الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
This study was conducted at Plant Pathology Lab. and Greenhouses of Botany Dept., Fac. of Agric., Moshtohor, Banha Univ.
and Virology Lab., Microbiology Dept., Fac. of Agric., Ain-Shams
Univ. During 2007/2008 and 2008/2009 growing seasons, different
tomato fields at Qalyoubia Governorate were surveyed for viruses
infections. Through, the assessment of disease incidence and severity,
Cucumber mosaic cucumovirus (CMV) was the dominant one among
the tomato viruses in the surveyed fields. Identification of isolated
virus (CMV) was achieved using host range, transmissition, stability
in sap, inclusion bodies and confirmed via Dot blot immunoassay
(DBIA). Obtained results dealing CMV confirmation was completely
agreement with the previous confidential recording. Therefore, many
experiments were successively to deducing if induction of systemic
acquired resistance against CMV was successfully achieved under
greenhouse and open field of tomatoes using four biotic inducers or
not.The effects of four inducers (three botanical extracts and
kombucha filtrate) in induction systemic resistance (SAR) in tomato
plants against CMV were detected via study the histopathological;
biochemical [dealing antiviral proteins (protein content, qualitative
protein, activity and isozyme of peroxidase and polyphenol oxidase)]; phytochemically [salicylic acid level, chlorophyll, phenols, total
amino acids, total carbohydrate contents] changes and detection
molecular marker of PRs gene. Virus infectivity was biologically
measured (disease incidence and severity and concentration of virus).
Firstly, SAR induction was check after performed the following
experiments which summarized as:
1- Histopathological changes in tomato leaves sprayed with
biotic inducers, tissue alterations were observed as progressive
increase in lignin accumulation in epidermal cells, number of
hairs, thickness of blade, number of xylem arms and phloem
layers. The alterations included, also, tissue-shrinkage, intense
staining, and precipitation of lignin in sub stomatal cavity,
mesophyll cell showing folding and layering of cell wall and
remains of host palisade cell walls.
2- Antiviral proteins as indicate on elicitations by inducers were
assessed after 7 days from spraying via protein content,
patterns, activities which markedly increased in treated tomato
plants than non-treated ones. In this concern, kombucha
filtrate was superior, but mixture extracts was the lowest
compared with healthy control. After 25 days from spraying
with inducers and inoculated with CMV, also plants treated
with kombucha filtrate produced the highest values of
proteins, while lowest produced as response to C. inerme
extract compared with healthy ones. Electrophoretic for
proteins using SDS-PAGE showed new protein bands with
molecular weight previously known for antiviral proteins were elicited by M. jalapa, C. inerme extracts and kombucha
filtrate. Peroxidase (POD) were markedly increased as result
to mixture extracts treatment, while polyphenol oxidase (PPO)
increased as result to M. jalapa extract treatment when tomato
plants were sprayed with inducers post-inoculation with CMV.
Kombucha filtrate elicited peroxidase isozyme in tomato noninoculated
with CMV, followed by the mixture extracts, while
post-inoculation M. jalapa extract was induced highest
activity of POD and lowest increase caused by C. inerme
extract. Polyphenol oxidase isozyme was highly activated with
M. jalapa extract, followed by C. inerme extract, then the
mixture of them.
3- Total salicylic acid was quantitatively determined in the
tomato plant sprayed with bioelicitors pre-inoculated with
CMV. SA was increased in the treated plants than non-treated,
and HPLC showed high levels of SA were elicited via
kombucha filtrate, followed by C. inerme, M. jalapa, and the
mixture extracts.
4- Photosynthetic pigments content (as Chlorophyll a, b plus
carotenoids) were reduced, generally in infected plants than
healthy ones. But, when tomato treated with the tested
elicitors pre-inoculation, Chl a, Chl b and carotenoids were
increased as result to spraying with M. jalapa, C. inerme,
mixture extracts and kombucha filtrate. The same trend was
observed when inoculated plants were treated with the same
order of elicitors.5- Phenols contents, was increased in the non-inoculated plants and treated with biotic inducers. The highest increase of total,
free and conjugate phenols were induced by M. jalapa extract
and kombucha filtrate, while lowest increase were recorded by
C. inerme and mixture extracts compared with control. Postinoculation,
all phenol contents were increased as response to
treatments with M. jalapa, mixture extracts, kombucha filtrate
and C. inerme, respectively.
