الفهرس 
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Abstract
The present study aimed to achieve the following objectives:
1. Confirmation of viruses.
2. Preparation and characterization of Chitosan Nanoparticles.
3. Application of nanoparticles in vitro
4. Application of nanoparticles in vivo
The results might be summarized as follows:
PART I
1. Confirmation of viruses
1.1 Potato virus Y (PVY)
1.1.1 Confirmation of the Virus Isolate Biologically
Symptoms developed on the five host plants (Datura metel,
Datura stramonium, Nicotiana tabacum cv.White Burley, N. tabacum cv.
Samsun, Nicotiana glutinosa, Solanum lycopersicum and Solanum
tuberosum) were used for biological confirmation of potato virus Y.
Datura metel showed mottling, vein clearing and crinkle while
Datura stramonium showed no symptomes. N. tabacum cv.White Burley
showed mosaic, veinal necrosis while N. tabacum cv. Samsun showed
severe mosaic, vein clearing and Nicotiana glutinosa showed severe mosaic.
Solanum lycopersicum showed mosaic and Solanum tuberosum showed
mosaic, necrosis, deformation and leaf narrow
1.1.2. Confirmation of the Virus Isolate Serologically
PVY has detected by DAS-ELISA. The positive result was record
by ELISA reader and compared with negative control.
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1.1.3. Confirmation of the Virus Isolate Molecularly
1.1.3.1 One Step RT-PCR
RT-PCR was carry out using Plant Total RNA Mini Kit from RBC
and primer specific sites for the viral coat protein gene of PVY with an
expected size of 800 bp on agarose gel electrophoresis.
1.1.4. Virus morphology
The morphology of viral particle of PVY showed difference in the
morphological shape by electron micrograph using negative staining
method. The Examination showed viral particles with a clear modal
length of 650 nm and 11nm wide.
1.2. Tobacco mosaic virus (TMV)
1.2.1. Confirmation of the Virus Isolate Biologically
Symptoms developed on the five host plants (Datura metel,
Datura stramonium, Chenopodium quinoa, Chenopodium amaranticolor,
Nicotiana glutinosa, N. tabacum cv. Samsun and Solanum lycopersicum)
were used for biological confirmation of potato virus Y.
Datura metel, Datura stramonium, Chenopodium quinoa,
Chenopodium amaranticolor, Nicotiana glutinosa showed local lesion
symptoms after 4 day from mechanically inoculation while N. tabacum
cv. Samsun and Solanum lycopersicum showed severe mosaic after 15 day
from mechanically inoculation.
1.2.2. Confirmation of the Virus Isolate Serologically
TMV has detected by DAS-ELISA. The positive result 0.973 O.
D was record by ELISA reader and compared with negative control 0.084
O.D.
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1.2.3. Confirmation of the Virus Isolate Molecularly
1.2.3.1 One Step RT-PCR
RT-PCR was carry out using Plant Total RNA Mini Kit from RBC
and primer specific sites for the viral movement protein gene of TMV
with an expected size of 422bp on agarose gel electrophoresis.
1.2.4. Virus morphology
The morphology of viral particle of TMV showed difference in the
morphological shape by electron micrograph using negative staining
method. The Examination showed rod shaped of viral particles. viral
particles with a clear modal length of 300 x 15 nm.
PART II
Preparation and characterization of Chitosan Nanoparticles
1. Size, Zeta Potential, and Morphology of Chitosan Nanoparticles
The chitosan nanoparticles prepared in the experiment exhibit a
white powdered shape, and are insoluble in water. The mean size and size
distribution of each batch of nanoparticle suspension was analyzed using
the Zeta-sizer analysis. The size distribution profile, represents a typical
batch of nanoparticles with a mean diameter of 29 nm and a
polydispersity index < 0.371.
Zeta potential that is surface charge. It was noticed that the
surfaces of chitosan nanoparticles have a positive charge about 33.5mV.
Chitosan nanoparticles were observed by the transmission electron
microscope and it was less than 20 nm in diameter with smooth and
spherical in shape.
