34.2%), G at 4.0% (27.8%) and G at 0.5% (27.5%)
without significant differences between the latter two treatments. Regarding
method/treatment interactions, the IR+SS/BP at 0.5% produces the highest increase
75
(88.9%) followed by IR/BP at 4.0% (86.4%) and IR+SS/G at 0.5% (55.0%) whereas,
the lowest significant increase SS/G at 0.5% (23.8%). On the contrary, the RDW
produced by IR/G at 0.5%, IR/BP at 0.5%, IR/BP at 0.5%, SS/BP at 0.5% and
IR+SS/BP at 4.0% were not significantly varied when compared with the untreated
control (Fig., 29a).
4.5.3.7.2.
Salicylic acid and riboflavin
The data in Table (17b) indicated that, the dry weight of roots (g)/plant (RDW)
was significantly affected by tested application methods, inducer treatments as well
as by the interaction in between. The IR method recorded the highest significant
increase in the RDW (7.7 g) followed by IR+SS method (6.4 g) and SS method (6.0
g), respectively with clear significant differences in between. However, all tested
inducer treatments significantly increased comparing to the untreated control. In this
respect, SA at 0.1mM induces the highest significant increase (59.9%) followed by R
at 0.1mM (40.8%), R at 10.0mM (39.5%) and SA at 10.0mM (37.0%) respectively.
Regarding interactions, IR/SA at 10.0mM recorded the highest significant increase
(95.3%) followed by IR/R at 10.0mM (92.9%). Whereas, SS/SA at 10.0mM, SS/R at
0.1mM and IR+SS/R at 10.0mM showed no significant effect on the RDW compared
with the untreated control (Fig., 29b).
Table 17a - Effect of garlic (G) and black pepper (BP) extracts at 0.5 % 4.0% using
different application methods on root dry weight (g.)/plant of tomato Carolina Gold
cv. under stress of infection with FOL isolate A (two-months after inoculation)
Dry weight of roots
** Increase %
(g.)/plant
Treatments
Mean
Mean
*
IR+S
SS
IR+SS
IR
SS
IR
S
5.1 6.1
7.7
3.8 23.8 55.0
G extract at 0.5%
6.31
27.5
7.4 6.5
5.0
50.4 31.1
1.9
G extract at 4.0%
6.33
27.8
9.4
6.2
7.4
88.9
BP extract at 0.5% 5.3 5.3
6.64
34.2
9.2 6.5
5.3
86.4 30.7
6.3
BP extract at 4%
6.99
41.1
Control
5.0 5.0
5.0
0.0
0.0
0.0
4.95
0.0
(untreated)
Mean
6.4 5.9
6.5
29.4 18.6 30.4
L.S.D. at 5% for:
Methods
NS
Treatments
0.38
Interaction
1.13
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
76
Table 17b - Effect of salicylic acid (SA) and riboflavin (R) at 0.1 and 10.0mM using
different application methods on root dry weight (g.)/plant of tomato Carolina Gold
cv. under stress of infection with FOL isolate A (two-months after inoculation)
Dry weight of roots
** Increase %
(g.)/plant
Treatments
Mean
Mean
IR+S
* IR SS
IR+SS
IR
SS
S
9.7
6.6
7.5
95.3 33.1 51.3
SA at 0.1mM
7.9
59.9
7.7
5.9
6.7
56.2 19.7 35.3
SA at 10.0mM
6.8
37.0
6.5
6.9
7.5
30.6 40.3 51.4
R at 0.1mM
7.0
40.8
9.5
5.8
5.4
92.9 16.6
9.2
R at 10.0mM
6.9
39.5
Control
5.0
5.0
5.0
0.0
0.0
0.0
5.0
0.0
(untreated)
Mean
7.7
6.0
6.4
55.0 21.9 29.4
L.S.D. at 5% for:
Methods
0.22
Treatments
0.36
Interaction
1.08
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
Fig. 29a - Effect of garlic (G) and black
pepper (BP) extracts at 0.5 % 4.0% using
different application methods on root dry
weight increase % of tomato Carolina
Gold cv. under stress of infection with
FOL isolate A (two-months after
inoculation)
Fig. 29b - Effect of salicylic acid (SA)
and riboflavin (R) at 0.1 and 10.0mM
using different application methods on
root dry weight increase % of tomato
Carolina Gold cv. under stress of
infection with FOL isolate A (twomonths after inoculation)
4.5.3.8. Root length (cm) per plant
4.5.3.8.1. Garlic and black pepper extracts
The data in Tables (18a) indicated that, the root length (cm)/plant (RL) was
significantly affected by tested application methods, inducer treatments as well as by
77
the interaction in between. The IR method recorded the highest significant increase in
the RL (21.7 cm) followed by SS method (19.9 cm) and IR+SS method (18 cm),
respectively. All tested inducer treatments induced significant increases in the RL
comparing to the untreated control. The highest significant increase was produced by
BP at 0.5% (41.6%) and BP at 4.0% (40.5%) followed by G at 4.0% (32.6%) and G
at 0.5% (25.4%), respectively in relation to the untreated control. Concerning
method/treatment interactions, SS/G at 0.5% induced the highest significant increase
in the RL (64.5%) followed by IR/BP at 0.5% (57.0%), SS/BP at 4.0% (53.8%) and
IR/BP at 4.0% (48.4%) while, the lowest significant increase was produced by
IR+SS/G at 4.0% (17.2%) comparing to the untreated control. On the other hand, the
RL was not significantly affected by SS/G at 0.5% and IR+SS/G at 0.5% comparing
to the untreated control (Fig., 30a).
4.5.3.8.2. Salicylic acid and riboflavin
The data in Tables (18b) indicated that, the root length (cm)/plant (RL) was
significantly affected by tested application methods, inducer treatments as well as by
the interaction in between. The SS method recorded the highest significant increase in
the RL (21.3 cm) followed by IR and IR+SS methods (19.5 cm). All tested inducer
treatments induced significant increases in the RL comparing to the untreated control.
The highest significant increase was produced by SA at 10.0mM (53.0%), SA at
0.1mM (36.2%), R at 10.0mM (30.5%) and R at 0.1mM (28.8%), respectively
without significant difference between the latter two treatments comparing to the
untreated control. Except IR/R at 0.1mM and SS/R at 10.0mM, all other
method/treatment interactions increased RL comparing to the untreated control. In
this respect, the highest increase was produced by SS/SA at 10.0mM (67.7%) and
SS/R at 0.1mM (65.6%) whereas; the lowest significant increase was induced by
IR+SS/R at 0.1mM (21.5%). However, IR/R at 0.1mM and SS/R at 10.0mM showed
no significant effect on the RL comparing to the untreated control (Fig., 30b).
Table 18a - Effect of garlic (G) and black pepper (BP) extracts at 0.5 % 4.0% using
different application methods on root length (cm.)/plant of tomato Carolina Gold cv.
under stress of infection with FOL isolate A (two-months after inoculation)
Root length (cm.)/plant
** increase %
Treatments
Mean
Mean
* IR
SS
IR+SS
IR SS IR+SS
25.5
16.5
16.3
5.4
G extract at 0.5%
19.4 64.5 6.5
25.4
20.0
23.5
18.2
G extract at 4.0%
20.6 29.0 51.6 17.2
32.6
24.3
20.0
21.5
BP extract at 0.5%
21.9 57.0 29.0 38.7
41.6
23.0
23.8
18.5
BP extract at 4%
21.8 48.4 53.8 19.4
40.5
15.5
15.5
15.5
0.0
Control (untreated)
15.5 0.0 0.0
0.0
Mean
21.7
19.9
18.0
39.8 28.2 16.1
L.S.D. at 5% for:
Methods
0.48
Treatments
0.80
78
Interaction
2.40
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
Table 18b - Effect of salicylic acid (SA) and riboflavin (R) at 0.1 and 10.0mM using
different application methods on root length (cm.)/plant of tomato Carolina Gold cv.
under stress of infection with FOL isolate A (two-months after inoculation)
Root length (cm.)/plant
** increase %
Treatments
Mean
Mean
* IR
SS
IR+SS
IR SS IR+SS
23.0
22.3
18.0
SA at 0.1mM
21.1 48.4 44.1 16.1
36.2
21.7
26.0
23.5
SA at 10.0mM
23.7 39.8 67.7 51.6
53.0
15.9
25.7
18.8
R at 0.1mM
20.1 2.4 65.6 21.5
29.8
21.7
17.2
21.8
R at 10.0mM
20.2 39.8 10.8 40.9
30.5
15.5
15.5
15.5
0.0
Control (untreated)
15.5 0.0 0.0
0.0
Mean
19.5
21.3
19.5
26.1 37.6 26.0
L.S.D. at 5% for:
Methods
0.41
Treatments
0.68
Interaction
2.03
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
Fig. 30a - Effect of garlic (G) and black
Fig. 30b - Effect of salicylic acid (SA)
pepper (BP) extracts at 0.5 % 4.0% using
and riboflavin (R) at 0.1 and 10.0mM
different application methods on root
using different application methods on
length increase % of tomato Carolina
root length increase % of tomato Carolina
Gold cv. under stress of infection with
Gold cv. under stress of infection with
FOL isolate A (two-months after
FOL isolate A (two-months after
inoculation)
inoculation)
4.5.3.9.
4.5.3.9.1.
Root volume (cm3) per plant
Garlic and black pepper extracts
79
The data in Tables (19a) indicated that, the root volume (cm3)/plant (RV) was
significantly affected by tested application methods, inducer treatments as well as by
the interaction in between. The three tested application methods were significantly
varied in this respect. The IR recorded the highest significant increase in the RV
(18.4 cm3) followed by IR+SS (17.6 cm3) and SS method (17.1 cm3), respectively
without significant difference between the latter two methods. All tested inducer
treatments significantly increased the RV comparing to the untreated control. The
highest significant increase was produced by using BP at 4.0% (60.0%) followed by
G at 4.0% (43.0%), G at 0.5% (30.8%) and BP at 0.5% (29.2%) without significant
differences between the latter two treatments compared to the untreated control. The
same data proved that, most tested method/treatment interactions significantly
increased RV comparing to the untreated control. The highest significant increase in
the RV was induced by using IR/BP at 4.0% (92.5%) followed by IR/G at 4.0%
(72.5%) and IR+SS/G at 0.5% (68.8%), respectively while the lowest significant
increase was induced by IR+SS/G at 0.5% (21.5%. On the other hand, using IR/G at
0.5%, IR/BP at 0.5%, SS/G at 0.5% showed no significant differences in the RV
when compared with the untreated control (Fig., 31a).
4.5.3.9.2. Salicylic acid and riboflavin
The data in Tables (19b) indicated that, the root volume (cm3)/plant (RV) was
significantly affected by tested application methods, inducer treatments as well as by
the interaction in between. The IR method recorded the highest significant RV (19.2
cm3) followed by SS method (18.3 cm3) and IR+SS (16.6 cm3), respectively. All
tested inducer treatments significantly increased the RV comparing to the untreated
control. The highest significant increase was produced by using R at 10.0mM
(59.2%) followed by SA at 0.1mM (47.9%), SA at 10.0mM (36.3%) and R at 0.1mM
(32.1%), respectively. Also, the RV was significantly increased by all tested
method/treatment interactions. IR/R at 10.0mM was the best interaction, increased
the RV by 92.5% followed by IR/SA at 0.1mM (75.0%) and SS/R at 10.0mM
(55.5%) whereas, the lowest significant increase was produced by IR+SS/R at 0.1mM
(27.5%) compared to the untreated control (Fig., 31b).
Table 19a - Effect of garlic (G) and black pepper (BP) extracts at 0.5 % 4.0% using
different application methods on root volume (cm3)/plant of tomato Carolina Gold cv.
under stress of infection with FOL isolate A (two-months after inoculation)
Root volume
** Increase %
(cm.3)/plant
Mea
Mea
Treatments
n
n
IR+S
* IR
SS
IR+SS
IR SS
S
17.
14.2
15.7
22.5
68.8
G extract at 0.5%
17.4 6.2
30.8
5
72. 35.
23.0
18.0
16.2
21.5
G extract at 4.0%
19.1
43.0
5
0
17.7
18.3
BP extract at 0.5% 15.7
17.2 17. 32. 37.5
29.2
80
BP extract at 4%
25.7
20.7
17.7
21.3
Control
(untreated)
13.3
13.3
13.3
13.3
Mean
18.4
17.1
17.6
5
92.
5
5
55.
0
0.0
37.
8
32.5
60.0
0.0
0.0
0.0
28.
0
32.1
L.S.D. at 5% for:
Methods
0.58
Treatments
0.96
Interaction
2.88
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
Table 19b - Effect of salicylic acid (SA) and riboflavin (R) at 0.1 and 10.0mM using
different application methods on root volume (cm3)/plant of tomato Carolina Gold cv.
under stress of infection with FOL isolate A (two-months after inoculation)
Root volume
** Increase %
(cm.3)/plant
Mea
Mea
Treatments
n
IR+S
n
* IR
SS
IR+SS
IR SS
S
75. 37.
23.3
18.3
17.5
31.3 47.9
SA at 0.1mM
19.7
0
5
26. 50.
16.8
20.0
17.7
32.5 36.3
SA at 10.0mM
18.2
3
0
25. 40.
16.7
18.7
17.5
31.3 32.1
R at 0.1mM
17.6
0
0
92. 57.
25.7
21.0
17.0
27.5 59.2
R at 10.0mM
21.2
5
5
13.3
13.3
13.3
0.0
Control (untreated)
13.3 0.0 0.0
0.0
43. 37.
Mean
19.2
18.3
16.6
24.5
8
0
L.S.D. at 5% for:
Methods
0.59
Treatments
0.98
Interaction
2.93
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
81
Fig. 31a - Effect of garlic (G) and black
pepper (BP) extracts at 0.5 % 4.0% using
different application methods on root
volume increase % of tomato Carolina
Gold cv. under stress of infection with
FOL isolate A (two-months after
inoculation)
Fig. 31b - Effect of salicylic acid (SA)
and riboflavin (R) at 0.1 and 10.0mM
using different application methods on
root volume increase % of tomato
Carolina Gold cv. under stress of
infection with FOL isolate A (twomonths after inoculation)
4.5.3.10. Fruit yield (g) per plant
4.5.3.10.1. Garlic and black pepper extracts
The data in Tables (20a) and Fig., (32a) indicated that, the weight of fruit yield
(g)/plant (WFY) was significantly affected by tested application methods, inducer
treatments as well as by the interaction in between. The three tested application
methods were significantly varied in this respect. The SS and IR+SS methods were
significantly better for increasing WFY (264.1-264.7 g/plant) comparing to the IR
method (249.2 g/plant). All tested inducer treatments significantly increased the
WFY comparing to the untreated control. In this respect, the highest significant
increase was produced by G at 4.0% (142.6%) and BP at 4.0% (142.2%) without
significant differences between them followed by and BP at 0.5% (135.6%).
However, the lowest significant increase in the WFY was produced by using G at
0.5% (39.7%) comparing with the untreated control. All tested method/treatment
interactions increased the WFY compared to the untreated control. In this regard, the
highest significant increase was produced by IR+SS/BP at 0.5% (545.9 g/plant)
which was increased it by 304.3% over the untreated control (135. g/plant). The
following interactions were: SS/BP at 4.0% (425.8 g), IR/G at 4.0% (410.2 g) and
SS/G at 4.0% (355.0 g) which increased by 215.3, 203.7 and 162.9%, respectively.
However, the lowest significant increase in the WFY was induced by using SS/G at
0.5% (170.3 g/plant) and IR/BP at 0.5% (174.3 g/plant) which were higher than the
untreated control by 26.1 and 29.1%, respectively.
4.5.3.10.2. Salicylic acid and riboflavin
The data in Tables (20b) indicated that, the weight of fruit yield (g)/plant
(WFY) was significantly affected by tested application methods, inducer treatments
as well as by the interaction in between. The IR+SS method recorded the highest
significant WFY (308.3 g/plant) followed by SS method (221.2 g/plant) comparing to
the IR method (199.7 g/plant). All tested inducer treatments significantly increased
82
the WFY comparing to the untreated control. In this respect, the highest significant
increase was produced by R at 10.0mM (289.7 g/plant) followed by SA at 10.0mM
(275.5 g/plant), R at 0.1mM (265.9 g/plant) and SA at 0.1mM (249.3 g/plant). These
inducer treatments increased WFY by 114.5, 104.0, 96.9 and 84.6%, respectively
over the untreated control. As for tested method/treatment interactions, IR+SS/R at
10.0mM was the best of all, increased the WFY to 416.3 g/plant followed by
IR+SS/R at 0.1mM (383.9 g g/plant), IR+SS/SA at 10.0mM (344.8 g/plant), IR/SA at
0.1mM (298.6 g/plant), SS/SA at 10.0mM (290.9 g/plant), respectively whereas, the
lowest significant increases were produced by SS/R at 10.0mM (266.8 g/plant) and
IR+SS/SA at 0.1mM (261.4 g/plant). These interactions increased the WFY by 208.3,
184.3, 155.3, 121.1, 115.4, 97.6 and 93.6%, respectively compared to the untreated
control. On the other hand, the observed increases in the WFY produced by IR/SA at
10.0mM, IR/R at 0.1 and 10.0mM, SS/SA at 0.1mM and SS/R at 0.1mM were not
significantly varied when compared to the untreated control (Fig., 32b).
4.6.
Effect of application methods of tested inducers treatments on leaf
pigments under stress of infection with the tomato Fusarium wilt
4.6.1. Garlic and black pepper extracts
4.6.1.1. Chlorophyll a
The data in Table (21a) indicated that using the tested plant extracts with
immersing root (IR) method produces higher amount of chlorophyll a (0.53 mg) than
spraying shoot (SS) method (0.44 m) whereas the IR+SS method was in between
(0.495 mg). All tested inducer treatment showed conspicuous increases in the
amounts of chlorophyll a. In this respect, G at 0.5% recorded the highest increase
(90.4%) followed by BP at 4.0% (67.3%), BP at 0.5% (62.9%) and G at 4.0%
(59.4%), respectively comparing to the untreated control (0.313 mg). Using the IR/G
at 0.5% recorded the highest increase in the amount of chlorophyll a (121.1%)
followed by IR+SS/G at 0.5% (92.3%), IR+SS/BP at 4.0% (84.7%), IR/G at 4.0%
(82.7%) and SS/BP at 0.5% (81.8%), respectively. The lowest increase in the
amounts of chlorophyll a was produced by SS/R at 4.0% (24.9%), SS/G at 4.0%
(44.7%) and IR+SS/0.5% (45.7%) compared to the untreated control (Fig., 33a).