6- Total RNA values (μg/g) were high in the non-inoculated but
treated tomato leaves with kombucha filtrate, M. jalapa
extract, followed by C. inerme extract then mixture extracts.
7- Molecular marker for SAR detection was achieved using RTPCR
to amplify of the PR-1a gene which elicited with
bioinducers in the tomato plants pre-inoculated. PR-1a gene
was isolated and molecularly sequenced and identified
compared with the related genes in the Gen-Bank.
8- Virus infectivity was determined to insure that systemic
acquired resistance is achieved. Reduction in the disease
severity percentage was recorded as result to spraying tomato
plants with bioelicitors (M. jalapa extract, followed by C.
inerme extract, kombucha filtrate then mixture extracts) then
inoculated with CMV. Also, concentration of the virus was
biologically assayed as means of local lesions.Secondly, after insure that the tested inducers were elicited
systemic acquired resistance against CMV in tomato plants under
greenhouse conditions, another experiments were performed using the
tested elicitors as biocontrol agents spraying on inoculated plants and
results were summarized as:
1- Histopathological changes as response to SAR induction was
examined in the inoculated tomato leaves and sprayed with
bioelicitors using light microscope. Generally, noticed that
treated plants were stronger in their growth than non-treated
plants as result to the increase in lignin precipitation,
numbers of xylem arms, phloem layers, skin hairs and
increasing thickness of cell wall, and blade. Infected plants
showed plasmolysis in the mesophyll cells, cell walls collapsed
and plastids become deformed and swollen a loss of orientation
along the inner cell wall. These alterations were intensified
with progressive tissue-shrinkage and desiccation causing the
walls of the palisade and spongy parenchyma to fold in a
layering fashion as well as reduction in vascular bundles.
2- Antiviral proteins as one of the protein contents and product
of induction process were increased in the inoculated plants
especially when sprayed with kombucha filtrate, while lowest
increase due to mixture extracts. After 7 days of spraying,
protein bands via variability analysis appeared 12 protein
bands, 11 in tomato plants treated with M. jalapa extract, 10 by
C. inerme extract and 8 for both mixture extracts and kombucha
filtrate, while non-treated and infected plants gave only 4 and 7 protein fractions. Meanwhile, after 25 days proteins content and
enzymes activity were markedly increased as result to
spraying with M. jalapa extract, lowest increase via C. inerme
extract compared to control. Variability analysis appeared 8
protein bands, 7 in mixture extracts, 6 in C. inerme extract,
and 5 in both M. jalapa extract and kombucha filtrate
treatments. Highest peroxidase and its isozyme activities was
induced by M. jalapa extract, and lowest by C. inerme extract
after 7 days of spraying. After 25 days, kombucha filtrate
induced highest peroxidase activity, followed by M. jalapa
extract, mixture extract then C. inerme extract. Peroxidase
isozyme activity was arranged as treatments of M. jalapa, C.
inerme, mixture extracts and kombucha filtrate, while
polyphenole oxidase isozyme was highest activity in
kombucha filtrate treatment, followed by M. jalapa extract,
then lowest increase with other treatments. After 7 days,
polyphenole oxidase activity was similar to peroxidase
isozyme, while after 25 days polyphenole oxidase isozyme
was highest activity in M. jalapa extract treatment, followed
by kombucha filtrate, mixture extracts, but decreased in C.
inerme than control. Variability analysis of polyphenole
oxidase isozyme showed 5, 3, 5 and 4 polypeptide bands of M.
jalapa, C. inerme, the mixture extracts and kombucha filtrate,
respectively. The result after 7 and 25 days, highest level of
protein genetic markers induced by M. jalapa extract
followed by C. inerme and mixture extracts, while kombucha filtrate induced low level of protein genetic
markers.