1.2. Infrared Spectroscopy
Chitosan nanoparticles were prepared between chitosan and
tripolyphosphate. FTIR studies of chitosan and chitosan nanoparticles
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were performed to characterize the chemical structure of nanoparticles. A
band at 3429cm 1 corresponds to the combined peaks of the NH2 and OH
group stretching vibration in chitosan. The band at 1633cm 1 is attributed
to the CONH2 group. A shift from 3429 to 3431cm 1 is shown, and the
peak is sharper in the chitosan nanoparticles, which appear the hydrogen
bonding is enhanced. The intensities of (NH2) band at 1550cm and
(CONH2) band at 1637cm, which can be observed clearly in pure
chitosan, decrease dramatically, and two new sorption bands at 1633 and
1428cm 1 appear which shows that the ammonium groups are crosslinked
with tripolyphosphate molecules.
Part III
Application of nanoparticles in vitro
1. Effect of the nanoparticles on virus morphology
The morphology of viral particles of PVY and TMV showed
difference in morphological shape by electron micrograph using negative
staining method. TEM examination was done on PVY and PVY treated
with silver nanoparticles, PVY and PVY treated with chitosan
nanoparticles, TMV and TMV treated with silver nanoparticles, TMV and
TMV treated with chitosan nanoparticles. The results observed that silver
and chitosan nanoparticles bind at a limit site on virus particles.
2. Effect of nanoparticles on virus infectivity
2.1. Effect of nanoparticles on PVY infectivity
After 21 and 28 days of inoculation on D. metel plants. The
chitosan nanoparticles, silver nanoparticles mixed with virus crude sap
due to reduction of virus infectivity. The results of D.metel plants which
mechanically inoculated with virus only give infection rate 100% whereas
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all plants were reaction with ELISA. The symptoms were Mosaic, vein
clearing, crinkle and leaf cup shape.
The results of D. metel plants which mechanically inoculated with
mixture of 2 g/L of chitosan nanoparticles and PVY suspension after 21
and 28 day were no symptoms and not reaction with ELISA test.
The results of D. metel plants which mechanically inoculated with
mixture of 50 ppm of silver nanoparticles and PVY suspension due to
reduction of symptoms with ratio 60%. The symptoms were vein clearing
and leaf cup shape. It is reaction with ELISA test.
2.2. Effect of nanoparticles on TMV infectivity
After 6 days of inoculation on N.glutinosa plants. The chitosan
nanoparticles, silver nanoparticles mixed with virus crude sap due to
reduction of virus infectivity.
The half leaf treated with TMV only showed 47.5 local lesions
while the half leaf treated with mixture of chitosan nanoparticle and TMV
suspension showed 16.5 local lesions. The reduction of viral activity
expressed by local lesions was 65.3% on N. glutinosa.
The half leaf treated with TMV only showed 65.75 local lesions
while the half leaf treated with mixture of silver nanoparticle and TMV
suspension showed 39.5 local lesions. The reduction of viral activity
expressed by local lesions was 40% on N. glutinosa. This finding suggests
that the chitosan nanoparticles are effective antiviral agent
PART IV
Application of nanoparticles in vivo
1. Application of nanoparticles as an antiviral agent
A) Silver nanoparticles
1.1. Virus infectivity
All concentrations were able to reduce virus infectivity after 25
days of inoculation in potato plants treated with silver nanoparticles preinoculation.
Data indicate that the potato plants treated with concentration of
50 ppm silver nanoparticles display a higher significant reduction in the
PVY virus infectivity than other concentrations. The highest percentage
reduction of virus infectivity with concentration of 50 ppm was 39.5 %.
This is followed by concentration of 55 ppm. While the less reduction
percentage achieved with concentration of 40 ppm 18.7% and 45 ppm
24.3 compared to the infected control.
1.2. Disease severity
Data indicate that all concentrations were able to reduce disease
severity after 25 days of inoculation in potato plants treated with silver
nanoparticles pre-inoculation.
Data indicate that concentration of 50 ppm showed the highest
significant reduction percentage of disease severity 51.2% followed by
concentration of 55 ppm 47.5% and 60 ppm 42.6%. However,
concentration of 40 ppm had the lowest significant percentage of disease
severity 31.1% compared to the infected control.
1.3. Virus concentration
At 21 days of inoculation, concentration of 60 ppm for silver
nanoparticles showed the lowest significant concentration of virus
replication followed by concentration of 55 ppm, 50 ppm.
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1.4. Reverse transcriptase - Polymerase chain reaction (RT-PCR)
The resulet showed that the virus found in all plants treated with
concentrations of silver nanoparticles and inoculated with PVY. RNA
amplification produced band with expected size 800 bp on agarose gel.