4.6.1.2. Chlorophyll b
The data in Table (21b) and Fig., (33b) indicated that, using the IR method
recorded the highest amount of chlorophyll b (0.606 mg) followed by the IR+SS
method (0.564 mg) and the SS method (0.396 mg), respectively. As for inducer
treatments, the highest increase in the chlorophyll b was recorded by G at 0.5%
(104.9%), followed by BP at 4.0% (61.3%), G at 4.0% (58.2%) and BP at 0.5%
(57.6%), respectively comparing to the untreated control which recorded 0.345 mg/g
fresh weight. All tested interactions between methods and inducer treatments
increased chlorophyll b content. Using the IR/G at 0.5% recorded the highest increase
in the chlorophyll b (176.5%) followed by IR+SS/G at 0.5% (109.9%), IR/BP at
4.0% (96.8%) and IR/G at 4.0% (85.5%) whereas, the lowest increase was induced
by IR+SS/G at 4.0% (23.2%), SS/BP at 4.0% (26.7%) and SS/G at 0.5% (28.4%),
respectively compared to the untreated control (0.345 mg).
83
Table 20a - Effect of garlic (G) and black pepper (BP) extracts at 0.5 % 4.0% using
different application methods on weight of fruit yield (g.)/plant of tomato Carolina
Gold cv. under stress of infection with FOL isolate A (two-months after inoculation)
Fruit yield (g.)/plant
** Increase %
Treatments
Mean
Mean
* IR
SS IR+SS
IR
SS IR+SS
196.9 170.3 198.8 188.7 45.8 26.1
47.2
G extract at 0.5%
39.7
410.2 355.0 217.7 327.6 203.7 162.9 61.2 142.6
G extract at 4.0%
BP extract at 0.5% 174.3 234.3 545.9 318.2 29.1 73.5 304.3 135.6
329.7 425.8 225.8 327.1 144.1 215.3 67.2 142.2
BP extract at 4%
0.0
0.0
Control (untreated) 135.0 135.0 135.0 135.0 0.0
0.0
Mean
249.2 264.1 264.7
84.6 95.6
96.0
L.S.D. at 5% for:
Methods
3.01
Treatments
5.02
Interaction
15.05
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
Table 20b - Effect of salicylic acid (SA) and riboflavin (R) at 0.1 and 10.0mM using
different application methods on weight of fruit yield (g.)/plant of tomato Carolina
Gold cv. under stress of infection with FOL isolate A (two-months after inoculation)
Fruit yield (g.)/plant
** Increase %
Treatments
Mean
Mean
* IR
SS IR+SS
IR
SS IR+SS
298.6 188.0 261.4 249.3 121.1 39.2
93.6
SA at 0.1mM
84.6
190.8 290.9 344.8 275.5 41.3 115.4 155.3 104.0
SA at 10.0mM
188.2 225.5 383.9 265.9 39.4 67.0 184.3 96.9
R at 0.1mM
186.0 266.8 416.3 289.7 37.7 97.6 208.3 114.5
R at 10.0mM
0.0
0.0
Control (untreated) 135.0 135.0 135.0 135.0 0.0
0.0
Mean
199.7 221.2 308.3
47.9 63.8 128.3
L.S.D. at 5% for:
Methods
19.29
Treatments
32.15
Interaction
96.44
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
84
Fig. 32a - Effect of garlic (G) and black
Fig. 32b - Effect of salicylic acid (SA)
pepper (BP) extracts at 0.5 % 4.0% using
and riboflavin (R) at 0.1 and 10.0mM
different application methods on weight
using different application methods on
of fruit yield increase % of tomato
weight of fruit yield increase % of tomato
Carolina Gold cv. under stress of
Carolina Gold cv. under stress of
infection with FOL isolate A (twoinfection with FOL isolate A (twomonths after inoculation)
months after inoculation)
Table 21a - Effect of garlic and black pepper extracts used as resistance inducers at
0.5 and 4.0% concentrations using different application methods* on chlorophyll a
(mg/g fresh weight) and % increase comparing to the control after two month of
inoculation with FOL isolate A
Chlorophyll a (mg/g fresh weight)
Black pepper
Application method Garlic extract at
Control Mean
0.5%
4.0%
0.5%
4.0%
0.692
0.572
0.472
0.602
0.313
IR*
0.530
0.456
0.472
0.569
0.391
0.313
SS
0.440
0.64
0.453
0.489
0.578
0.313
IR+SS
0.495
Mean
0.596
0.499
0.510
0.524
0.313
0.488
** Increase %
121.1
82.7
50.8
92.3
0.0
IR
45.7
50.8
81.8
24.9
0.0
SS
104.5
44.7
56.2
84.7
0.0
IR+SS
Mean
90.4% 59.4% 62.9%
67.3%
0.0%
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
4.6.1.3. Total chlorophyll
The data in Table (21c) indicated that, using the IR method recorded the highest
amount of total chlorophyll (1.161 mg) followed by the IR+SS method (1.011 mg)
and the SS method (0.911 mg), respectively. All tested inducer treatment showed
conspicuous increase in the amounts of total chlorophyll. The highest increase was
recorded by G at 0.5% (98.0%) followed by BP at 4.0% (64.1%), BP at 0.5% (60.1%)
and G at 4.0% (58.8%), respectively comparing to the untreated control which
85
recorded 0.658 mg/g fresh weight. All tested interactions increased total chlorophyll
content. In this regard, IR/G at 0.5% recorded the highest increase (150.2%) followed
by IR+SS/G at 0.5% (107.3%), IR/BB at 4.0% (94.7%) and IR/G at 4.0% (84.2%)
whereas, the lowest increase was produced by SS/BP at 4.0% (25.8%) comparing to
the untreated control (Fig., 33c).
Table 21b - Effect of garlic and black pepper extracts used as resistance inducers at
0.5 and 4.0% concentrations using different application methods* on chlorophyll b
(mg/g fresh weight) and % increase comparing to the control after two month of
inoculation with FOL isolate A
Chlorophyll b (mg/g fresh weight)
Black pepper
Application method Garlic extract at
Control Mean
0.5%
4.0%
0.5%
4.0%
0.954
0.64
0.536
0.679
0.345
IR*
0.954
0.443
0.572
0.559
0.437
0.345
SS
0.443
0.724
0.425
0.536
0.553
0.345
IR+SS
0.724
Mean
0.707
0.546
0.544
0.556
0.345
0.707
** Increase %
176.5
85.5
55.4
96.8
0.0
IR
28.4
65.8
62.0
26.7
0.0
SS
109.9
23.2
55.4
60.3
0.0
IR+SS
Mean
104.9
58.2
57.6
61.3
0.0
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
Table 21c - Effect of garlic and black pepper extracts used as resistance inducers at
0.5 and 4.0% concentrations using different application methods* on total
chlorophyll (mg/g fresh weight) and % increase comparing to the control after two
month of inoculation with FOL isolate A
Total chlorophyll (mg/g fresh weight)
Black pepper at
Application method Garlic extract at
Control Mean
0.5%
4.0%
0.5%
4.0%
1.646
1.212
1.008
1.281
0.658
IR*
1.161
0.899
1.044
1.128
0.828
0.658
SS
0.911
1.364
0.878
1.025
1.131
0.658
IR+SS
1.011
Mean
1.303
1.045
1.054
1.080
0.658
1.028
** Increase %
150.2
84.2
53.2
94.7
0.0
IR
36.6
58.7
71.4
25.8
0.0
SS
107.3
33.4
55.8
71.9
0.0
IR+SS
Mean
98.0
58.8
60.1
64.1
0.0
86
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
Fig. 33a - Effect of garlic and black
Fig. 33b - Effect of garlic and black
pepper extracts used as resistance
pepper extracts used as resistance
inducers at 0.5 and 4.0% concentrations
inducers at 0.5 and 4.0% concentrations
using different application methods* on
using different application methods* on
chlorophyll a increase % comparing to
chlorophyll b increase % comparing to
the control after two month of inoculation the control after two month of inoculation
with
with
FOL isolate A
FOL isolate A
Fig. 33c - Effect of garlic and black pepper extracts used as resistance inducers at 0.5
and 4.0% concentrations using different application methods* on total chlorophyll
increase % comparing to the control after two month of inoculation with FOL isolate
A
4.6.2. Salicylic acid and riboflavin
4.6.2.1. Chlorophyll a
Regardless inducer treatments, the data in Table (22a) indicated that the
combined (IR+SS) application method produces the highest amounts of chlorophyll a
(0.549 mg) followed by the IR method (0.491 mg) and SS method (0.295 mg),
respectively. Using R at 0.1 and 10.0mM caused the highest and lowest increase in
average amounts of chlorophyll a i.e. 83.2 and 23.2%) while, SA at 0.1 and 10.0mM
87
increased it by 54.1 and 50.5%, respectively comparing to the untreated control (0.313
mg). As for method/treatment interactions, using the IR+SS/R at 0.1mM recorded the
highest increase in the chlorophyll a (146.6%) followed by R+SS/SA at 0.1mM (108.3)
IR/R at 0.1mM (99.7%), respectively. The lowest increase in the amounts of
chlorophyll a was produced by SS/R at 0.1mM (3.2%) whereas using SS/SA at
0.1mM, SS/SA at 10.0mM and SS/R at 10.0mM decreased the chlorophyll a content
by 7.0, 9.9 and 14.4%, respectively compared to the untreated control Fig., 34a).
4.6.2.2. Chlorophyll b
The data in Table (22b) indicated that, using the IR+SS method recorded the
highest amount of chlorophyll b (0.568 mg) followed by the IR method (0.528 mg)
and the SS method (0.311 mg), respectively. Regardless application methods all
tested inducer treatments increased chlorophyll b content comparing to the untreated
control which recorded 0.345 mg/g fresh weight. In this respect, the highest increase
was recorded by R at 0.1mM (70.6%) followed by SA at 0.1mM (54.5%), SA at
10.0mM (43.0%) and R at 10.0mM (11.6%), respectively. Using IR+SS/SA at
0.1mM recorded the highest increase (117.1%) followed by IR+SS/R at 0.1mm
(115.9%), IR/R at 0.1mM (94.2%) and IR/SA at 10.0mM (79.4%) whereas the lowest
increase was produced by SS/R at 0.1mM (1.7%). However, SS/SA at 0.1mM, SS/SA
at 10.0mM and SS/R at 10.0mM decreased amount of chlorophyll b by 13.0, 13.9 and
23.8%, respectively compared to the untreated control (Fig., 34b).
Table 22a - Effect of salicylic acid (SA) and riboflavin (R) used as resistance
inducers at 0.1 and 10.0mM concentrations using different application methods* on
chlorophyll a (mg/g fresh weight) and % increase comparing to the control after two
month of inoculation with FOL isolate A
Chlorophyll a (mg/g fresh weight)
Salicylic acid at
Riboflavin at
Application method
Control Mean
0.1mM 10.0mM 0.1mM 10.0mM
0.504
0.576
0.625
0.437
0.313
IR*
0.491
0.291
0.282
0.323
0.268
0.313
SS
0.295
0.652
0.555
0.772
0.452
0.313
IR+SS
0.549
Mean
0.482
0.471
0.573
0.386
0.313
0.445
** Increase %
61.0
84.0
99.7
39.6
0.0
IR
-7.0
-9.9
3.2
-14.4
0.0
SS
108.3
77.3
146.6
44.4
0.0
IR+SS
Mean
54.1
50.5
83.2
23.2
0.0
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
4.6.2.3. Total chlorophyll
The data in Table (22c) indicated that the IR+SS method recorded higher amount
of total chlorophyll (1.116 mg) than the IR method (1.019 mg) and the SS method
88
(0.607 mg), respectively. Regardless application methods, R at 0.1mM recorded the
highest increase in the total chlorophyll (76.6%) followed by SA at 0.1mM (54.3%), SA
at 10.0mM (46.6%) and R at 10.0mM (17.1), respectively comparing to the untreated
control (0.658 mg). As for method/treatment interactions, IR+SS/R at 0.1mM recorded
the highest increase in the total chlorophyll content (130.5%) followed by IR+SS/SA at
0.1mM (112.9%) and IR/R at 0.1mM (1.295 mg) while, the lowest increase was
produced by SS/R at 0.1mM (2.1%). However, SS/SA at 0.1mM, SS/SA at 10.0mM and
SS/R at 10.0mM decreased amount of total chlorophyll by 10.2, 12.0 and 19.3%,
respectively compared to the untreated control (Fig., 34c).
Table 22b - Effect of salicylic acid (SA) and riboflavin (R) used as resistance
inducers at 0.1 and 10.0mM concentrations using different application methods* on
chlorophyll b (mg/g fresh weight) and % increase comparing to the control after two
month of inoculation with FOL isolate A
Chlorophyll b (mg/g fresh weight)
Application method
Salicylic acid at
Riboflavin at
Control Mean
0.1mM 10.0mM 0.1mM 10.0mM
0.55
0.619
0.67
0.457
0.345
IR*
0.528
0.3
0.297
0.351
0.263
0.345
SS
0.311
0.749
0.564
0.745
0.435
0.345
IR+SS
0.568
Mean
0.533
0.493
0.589
0.385
0.345
0.469
** Increase %
59.4
79.4
94.2
32.5
0.0
IR
-13.0
-13.9
1.7
-23.8
0.0
SS
117.1
63.5
115.9
26.1
0.0
IR+SS
Mean
54.5
43.0
70.6
11.6
0.0
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
Table 22c - Effect of salicylic acid (SA) and riboflavin (R) used as resistance
inducers at 0.1 and 10.0mM concentrations using different application methods* on
total chlorophyll (mg/g fresh weight) and % increase comparing to the control after
two month of inoculation with FOL isolate A
Total chlorophyll (mg/g fresh weight)
Application method
Salicylic acid at
Riboflavin at
Control Mean
0.1mM 10.0mM 0.1mM 10.0mM
1.054
1.195
1.295
0.894
0.658
IR*
1.019
0.591
0.579
0.674
0.531
0.658
SS
0.607
89
1.401
1.119
1.517
IR+SS
Mean
1.015
0.964
1.162
** Increase %
60.2
81.6
96.8
IR
-10.2
-12.0
2.4
SS
112.9
70.1
130.5
IR+SS
Mean
54.3
46.6
76.6
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
0.887
0.771
0.658
0.658
35.9
-19.3
34.8
17.1
0.0
0.0
0.0
0.0
1.116
0.914
Fig. 34a - Effect of salicylic acid (SA)
Fig. 34b - Effect of salicylic acid (SA)
and riboflavin (R) used as resistance
and riboflavin (R) used as resistance
inducers at 0.1 and 10.0mM
inducers at 0.1 and 10.0mM
concentrations using different application concentrations using different application
methods* on chlorophyll a increase %
methods* on chlorophyll b increase %
comparing to the control after two month comparing to the control after two month
of inoculation with FOL isolate A
of inoculation with FOL isolate A
Fig. 34c - Effect of salicylic acid (SA) and riboflavin (R) used as resistance inducers
at 0.1 and 10.0mM concentrations using different application methods* on total
chlorophyll increase % comparing to the control after two month of inoculation with
FOL isolate A
90
4.7.
Effect of application methods of tested inducers treatments on
phenols content under stress of infection with the tomato Fusarium wilt
4.7.1. Garlic and black pepper extracts
4.7.1.1. Free phenols content
The data in Table (23a) and Fig., (22a) found that, the free phenol content
(mg/100 g fresh weight) was affected differently by the tested application methods,
inducer treatments as well as by the interaction in between. As for application
methods, the IR+SS method recorded the highest average of free phenols (2.313 mg)
while the IR and SS methods recorded 1.39 and 2.24 mg, respectively. All tested
inducer treatments increased the free phenols content (mg catechol/100 g fresh
weight) comparing to the untreated control. The highest increase was induced by G at
0.5% (149.3%), BP at 4.0% (127.3%), G at 4.0% (119.0%) and BP at 0.5% (106.9%),
respectively in relation to the untreated control which recorded (0.988 mg catechol
/100 g fresh weight). As for interactions, IR+SS/G at 0.5% recoded the highest
increase in the free phenols content (328.4%) followed by SS/G at 4.0% (208.8%)
while, the lowest increase was induced by IR/G at 0.5% (21.4%) comparing with the
untreated control (Fig., 35a).
Table 23a - Effect of garlic and black pepper extracts used as resistance inducers at
0.5 and 4.0% concentrations using different application methods* on the free phenols
content (mg catechol /100 g fresh weight) and % increase comparing to the control
after two month of inoculation with FOL isolate A
Free phenols content (mg/100 g fresh
weight)
Application
Contro Mea
method
Garlic extract at
Black pepper
l
n
0.5%
4.0%
0.5%
4.0%
1.199
1.675
1.623
1.464
0.988 1.390
IR*
1.958
3.051
2.575
2.628
0.988 2.240
SS
4.233
1.764
1.934
2.646
0.988 2.313
IR+SS
Mean
2.463
2.163
2.044
2.246
0.988 1.981
** Increase %
21.4
69.5
64.3
48.2
0.0
IR
98.2
208.8
160.6
166.0
0.0
SS
328.4
78.5
95.7
167.8
0.0
IR+SS
Mean
149.3
119.0
106.9
127.3
0.0
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
4.7.1.2. Conjugated phenols content
The data in Table (23b) and Fig., (35b) showed that, the conjugated phenols
content (mg/100 g fresh weight) was affected differently by the tested application
methods, inducer treatments as well as by the interaction in between. As for
application methods, SS method recorded the highest average of conjugated phenols
(6.657 mg) followed by IR+SS method (6.569 mg) and IR method (5.463 mg),
91
respectively. All tested inducer treatments increased the conjugated phenols content.
In this respect, BP at 4.0% recorded the highest increase (147.5%) followed by G at
4.0% (104.4%), G at 0.5% (53.5%) and BP at 0.5% (2.3%), respectively comparing
to the untreated control. Also, all interactions increased the conjugated phenols
content comparing to the untreated control. In this respect, the highest increase was
recorded by using SS/BP at 4.0% (210.9%), IR+SS/BP at 4.0% (187.3%) and
IR+SS/G at 4.0% (148.8%) while IR/BP at 0.5%, SS/BP at 0.5% and IR+SS/BP at
0.5% recorded the lowest increase in the conjugated phenols (1.8-2.9%) followed by
IR+SS/G at 0.5% (13.4%) comparing to the untreated control.
