الفهرس 
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Abstract
known to affect and to endanger the pome fruit worldwide, they may have devastating effects on pome fruit longevity, and on the quality and quantity of the crop. The present work aimed to study several aspects of some pome fruit viruses (ApMV and ASGV) including disease distribution in some Egyptian regions, identification and characterization of the Egyptian isolated strains by host range, symptomatology, serological tests, and mode of transmissions biologically by vectors, in addition to their sequencing. The study also extended to the histopathological changes, and the effect of
viral infection on some plant components, i.e. chlorophyll, total phenols
total indols, total amino acids and total sugars. An attempt to eliminate
ApMV and ASGV using thermotherapy with meristem tip culture.
Moreover, some compounds were used as antiviral, in addition to
determination the peroxidase and polyphenoloxidase activities and protein
profile.
The obtained results of the present study could be summarized as
follows:
1. Apple mosaic virus (ApMV) is one of the oldest known and most
widespread apple viruses. Apple trees infected with apple mosaic
virus develop pale to bright cream spots on spring, Leaves as they
expand, these spots may become necrotic after exposure to summer
sun and heat.
2. Apple stem grooving virus (ASGV), the type member of the genus
Capillovirus. ASGV is the causal agent of decline and graft union
necrosis diseases in Malus rootstock. ASGV infection is frequently
symptomless in apple cultivars. Our results demonstrated the
presence of two viruses, (ApMV) and (ASGV).
3. The aforementioned viruses (ApMV and ASGV) used in this study in
three successive seasons (2009-2010, 2010-2011 and 2011-2012)
among four governorates: Kalyobiya, Monofiya, El-Beheira, and
Ismailia. Different symptoms varied from mosaic, chlorosis, and
SUMMARY 195
crime spots, and abnormalities (swelling) budunion were generally
observed on the infected trees during the survey, in addition
symptomless trees which also were observed. These collected
samples were thereafter tested in the serological laboratory using
DAS-ELISA technique.
4. In the first season (2009-2010), ApMV incidence among the five
governorates was highest in Kalyobiya 20.00% in infected apple trees
and 13.33% in infected pear trees, as old orchards, while ASGV
incidence was 15.00% in apple trees, and 13.33% in pear trees
5. In the second season the infection percentages were increased to be
21.42% in infected apple trees and 20.00% in infected pear trees, as
old orchards, while ASGV incidence was 18.57% in apple trees, and
16.66% in pear trees.
6. In the third season the infection percentages were increased to be
24.39% in infected apple trees and 20.0% in infected pear trees, as
old orchards, while ASGV incidence was 17.07% in apple trees, and
24.00% in pear trees.
7. The results indicated that the mean percentages of ApMV infection
were 11.62%, in infected apple trees and 9.25% in infected pear
trees, while the mean percentages of ASGV infection were 8.61%
infected apple trees and 9.15% in infected pear trees, in all over the
three seasons in the five Governorates.
8. In general, from a total of 800 apple samples collected during the
three seasons, 11.62% of samples were ApMV positive, and 9.25%
were ASGV positive. While from a total of 557 pear samples
collected during the three seasons, 8.43% of samples were ApMV
positive, and 8.97% were ASGV positive.
9. In some orchards, ApMV and ASGV were detected either alone or in
dual infection with each other. Such results were confirmed using
specific Pab antiserum. In general the mix infection rates were
estimated by DAS-ELISA as following; 3.25% and 2.51% in apple
and pear trees respectively.
SUMMARY 196
10. Generally, the infection of either ApMV or ASGV by visual
examination or using DAS-ELISA test was increased in second
season and the third season compared with the first season.
11. Regards isolation and identification of the two virus isolates the host
range of ApMV was isolated on healthy seedlings of Malus rootstock
by grafting inoculated. These results were confirmed by DAS-ELISA.
On the other hand Citrus excelza is the differentiate host plant for ASGV
and gave positive reaction with specific symptoms (Tattered leaf), while
it gave no reaction with ApMV.