However, there were differences in the density of each amplicon but the
ampilicon produced from the 50 mg /l treatment was less dense.
1.5. Plant growth parameters
Result showed that treatment with 50 ppm increased the fresh
weight of inoculated-treated potato plants significantly as compared with
infected ones. PVY infection reduced the fresh weight of control plants at
25-day 32.6 g compared to healthy plants 67.5 g.
Effect of virus infection on dry weight was clear of control plants
3.6 g/ plant, while 50 ppm treatment increased the dry weight under
infection condition significantly to 5.7 g. treatment without inoculation
increased the dry weight to 7.5 g for 50 ppm treatment. This increasing
was significant higher than healthy plants 6.5 g.
Data showed that treatment with 50 ppm increased the leaf area of
inoculated-treated potato plants significantly as compared with infected
plants. PVY infection reduced the leaf area of control plants at 25-day 7.1
cm compared to healthy plants 13.7 cm. while 50 ppm treatment
increased the leaf area under infection condition to 11.7 cm. treatment
without inoculation increased the leaf area to 17.6 for 50 ppm treatment.
Effect of virus infection on tuber weight was clear of control
plants 9.1 g/ plant, while 50 ppm treatment increased the tuber weight
under infection condition significantly to 17.3 g. treatment without
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inoculation increased the tuber weight to 30.3 g for 50 ppm treatment.
This increasing was significant higher than healthy plants 27.5 g.
1.6. Antioxidant enzyme activites
1.6.1. Peroxidase (PO) activity
Data indicted that the activites of PO at 14 days of inoculation was
non-significant higher with concentration of 60 ppm of silver
nanoparticles. While, the lowest PO activity was at concentration of 40
ppm compared to control.
1.6.2. Polyphenoloxidase (PPO) activity
Data indicted that the activites of PO at 14 days of inoculation was
significant higher with concentration of 60 ppm of silver nanoparticles.
While, the significant lowest PO activity was at concentration of 40 ppm
compared to control.
1.7. Total Phenol contents
Treatment (60 ppm) was the most effective on accumulation of
phenol content compared to the other treatments. This is followed by
Treatment (45 ppm). All concentrations resulted in the significant
increase of phenol content at 14 days of inoculation.
1.8. Total amino acid contents
In this analysis, amino acids content in potato leaves treated with
silver nanoparticles and inoculated with PVY was increased comparing
with healthy and inoculated controls significantly. After 14 days of
applying AgNPs, treatment (50 ppm) was the most effective on
significant increase of amino acids content compared to the other
treatments. This is followed by treatment (45 ppm). All concentrations
resulted in the increase of amino acids content at 14 days of inoculation.
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1.9. Photosynthetic pigments
Chlorophyll A, B and carotenoids contents were determined afar
14 days from inoculation to detect the effect of treatments with silver
nanoparticles on those pigments in potato plants.
In general, the results showed that PVY infection significantly
reduced chlorophyll A, B, carotenoids compared to those of healthy
plants. Treatment of plants with AgNPs increased chlorophyll A, B,
carotenoids contents. Data showed that treatment (50 ppm) was the most
effective on increase of Chlorophyll A, B and carotenoids contents
compared to the other treatments. This is followed by treatment (45 ppm).
1.10. Total silver in potato tuber
In this analysis, total silver in potato tuber treated with silver
nanoparticles and inoculated with PVY was determined. After 14 days of
applying AgNPs, all concentrations was Less than the limit (10 ppm). The
differences in treatment are significant.
B) chitosan nanoparticles
1. Virus infectivity
All concentrations of ChNPs were able to reduce virus infectivity
after 25 days of each pre and post virus inoculation in potato plants
treated with ChNPs compared with virus inoculated plant without ChNPs
treated. Potato plants treated with ChNPs pre and post PVY inoculation
not appeared symptoms while virus inoculated plants without ChNPs
treatment showed mild symptoms (vein clearing and mild mosaic).
This result was confirmed by DAS-ELISA test using PVY specific
poly clonal antibody and PCR test using PVY coat protein gene.
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2. Virus concentration
Result of ELISA test demonstrated that all treatments reduced the
virus concenration signficantly compared to the control. At 21 days of
inoculation, all concentration of chitosan nanoparticles to pre-inoculation
and post-inoculation showed the lowest concentration of virus replication
followed by concentration of control.