4.7.1.3. Total phenols content
The data in Tables (23c) and Fig., (22c) proved that, the SS and IR+SS methods
recorded the higher total phenols i.e. 8.897 and 8.882 mg more than the IR method
(6.854 mg). All tested inducer treatments increased the total phenols content
comparing to the untreated control. The highest increase was recorded by BP at
10.0mM (143.4%) followed by G at 4.0% (107.3%), G at 0.5% (73.0%) and BP at
0.5% (23.6%), respectively. As for interactions, the highest increase was recorded by
using SS/BP at 4.0% (201.8%) followed by IR+SS/BP at 4.0% (183.3%) and
IR+SS/G at 4.0% (134.5%) whereas, the lowest increase was recorded by IR/BP at
0.5% (14.5%) comparing to the untreated control (Fig., 35c).
Table 23b - Effect of garlic and black pepper extracts used as resistance inducers at
0.5 and 4.0% concentrations using different application methods* on the conjugated
phenols content (mg catechol /100 g fresh weight) and % increase comparing to the
control after two month of inoculation with FOL isolate A
Conjugated phenols content (mg/100 g fresh
weight)
Application
Contro
Mean
method
Garlic extract at
Black pepper
l
0.5%
4.0%
0.5%
4.0%
6.482
7.484
3.925
5.565
3.857 5.463
IR*
6.901
6.569
3.968
11.992
3.857 6.657
SS
4.374
9.595
3.941
11.08
3.857 6.569
IR+SS
Mean
5.919
7.883
3.945
9.546
3.857 6.230
** Increase %
68.1
94.0
1.8
44.3
0.0
IR
78.9
70.3
2.9
210.9
0.0
SS
13.4
148.8
2.2
187.3
0.0
IR+SS
Mean
53.5
104.4
2.3
147.5
0.0
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
92
Table 23c - Effect of garlic and black pepper extracts used as resistance inducers at
0.5 and 4.0% concentrations using different application methods* on the total phenols
content (mg catechol /100 g fresh weight) and % increase comparing to the control
after two month of inoculation with FOL isolate A
Total phenols content (mg/100 g fresh
weight)
Application
Contro Mea
method
Garlic extract at
Black pepper
l
n
0.5%
4.0%
0.5%
4.0%
7.681
9.159
5.548
7.029
4.845 6.852
IR*
8.859
9.62
6.543
14.62
4.845 8.897
SS
8.607
11.359
5.875
13.726
4.845 8.882
IR+SS
Mean
8.382
10.046
5.989
11.792
4.845 8.211
** Increase %
58.5
89.0
14.5
45.1
0.0
IR
82.8
98.6
35.0
201.8
0.0
SS
77.6
134.4
21.3
183.3
0.0
IR+SS
Mean
73.0
107.3
23.6
143.4
0.0
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
93
Fig. 35a - Effect of garlic and black
pepper extracts used as resistance
inducers at 0.5 and 4.0% concentrations
using different application methods* on
the free phenols content increase %
comparing to the control after two month
of inoculation
with FOL isolate A
Fig. 35b - Effect of garlic and black
pepper extracts used as resistance
inducers at 0.5 and 4.0% concentrations
using different application methods* on
the conjugated phenols content increase
% comparing to the control after two
month of inoculation with FOL isolate A
Fig. 35c - Effect of garlic and black pepper extracts used as resistance inducers at 0.5
and 4.0% concentrations using different application methods* on the total phenols
content increase % comparing to the control after two month of inoculation with FOL
isolate A
4.7.2. Salicylic acid and riboflavin
4.7.2.1. Free phenols content
The data in Table (24a) and Fig., (36a) found that, the free phenol content (mg/100
g fresh weight) was affected differently by the tested application methods, inducer
treatments as well as by the interaction in between. The IR+SS method recorded the
highest average of free phenols (2.235 mg) while the IR and SS methods recorded 1.76
and 1.915 mg, respectively. All tested inducer treatments increased the free phenols
content. The highest increase was induced by R at 10.0mM (226.1%) followed by SA at
10.0mM (125.5%), SA at 0.1mM (85.6% and R at 0.1mM (59.9%), respectively in
relation to the free phenols in the untreated control (0.988 mg). All interactions increased
the free phenols contents to different extents. Using IR+SS/R at 10.0mM recoded the
94
highest increase (292.7%) followed by IR/R at 10.0mM (196.4%) and SS/R at 10.0mM
(189.2%) while, the lowest increase was induced by IR/SA at 0.1mM and SS/R at
0.1mM (26.7%) comparing with the untreated control.
Table 24a - Effect of salicylic acid and riboflavin used as resistance inducers at 0.1
and 10.0mM concentrations using different application methods* on the free phenols
content (mg catechol /100 g fresh weight) and % increase comparing to the control
after two month of inoculation with FOL isolate A
Free phenols content (mg/100 g fresh
weight)
Application
Contro Mea
Salicylic acid at
Riboflavin at
method
l
n
0.1mM
10.0Mm
0.1mM
10.0Mm
1.252
2.099
1.534
2.928
0.988 1.760
IR*
2.557
1.922
1.252
2.857
0.988 1.915
SS
1.693
2.663
1.952
3.88
0.988 2.235
IR+SS
Mean
1.834
2.228
1.579
3.222
0.988 1.970
** Increase %
26.7
112.4
55.3
196.4
0.0
IR
158.8
94.5
26.7
189.2
0.0
SS
71.4
169.5
97.6
292.7
0.0
IR+SS
Mean
85.6
125.5
59.9
226.1
0.0
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
4.7.2.2. Conjugated phenols content
The data in Table (24b) and Fig., (36b) showed that, the conjugated phenols
content (mg/100 g fresh weight) was affected differently by the tested application
methods, inducer treatments as well as by the interaction in between. The IR method
recorded the highest average of conjugated phenols (8.558 mg) followed by IR+SS
method (8.228 mg) and SS method (6.773 mg), respectively. All tested inducer
treatments increased the conjugated phenols content. In this respect, R at 10.0mM
recorded the highest increase (293.2%) while SA at 10.0mM, SA at 0.1mM and R at
0.1mM increased it by 67.4-83.4% comparing to the untreated control. All interactions
increased the conjugated phenols content also. In this respect, the highest increase was
recorded by using IR+SS/R at 10.0mM (322.2%) followed by IR/R at 10.0mM
(294.7%), SS/R at 10.0mmM (262.8%), IR/R at 0.1mM (154.1%), and IR+SS/SA at
10.0mM (132.5%), while SS/SA at 0.1mM and IR+SS/R at 0.1mM increased the
conjugated phenols by 2.3-2.9% only comparing to the untreated control.
4.7.2.3. Total phenols content
The data in Table (24c) and Fig., (36c) proved that, the total phenols content
(mg/100 g fresh weight) was affected differently by the tested application methods,
inducer treatments as well as by the interaction in between. The IR+SS method
recorded the highest total phenols (10.463 mg) followed by IR method (10.318 mg)
and SS method (8.688 mg), respectively. All tested inducer treatments increased the
95
total phenols content comparing to the untreated control. The highest increase was
recorded by R at 10.0mM (279.5%) followed by SA at 10.0mM (92.0%), SA at
0.1mM (76.4%) and R at 0.1mM (65.8%), respectively. All interactions between
application methods and inducer treatments increased the conjugated phenols content
comparing to the untreated control. In this respect, the highest increase was recorded
by IR+SS/R at 10.0mM (316.2%) followed by IR/R at 10.0mM (274.7%), SS/R at
10.0mmM (247.8%), IR+SS/SA at 10.0mM (140.1%) and IR/R at 0.1mM (134.0%),
while the lowest increase was recorded by SS/SA at 0.1mM (34.2%) and SS/R at
0.1mM (41.4%) comparing to the untreated control.
Table 24b - Effect of salicylic acid and riboflavin used as resistance inducers at 0.1
and 10.0mM concentrations using different application methods* on the conjugated
phenols content (mg catechol /100 g fresh weight) and % increase comparing to the
control after two month of inoculation with FOL isolate A
Conjugated phenols content (mg/100 g
fresh weight)
Application
Control Mean
method
Salicylic acid at
Riboflavin at
0.1mM
10.0Mm
0.1mM 10.0Mm
8.125
5.782
9.802
15.225
3.857
IR*
8.558
3.944
6.475
5.597
13.992
3.857
SS
6.773
8.063
8.968
3.968
16.284
3.857
IR+SS
8.228
Mean
6.711
7.075
6.456
15.167
3.857
7.853
** Increase %
110.7
49.9
154.1
294.7
0.0
IR
2.3
67.9
45.1
262.8
0.0
SS
109.0
132.5
2.9
322.2
0.0
IR+SS
Mean
74.0
83.4
67.4
293.2
0.0
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
Table 24c - Effect of salicylic acid and riboflavin used as resistance inducers at 0.1
and 10.0mM concentrations using different application methods* on the total phenols
content (mg catechol /100 g fresh weight) and % increase comparing to the control
after two month of inoculation with FOL isolate A
Total phenols content (mg/100 g fresh
weight)
Application
Control Mean
method
Salicylic acid at
Riboflavin at
0.1mM 10.0Mm 0.1mM
10.0Mm
10.31
9.377
7.881
11.336
18.153
4.845
IR*
8
6.501
8.397
6.849
16.849
4.845
SS
8.688
10.46
9.756
11.631
5.92
20.164
4.845
IR+SS
3
96
Mean
8.545
9.303
8.035
** Increase %
93.5
62.7
134.0
IR
34.2
73.3
41.4
SS
101.4
140.1
22.2
IR+SS
Mean
76.4
92.0
65.8
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
18.389
4.845
274.7
247.8
316.2
279.5
0.0
0.0
0.0
0.0
9.823
Fig. 36a - Effect of salicylic acid and
Fig. 36b - Effect of salicylic acid and
riboflavin used as resistance inducers at
riboflavin used as resistance inducers at
0.1 and 10.0mM concentrations using
0.1 and 10.0mM concentrations using
different application methods* on the free
different application methods* on the
phenols content increase % comparing to
conjugated phenols content increase %
the control after two month of inoculation comparing to the control after two month
with FOL isolate A
of inoculation with FOL isolate A
Fig. 36c - Effect of salicylic acid and riboflavin used as resistance inducers at 0.1 and
10.0mM concentrations using different application methods* on the total phenols
content increase % comparing to the control after two month of inoculation with FOL
isolate A
4.8.
Total soluble protein “TSP” content
4.8.1. Garlic and black pepper extracts
97
The data in Tables (25a) and Fig., (37a) indicated that, the total soluble protein
“TSP” (mg/100 g fresh weight) was positively affected by tested application
methods, inducer treatments as well as by the interaction in between. The SS
application method recorded the highest average of TSP (26.82 mg) followed by the
SS method (19.94 mg) and the IR+SS method (17.88 mg), respectively. Regardless
application method, BP at 0.5% recorded the highest increase in the TSP (114.8%)
followed by G at 4.0% (110.3%), BP at 0.5% (73.5%) and G at 0.5% (56.6%),
respectively comparing to the untreated control which recorded 12.6 mg/100g fresh
weight. All method/treatment interactions increased TSP. The highest increase
recorded by SS/G at 4.0% (248.8%) followed by IR+SS/BP at 0.5% (129.4), SS/BP
at 0.5% (127.8%), SS/BP at 4.0% (120.6%), respectively. The remained interactions
increased TSP by 24.6-86.5% comparing to the untreated control.
Table 25a - Effect of Garlic and black pepper extracts used as resistance inducers at
0.5 and 4.0% concentrations using different application methods* on the TSP
(mg/100 g fresh weight) and % increase comparing to the control after two month of
inoculation with FOL isolate A
Total soluble protein (mg/100g fresh
weight)
Application
Black pepper
Control Mean
method
Garlic extract
extract
0.5%
4.0%
0.5%
4.0%
21.1
19.9
23.5
22.6
12.6
IR*
19.940
21.1
43.9
28.7
27.8
12.6
SS
26.820
17
15.7
28.9
15.2
12.6
IR+SS
17.880
Mean
19.733
26.500
27.033
21.867
12.600 21.547
** Increase %
67.5
57.9
86.5
79.4
0.0
IR
67.5
248.4
127.8
120.6
0.0
SS
34.9
24.6
129.4
20.6
0.0
IR+SS
Mean
56.6
110.3
114.6
73.5
0.0
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
4.8.2. Salicylic acid and riboflavin
The data in Table (25b) and Fig., (37b) indicated that, the IR application
method recorded the highest average of TSP (34.86 mg) followed by the IR+SS
method (27.96 mg) and the SS method (25.55 mg), respectively. As for inducer
treatment, R at 10.0mM recorded the highest increase in the TSP (289.4%) followed
by SA at 10.0mM (184.7%), R at 0.1mM (115.1%) and SA at 0.1mM (75.4%),
respectively comparing to the untreated control. All tested interactions increased
TSP. The highest increase was recorded by IR+SS/R at 10.0mM (338.9%) followed
by IR/SA at 10.0mM (352.4%), IR/R at 10.0mM (311.1%), SS/R at 10.0mM
(218.3%), IR/R at 0.1mM (143.7%) and IR+SS/SA at 10.0mM (108.7%),
98
respectively. The remained interactions increased TSP by 76.2-92.9% comparing to
the untreated control.
4.9.
Activity of the oxidative enzymes
4.9.1. Polyphenoloxidase (PPO) enzyme
4.9.1.1. Garlic and black pepper extracts
The data in Table (26a) and Fig., (38a) indicated that, the activity of
polyphenoloxidase (PPO) enzyme (O.D at 430nm/g FW/Min) was affected
differently by tested application methods, inducer treatments as well as by the
interaction in between. All tested inducer treatment increased PPO activity comparing
to the untreated control. Using G at 0.5% recorded the highest increase in the PPO
activity (27.6%) followed by BP at 4.0% (13.5%), BP at 0.5% (11.1%) and G at 4.0%
(5.0%), respectively. Most interactions, however, increased PPO activity but few
decreased it. The SS method recorded the highest average of PPO activity (44.3)
followed by the IR+SS method (38.5) and the IR method (38.2), respectively. In this
respect, SS/BP at 0.5% recorded the highest increase (46.7%) followed by IR+SS/BP
at 0.5% (31.5%), IR/G at 0.5% (30.4%), respectively.
Table 25b - Effect of salicylic acid and riboflavin used as resistance inducers at 0.1
and 10.0mM concentrations using different application methods* on the TSP
(mg/100 g fresh weight) and % increase comparing to the control after two month of
inoculation with FOL isolate A
Total soluble protein (mg/100g fresh
weight)
Application
Contro Mea
Salicylic
acid
at
Riboflavin
at
method
l
n
0.1mM 10.0Mm 0.1mM
10.0Mm
22.2
57
30.7
51.8
12.6
IR*
34.86
25.1
24.3
24
40.1
12.6
SS
25.22
19
26.3
26.6
55.3
12.6
IR+SS
27.96
Mean
22.10
35.87
27.10
49.07
12.60 29.35
** Increase %
76.2
352.4
143.7
311.1
0.0
IR
99.2
92.9
90.5
218.3
0.0
SS
50.8
108.7
111.1
338.9
0.0
IR+SS
Mean
75.4
184.7
115.1
289.4
0.0
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
99
Fig. 37a - Effect of Garlic and black
Fig. 37b - Effect of salicylic acid and
pepper extracts used as resistance inducers riboflavin used as resistance inducers at
at 0.5 and 4.0% concentrations using
0.1 and 10.0mM concentrations using
different application methods* on the TSP different application methods* on the TSP
increase % comparing to the control after increase % comparing to the control after
two month of inoculation with FOL isolate two month of inoculation with FOL isolate
A
A
4.9.1.2. Salicylic acid and riboflavin
The data in Table (26b) and Fig., (38b) indicated that, the activity of
polyphenoloxidase (PPO) enzyme (O.D at 430nm/g FW/Min) was affected differently
by tested application methods, inducer treatments as well as by the interaction in
between. The SS method recorded the highest average of PPO activity (52.1) followed
by the IR method (51.2) and the IR+SS method (39.6), respectively. As for inducer
treatments, the highest PPO activity was recorded by R at 10.0mM (55.5%) followed
by SA at 10.0mM (36.8%), R at 0.1mM (35.2%) and SA at 0.1mM (29.9%),
respectively. As for interactions, SS/R at 0.1mM recorded the highest increase (94.2%)
followed by IR/SA at 10.0mM (87.0%), SS/R at 10.0mM (82.3%), IR/R at 10.0mM
(71.5%) and IR+SS/SA at 0.1mM (48.6%), respectively. However, only IR+SS/R at
0.1mM, and IR+SS/SA at 10.0mM decreased PPO activity by 11.0 and 3.0%,
respectively compared to the untreated control. The remained interactions increased the
PPO activity by 4.4-29.8% except IR/BP at 0.5% and IR+SS/G at 4.0% which
decreased it by 44.8 and 27.3%, respectively compared to the untreated control.
4.9.2. Activity of peroxidase (POD) enzyme
4.9.2.1. Garlic and black pepper extracts
The data in Tables (27a) and Fig., (39a) indicated that, the activity of
peroxidase (POD) enzyme (420/min/g fresh weight) was affected differently by
tested application methods, inducer treatments as well as by the interaction in
between. The SS method recorded the highest average of POD activity (61.5)
followed by the IR+SS method (55.2) and the IR method (27.0), respectively. Using
BP at 0.5% recorded the highest POD activity (339.0%) followed by BP at 4.0%
(325.9%), G at 0.5% (311.1%) and G at 4.0% (299.3%), respectively. All tested
interactions increase POD activity comparing to the untreated control which recorded
100
13.5 (420/min/g fresh weight). In this respect, SS/G at 4.0% recorded the highest
increase (640.7%) followed by IR+SS/BP at 4.0% (595.6%), SS/G at 0.5% (411.1%),
IR+SS/BP at 0.5% (401.5%), SS/BP at 0.5% (397.8%), IR+SS/G at 0.5% (352.6%)
and SS/BP at 4.0% (328.9%) whereas the remained interactions increased it by 53.3217.8% comparing to the untreated control.
Table 26a - Activity of PPO enzyme (430/min/g fresh weight) in tomato leaves as
affected by inducer treatments (garlic and black pepper extracts at 0.5 & 4.0% conc.)
using application methods after two months from inoculation with FOL isolate A
Polyphenoloxidase activity (430/min/g
fresh weight)
Application
Contro
Mean
Black pepper extract
method
l
Garlic extract at
at
0.5%
4.0%
0.5%
4.0%
47.2
47.0
20.0
40.8
36.2
IR*
38.2
47.0
40.7
53.1
44.7
36.2
SS
44.3
44.4
26.3
47.6
37.8
36.2
IR+SS
38.5
Mean
46.2
38.0
40.2
41.1
36.2
40.3
** Increase %
30.4
29.8
-44.8
12.7
0.0
IR
29.8
12.4
46.7
23.5
0.0
SS
22.7
-27.3
31.5
4.4
0.0
IR+SS
Mean
27.6
5.0
11.1
13.5
0.0
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
4.9.2.2. Salicylic acid and riboflavin
The data in Tables (27b) and Fig., (39b) indicated that, the activity of
peroxidase (POD) enzyme (420/min/g fresh weight) was affected differently by
tested application methods, inducer treatments as well as by the interaction in
between. The SS method recorded the highest average of POD activity (79.5)
followed by the IR+SS method (53.5) and the IR method (50.4), respectively.