12. The apple and pear pome fruits naturally ApMV and ASGV infected
were used as a source of ApMV-EG and ASGV-EG isolates. The
ApMV-EG and ASGV-EG isolates were tested and propagated on
Cucumis sativus which is consider differentiate host plant and gave
systemic reaction with ApMV and N benthemiana gave systemic
reaction with ASGV.
13. The general outlook at the results indicated that ApMV and ASGV have
a wide host range between members of the family Solanacea (N.
glutinosa, N. benthamiana), Legumine (Ph. vulgaris), Chenopodiaceae
(Ch. quinoa & Ch. amaranticolor), Cucurbitaceae, and Amarantheaceae.
14. from 16 tested host plants,(ApMV) infected 15 hosts,chlorotic local
lesions appeared on one host (Ch. quinoa). chlorotic local lesions,
systemic mottle on N. gloutinosa, Chlorosis on N. benthemiana,
Mosaic, purple brown spots, chlorotic local lesions, systemic necrosis
on Ph. vulgaris and systemic infection in C. sativus.
15. For ASGV 16 hosts were infected from 14 tested host plants, (ASGV)
infected 5 hosts, systemic chlorotic local lesions appeared on (Ch.
quinoa and Ph. vulgaris). Necrotic local lesions, chlorotic on N
gloutinosa, Chlorosis on N benthemiana.
16. Both viruses were identified according to host range,
symptomatology, modes of transmission, serological reactions using
(DAS-ELISA) techniques, cytological changes.
17. Concerning the mechanical transmission, ApMV and ASGV were
transmitted using mechanical inoculation from the infected source
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plants to the indictor test plants. The results of mechanical
transmission were confirmed by ELISA.
18. In terms of serological diagnosis, DAS-ELISA, DTBIA and DBIA
were used to detect viruses in the infected tissues. DAS-ELISA
protocol was done for detection of the viruses. Infected samples gave
positive results when detected with ApMV and ASGV using specific
polyclonal antibodies. Samples with an absorbance of at least twice
that of control were considered positive.
19. Also the presence of ApMV and ASGV in the infected apple and pear
leaves was checked using both DTBIA and DBIA, Applying DTBIA
test was found to be sensitive to detect ApMV antigen in the infected
samples which gave clear purple color in positive reactions on the
tested trees, moreover no reaction with negative control. Applying
DTBIA test, for ASGV detection, using ASGV antigen in the samples
of apple and pear (leaves, petioles and stems) collected from cv.
Anna, and cv. LeCont and grafted trees ASGV gave a clear positive
reaction while no reaction with negative control.
20. It was clear that strong positive reaction was recorded with dots
applied from extracts of infected samples, whereas those extracted
from healthy ones remained green, these results were compared with
ELISA readings of the same samples.
21. The rootstocks were differed in response to ApMV-EG and ASGV-EG
isolates where Malus sylvestris, Maling Morten 106 (MM) ( Pyrus
communis and betialiopholia) , were indexed by graft inoculation with
two blind buds from ApMV and ASGV infected apple and pear plants.
The obtained results indicated that tested woody plants were differed in
response to ApMV and ASGV isolate. For ApMV Malus and
betialiopholia rootstocks appeared low response and gave mosaic
symptoms. While Maling Morten 106 (MM) and Pyrus communis
appeared hyper sensitivity for ApMV Malus and Maling Morten 106
(MM) appeared more sensitivity for ASGV than Pyrus communis and
betialiopholia. No symptoms were observed on negative control, the
results were confirmed by DAS-ELISA test using specific antiserum.
SUMMARY 198
22. About vector transmission Aphis pomi insects were used for virus’s
transmission, The results indicated that were un-capable to transmit
neither the ApMV nor ASGV after acquisition period about 15, 30 ,45
and 60 min only allowed to feed for 60 mins. These plants were tested
serologically by DAS-ELISA against specific ApMV and ASGV
antiserum and gave negative results. The obtained results showed that
ApMV and ASGV don’t transmit by vectors from infected plants to
healthy plants.