3. Reverse transcriptase - Polymerase chain reaction (RT-PCR)
Detection of potato virus Y (PVY) in plants treated with different
concentration of ChNPs pre and post inoculation by RT-PCR and 1%
agarose gel electrophoresis analysis and hundred ladder marker showed
that postive plants in all treatment. RNA amplification produced band
with expected size 800 bp on agarose gel. However, there were
differences in the density of each amplicon but the ampilicon produced
from the 2 g /l treatment was less in density.
4. Plant growth parameters
Data showed that treatment with 2 g/l increased the fresh weight
of inoculated-treated potato plants significantly to 65 g pre-inoculation
and 75.3 g post-inoculation as compared with infected ones. This
increasing was a little than healthy plants. However, PVY infection
reduced the fresh weight of control plants significantly at 25 days 48 g
compared to healthy plants 86 g. Treatment of post-inoculation showed an
increasing of fresh weight than that of pre-inoculation.
Effect of virus infection on dry weight was clear of control plants
6.2 g/ plant, while 2 g/l treatment increased the dry weight under infection
condition significantly to 9.6 g pre-inoculation and 10.9 g postinoculation.
Treatment of post-inoculation showed an increasing of dry
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weight than that of pre-inoculation significantly at 25 days of inoculation.
This increasing was higher than healthy plants 13 g.
Data showed that treatment with 2 g/l increased the leaf area of
inoculated-treated potato plants significantly to 14.4 cm pre-inoculation
and 17.2 cm post-inoculation as compared with infected ones. This
increasing was a little than healthy plants. However, PVY infection
reduced the leaf area of control plants significantly at 25 days 12.7 cm
compared to healthy plants 18.3 cm. Treatment of post-inoculation
showed an increasing of leaf area than that of pre-inoculation.
Effect of virus infection on tuber weight was clear of control
plants 8.7 g/ plant, while 2 g/l treatment increased the tuber weight under
infection condition significantly to 14 g pre-inoculation and 15.1 g postinoculation.
Treatment of post-inoculation showed an increasing
significantly of tuber weight than that of pre-inoculation at 25 days of
inoculation. This increasing was higher than healthy plants 17.3 g.
3. Antioxidant enzyme activites
3.1. Peroxidase (PO) activity
Data indicted that the activites of PO at 14 days of inoculation was
significant higher with concentration of 2 g/l of chitosan nanoparticles
compared to control. Treatment of post-inoculation showed an increasing
of PO activity than that of pre-inoculation.
3.2. Polyphenoloxidase (PPO) activity
Data indicted that the activites of PO at 14 days of inoculation was
significant higher with concentration of 2 g/l of chitosan nanoparticles
compared to control. Treatment of post-inoculation showed an increasing
of PPO activity than that of pre-inoculation.
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4. Total Phenol contents
Our results indicated that PVY increased levels of phenol content
significantly in inoculated plants compared with healthy ones. High
acculmulation of phenol content was observed at 14 days with silver
nanoparticles.
concentration of chitosan nanoparticles (2 g/l) was the most
effective on accumulation of phenol content compared to the other
concentration. Treatment of post-inoculation showed an increasing of
phenol content than that of pre-inoculation.
5. Total amino acid contants
In this analysis, amino acids content in potato leaves treated with
chitosan nanoparticles and inoculated with PVY was increased
significantly comparing with control. After 14 days of applying chitosan
nanoparticles, concentration of 2 g/l was the most effective on increase of
amino acids content compared to the other concentration. Treatment of
post-inoculation showed an increasing of amino acid content than that of
pre-inoculation. All concentrations resulted in the increase of amino acids
content at 14 days of inoculation.
6. Photosynthetic pigments
Chlorophyll A, B and carotenoids contents were determined afar
14 days from inoculation to detect the effect of treatments with chitosan
nanoparticles on those pigments in potato plants.
In general, the results showed that PVY infection significantly
reduced chlorophyll A, B and non-significantly carotenoids compared to
those of healthy plants. Treatment of plants with chitosan nanoparticles
increased chlorophyll A, B, carotenoids contents. Result showed that
concentrations of 2 g/l was the most effective on increase of Chlorophyll
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A, B and carotenoids contents compared to the other treatments.
Treatment of post-inoculation showed an increasing of plant pigments
content than that of pre-inoculation