Regardless application methods, SA at 0.1mM recorded the highest increase
(636.5%) followed by R at 10.0mM (432.8%), R at 0.1mM (363.2%) and SA at
10.0mM (331.4%), respectively comparing to the untreated control. The POD activity
was increased by all tested interactions. In this respect, SS/SA at 0.1mM increased
POD activity by 10 folds over than the untreated control followed by IR+SS/SA at
0.1mM (6.69 times), SS/R at 0.1mM (5.71 folds) and SS/R at 10.0mM (5.41 folds)
while the remained interactions increased the POD activity by 2.23 to 4.57 times over
that recorded by the untreated control.
101
Table 26b - Activity of PPO enzyme (430/min/g fresh weight) in tomato leaves as
affected by inducer treatments (salicylic acid and riboflavin at 0.1 & 10.0mM conc.)
using application methods after two months from inoculation with FOL isolate A
Polyphenoloxidase activity (430/min/g fresh
Application
weight)
Contro Mea
method
l
n
Salicylic acid at
Riboflavin at
0.1mM
10.0Mm
0.1mM
10.0Mm
45.3
67.7
44.6
62.1
36.2
IR*
51.2
42.0
45.8
70.3
66.0
36.2
SS
52.1
53.8
35.1
31.9
40.8
36.2
IR+SS
39.6
Mean
47.0
49.5
48.9
56.3
36.2
47.6
** Increase %
25.1
87.0
23.2
71.5
0.0
IR
16.0
26.5
94.2
82.3
0.0
SS
48.6
-3.0
-11.9
12.7
0.0
IR+SS
Mean
29.9
36.8
35.2
55.5
0.0
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
102
Fig. 38a - Activity of PPO enzyme in
tomato leaves as affected by inducer
treatments (garlic and black pepper
extracts at 0.5 & 4.0% conc.) using
application methods after two months
from inoculation
with FOL isolate A
Fig. 38b - Activity of PPO enzyme in
tomato leaves as affected by inducer
treatments (salicylic acid and riboflavin
at 0.1 & 10.0mM conc.) using application
methods after two months from
inoculation with FOL isolate A
Table 27a - Activity of POD enzyme (420/min/g fresh weight) in tomato leaves as
affected by inducer treatments (garlic and black pepper extracts at 0.5 & 4.0% conc.)
using application methods after two months from inoculation with FOL isolate A
Peroxidase activity (420/min/g fresh
weight)
Application
Garlic extract
Black pepper extract Control Mean
method
at
at
0.5%
4.0%
0.5%
4.0%
36.4
21.7
42.9
20.7
13.5
IR*
27.0
69.0
100.0
67.2
57.9
13.5
SS
61.5
61.1
40.0
67.7
93.9
13.5
IR+SS
55.2
Mean
55.5
53.9
59.3
57.5
13.5
47.9
** Increase %
169.6
60.7
217.8
53.3
0.0
IR
411.1
640.7
397.8
328.9
0.0
SS
352.6
196.3
401.5
595.6
0.0
IR+SS
Mean
311.1
299.3
339.0
325.9
0.0
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
103
Table 27b - Activity of POD enzyme (420/min/g fresh weight) in tomato leaves as
affected by inducer treatments (salicylic acid and riboflavin at 0.1 & 10.0mM conc.)
using application methods after two months from inoculation with FOL isolate A
Peroxidase activity (420/min/g fresh
Application
weight)
Contro Mea
method
l
n
Salicylic acid at
Riboflavin at
0.1mM 10.0Mm 0.1mM
10.0Mm
45.9
63.9
53.4
75.2
13.5
IR*
50.4
148.5
58.2
90.6
86.6
13.5
SS
79.5
103.9
52.6
43.6
54.0
13.5
IR+SS
53.5
Mean
99.4
58.2
62.5
71.9
13.5
61.1
** Increase %
240.0
373.3
295.6
457.0
0.0
IR
1000.0
331.1
571.1
541.5
0.0
SS
669.6
289.6
223.0
300.0
0.0
IR+SS
Mean
636.5
331.4
363.2
432.8
0.0
* IR = immersing roots, SS = spraying shoots
** Increase (%) = (treatment – control)/control X 100
Fig. 39a - Activity of POD enzyme in
tomato leaves as affected by inducer
treatments (garlic and black pepper
extracts at 0.5 & 4.0% conc.) using
application methods after two months
from inoculation with FOL isolate A
Fig. 39b - Activity of POD enzyme in
tomato leaves as affected by inducer
treatments (salicylic acid and riboflavin
at 0.1 & 10.0mM conc.) using application
methods after two months from
inoculation with FOL isolate A
4.10. Anatomical studies
Out of 18 anatomical characters investigated in tomato leaf petiole, 10, 5 and 7
characters in case garlic extracts, 14, 7 and 12 characters in case of black pepper
extracts were positively changed in the IR, SSS and IR+SS application methods,
respectively comparing to the untreated control (Tables 28 and Photo, 2). Number of
104
xylem vessels (NXV) in the vascular bundle as well as the width of the vascular
bundle (WVB) seemed to be correlated with the resistance against the Fusarium wilt
disease more than any other investigated anatomical structures of the tomato leaf
petiole. NXV recorded 80, 46 and 36 (in garlic extracts) and 65, 40 and 39 µm (in
black pepper extracts) for the IR, SS, and IR+SSS application methods recorded,
respectively comparing with NXV 32 in the untreated control. However, the WVB
recorded 1496.7, 968.4 and 1244.3 µm (in garlic extracts) and 1188.9, 1656, 1620.0
µm (in black pepper extracts) for the three application methods, respectively
comparing with 893.7 µm in case of the untreated control.
Table 28a - Effect of garlic extract used for immersing (I) roots, spraying (S) shoots
and I+S of tomato seedlings on the anatomical structure of leaf petiole after two
months treatment and inoculation with the tomato Fusarium wilt pathogen isolate A
Garlic extract
Black pepper extract
Contr
Anatomical character
IR+S
ol
IR
SS
S
11.7
13.5 11.7
10.8
13.5
11.7
Cuticle thick. (µm)
13.5
36.9
Epidermal layer thick.
35.1* *
37.8* 35.1* 22.7
36.9* 28.8
(µm)
Number of chollenchyma
3.5
4.0
3.5
5.5*
5.0
4.5
layers
5.0
180.
Chollenchma layers thick.
180.9 0
207.9 211.1 191.7 202.5 216.5
(µm)
Number of parenchyma
6.0*
4.0
4.0
5.0*
5.5*
5.5*
layers
4.0
331.2
Parenchyma layers thick. 367.2 331. 336.6 392.4
*
2*
*
*
286.2 *
(µm)
304.2
548.1 511. 544.5 603.5
533.7
*
2
*
*
477.9 *
Cortex thick. (µm)
520.7
111.
Outer phloem thick. in
(1)
71.1
6*
61.7
99.9* 81.0* 52.2
V.B. (µm)
74.7
Cambium thick. in V.B.
72.0* 39.6 45.9
59.4* 56.7
60.3* 59.0
(µm)
307.
501.3 482.4
395.6 *
*
429.3 446.4
Xylem thick. in V.B. (µm) 435.6 8
46.0
Number of xylem vessels in
80.0* *
36.0* 65.0* 40.0* 39.0* 32.0
V.B.
105
Largest vessels thick. in
V.B. (µm)
Inner phloem thick. in
V.B. (µm)
Length of Vascular Bundle
(µm)
Widest of V.B. (µm)
Number of pith layers
Pith layers thick. (µm)
75.4*
101.7
*
680.4
*
1496.
7*
14.0
1136.
7
3687.
3
65.7
78.3*
52.2
511.
2
968.
4*
17.0
1248
.3
3393
.9
86.0*
589.1
1244.
3*
13.0
1350.
0
3716.
1
66.6
117.0
*
777.6
*
1188.
9*
22.0
1710.
0*
4563.
9*
Whole section thick. (µm)
(1)
V.B. = Vascular bundle
* Positive changed character comparing to the control
54.9
93.6*
68.0
72.0*
692.1
*
1656.
0*
16.0
1287.
0
3699.
4
79.7*
64.8
621.5
1620.
0*
24.0*
2016.
0*
4423.
6*
644.9
893.7
22.0
1531.
8
3947.
4
Untreated control
Photo 2 - Anatomical structure of tomato leaf petiole after two months from
immersing (IR) roots, spraying (SS) shoots, and IR+SS of tomato transplants with
extracts of garlic (left) and black pepper (right) and inoculation with FOL isolate A.
Control (untreated)
5 DISCUSSION
Fusarium oxysporum is an abundant saprophyte in soil and organic matter and
occurs worldwide in the rhizosphere of many plant species. Two formae speciales of
this fungus are known to affect tomato, a crop plant of great economic importance.
Fusarium oxysporum f.sp. lycopersici (Fol) is the cause of a severe wilt disease,
whereas F. oxysporum f.sp. radicis-lycopersici (Forl) causes crown and root rot
[104]. Fusarium will of tomato caused by the vascular wilt pathogen Fusarium
oxysporum Schlechtend. Fr. f. sp. lycopersici (Sacc.) W. Q Snyder & H. N. Hans., is
a devastating disease that occurs in major tomato-growing regions of the world [209]
and [177]. Tomato wilt become one of a limiting factor in the production of
106
tomato and accounts for yield losses annually. It has become one of the most
prevalent and damaging diseases wherever tomatoes are grown intensively
because the pathogen persists indefinitely in infested soils. The use of resistant
varieties is the best strategy for disease control [184] and [177].
In the present work, nine isolates of Fusarium oxysporum were isolated from
tomato 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 threeseptate spores are more common. Chlamydospores, both smooth and rough walled,
are abundant and form terminally or on an intercalary basis. They are generally
solitary, but occasionally form in pairs or chains. The tested Fusarium oxysporum
isolates were significantly varied in their colony diameter and sporulation capacity.
Fusarium oxysporum is a soilborne fungus that includes both nonpathogenic and
pathogenic strains. Nonpathogenic strains of Fusarium oxysporum colonize the
cortex of plant roots without causing disease symptoms, whereas pathogenic strains
can move past the cortical tissue and invade the vascular tissue of susceptible hosts,
causing vascular wilt diseases. These pathogenic strains show a high level of host
specificity and are subdivided into formae speciales based on the plant species
attacked and into races based on the host cultivars attacked. F. oxysporum strains
pathogenic on tomato plants, Lycopersicon esculentum Miller) are classified into two
formae speciales, f. sp. lycopersici causing the vascular wilt disease of tomato and f.
sp. radicis-lycopersici causing Fusarium crown and root rot [20] and [79].
Plants of tomato cultivar Carolina Gold inoculated with most isolates of
Fusarium oxysporum manifested different degrees of wilt after 2 months from
inoculation. The vascular bundle of infected tomato plant showed dark lines in both
sides. Appearance of wilt symptoms and vascular discoloration in both stem and roots
of the inoculated tomato plants emphasized identification of tested isolates as
Fusarium oxysporum f. sp. lycopercici [10]. The used tomato cultivar Carolina Gold,
whoever, was reported to be resistant against Fusarium wilt race 1 and race 2 [114].
This browning of the vascular tissue is characteristic of the tomato Fusarium wilt
disease caused by F. oxysporum f. sp. lycopersici (FOL) and can be used for its
tentative identification [139]. The resistance of this tomato cultivar might be
overcome by new race of FOL. Many of the commercial tomato cultivars with
resistance to FOL race 1 planted in Taiwan displayed Fusarium wilt symptoms. The
vascular tissue was usually dark brown and discoloration extended to the apex. The
wilting became more extensive until plants collapsed and died [177]. Tomato plants
(cultivar Carolina Gold) inoculated with most isolates of the Fusarium wilt
particularly isolates A and G showed significant decreases in their growth characters
and weight of fruit yield/plant comparing with the non-inoculated control plants. The
plant height, fresh weight of stem and roots/plant of tomato plants were obviously
higher in the non-inoculated than those inoculated with the tomato wilt pathogen. The
107
tomato plants inoculated with pathogen gave lower total fruit weight per plant than
control (un-inoculated) plants [180].
The uses of resistant cultivars or rootstocks are the most reliable way to prevent
the diseases. In the present, the tested commercial and experimental tomato cultivars
were responded differently against inoculation with Fusarium oxysporum f. sp.
lycopersici (FOL). In this respect, the tomato cultivars Carolina Gold was the most
susceptible as it recorded the highest disease severity followed by Dona, EXP 4, EXP
5, and EXP 3. However, EXP 1 and EXP 2 cultivars were the most resistant against
FOL infection as they remained wilt disease-free looks like non-inoculated plants. In
fact, the FOL has three physiological races (1, 2, and 3) and are distinguished by their
specific pathogenicity on tester plants carrying dominant race-specific resistance
genes [41] and [171]. After reporting race 1 and race 2 [13], race 3 of FOL was
determined in Australia in 1978 [41] and [76]. The followed studies conducted in
several U.S.A. states and Mexico showed that the lack of I-genes, conferring
resistance to FOL races in commercially cultivated tomatoes, were concerned with
plant susceptibility [49]. Resistant tomato plants with the I-gene against race 1 were
used to control the disease in breeding varieties [31] and [82]. However, race 2
overcame the resistance of race 1-resistant cultivars, as reported in Korea and in
Ohio, USA [203]. Race 3 was also observed in Australia and Florida [82]. Resistance
to race 3 in Lycopersicon pennellii genotype harboring the I3 gene was found [132]
and [159]. Races of FOL could be distinguished by their differential virulence on
tomato cultivars containing different dominant resistance genes [132]. Therefore, the
use of resistant varieties is suggested as the best strategy for disease control,
compared to biological control measurements [12]. The 3 FOL physiological races
are distinguished by their specific pathogenicity to different tomato cultivars. But
[96] showed that, even if an isolate shows the properties of a specific race, it can
genetically be different and may have the tendency to change its genetic properties.
Race1 and race2 are distributed throughout the world, whereas race3 has a more
limited geographic distribution [95]. In addition to formally report about the presence
of FOL races, some genetic changes on the pathogen in Turkey are also been
reported. All investigated growth characters and fruit yield/plant of most tested
tomato cultivars were significantly lower in inoculated than un-inoculated plants
particularly Carolina Gold and Dona cultivars. The yield (weight of fruits/plant) of
plants inoculated with FOL was significantly lower than the healthy un-inoculated
plants [109] and [33]. The fresh weight was significantly lower in the wilted tomato
plants than healthy (un-inoculated) ones [215]. In term of weight of fruit yield/plant,
the moderately resistant EXP4 was significantly better than the high resistant
cultivars EXP1 or EXP2 then EXP 4 could be subjected to further tests to introduce it
as new commercial cultivar in Kazakhstan.
The present investigation aimed to evaluate garlic extract, black pepper extract as
natural inducers and salicylic acid and riboflavin as chemical inducers on the in vitro
growth and sporulation of the most virulent isolate of Fusarium oxysporum f.sp.
lycopersici (FOL). The in vitro growth and sporulation of tested FOL isolate was
completely inhibited by the aqueous garlic (G) extract at concentration ≥ 2%,
riboflavin (R) and salicylic acid (SA) at concentrations of 5 and 10 mM, respectively
108
while, the black pepper (BP) extract was the least effective in this respect as the fungus
could grow and produced appreciable number of spores even at its highest tested
concentration (4.0%). On the opposite side, the in vitro FOL growth was relatively
enhanced while its sporulation was reduced by by R at 0.1 and 0.5 mM. These results
suggested that some of tested inducer treatments might be beneficial for inducing
resistance against tomato wilt caused by Fusarium oxysporum f.sp. lycopersici. In fact,
the inhibitory effects of several plant extracts and safe chemicals on the in vitro growth
of different plant pathogens were investigated by many investigators. It was found that
sclerotial germination of onion pathogen was less after soaking in salicylic acid than in
either phenol or garlic acid. Increasing concentration of the phenolic compounds in the
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 and
phemol [50 and 100 ppm] [170].
The antimicrobial activity of different concentrations [50, 100, 200, 300 and
500 ml/l] of essential oil extracts of three type of onions and garlic against two
bacteria, Staphylococcus aureus, Salmomella enteritidis, and three fungi, Aspergillus
niger, Penicillium cyclopium and Fusarium oxysporum. With garlic extract, high
inhibitory activity was observed for all tested concentrations. The fungus F.
oxysporum showed the lowest sensitivity towards essential oil extracts, whereas A.
niger and P. cyclopium were significantly inhibited particularly at low
concentrations. Conclusively, where seasoning is desired, essential oil extracts of
onions and garlic can be used as natural antimicrobial additives for incorporating in
various food products [28]. The garlic extract was the most effective against
Fusarium oxysporum f. sp. lycopersici [1]. Garlic extracts decreased sporulation of
Fusarium oxysporum f. sp. lycopersici with increasing concentration from 5% to 30%
[9]. The extract of garlic partially inhibited the growth of A. tamarii and P. commune.
However, it inhibited completely the growth of P. implicatum and E. nidulans at the
doses of 0.5 and 1%. Oregano partially inhibited all mould species, significantly
reducing the growth of colonies, especially of E. nidulans [93.3%] [55].
Garlic (G) and black pepper (BP) extracts at 0.5 & 4.0% concentrations,
salicylic acid (SA) and riboflavin (R) at 0.1 & 10.0mM concentrations were used for
controlling Fusarium wilt disease under glasshouse conditions. Three application
methods namely immersing root (IR), spraying shoots (SS) and IR+SS methods were
used for treating tomato seedling (Carolina Gold cv.) with each inducer treatment. All
treatments were carried out before inoculation with FOL. Effects of tested natural and
chemical inducers on % wilted plants, wilt disease severity (DS), their impacts on
certain plant growth parameters and biochemical changes (phenols, total soluble
protein, chlorophyll, activity of certain oxidative enzymes) were determined.
As for plant extracts, the highest concentration (4.0%) of G and BP was
significantly better than the lowest concentration (0.5%) for reducing % wilted plants
and wilt disease severity (DS) comparing to the untreated inoculated control.