3- Photosynthetic pigments content (as Chlorophyll a, b plus
carotenoids) were reduced, generally in infected plants than
healthy ones. But, when tomato treated with the tested
elicitors pre-inoculation, Chl a, Chl b and carotenoids were
increased as result to spraying with M. jalapa, C. inerme,
mixture extracts and kombucha filtrate. The same trend was
observed when inoculated plants were treated with the same
order of elicitors.
4- Total phenols, was increased in the non-inoculated plants and
treated with biotic inducers. The highest increase of total, free
and conjugate phenols were induced by M. jalapa extract and
kombucha filtrate, while lowest increase were recorded by C.
inerme and mixture extracts compared with control. Postinoculation,
all phenol contents were increased as response to
treatments with M. jalapa, mixture extracts, kombucha filtrate
and C. inerme, respectively.
5- Total free amino acids content were determined in inoculated
tomato leaves then sprayed with bioagents. After 7 days from
spraying, M. jalapa extract and kombucha filtrate recorded the
highest amount of total amino acids, followed by mixture and
C. inerme extracts. While, after 25 days from spraying, M.
jalapa extract and kombucha filtrate produced the highest
amount of total amino acids, followed by C. inerme and
mixture extracts compared with control.
Summary and Conclusions
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6- Total carbohydrate content was increased after 7 days from
spraying inoculated tomato leaves with kombucha filtrate and
M. jalapa extract, followed by mixture and C. inerme extracts.
After 25 days, M. jalapa and C. inerme extracts produced the
highest increase in total carbohydrates content, followed by
mixture extracts and kombucha filtrate compared with control.
7- Virus infectivity was determined as indicator of control.
Reduction in the disease severity percentage was recorded as
result to spraying tomato plants with M. jalapa extract,
followed by C. inerme extract, kombucha filtrate then mixture
extracts compared with control. Also, concentration of the
virus was biologically assayed as means of local lesions.
Highest inhibition of virus infectivity due to M. jalapa extract,
kombucha filtrate, mixture extracts and C. inerme extract
compared with control.
Summary and Conclusions
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CONCLUSIONS
The objectives of this study were isolation and identification of the
most frequently and economically viruses causing serious losses in
tomato crop in the different location of Qalyoubia Governorate,
evaluating some medicinal plant extracts and kombucha filtrate as biotic
inducers to induction systemic acquired resistance in the tomato plants
against CMV and using more effective bioinducers as bioelicitors for
control viruses infection via induction ’pathogenesis-related’ (PR-1a)
genes.
Target virus was chosen according to its more frequently and
severity among the isolated viruses in these locations at the winter season
from the study year. Isolated virus was confirmed biologically and
serologically assays. Extracts of two medicinal plants (Clerodendrum
inerme L. Gaertn and Mirabilis jalapa L.) and were individually or in
mixture in addition to kombucha filtrate were evaluated as bioinducers.
All the four inducers were successfully in the induction of systemic
acquire resistance (SAR) in the uninoculated tomato plants and sprayed
with (50% v/v) of inducers.
Tested bioinducers were used as biocontrol to inhibiting the virus
infection of tomato plants as spraying every 15 days under greenhouse
conditions. Pathogenesis-related (PR-1a) gene was molecularly isolated
and identified via sequencer which compared with those recorded in the
Gen-Bank.
In conclusion, using medicinal extracts and other natural inducers
were promise with good systemic acquired resistance against the great
numbers of plant pathogens. In future, induction of resistance can be
done cheaply and easily using natural substances.
Summary and Conclusions
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RECOMMENDATIONS
This study can be recommended, for obtained healthy tomato
plants and reduced crop losses, with the following:
1- Periodicity explore the tomato plants from sowing date until
harvested and eliminate any plants exhibited virus-like
symptoms and burn them.
2- Surrounded the small cultivated area (for seed production or
breeding program searches) with enclosure of Mirabilis
jalapa L. plants as an embellishment plant which release
volatile substances work as antifeedant for numbers of pest
insects (as virus vectors).
3- Soak the root system of tomato seedlings in the 50% of the
following inducers, and spraying tomato plants every 15
days with 50% of water extracts of both Clerodendrum
inerme and Mirabilis jalapa or their mixture, or kombucha
filtrate from transplanting date to harvest.