However, G at 0.5 and 4.0%, BP at 0.5 and 4.0% increased plant height, number of
leaves/plant, fresh weight of leaves/plant, dry weight of leaves/plant, stem fresh
weight/plant, root fresh weight/plant, root dry weight/plant, root length, root volume,
weight of fruit yield/plant comparing to the untreated control. Similarly, SA at 0.1
109
and 10.0mM and R at 0.1 and 10.0mM decreased % wilted plants and wilt disease
severity meanwhile increased plant height, number of leaves/plant, fresh weight of
leaves/plant, dry weight of leaves/plant, stem fresh weight/plant, root fresh
weight/plant, root dry weight/plant, root length, root volume, weight of fruit
yield/plant comparing to the untreated inoculated control. These results declared that
the tomato plants treated with G or BP extracts at 0.5 and 4.0% concentrations or SA
or R at 0.1 or 10.0mM concentrations rendered plants healthier then their vegetative
growth parameters and fruit yield production were increased.
Regarding the interactions between application method and different inducer
treatments, the present results indicated that, IR/G and SS/G at 4.0% and SS/BP at
4.0% completely prevented wilt disease infection followed by IR/SA at 0.1mM,
IR/SA at 10.0mM, SS/SA at 10.0mM and IR+SS/R at 10.0mM, IR/R at 10.0mM,
IR+SS/R at 0.1mM and IR+SS/SA at 10.0mM, respectively comparing with the
control. However, the investigated vegetative plant growth parameters were fond to
be responded differently against tested method/inducer treatment interactions. For
example, IR+SS/BP at 4.0 and IR/G at 0.5% were the best for increasing the plant
height and number of leaves/plant while, IR/BP at 0.5%, SS/G at 4.0% and SS/BP at
0.5% in case of plant height and IR/G at 0.5% in case of number of leaves/plant were
not significantly varied when compared with their respective untreated control. Also,
IR/SA at 0.1mM resulted in significant increase in plant height whereas IR/R at
0.1mM, SS/R at 10.0mM and IR+SS/SA at 0.1mM showed no significant effects on
the plant height when compared with the untreated control. Also, using IR+SS/R at
10.0mM recorded the highest increase in the number of leaves/plant followed by
SS/SA at 0.1mM while, IR/R at 0.1mM recorded the lowest significant increase
comparing to the control. All tested method/treatment interactions significantly
increased the root fresh weight/plant and weight of fruit yield/plant compared to the
untreated control. The highest significant increase in fruit yield/plant was produced
by IR+SS/BP at 0.5% followed by SS/BP at 4.0%, IR/G at 4.0% and SS/G at 4.0%
comparing to the untreated control which produces only 135 g per plant.
In fact, the plant natural resistance to potential parasites is regulated by two
fundamental mechanisms: the “nonhost” and the “gene-for-gene” resistance,
respectively. The latter is relevant when a cultivar resistant (R) gene product
recognizes an avirulence gene product in the attacking pathogen and triggers an array
of biochemical reactions that halt the pathogen around the site of attempted invasion.
To cope with virulent pathogens, plants may benefit by some temporary immunity
after a challenge triggering such an array of defense reactions, following a localized
necrotizing infection as a possible consequence of a hypersensitive response (HR).
This process, mediated by accumulation of endogenous salicylic acid (SA), is called
systemic acquired resistance (SAR) and provides resistance, to a certain extent even
against unrelated pathogens, such as viruses, bacteria, and fungi, for a relatively longlasting period. SAR may be more potently activated in plants pretreated with
chemical inducers, most of which appear to act as functional analogues of SA. This
review summarizes the complex aspects of SAR as a way to prevent crop diseases by
activating the plants' own natural defenses. The following outline is taken: (1)
introduction through the historical insight of the phenomenon; (2) oxidative burst,
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which produces high levels of oxygen reactive species in a way similar to the
inflammation state in animals and precedes the HR to the pathogen attack; (3) SAR
as a coordinate action of several gene products leading to the expression of defenses
well beyond the time and space limits of the HR; (4) jasmonic acid (JA) and ethylene
as other endogenous factors mediating a different pahway of induced resistance; (5)
pathogenesis related proteins (PR proteins) de novo synthesized as specific markers
of SAR; (6) exogenous inducers of SAR, which include both synthetic chemicals and
natural products; (7) the pathway of signal transduction between sensitization by
inducers and PR expression, as inferred by mutageneses, a process that is still, to a
large extent, not completely elucidated; (8) prospects and costs; (9) final remarks on
the state-of-the-art of the topic reflecting the chemical view of the author, based on
the more authoritative ones expressed by the authors of the reviewed papers [80].
Because of hazards of pesticides in general, and fungicides in specific, on
public health and environmental balance, a relatively recent direction of pest control
management was introduced. The so called "induced resistance" is a promising
modern approach with a broad spectrum in plant disease control. It could be induced
in plants by applying chemical elicitors [157]. Chemical elicitors (inducers) have
been used to predispose the defense mechanisms in plants against diseases. Several
investigators [199], [216], [56] and [8] and many others used different inducers like
salicylic, benzoic, citric and oxalic acids beside ribavirin. On the other hand, induced
resistance may also affect other growth parameters; chlorophyll content, plant
growth, accumulation of antifungal compounds and increasing activity of oxidative
enzymes [183], [90], [216], [125] and [70]. Induced systemic resistance (ISR) of
plants against pathogens is a widespread phenomenon that has been intensively
investigated with respect to the underlying signalling pathways as well as to its
potential use in plant protection. Elicited by a local infection, plants respond with a
salicylic-dependent signaling cascade that leads to the systemic expression of a broad
spectrum and long-lasting disease resistance that is efficient against fungi, bacteria
and viruses. Changes in cell wall composition, de novo production of pathogenesisrelated-proteins such as chitinases and glucanases, and synthesis of phytoalexins are
associated with resistance, although further defensive compounds are likely to exist
but remain to be identified [92]. Treating tomato plants infected by Fusarium
oxysporum by spraying 3 times with JA and SA significantly reduced % of disease
incidence. JA had the highest effective (92% efficiency). Growth rate (shoot and
root) markedly inhibited in tomato plants in response to Fusarium wilt disease as
compared with healthy control. JA and SA were more pronounced in increasing
tomato growth especially when applied together. Reduction in total chlorophyll in
infected leaves significantly decreased in plants treated with SA. Also, total soluble
sugars, free amino acids and total soluble proteins increased in both leaves and roots
of JA or SA- treated plants as compared with infected control. Results revealed that
induction in the uptake of nutrients could be responsible for increasing susceptibility
of tomato plants to Fusarium wilt disease. On the other hand, infection with F.
oxysporum markedly altered hormonal balance (IAA, GA, ABA, zeatin and zeatinriboside) in leaves and roots of tomato plants. Thus, ABA was accumulated while
levels of IAA, GA and cytokinins markedly reduced in infected plants. Actually,
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results revealed that increase in disease incidence and decrease in growth of Fusarium
- tomato plants could be a morphological expression of the hormonal imbalance [64].
The induced resistance is a non-specific form of disease resistance in plants that
can act against a wide range of pathogens, and can be activated by several non specific
inducers, known also as elicitors [204]. Some elicitors have systemic activity
(induction of generalized resistance) while others only induce local resistance, at the
site of application [148]. There are now a variety of commercial resistance inducers
available, some of which are registered in Italy for use on grapevine as plant protectant
products. Efforts to make this type of resistance useful have taken several routes. One
approach has been to search for chemicals (both natural and synthetic) that can activate
disease resistance in plants just like a pathogen infection. These compounds, several of
which have been tested on vegetable crops, are not directly toxic to pathogens, but
rather cause expression of the genes that plants naturally use for defense against
infection. In studies at MSU, several synthetic resistance-activating compounds have
been used under field conditions to induce resistance in cucumber against angular leaf
spot and in soybean against white mold. These results are encouraging, as they have
demonstrated the potential to use induced resistance to control disease.
Actually, the natural plant extracts may provide an alternative to fungicides.
Allium genus revered to possess anti-bacterial and anti-fungal activities and include
the powerful antioxidants, sulfur and other numerous phenolic compounds which
arouse significant interests [30], [200], [213], [151], [91], [117], [163], [83], [28],
[85] and [217]. The inhibitory activity of garlic [Allium sativum L.], onion [Allium
cepa L.] and leek [Allium porrum L.] extracts [aqueous, acetone and ethyl alcohol]
against mould has been reported by numerous authors. It has also been observed that
alliicin, thiosulfonates and other compounds show fungistatic activities against
several fungi [210], [81], [200], [85], [18] and [91]. Similarly, ajoene compound
which is a derivative of alliicin and obtained from garlic with ethyl alcohol extraction
is very inhibitory against A. niger, Candida albicans and Paracoccidioides
brasiliensis [144]. Ajoene compound from garlic have stronger antifungal activity
than alliicin. Ajoene damages the cell walls of fungi [214]. Activity of the garlic
extract may be due to sulfur-containing compounds such as ajoene or allicin. Sprays
with the aqueous garlic extracts have antibiotic and antifungal properties and will
suppress a number of plant diseases, including powdery mildew on cucumbers and, to
some extent, black spot on roses. Garlic extracts controlled diseases such as mildew,
rusts, fruit rots, blights, and black spot [154]. Activity may be due to sulfurcontaining compounds such as ajoene or allicin. Garlic releases fungicidal chemicals
into the soil. Garlic extract shows high inhibitory activity against Aspergillus niger,
Penicillium cyclopium and Fusarium oxysporum for all tested concentrations i.e. 50,
100, 200, 300 and 500 ml/l [28].
The effect of crude extracts of neem [Azadirachta indica] leaf, neem seed and
garlic [Allium sativum] at concentrations ranging from 5% to 30% of the material was
assessed in 100 ml of Potato Dextrose Agar on mycelial growth of Fusarium
oxysporum f. sp. lycopersici. All the extracts inhibited mycelial growth at various
levels. The garlic extracts decreased sporulation with increasing concentration and
cultures grown on extract amended agar plates remained viable [9]. The acetone
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extracts of the bark-compost possessed strong antifungal activity against Fusarium
oxysporum f. sp. cucumerinum [106]. The Fusarium wilt disease on many crops could
be controlled by using plant extracts. Infection with Fusarium wilt disease (Fusarium
oxysporum) on Papaya seedlings may reach about 70% in some nurseries. This
disease was controlled by using plant extracts of three plants species; neem
[Azadirachta indica], masqit [Prosopis jaliflora], and milk–giant [Calotropis
procera]. The effect of aqueous extract of these plant extracts on fungal growth was
tested under laboratory conditions. The effect of adding the extracts to the soil
planted with infected seedlings was also tested under greenhouse conditions. The
results indicated that plant extract inhibited the fungal growth which reached 56.9,
55.8 and 77.0% with milk-giant, neem and masqit, respectively. The extract of milkgiant gave a better effect than other extracts. The results also showed that plant
extracts decreased disease incidence of the seedlings to 9.2% for milk-giant; 17% for
masqit and 18.6% for neem. This data emphasized the efficiency of plant extracts in
controlling the papaya wilt disease due to the toxic effect of sulfur and the amino
acids content of the leaves on the fungus [166].
Also, several biologically important phytochemicals have been extracted from
Piper nigrum plants [137], [112] and [25]. Alkaloids in fruits of P. nigrum ranges
from 4 to 5% [53]. The combinations of extracts of pepper and mustard, the cassia
extract alone and the essential oil of clove suppress the development of Fusarium
oxysporum in melon [34]. Plant extracts of six plant species, cloves [Dianthus
caryophyllus], cinnamon [Cinnamum zeylamicum], thyme [Thymus vulgaris L.]
fenugreek [Trigonella fonicum], amme [Ammi visnagal], black pepper [Piper nigrum]
and three essential oils, geranium [Pelargonium gravedens], black cumin seeds
[Nigella sativa L.] and blue gum [Eucalyptus globulus] were evaluated for their
antifungal effect on the mycelial growth, incidence and disease severity of onion neck
rot disease [Botrytis allii]. The antifungal properties of clove extract were more
effective than black pepper on inhibiting mycelial growth and disease incidence [4].
Aqueous extracts of 15 plant species were tested against onion white rot fungus
Sclerotium cepivorum that was grown in potato dextrose agar culture. Eeach extract
presented a fungicidal effect, at a concentration of 5%, when applied on allspice
[Pimenta dioica] and clove [Syzygium aromaticum]. Only clove extract retained its
effect at a concentration of 1%, while allspice lost it at 3%. Cinnamon [Cinnamomum
zeylanicum] and yam bean [Pachy erosus] extracts produced total inhibition of
sclerotial production besides a poor mycelial growth. Different types of interactions
were present when the extracts were mixed: all combinations presented a lost of
fungicidal effect [antagonistic effect], including allspice extract; a retained fungicidal
effect [single fungicidal effect] occurred in most clove mixtures and in the
combination of clove and black pepper [Piper nigrum] the retained fungicidal effect
was even below the minimal lethal dose [synergistic effect]. The combination of
extracts showed that the effect of each plant extract could be modified by the
reactions of the complex mixture of plant compounds [140].
Piper nigrum, commonly known as ``Black-pepper``, has gained a global
consideration because of its volume in the spice industry. This plant has shown great
potential for the discovery of novel biologically active compounds and need for
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techniques to enhance the production of high quality consistent plant material for
feasible accumulation of metabolites [2]. Infection with FOL significantly reduced
the crop yield and quality. Several plant extracts were found to be highly effective on
different isolates of Fusarium wilt in the laboratory, and were tested with other
control methods on two tomato varieties artificially inoculated with the Fusarium wilt
fungus. Results showed that these extracts reduced wilt infection rate 49 days after
planting on both tested varieties. The most effective treatment after the fungicide
Tachigaren was garlic extract [184] and [6]. The fresh weight of plant stem, number
and weight of tomato fruits were significantly lower in tomato plants inoculated than
those non-inoculated with the wilt pathogen “Fusarium oxysporum f.sp. lycopersici”
[180]. The antifungal properties of cloves extract was more effective than black
pepper on inhibiting mycelial growth and disease incidence of onion neck rot disease
caused by Botrytis allii [4]. [140] tested the aqueous extracts of 15 plant species
including black pepper [Piper nigrum] against onion white rot fungus Sclerotium
cepivorum that was grown in potato dextrose agar culture. Each extract presented a
fungicidal effect, at a concentration of 5%. Piper nigrum (Black-pepper), shown great
potential for the discovery of novel biologically active compounds and need for
techniques to enhance the production of high quality consistent plant material for
feasible accumulation of metabolites [2].
As for safe chemicals, riboflavin which is known as vitamin B2 might promotes
fungal growth at low concentrations. Both riboflavin and nicotinic acid accelerated
the accumulation of carbohydrates and fat in the mycelium. Nicotinic acid and to a
less extent riboflavin enhanced sugar and nitrogen absorption and the rate of building
up of cellular material in consequence. Both riboflavin and nicotinic acid accelerated
the accumulation of carbohydrates and fat in the mycelium [145]. Riboflavin caused
a fungicidal reaction to all the fungi tested in Czapek's medium containing Lmethionine. The fungicidal effect of the riboflavin-methionine-light combination
occurred at concentrations of riboflavin and methionine less than 1.33 μM and 0.5
mM, respectively. Riboflavin at 1.0 mM concentration did not affect the growth of
pathogens causing chickpea Fusarium wilt and charcoal rot diseases [169]. SA
completely inhibited the mycelial development of FOL in vitro at concentrations
from 0.6 mM to 1.0 mM [201] and [147]. The mycelial growth of FOL was not
significantly affected by salicylic acid [127].
The mechanism of riboflavin-IR and defense responses in rice against
Rhizoctonia sheath diseases was studied by [195]. Riboflavin-IR can be linked to the
induction of defense pathways leading to formation of structural barriers such as lignin
in rice plants. Using riboflavin as a plant defense activator can be a new, simple, and
environmentally safe strategy to control Rhizoctonia sgeath diseases of rice. The
lowest concentration of riboflavin tested (0.01 mM) had the best effect on induction of
resistance against R. solani and R. oryzae-sativae, the causal agents of sheath blight
and sheath spot of rice, respectively. Riboflavin did not have any direct effect on the
growth of fungi in vitro. Also, at concentrations necessary fo induction of resistance
(0.01 to 2 mM), no macroscopic or microscopic cell death in rice was observed [195].
Therefore, riboflavin is able to activate resistance mechanisms in rice, like dicots, in a
hypersensitive response (HR)-independent manner. Production of hydrogen peroxide
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was detected at 12 h after inoculation using DAB staining method. Expression of
cationic rice peroxidase, POC 1, was induced at 18 h after inoculation in riboflavin
treated rice plants. The expression in control plants was lower than that observed in
treated plants. A correlation was found between induction of resistance by riboflavin
and up regulation of DOC 1 gene. A variety of roles have been proposed for the
involvement of peroxidases in the defense response [94]. One possible role is the
generation of reactive oxygen species (ROS) by peroxidase–oxidative activity. The
fact that the production of hydrogen peroxide was upstream of induction of POC 1
gene expression, ruled out the possibility of involvement of POC1 in the generation of
ROS in these interactions. Another possible function of peroxidases in the formation
of structural barriers such as cell wall enhancement and deposition of cell wall
apposition, both of which can be involved in the polymerization of lignin or suberin,
the cross-linking of wall glycoproteins or polysaccharides, and the apposition of
antimicrobial phenols. Lignin was detected in riboflavin treated plants. Therefore,
riboflavin-IR can be linked to the induction of defense pathways leading to formation
of structural barriers in rice plants [129].
The powdery mildew (Sphaerotheca fuliginea Pollacci) infection in cucumber
was significantly reduced by foliar application of a mixture of riboflavin and
methionine (RM). The effects of fungicidal activity on leaves applied with RM were
detected through restriction of progress of colonies and disease severity compared
with control plants. The initial response to foliar application of RM was abrupt
generation of hydrogen peroxide in the leaves of cucumber plants. Activities of
antioxidant enzymes such as SOD and POD were abruptly increased by foliar
application of RM. However, activities of antioxidant enzymes in control plants were
increased with disease development 9 d after pathogen inoculation. Cucumber leaves
have six major SOD isoforms. When plants were foliar-applied with RM, densities of
three SOD isozyme bands at SOD-1, SOD-2, and SOD-3 were increased 3 d after
foliar application. Leaves of cucumber plants have three major POD isozyme bands.
Densities of three POD isozyme bands were increased 3 d after foliar application with
RM. Four major PPO isozyme bands were determined in cucumber leaves. Though
the overall banding patterns of PPO in control and RM-applied plants were similar,
the band profiles in leaves applied with RM were characterized by high densities of
the three major isoforms. Activities of PPO in leaves applied with RM increased
rapidly during the 3 d after foliar application, and then remained relatively constant
for 15 d. Although activities of PPO in the leaves of control plants also abruptly
increased after 9 d, it was lower than those of RM-applied plants during the whole
time. The difference in lignin content between control and RM-applied plants was
detected 9 d after foliar application; it was high in leaves applied with RM [107].
[121] investigated the effects of riboflavin on defense responses and secondary
metabolism in tobacco [Nicotiana tabacum cv. NC89] cell suspensions and the
effects of protecting tobacco seedlings against Phytophthora parasitica var.
nicotianae and Ralstonia solanacearum. Defense responses elicited by riboflavin in
tobacco cells included an oxidative burst, alkalinization of the extracellular medium,
expression of 4 defense-related genes with different kinetics and intensities, and
accumulation of 2 total phenolic compounds, scopoletin and lignin. When applied to
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tobacco plants challenged by P. parasitica and R. solanacearum, riboflavin treatment
resulted in 47.9% and 48.0% protection, respectively. These results suggest that
riboflavin can both induce a series of defense responses and secondary metabolism in
cell suspensions and protect tobacco against P. parasitica and R. solanacearum.
Salicylic acid (SA) is a naturally occurring phenolic in many plants and has been
shown to function as a signal compound initiating plant defense systems in response
to stress, with some reporting responses such as reduced transpiration and enhanced
adventitious root initiation [124]. Soil drenches and foliar applications of SA resulted
in enhanced tolerance of bean (Phaseolus vulgaris L.) and tomato (Lycopersicon
esculentum Mill.) to heat, chilling, and drought stresses [175]. Foliar application of
SA before 48 h of exposure to 40°C followed by 3 d of UV-B stress resulted in 30%
greater maintenance of canopy photochemical activity of Agrostis palustris Huds
[174]. The effect of salicylic acid [SA] and Glomus etunicatum [GE] on plant
development of tomatoes and infection potential of wilt disease Fusarium oxysporum
f.sp lycopersici [FOL] were studied. The effects of different SA concentrations on
mycelial development of FOL were tested in vitro and two concentrations of SA and
GE were included in pot experiment. SA completely inhibited the mycelial
development of FOL in vitro at concentrations from 0.6 mM to 1.0 mM and ED50
value was found as 0.51 mM. GE could increase dry weight of plant, length of shoot
and root growth irrespective whether FOL infected the tomato plants. The root
colonization by GE was determined as 62.3% when the FOL was absent and as
53.2% when the plants were infected. However, in different combinations of GE and
SA, the root colonization was determined between 19.1 and 34.2%. In pot
experiments, the combination of GE and 1 mM SA had the highest effect on infection
of Fusarium wilt and disease severity was reduced by 70%. Results indicate that GE
increases the growth of tomato plants, and could be used against Fusarium wilt of
tomato [147]. While SA is effective against the pathogen, the root colonization of GE
is, however, affected negatively by SA. Soaking sesame seeds in filtrated and
autoclaved garlic extracts decreased the charcoal rot disease severity to 3.3 and
20.0% and increased the healthy plants to 83.3 and 33.3%, respectively compared
with check [soaked in water] which recorded 23.3 and 26.7% for both parameters,
respectively. Soaking sesame seeds in 2, 4 and 8mM salicylic acid decreased charcoal
rot rotted sesame plant to 3.3, 0, 0% and increase healthy plants to 90, 96.7 and 90%,
increased peroxidase activity to 1.97, 1.44 and 1.18, polyphenoloxidase activity to
1.51, 1.36 and 1.26 and catalase activity to 2.7, 2.6 and 2.46 comparing to check
plants which recorded 0.63, 0.62 and 1.83 for the three oxidative enzymes
respectively. Also, the free phenols increased to 13.3, 10.7 and 9.6 mg/gm fresh
weight, conjugated phenols to 3.7, 0.4 and 0.6 mg/gm fresh weight and total phenols
to 17.0, 11.1 and 10.2 mg/gm fresh weight at the 3 SA concentrations, respectively
compared with 6.2, 0.6 and 6.8 in check plants [61].
The influence of salicylic acid (SA) doses of 50 and 250 μM, for a period of up
to 7 days, on selected physiological aspects and the phenolic metabolism of
Matricaria chamomilla plants. SA exhibited both growth-promoting (50 μM) and
growth-inhibiting (250 μM) properties, the latter being correlated with decrease of
chlorophylls, water content and soluble proteins. In terms of phenolic metabolism, it
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seems that the higher SA dose has a toxic effect, based on the sharp increase in
phenylalanine ammonia-lyase (PAL) activity (24 h after application), which is
followed by an increase in total soluble phenolics, lignin accumulation and the
majority of the 11 detected phenolic acids. Guaiacol-peroxidase activity was elevated
throughout the experiment in 250 μM SA-treated plants. In turn, some responses can
be explained by mechanisms associated with oxidative stress tolerance; these mitigate
acute SA stress (which is indicated by an increase in malondialdehyde content).
However, PAL activity decreased with prolonged exposure to SA, indicating its
inhibition. Accumulation of coumarin-related compounds (umbelliferone and
herniarin) was not affected by SA treatments, while (Z)- and (E)-2-β-Dglucopyranosyloxy-4-methoxycinnamic acids increased in the 250 μM SA-treated
rosettes. Free SA content in the rosettes increased significantly only in the 250 μM
SA treatment, with levels tending to decrease towards the end of the experiment and
the opposite trend was observed in the roots [115] and [168].
The intensity and timing of the reactive oxygen species (ROS) formation, lipid
peroxidation and expression of antioxidant enzymes as initial responses of tomato
(Solanum lycopersicum L.) against the invading necrotrophic pathogen Fusarium
oxysporum f. sp. lycopersici were investigated. The concentration of hydrogen
peroxide (H2O2) was 2.6 times higher at 24 h post-inoculation (hpi) and lipid
peroxidation was 4.4 times higher at 72 hpi in the extracts of inoculated roots than in
the control. An increase in total phenolic content was also detected in inoculated roots.
The activities of the antioxidative enzymes, viz., superoxide dismutase (SOD), catalase
(CAT), guaiacol peroxidase (GPX) and ascorbate peroxidase (APX), increased in
response to pathogen inoculation. SOD activity at 48 hpi in inoculated roots was 2.9
times that in the control. CAT activity showed a decrease after 24 hpi and the increase
in activities of GPX and APX was insignificant after 24 hpi in the inoculated roots. The
oxidative burst generated in the interaction between tomato and F. oxysporum f. sp.
lycopersici may be an early first line of defense by the host mounted against the
invading necrotrophic pathogen. However, seemingly less efficient antioxidative
system (particularly the decrease of CAT activity after 24 hpi) leading to sustained
accumulation of ROS and the observed higher rate of lipid peroxidation indicate that
the biochemical events are largely in favour of the pathogen, thus making this host–
pathogen interaction a compatible combination. It is discussed that the oxidative burst
served as a weapon for the necrotrophic pathogen because the antioxidative system
was not strong enough to impede the pathogen ingress in the host [126].
The 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 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. The salicylic acid-treated tomato
plants challenged with FOL exhibited significantly reduced vascular browning and
leaf yellowing wilting. The mycelial growth of FOL was not significantly affected by
salicylic acid. None of the three concentrations of SA tested, viz., 100 μM, 200 μM
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and 300 μM were found to inhibit mycelial growth of FOL significantly as compared
to control. Significant increase in basal level of salicylic acid in noninoculated plants
indicated that tomato root system might have the capacity to assimilate and distribute
salicylic acid throughout the plant. The results indicated that the induced resistance
observed in tomato against FOL might be a case of salicylic acid-dependent systemic
acquired resistance. Tomato plants grown hydroponically were exogenously fed with
SA through roots and leaves, and then challenged with FOL after two days, i.e. 48 h
of last SA application. The percent of vascular browning and leaf yellowing wilting
caused by by FOL on tomato plants was markedly reduced when plants were grown
in presence of 200 μM SA. Tomato plants inoculated with FOL conidia, but not
receiving 200 μM SA treatment through roots, exhibited typical vascular browning
and leaf yellowing wilting, while the SA-treated plants showed less than 25%
vascular browning and leaf yellowing wilting after 4 weeks of the experiment.
Similarly, the foliar application of 200 μM SA on the hydroponically grown tomato
plants significantly affected infection and wilt development by FOL on tomato plants.
The tomato plants inoculated with FOL conidia, but not receiving 200 μM SA
treatment as foliar spray, exhibited characteristic vascular browning and leaf
yellowing wilting, while the SA-treated plants showed less than or equal to 25%
vascular browning and leaf yellowing wilting after 4 weeks of the experiment [127].
In fact, the treatments increased photosynthetic pigments which in turn
increased carbohydrate content in plant tissues. Carbohydrates are the main
repository of photosynthetic energy, they comprise structurally polysaccharide of
plant cell walls, principally cellulose, hemicelluloses and pectin that consider a
barrier against plant pathogens invasion and phenolic compounds are associated with
structural carbohydrates, which play a major and important role in plant defense [87].
In addition, the enhancement in chlorophyll content is resulting from stimulating
pigment formation and increasing the efficacy of photosynthetic apparatus with a
better potential for resistance as well as decreasing photophosphorylation rate, which
occurred after infection [16]. In this connection, the adaptation of plants to biotic and
abiotic stress is due to the stimulation of protective biochemical systems and
synthesis of secondary metabolites such as phenolics [162]. The increase in seed oil
content may be due to the improvement in photosynthetic pigments since there is a
relationship between photosynthesis processes and oil biosynthesis during seed
development in terms of inducing sucrose translocation [187]. It was found that all
tested chemicals decreased damping-off and charcoal rot diseases and at the same
time enhanced the vegetative growth and increased the enzymatic activity, total
phenols and chlorophyll contents. Besides, these chemicals are saving for both
environment and public health.
All tested inducers (plant extracts and safe chemicals) increased
polyphenoloxidase (PPO) activity comparing to the untreated control. Using G
extract at 0.5% recorded the highest increase in the PPO activity followed by BP
extract at 4.0%, BP extract at 0.5% and G extract at 4.0%, respectively. Most
method/plant extract interactions, however, increased PPO activity but few decreased
it. In this respect, SS/BP at 0.5% recorded the highest increase followed by
IR+SS/BP at 0.5%, IR/G at 0.5%, respectively. Also, all safe chemical inducer
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treatments, increased PPO activity, R at 10.0mM was the most effective in this
respect followed by SA at 10.0mM, R at 0.1mM and SA at 0.1mM, respectively. As
for interactions, SS/R at 0.1mM recorded the highest increase followed by IR/SA at
10.0mM, SS/R at 10.0mM, IR/R at 10.0mM and IR+SS/SA at 0.1mM, respectively.
However, only IR+SS/R at 0.1mM, and IR+SS/SA at 10.0mM decreased PPO
activity compared to the untreated control.
Many plant phenolic compounds are known to be antimicrobial, function as
precursors to structural polymers such as lignin, or serve as signal molecules [89].
Significant increase in phenolic content was positively proportional to the degree of
plant resistance against the pathogens, [5]. Results in the present study clearly
indicate that the formation of total soluble phenols was slowed down in infected
control compared with plants which were inoculated with FOL and treated with
different inducer treatments. However, there was a greater reduction in the phenolic
content in plants infected with Fusarium oxysporium f. sp. lycopersici (FOL).
Application of riboflavin or salicylic acid treatments stimulated the formation of free,
conjugated and total soluble phenols. All phenol fractions markedly increased in
leaves of all treated tomato plants. A positive correlation was observed between the
accumulation of phenolic compounds and salicylic acid [84], [15], [64] and [5].
The present results revealed that, all treatments of plant extracts increased the
free, conjugated and total phenols content in tomato leaves comparing to the
untreated control. As for free phenols, the highest % increase was induced by G at
0.5% followed by BP at 4.0%, G at 4.0% and BP at 0.5%, respectively in relation to
the untreated control. As for interactions, IR+SS/G at 0.5% recoded the highest
increase in the free phenols content followed SS/G at 4.0% while, the lowest increase
was induced by IR/G at 0.5% comparing with the untreated control. Also, the
conjugated phenols content was increased. Using BP at 4.0% recorded the highest
increase in the conjugated phenols content (147.5%) followed by G at 4.0%, G at
0.5% and BP at 0.5%, respectively comparing to the untreated control. Also, all
interactions increased the conjugated phenols content comparing to the untreated
control. In this respect, the highest increase was recorded by using SS/BP at 4.0%,
IR+SS/BP at 4.0% and IR+SS/G at 4.0% while IR/BP at 0.5%, SS/BP at 0.5% and
IR+SS/BP at 0.5% recorded the lowest increase in the conjugated phenols followed
by IR+SS/G at 0.5% comparing to the untreated control. Regarding the total phenols
content, the highest increase was recorded by BP at 10.0mM followed by G at 4.0%,
G at 0.5% and BP at 0.5%, respectively. As for interactions, the highest increase was
recorded by using SS/BP at 4.0% followed by IR+SS/BP at 4.0% and IR+SS/G at
4.0% whereas, the lowest increase was recorded by IR/BP at 0.5% comparing to the
untreated control. Also, all used safe chemical inducer treatments i.e. SA and R at 0.1
and 10.0mM concentrations increased the free phenols content. The highest increase
was induced by R at 10.0mM followed by SA at 10.0mM, SA at 0.1mM and R at
0.1mM, respectively in relation to the free phenols in the untreated control. All
interactions increased the free phenols contents to different extents. Using IR+SS/R
at 10.0mM recoded the highest increase followed by IR/R at 10.0mM and SS/R at
10.0mM while, the lowest increase was induced by IR/SA at 0.1mM and SS/R at
0.1mM comparing with the untreated control. The used chemical inducer treatments
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increased the conjugated phenols content. In this respect, R at 10.0mM recorded the
highest increase comparing to the untreated control. All interactions increased the
conjugated phenols content also. In this respect, the highest increase was recorded by
using IR+SS/R at 10.0mM followed by IR/R at 10.0mM, SS/R at 10.0mmM, IR/R at
0.1mM, and IR+SS/SA at 10.0mM comparing to the untreated control. All chemical
inducer treatments increased the total phenols content comparing to the untreated
control. The highest increase was recorded by R at 10.0mM followed by SA at
10.0mM, SA at 0.1mM and R at 0.1mM, respectively. All interactions between
application methods and chemical inducer treatments increased the conjugated
phenols content comparing to the untreated control. In this respect, the highest
increase was recorded by IR+SS/R at 10.0mM followed by IR/R at 10.0mM, SS/R at
10.0mmM, IR+SS/SA at 10.0mM and IR/R at 0.1mM comparing to the untreated
control.
All tested inducer treatments increased the total soluble proteins, under stress
of infection with Fusarium oxysporium f. sp. lycopersici (FOL) compared with the
untreated control. Using BP at 0.5% recorded the highest increase in the total soluble
protein “TSP” followed by G at 4.0%, BP at 0.5% and G at 0.5%, respectively
comparing to the untreated control. All method/treatment interactions increased TSP.
The highest increase recorded by SS/G at 4.0% followed by IR+SS/BP at 0.5%,
SS/BP at 0.5%, SS/BP at 4.0%, respectively comparing to the untreated control. As
for chemical inducer treatment, R at 10.0mM recorded the highest increase in the
TSP followed by SA at 10.0mM, R at 0.1mM and SA at 0.1mM, respectively
comparing to the untreated control. All tested interactions for safe chemicals
increased TSP. The highest increase was recorded by IR+SS/R at 10.0mM followed
by IR/SA at 10.0mM, IR/R at 10.0mM, SS/R at 10.0mM, IR/R at 0.1mM and
IR+SS/SA at 10.0mM, respectively comparing to the untreated control. These
findings are supported by the results of [64], [5] and [60] reported that close
correlation between the levels of total soluble proteins in response to SA pointed to
exert their action mechanism upon DNA- RNA synthesizing protein machinery at
transcriptional and/or translocational levels with magnitudes. Finally, the tested
inducer treatments had effective to overcome the wilt disease symptoms mediated by
restoring the metabolic alterations imposed by infection. The observed decrease in
the protein content in tomato tissues as a result of pathogen infection may be due to
some activities related to a hypersensitive response [44]. SA elicitor increased total
proteins in infected tomato plants than the untreated control [97].
One of the most important indicators of physiological activity is the rate of
photosynthesis, which is related to the chlorophyll content of plants. The present
results revealed that the tomato plants treated with tested plant extracts or safe
chemicals and inoculated with FOL showed conspicuous increase in the amounts of
total chlorophyll. The highest increase was recorded by G at 0.5% followed by BP at
4.0%, BP at 0.5% and G at 4.0%, respectively comparing to the untreated control. All
tested interactions increased total chlorophyll content. IR/G at 0.5% recorded the
highest increase followed by IR+SS/G at 0.5%, IR/BB at 4.0% and IR/G at 4.0%
comparing to the untreated control. As for safe chemicals, R at 0.1mM recorded the
highest increase in the total chlorophyll followed by SA at 0.1mM, SA at 10.0mM
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and R at 10.0mM, respectively comparing to the untreated control. As for
method/treatment interactions, IR+SS/R at 0.1mM recorded the highest increase in
the total chlorophyll content followed by IR+SS/SA at 0.1mM and IR/R at 0.1mM.
However, SS/SA at 0.1mM, SS/SA at 10.0mM and SS/R at 10.0mM decreased
amount of total chlorophyll compared to the untreated control. The observed
reduction in chlorophylls in tomato leaves as a result of Fusarium wilt disease may be
a consequence of the fungal effect on the release of transported toxins which leads to
the liberation of reactive oxygen species. It was found that toxins which produced by
Fusarium were induced inhibition of chlorophyll biosynthesis [7]. Also, decreased in
biomass and chlorophyll content in tomato plants might results from high level of
lipid peroxidation mediating cell damage in tomato tissues [63]. In general, the
chlorophyll content was conspicuously higher in leaves of tomato plants treated with
different tested inducers, this finding was in agreement with [88], [64], [93], [5] and
[97]. The high chlorophyll content in inducer-treated plants could be attributed to its
stimulatory effect on rubisco (ribulose 1, 5-bisphosphate carboxylase) activity [111].
The oxidative enzymes i.e. peroxidase (POD) and polyphenoloxidase (PPO) are
important in the defense mechanism against pathogens, through their role in the
oxidation of phenolic compounds to quinines, causing increasing in antimicrobial
activity. Therefore, they may be directly involved in stopping pathogen development
[155], [179] and [133]. The present results showed that, the garlic (G) and black
pepper (BP) which tested at 0.5 and 4.0% concentrations as resistance inducer
treatments increased activities of PPO and POD enzymes to different extents in
leaves of treated tomato plants comparing to the untreated control. Using BP at 0.5%
recorded the highest % increase in POD activity followed by BP at 4.0%, G at 0.5%
and G at 4.0%, respectively whereas, the SS/G at 4.0% was the most effective
interaction for increasinf POD activity followed by IR+SS/BP at 4.0%, SS/G at 0.5%,
IR+SS/BP at 0.5%, SS/BP at 0.5%, IR+SS/G at 0.5% and SS/BP at 4.0% comparing
to the untreated control. Regarding safe chemical treatments, SA at 0.1mM recorded
the highest % increase in POD activity followed by R at 10.0mM, R at 0.1mM and
SA at 10.0mM, respectively comparing to the untreated control. The POD activity
was increased by all tested method/chemical interactions. In this respect, SS/SA at
0.1mM increased POD activity by 10 folds over than the untreated control followed
by IR+SS/SA at 0.1mM (6.69 times), SS/R at 0.1mM (5.71 folds) and SS/R at
10.0mM (5.41 folds) over that recorded by the untreated control.
Using G at 0.5% recorded the highest increase in the PPO activity followed by
BP at 4.0%, BP at 0.5% and G at 4.0%, respectively. Most interactions, however,
increased PPO activity, SS/BP at 0.5% recorded the highest increase followed by
IR+SS/BP at 0.5%, IR/G at 0.5%, respectively compared to the untreated control. As
for chemical inducer treatments, the highest PPO activity was recorded by R at
10.0mM followed by SA at 10.0mM, R at 0.1mM and SA at 0.1mM, respectively. As
for interactions, SS/R at 0.1mM recorded the highest increase followed by IR/SA at
10.0mM, SS/R at 10.0mM, IR/R at 10.0mM and IR+SS/SA at 0.1mM, respectively.
However, only IR+SS/R at 0.1mM, and IR+SS/SA at 10.0mM decreased PPO
activity to some extent compared to the untreated control. In fact, the activity of POD
was sharply increased in response to foliar spray of salicylic acid [65] and [127]. The
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present results were in agreement with [72] and [103], since they reported that the
activity of both polyphenoloxidase and peroxidase increased after the treatment with
Trichoderma harzianum, and also with [64] and [126], since they showed that the
activity of POD was sharply increased in response to foliar spray of salicylic acid.
Spraying of salicylic acid enhanced the activity of the enzyme in trichoderma
seedling root dipping treatment not in soil applied treatment. The last obtained result
was supported by [77]. All of their resistance inducer treatments increased the
activity of PPO and POD relative to the infected control. T2 treatment was more
effective in increasing the activity of the enzymes. Combination of salicylic acid with
trichoderma as seedling root dipping (T2) and thiophanate methyl with its half
recommended rate recorded a higher PPO and POD activity since it reached to 305%
for the first one and 315% for the second relative to the infected control.
Combinations of biocontrol agents and resistance inducer could provide promising
integrated alternatives in suppression of Fusarium wilt disease of tomato plants due to
number of mechanisms involved. So, a new approach for the management of
biological control of Fusarium wilt disease of tomato plants depends on the activation
of the defense of the plant against pathogen (Systemic resistance) by salicylic acid as
inducer and suppression of the fungal pathogenicity by applying Trichoderma
harzianum as bicontrol agent [97].
The mechanisms of cultivar responses against Fusarium wilt pathogens require
further studies. The possible causes of wilting in tomato plants infected with
Fusarium oxysporum f.sp. lycopersicii were examined. Determinations of leaf water
potential and solute potential showed that the wilting was due to water stress. The
diffusive resistance of leaves to water vapor loss in infected plants was as high as or
higher than the resistance in healthy plants at a given leaf water potential, and it was
concluded that an alteration in transpirational behavior did not cause water stress to
occur in infected plants. Measurements of water flow through excised root systems
indicated that infection did not increase the resistance of roots to water flow. When
water was forced through stem segments the resistance of infected segments was
found to be several times the resistance of healthy segments. However, accurate
estimation of xylem resistance in infected plants was impossible by this technique
because the resistance of infected segments decreased markedly as water flow
occurred. Apparently, a major portion of the resistance in infected xylem can be
attributed to material that was removed by abnormally high rates of water flow. In
general, the results confirm the hypothesis that a high xylem resistance to water flow
is the sole cause of the wilting which characterizes Fusarium wilt [58].
The anatomical studies in petioles of the fifth leaf of tomato plant treated by
immersing their roots (IR), spraying their shoots (SS) or IR+SS by garlic or black
pepper extracts at 4.0% concentration and inoculated with FOL showed induced
positive changes in the water conductive elements particularly xylem vessels and
width of the vascular bundles in tomato plants treated with G or BP extracts
compared with untreated control. These positive changes might involve in the
induced systemic resistance which lead to resist or delay development of the
Fusarium wilt disease in tomato plants. Number of xylem vessels (NXV) in the
vascular bundle as well as the width of the vascular bundle (WVB) seemed to be
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correlated with the resistance against the Fusarium wilt disease more than any other
investigated anatomical structures of the tomato leaf petiole. Thus, the garlic or black
pepper extracts for treating tomato seedlings before transplanting 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. In fact, the
functional water-conducting system, the tracheary elements of the xylem, is required
to sustain plant growth and development. [99]. The enlarged number of xylem vessels
and width of the vascular bundles caused by the garlic and black pepper extracts
might be considered as a probable induced defense mechanism against the tomato
Fusarium wilt. It is interest to state that neither conidia nor mycelia of the tomato
Fusarium wilt pathogen were detected in leaf petioles of treated and untreated tomato
plants. Such findings agree with [150] who 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, the absence of conidia in
advance of mycelium in the xylem vessel elements is probably the primary reason for
the success of culture indixing as a control 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 [113].
SUMMARY
Tomato (Lycopersicon esculentum Mill.) is one of the world’s most important
crops due to the high value of its fruits both for fresh market consumption and in
numerous types of processed products. One of the main constraints to tomato
cultivation is damage caused by pathogens, including viruses, bacteria, nematodes
and fungi, which cause severe losses in production. The soil-borne fungus Fusarium
oxysporum f. sp. radicis-lycopersici (FORL) causes Fusarium crown and root rot of
tomato, often referred to as ‘crown rot’. Fusarium oxysporum f. sp. lycopersici (FOL)
inhabits most tomato-growing regions worldwide, causing tomato production yield
losses. FOL has an extensive presence in all continents and become one of a limiting
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factor in the production of tomato and accounts for yield losses annually. It has
become one of the most prevalent and damaging diseases wherever tomatoes are
grown intensively because the pathogen persists indefinitely in infested soils. FOL
attacks only certain tomato cultivars. New races of Fusarium oxysporum f. sp.
lycopersici could develop through spontaneous random mutation or genetic
recombination. Because F. oxysporum is an imperfect fungus, parasexual
recombination is the only mechanism by which re-assortment of genetic material can
occur. Heterokaryon formation, a prerequisite for parasexual recombination, has been
demonstrated in several formae speciales of F. oxysporum, including F. o.
lycopersici.
This work was carried out in Almaty (Kazakhestan) and mainely aimed to:
isolatation and identification of the tomato Fusarium wilt fungus, testing pathogenic
ability of the isolated fungi to tomato Carolina Gold cultivar, evaluating responses of
some commercial and new experimental tomato cultivars against infection with the
most virulent isolate, evaluating effects of different treatments of resistance inducers
(plant extracts and safe chemicals) against the in vtro growth and sporulation of the
tomato Fusarium wilt fungus, evaluating the in vivo effectes of the these inducer
treatments on the wilt disease incidence, plant growth and fruit yield, biochemical
plant constituents (leaf pigments, phenols content, and total soluble protein), the
oxidative enzymes polyphenol pxidase and peroxidase in plant tissues and the
anatomical structure of leaf petiole in the treated and untreated tomato plants under
stress of infection with the tomato Fusarium wilt. The most important results could be
summarized as following:
1. Nine isolates of Fusarium were isolated from wilted tomato plants grown under
glasshouse condition at different location of Almaty, Kazakhstan. All isolates
formed colonies with conidia and mycelia with morphological characteristics
typical of F. oxysporum. The in vitro growth and sporulation of these isolates
were significantly varied
2. All F oxysporum isolates caused different degrees of wilt disease symptoms and
vascular discoloration and showed negative effects on plant height, number of
leaves/plant, fresh weight of leaves/plant, fresh weight of stem/plant, fresh weight
of roots/plant, and root length and fruit yield/plant of the Carolina Gold tomato
cultivar. This was the first record for presence of tomato wilt caused by Fusarium
oxysporum f. sp. lycopersici (FOL) in Kazakhstan.
3. Two commercial tomato cultivars (Carolina Gold and Dona) and five new
experimental cultivars (EXP8340 (EXP1), EXP8355 (EXP2), EXP8416 (EXP3),
EXP8420 (EXP4) and EXP8576 (EXP5) were evaluated against infection with
FOL isolate A under glasshouse conditions of Almaty, Kazakhstan. These cultivars
could be classified as: 1) Susceptible (Carolina Gold and Dona), 2) High resistant
(EXP 1 and EXP 2) and moderate resistant (EXP3, EXP5 and EXP4). Plant growth
characters and fruit yield/plant were significantly lower in FOL inoculated than uninoculated plants particularly in Carolina Gold and Dona cultivars. In term of early
fruit yield production, the moderately resistant EXP4 was significantly better than
the high resistant cultivars EXP1 or EXP2 then it could be subjected to further tests
to introduce it as commercial cultivar in Kazakhstan.
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4. The garlic (G) extract showed the highest inhibitory effect on the FOL radius growth
followed by black pepper (BP) extract, riboflavin (R) and salicylic acid (SA) which
reduced the FOL radius growth by 55.41, 35.03, 32.99 and 26.4%, respectively. The
reduction in growth was increased successfully as inducers concentrations increased.
The radius growth was completely inhibited (100.0% inhibition) by G at ≥2.0%, R at
≥5mM or SA at 10.0mM. However, growth was not significantly affected by SA at
0.1mM meanwhile significantly enhanced by using R at 0.1mM or 0.5mM
comparing to the untreated control medium. The BP at 4.0% concentration reduced
growth by 66.5% comparing to the untreated control.
5. The G extract, R and SA were significantly equal and recorded the highest
inhibitory effect against the FOL sporulation decreasing it by 59.84, 59.03 and
58.47%, respectively comparing to the untreated control. Sporulation was
significantly decreased as inducer’s concentration increased. Spore production
was completely inhibited by G at ≥ 2.0%, R ≥ 5.0mM and SA at 10.0mM. The BP
extract significantly decreased sporulation (66.5%) comparing to the untreated
control but it allowed it and produces appreciable number of spores even at at
4.0% (0.471x106spores/ml) concentration comparing to the untreated control
(1.451 x106spores/ml).
Three application methods namely immersing roots (IR), spraying shoots (SS)
and combination together (IR+SS) were used for treating seedlings (4-weeks old)
of the tomato cultivar Carolina gold with different inducer treatments i.e. G and
BP extracts at 0.5 and 4.0%, SA and R at 0.1 and 10.0mM before transplanting in
pots under glasshouse conditions. Each seedling was inoculated, one week after
planting with 20 ml of spore suspension (106spores/ml) of the FOL isolate A.
After two months, all treatments were evaluated for percentage of wilted plants,
wilt disease severity, plant height, number of leaves/plant, fresh and dry weight of
leaves/plant, stem fresh weight/plant, fresh and dry weights of roots/plant, root
length and volume/plant and fruit yield/plant. Effect of tested treatments on
biochemical constituents i.e. leaf chlorophylls, phenols content, total soluble
protein content, activities of the oxidative enzymes polyphenoloxidase and
peroxidase were also investigated.
6. BP at 4.0% and G at 4.0% were the most effective, decreasing % wilted plants by
86.7 and 80.0% whereas, G at 0.5% was the least effective, decreased % wilted
plants by 46.7% comparing to the untreated control. Using IR/G and SS/G at
4.0% in addition to SS/BP at 4.0% were the most effective as disease infection
was completely suppressed (100.0% reduction) followed by IR/BP at 4.0%,
IR+SS/BP at 4.0% and IR+SS/BP at 0.5% (80.0% reduction) while, IR/BP at
0.5% decreased wilt infection only by 20.0% comparing to the untreated control.
7. SA at 10.0mM and R at 10.0mM reduced % wilted plants by 73.3% followed by
SA at 0.1mM (60.0%) and R at 0.1mM (53.3%) compared with the control
treatment. Using SS and IR+SS with SA or R at 10.0mM decreased % wilted
plants by 80.0% whereas, SS/SA at 0.1mM and IR/R at 0.1mM decreased it by
40.0% comparing to the untreated control.
8. BP extract at 4.0% decreased the wilt disease severity (DS) by 94.1% followed by
G at 4.0% (84.3%), G at 0.5% (62.7%), and BB at 0.5% (60.8%), respectively
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comparing to the untreated control. Using IR/G at 4.0%, SS/G at 4.0% and SS/BP
at 4.0% completely suppressed disease development (100.0% reduction in DS)
followed by IR/BP at 4.0% and IR+SS/BP at 0.5% (94.1% reduction), SS/BP at
0.5% (70.6% reduction) whereas IR/BP at 0.5% decreased the DS by 17.6%
comparing with the control.
9. SA at 10.0mM decreased the DS by 86.3% followed by R at 10.0mM (74.5%),
SA at 0.1mM (72.5%) and R at 0.1mM (70.6%), respectively. The highest
significant reduction in DS was recorded by IR/SA at 0.1mM (94.1%) followed
by IR/SA at 10.0mM, SS/SA at 10.0mM and IR+SS/R at 10.0mM (88.2%
reduction), IR/R at 10.0mM, IR+SS/R at 0.1mM and IR+SS/SA at 10.0mM
(82.4% reduction), IR/R at 0.1mM and IR+SS/SA at 0.1mM (70.6% reduction),
respectively comparing with the control.
10.G at 0.5% recorded the highest plant height (163.9 cm) followed by BP at 4.0%
(150.5 cm), G at 4.0% (137.6 cm) and BP at 0.5% (136.6 cm). Using IR+SS/BP
at 4.0 increased plant height by 40.8% (174.8 cm) followed by IR/G at 0.5%
which increased plant height by 38.7% (172.2 cm) while, IR/BP at 0.5%
increased plant height by 7.4% (133.3 cm). However, IR/BP at 0.5%, SS/G at
4.0% and SS/BP at 0.5% showed no significant effects on the plant height when
compared with the untreated control.
11.SA at 0.1mM recorded the highest plant height (162.2 cm) followed by R at
10.0mM (149.1cm), SA at 10.0mM (148.6 cm) and R at 0.1mM (129.3 cm)
comparing to the untreated control (124.2 cm). Using IR/SA at 0.1mM increased
plant height by 53.4% (190.5cm) comparing to the control. However, IR/R at
0.1mM, SS/R at 10.0mM and IR+SS/SA at 0.1mM showed no significant effects
on the plant height when compared with the untreated control.
12.G at 0.5% recorded the highest number of leaves (NL) per plant (15.4) followed
by BP at 4.0% (15.2), G at 4.0% (14.1) and BP at 0.5% (12.8) comparing to the
untreated control (9.0). Using IR+SS/BP at 4.0% increased NL by 77.8%
followed by IR/G at 0.5% and SS/G at 0.5% (74.1%). However, the NL produced
by IR/G at 0.5% interaction (9.5) was not significantly varied when compared
with the untreated control.
13. R at 10.0mM recorded the highest NL (15.3) while, R at 0.1mM recorded the lowest
NL (15.2), both treatments increased NL by 70.4 and 58.0%, respectively comparing
to the untreated control. Using IR+SS/R at 10.0mM recorded the highest increase in
the NL (90.7%) followed by SS/SA at 0.1mM (74.1%) while, IR/R at 0.1mM
recorded the lowest significant increase (42.6%) comparing to the control.
14.BP at 4.0% and G at 0.5% increased the fresh weight of leaves (g) per plant
(FWL) by 49.2 and 47.9%, respectively whereas, G at 4.0% increased it by 33.1%
comparing to the control (65.4g/plant). Using IR+SS/BP at 0.5% recorded the
highest increase in the FWL (87.1%) followed by IR/BP at 4.0% (85.1%), SS/G
at 0.5% (65.6%) and IR/G at 4.0% (59.6%) whereas, SS/BP at 0.5% increased it
by 14.4% comparing to the untreated control. The increases in FWL caused by
IR+SS/G at 4.0% (12.0%) and IR/BP at 0.5% (8.5%) were not significantly
varied when compared to the untreated control.
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15. SA at 0.1mM was the best, increased FWL by 65.3% followed by R at 10.0mM
(51.6%), SA at 10.0mM (39.6%) and R at 0.1mM (30.8%), respectively comparing
to the untreated control. Using IR/R at 0.1mM, IR/SA 0.1mM, SS/SA at 0.1mM and
SS/R at 0.1mM were the best of all which increased the FWL by 84.1, 83.0, 77.6 and
75.6% comparing to the control. The lowest significant increase in the FWL was
induced by IR+SS/SA at 0.1mM (35.3%) whereas, many interactions i.e. IR/SA at
10.0mM, IR/R at 0.1mM, SS/R at 10.0mM and IE+SS/R at 0.1mM showed no
significant effect in this respect comparing to the untreated control.
16.BP at 0.5% (27.54 g/plant) and G at 0.5% (27.44 g/plant) were the best, increased
the dry weight of leaves per plant (DWL) by 35.5 and 35.0%, respectively
whereas the lowest significant increase was induced by G at 4.0% (19.6%)
comparing to the untreated control. Using SS/BP at 0.5% induces the highest
increase in the DWL (93.5%) followed by SS/G at 0.5% (62.0%), IR/BP at 4.0%
(53.2%) while, the lowest significant increases i.e. 13.2 and 14.2% were induced
by G and BP at 4.0%, respectively comparing to the untreated control. On the
other hand, the increases in the DWL/plant caused by IR/BP at 0.5%, SS/BP at
0.5%, IR+SS/G at 0.5 and 4.0% IR+SS/BP at 4.0% were not significantly varied
comparing to the untreated control.
17.SA at 0.1mM induces the highest significant increase in DWL (57.4%) followed
by R at 10.0mM (42.2%), SA at 10.0mM (24.6%) and R at 0.1mM (19.7%),
respectively comparing to the untreated control. Using SS/SA at 0.1mM induces
the highest increase in the DWL (99.5%) followed by IR/R at 10.0mM (72.2%)
and IR/SA at 0.1mM (71.1%) while, the lowest significant increase was induced
by SS/SA at 10.0mM (18.2%) comparing to the untreated control. Some
interactions i.e. IR/SA at 10.0mM, IR/R at 0.1mM, SS/R at 10.0mM, IR+SS/SA
at 0.1mM and IR+SS/R at 0.1mM showed no significant effect on the DWL if
compared with the untreated control.
18.G at 0.5% increased stem fresh weight (SFW) by 26.5% followed by BP at 0.5%
(21.5%), BP at 4.0% (10.7%) and G at 0.5% (8.1%), respectively comparing to
the control. Using IR+SS/BP at 0.5% increased SFW by 57.7% followed by
IR+SS/G at 0.5%, IR/G at 0.5%, SS/G at 0.5%, IR/G at 4.0%, IR/BP at 4.0%,
IR+SS/BP at 4.0% and SS/BP at 0.5% which increased it by 29.3, 25.7, 24.4,
19.9, 12.0, 11.1 and 9.0%, respectively comparing to the control. The remained
interactions showed no significant differences on the SFW when compared with
the untreated control.
19.SA at 0.1mM induces the highest significant increase in the SFW (46.2%)
followed by SA at 10.0mM (24.6%), R at 10.0mM (22.2%), and R at 0.1mM
(6.1%), respectively. Using SS/SA at 0.1MM causes the highest increase (72.3%)
followed by IR/SA at 0.1mM (62.6%) and IR/R at 10.0mM (61.1%) whereas the
lowest significant increase was induced by SS/R at 0.1mM (13.1%). On the other
hand, IR/SA at 10.0mM, IR/R at 0.1mM, SS/R at 10.0mM, SS/R at 10.0mM and
IR+SS/R at 10.0mM showed no significant differences in the SFW when
compared with the untreated control.
20.G at 0.5% induces the highest increase in the root fresh weight (RFW) i.e. 26.8%
followed by BP at 4.0% (24.5%), G at 4.0% (20.5%) and BP at 0.5% (19.2%),
127
respectively comparing to the untreated control. All interactions between methods
and inducer treatments showed significant increase in the RFW comparing to the
untreated control. In this regard, IR+SS/G at 0.5 and 4.0% caused the highest
(50.2%) and lowest significant increase (11.4%), respectively.
21. SA at 0.1mM recorded the highest increase in the RFW (35.1%) followed by R at
10.0mM (26.7%), SA at 10.0mM (17.2%) and R at 0.1mM (12.2%), respectively
comparing to the untreated control. Using IR/R at 10.0mM was the best in this
respect (74.3%) followed IR/SA at 0.1mM (69.9%) while, the lowest significant
increase (15.1%) was recorded by IR+SS/SA at 0.1mM compared with the untreated
control. On the other hand, IR/R at 10.0mM, SS/SA at 10.0mM, SS/R at 10.0mM,
IR+SS/SA at 10.0mM, IR+SS/R at 10.0mM and IR+SS/R at 10.0mM showed no
significant differences in the RFW when compared with the control treatment.
22.BP at 4.0% induces the highest increase in the root dry weight (RDW) i.e. 41.1%
followed by BP at 0.5% (34.2%), G at 4.0% (27.8%) and G at 0.5% (27.5%)
without significant differences between the latter two treatments. Using
IR+SS/BP at 0.5% produces the highest increase (88.9%) followed by IR/BP at
4.0% (86.4%) and IR+SS/G at 0.5% (55.0%) whereas, the lowest significant
increase SS/G at 0.5% (23.8%). On the contrary, the RDW produced by IR/G at
0.5%, IR/BP at 0.5%, IR/BP at 0.5%, SS/BP at 0.5% and IR+SS/BP at 4.0% were
not significantly varied when compared with the untreated control.
23.SA at 0.1mM induces the highest significant increase in the RDW (59.9%)
followed by R at 0.1mM (40.8%), R at 10.0mM (39.5%) and SA at 10.0mM
(37.0%) respectively. Regarding interactions, IR/SA at 10.0mM recorded the
highest significant increase (95.3%) followed by IR/R at 10.0mM (92.9%).
Whereas, SS/SA at 10.0mM, SS/R at 0.1mM and IR+SS/R at 10.0mM showed no
significant effect on the RDW compared with the untreated control.
24. The highest significant increase in the root length (RL) was produced by BP at 0.5%
(41.6%) and BP at 4.0% (40.5%) followed by G at 4.0% (32.6%) and G at 0.5%
(25.4%), respectively in relation to the untreated control. Using SS/G at 0.5%
induced the highest significant increase in the RL (64.5%) followed by IR/BP at
0.5% (57.0%), SS/BP at 4.0% (53.8%) and IR/BP at 4.0% (48.4%) while, the lowest
significant increase was produced by IR+SS/G at 4.0% (17.2%) comparing to the
untreated control. On the other hand, the RL was not significantly affected by SS/G
at 0.5% and IR+SS/G at 0.5% comparing to the untreated control.
25.SA at 10.0mM causes the highest increase in the RL (53.0%) followed by SA at
0.1mM (36.2%), R at 10.0mM (30.5%) and R at 0.1mM (28.8%) comparing to
the untreated control. Except IR/R at 0.1mM and SS/R at 10.0mM, all other
method/treatment interactions increased RL comparing to the untreated control.
The highest increase was produced by SS/SA at 10.0mM (67.7%) and SS/R at
0.1mM (65.6%) whereas, the lowest significant increase was induced by IR+SS/R
at 0.1mM (21.5%). However, IR/R at 0.1mM and SS/R at 10.0mM showed no
significant effect on the RL comparing to the untreated control.
26. BP at 4.0% induces the highest significant increase in the root volume (RV) i.e.
60.0% followed by G at 4.0% (43.0%), G at 0.5% (30.8%) and BP at 0.5% (29.2%)
compared to the untreated control. Using IR/BP at 4.0% increased RV by 92.5%
128
followed by IR/G at 4.0% (72.5%) and IR+SS/G at 0.5% (68.8%), respectively
while the lowest significant increase was induced by IR+SS/G at 0.5% (21.5%. On
the other hand, using IR/G at 0.5%, IR/BP at 0.5%, SS/G at 0.5% showed no
significant differences in the RV when compared with the untreated control.
27. R at 10.0mM causes the highest increase in the RV (59.2%) followed by SA at
0.1mM (47.9%), SA at 10.0mM (36.3%) and R at 0.1mM (32.1%), respectively. All
tested method/treatment interactions significantly increased RV. IR/R at 10.0mM
was the best interaction, increased the RV by 92.5% followed by IR/SA at 0.1mM
(75.0%) and SS/R at 10.0mM (55.5%) whereas, the lowest significant increase was
produced by IR+SS/R at 0.1mM (27.5%) compared to the untreated control.
28.All tested inducer treatments significantly increased the weight of fruit yield
(g)/plant (WFY) comparing to the untreated control. G at 4.0% and BP at 4.0%
produced the highest increase (142.2-142.6%) followed by BP at 0.5% (135.6%)
and G at 0.5% (39.7%) comparing with the untreated control (135.0 g/plant).
Using IR+SS/BP at 0.5% produces the highest WFY (545.9 g/plant) followed by
SS/BP at 4.0% (425.8 g), IR/G at 4.0% (410.2 g), SS/G at 4.0% (355.0 g), SS/G
at 0.5% (170.3 g/plant) and IR/BP at 0.5% (174.3 g/plant), respectively.
29.R at 10.0mM produced the highest WFY (289.7 g/plant) followed by SA at
10.0mM (275.5 g/plant), R at 0.1mM (265.9 g/plant) and SA at 0.1mM (249.3
g/plant), respectively compared to the untreated control (135.0 g/plant). Using
IR+SS/R at 10.0mM increased WFY to 416.3 g/plant followed by IR+SS/R at
0.1mM (383.9 g g/plant), IR+SS/SA at 10.0mM (344.8 g/plant), IR/SA at 0.1mM
(298.6 g/plant), SS/SA at 10.0mM (290.9 g/plant), respectively whereas, the
lowest significant increases were produced by SS/R at 10.0mM (266.8 g/plant)
and IR+SS/SA at 0.1mM (261.4 g/plant). On the other hand, the observed
increases in the WFY produced by IR/SA at 10.0mM, IR/R at 0.1 and 10.0mM,
SS/SA at 0.1mM and SS/R at 0.1mM were not significantly varied when
compared to the untreated control.
30.Using the IR+SS/R at 0.1mM recorded the highest amount of chlorophyll a
(0.772 mg) followed by IR/G at 0.5% (0.692 mg), IR+SS/SA at 0.1mM (0.652
mg), IR+SS/G at 0.5% (0.64 mg) and IR/BP at 4.0% (0.602 mg), respectively.
The lowest increase in the amounts of chlorophyll a was produced by SS/R at
0.1mM (0.323) whereas those produced by SS/SA at 0.1mM (0.291 mg), SS/SA
at 10.0mM (0.282 mg) and SS/R at 10.0mM (0.268 mg) were obviously lower
than that produced by the untreated control (0.313 mg).
31.Using IR/G at 0.5% recorded the highest increase in the amount of chlorophyll b
(0.0.954 mg) followed by IR+SS/SA at 0.1mM (0.749 mg), IR+SS/R at 0.1mm
(0.745 mg), and IR+SS/G at 0.5% (0.724 mg), respectively whereas, the lowest
increase in amount of chlorophyll b (0.351 mg) was produced by SS/R at 0.1mM.
However, SS/SA at 0.1mM, SS/SA at 10.0mM and SS/R at 10.0mM decreased
amount of chlorophyll b to 0.300, 0.297 and 0.263 mg, respectively compared to
the untreated control (0.345 mg).
32.The total chlorophyll was increased by most investigated method/treatment
interactions. In this regard, IR/G at 0.5% recorded the highest amount (1.646 mg)
followed by IR+SS/SA at 0.1mM (1.401 mg), IR+SS/G at 0.5% (1.364 mg), and
129
IR/R at 0.1mM (1.295 mg), respectively. The lowest increase in the amount of
total chlorophyll (0.674 mg) was produced by SS/R at 0.1mM whereas SS/SA at
0.1mM, SS/SA at 10.0mM and SS/R at 10.0mM were decreased amount of total
chlorophyll to 0.591, 0.579 and 0.531 mg, respectively compared to the untreated
control (0.658 mg).
33. The free phenols content was increased by all tested inducer treatments
comparing to the untreated control. The highest increase was induced by using R
at 10.0mM (226.1%) followed by G at 0.5% (149.3%), SA at 10.0mM (127.3%),
G at 4.0% (125.5%), BP at 0.5% (106.9%), SA at 0.1mM (85.6% and R at 0.1mM
(59.9%), respectively. The free phenols content recorded in the untreated control
was (0.988 mg). Among tested interactions, IR+SS/G at 0.5% recoded the highest
increase in the free phenols content (328.4%) followed by IR+SS/R at 10.0mM
(292.7%) and SS/G at 4.0% (208.8%) while, the lowest increase was induced by
IR/G at 0.5% (21.4%), IR/SA at 0.1mM and SS/R at 0.1mM (26.7%) comparing
with the untreated control.
34. The conjugated phenols content was increased by all tested inducer treatments
comparing to the untreated control. In this respect, R at 10.0mM recorded the
highest increase (293.2%) followed by BP at 4.0% (147.5%), G at 4.0%
(104.4%), SA at 10.0mM (83.4%), SA at 0.1mM (67.4%), R at 0.1mM (67.4%),
G at 0.5% (53.5%) and BP at 0.5% (2.3%), respectively. As for interactions
between application methods and inducer treatments, the highest increase was
recorded by IR+SS/R at 10.0mM (322.2%) followed by IR/R at 10.0mM
(294.7%), SS/R at 10.0mmM (262.8%), SS/BP at 4.0% (210.9%), IR+SS/BP at
4.0% (187.3%), IR/R at 0.1mM (154.1%), IR+SS/G at 4.0% (148.8%) and
IR+SS/SA at 10.0mM (132.5%), while IR/BP at 0.5%, SS/BP at 0.5% and
IR+SS/BP at 0.5%, IR+SS/R at 0.1mM and SS/SA at 0.1mM recorded the lowest
increase in the conjugated phenols (1.8-2.9%) followed by IR+SS/G at 0.5%
(13.4%) comparing to the untreated control.
35. All tested inducer treatments increased the total phenols content (23.6-279.5%)
comparing to the untreated control. The highest increase was recorded by R at
10.0mM (279.5%) followed by BP at 4.0% (143.4%), G at 4.0% (107.3%), SA at
10.0mM (92.0%), SA at 0.1mM (76.4%), G at 0.5% (73.0%), R at 0.1mM
(65.8%) and BP at 0.5% (23.6%), respectively. All interactions between
application methods and inducer treatments increased the conjugated phenols
content (14.5-316.2%) comparing to the untreated control. In this respect, the
highest increase was recorded by using IR+SS/R at 10.0mM (316.2%) followed
by IR/R at 10.0mM (274.7%), SS/R at 10.0mmM (247.8%), SS/BP at 4.0%
(201.8%), IR+SS/BP at 4.0% (183.3%), IR/SA at 10.0mM (140.1%), IR+SS/G at
4.0% (134.4%) and IR/R at 0.1mM (134.0%), while lowest increase was recorded
by IR/BP at 0.5% (14.5%), IR+SS/BP at 0.5% (21.3%) and IR+SS/R at 0.1mm
(22.2%) comparing to the untreated control.
36.Regardless application method, R at 10.0mM, SA at 10.0mM, G at 4.0%, R at
0.1mM, BP at 0.5%, G at 0.5%, SA at 0.1mM and BP at 4.0% increased average
of total soluble protein (TSP) by 245.7, 119.4, 105.4, 104.7, 62.0, 56.6, 53.5 and
47.3%, respectively comparing to the untreated control. Most method/treatment
130
interactions increased TSP comparing to the untreated control. The highest
increase in the TSP was recorded by IR+SS/R at 10.0mM (339.5%) followed by
SS/G at 4.0% (248.8%), SS/R at 10.0mM (218.6%) and IR/SA at 10.0mM
(209.3%), respectively. However, IR/BP at 4.0% only decreased TSP by 72.1%
comparing to the untreated control.
37.All tested inducer treatment increased PPO activity comparing to the untreated
control. Using R at 10.0mM, SA at 10.0mM, R at 0.1mM, SA at 0.1mM, G at
0.5%, BP at 4.0%, BP at 0.5% and G at 4.0% increased PPO activity by 55.4,
36.7, 34.9, 29.7, 27.4, 13.4, 10.9 and 4.9%, respectively. Most interactions
between application methods and inducer treatments increased PPO activity (4.4
to 93.9%) and few interactions decreased it (-3.2 to -45.0%) comparing to the
control treatment. In this respect, recorded the highest increase was recorded by
SS/R at 0.1 mM (93.9%) followed by IR/SA at 10.0 mM (86.9%), SS/R at 10.0
mM (82.1%), IR/R at 10.0 mM (71.4%), IR+SS/SA at 0.1 mM (48.4%),
respectively while, only IR/BP at 0.5%, IR+SS/G at 4.0%, IR+SS/R at 0.1 mM,
and IR+SS/SA at 10.0 mM decreased PPO activity by 45.0, 27.5, 12.0 and 3.2%,
respectively compared to the untreated control.
38.Using SA at 0.1mM, R at 10.0mM, R at 0.1mM, BP at 0.5%, SA at 10.0mM, BP
at 4.0%, G at 0.5% and G at 4.0% increased the PRO activity by 6.39, 4.37, 3.64,
3.4, 3.32, 3.27, 3.12, 3.0 folds over than the untreated control. However, SS/SA at
0.1mM, IR+SS/SA at 0.1mM, SS/G at 4.0%, IR+SS/BP at 4.0%, SS/R at 0.1mM
and SS/R at 10.0mm were the most effective, increasing PRO activity by 10.03,
6.72, 6.43, 5.97, 5.73, 5.43 times, respectively over than the untreated control.
While IR/BP at 4.0% and IR/G at 4.0% increased the PRO activity only by 0.54
and 0.61 times over that the untreated control.
39. The anatomical studies for petioles of the fifth leaf of tomato plant treated by
immersing their roots (IR), spraying their shoots (SS) or IR+SS by garlic or black
pepper extracts at 4.0% concentration and inoculated with FOL showed induced
positive changes in the water conductive elements particularly xylem vessels and
width of the vascular bundles in tomato plants treated with G or BP extracts
compared with untreated control. These positive changes might involve in the
induced systemic resistance which lead to resist or delay development of the
Fusarium wilt disease in tomato plants.
CONCLUSION
In the present work, wilted tomato plants were repeatedly observed among
tomato plants grown under greenhouse conditions at Almaty, Kazakhstan. The
tomato wilt disease was found to be caused by Fusarium oxysporum f. sp. lycopersici
(FOL) according to isolation trials, morphological characteristics of the isolated fungi
and their pathogenic abilities in addition to the external and internal disease
symptoms they are caused. Infection with FOL caused considerable reduction in most
of the investigated plant growth characters in addition to great loss in tomato fruit
yield production. The highest reduction in plant growth consequently fruit yield
production was associated with the most susceptible tomato cultivars. The new
experimental tomato cultivars, in general, were more resistant against the FOL
131
infection than the commercial tomato cultivars. Using some plant extracts (garlic and
black piper) and safe chemicals (salicylic acid and riboflavin) each at a known
concentration could be suppressed the in vitro growth and sporulation of FOL. Some
in vitro treatments were selected as resistance inducers to treat tomato seedling at
time of planting by either immersing their roots (IR) and/or spraying their shoots (SS)
as application methods under stress of the Fusarium wilt pathogen. After two months
from treatment, most of the selected resistance inducer treatments resulted in
successful control of the tomato wilt disease incidence and improved tomato fruit
yield production under greenhouse conditions.
The recommendations:
1) Instead of the susceptible commercial cultivars namely Carolina Gold and
Dona, we recommended to use the “resistant” new experimental tomato
cultivars namely Exp1, Exp2 and Exp3 (provided by the seed company Rijk
Zwaan Ltd. - Uzbekistan) for suppressing Fusarium wilt infection and
increased yield productivity.
2) Treating tomato transplants with: 1) Spraying shoots (SS) with black piper
extract at 4% concentration, 2) Immersing roots (IR) with garlic extract at 4%
concentration or 3) or spraying shoots (SS) with garlic extract at 4%
concentration to suppressing the FOL infection and increasing fruit yield
production by more than 300, 200 and 160%, respectively compared to the
untreated control. However, the combined method (IR+SS) using riboflavin at
10mM reduced wilt disease severity by more than 88% and increased fruit
yield production by more than 200% compared to the untreated control.
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