23. ApMV and ASGV could be transmitted through seeds of C. sativus
and, Ph. vulgaris, gave different transmission percentages according
to the virus and the type of plant. These percentages were ranged
from (6%) in C. sativus for ApMV, while ASGV don’t transmitted
through seed of C. sativus. Regards Ph. vulgaris, the percentages of
transmissions were 10% and 13% for ApMV and ASGV respectively.
These results were confirmed by ELISA.
24. Fresh leaf tissues of infected apple and pear were extracted using
RNeasy Plant Mini Kit (Qiagene, Inc). The obtained extraction was
amplified by PCR. The amplified PCR fragments were used for cloning,
sequencing and comparing the sequence with other of those sequencing
available in GenBank. The obtained results demonstrated the successful
detection of ApMV by amplification of the unique 262bp which report
the accuracy of ApMV identification in RT-PCR assays using DNA
primers for coat protein region. The results also demonstrated the
successful application of RT-PCR assays for the detection of coat protein
region of ASGV in apple and pear trees using ASGV specific primers. A
specific product of (524 bp.). Fragments of the expected size, 262 bp
ApMV cDNA and 524 bp ASGV cDNA amplified products were
directly cloned into pCR™ TOPO vector using TA™ Cloning system
(Invitrogen, Carlsbad, CA). The recombinant plasmid was successfully
transformed into E. coli (Xl1blue strain). Recombinant plasmids which
had correct inserts of ApMV (262 bp) and ASGV (524 bp) were
validated using PCR colony and restriction analysis then applied for
nucleotide sequence. Nucleotide sequence analysis for 262 bp.
SUMMARY 199
Amplified fragment from coat protein gene of ApMV genome showed
homology ranged from 100% with ApMV in Czech Republic, 97%
similarity with ApMV strain in Ukraine 95% similarity with ApMV
strain IN Turkey was observed, and 92% of similarity of ApMV with
Korea Strain, available in GenBank. Multiple sequence alignment of the
524 bp from ASGV 3´ non-coding region isolated from Egypt with some
ASGV isolates available in the GenBank indicated that the 3´ noncoding
region sequences of ASGV isolated from Egypt shared 92% with
ASGV isolate in Brazil and China, 90% with ASGV isolate in Taiwan
and USA and 88% with ASGV isolate in Czech republic.
25. Comparison between bases composition of complete genome sequence
for ApMV (Egyptian isolate) and four ApMV isolates available in the
GenBank, and also comparison between ASGV (Egyptian isolate) and
five ASGV isolates available in the GenBank were done to determine
the relationship with other recommended ApMV-EG and ASGV-EG
registered in GenBank. A similarity to the other sequences of ApMV-EG
ranged from 100% to 92% while in case of ASGV-EG. A similarity to
the other sequences ranged from 92% to 88% with other isolates
available in GenBank.
26. Concerning cytological effects of ApMV and ASGV infection, light
microscopy examination revealed that amorphous cytoplasmic inclusion
bodies in epidermal stripes of leaves samples, taken from the lower
surface of systemically ApMV and ASGV infected N. benthemiana, 20
days after inoculation. It was observed that cytoplasmic amorphous
inclusions formed in infected leaves, seemed to be attached to the
nucleus from one or two sites, while these inclusions have never been
observed in the epidermal stripes of healthy leaves.
27. Semi thin sections of ApMV in infected and healthy apple cv. (Anna)
leaves were examined using light microscopy. In the present study,
examination ultrathin section of infected apple leaf, showing the
mesophyll cells with relative narrow space. The mesophyll cells are
elongated, chloronchyma with uniformly wide cell wall and contained
nuclous, chloroplast and mitochondria. The size of spongy cells was
SUMMARY 200
small characterized with reduced intracellular space in some parts of the
section and cells became compacted in comparison with tissues of
healthy leaves and regular palisade cells were observed in some parts of
the healthy section, while irregular ones were observed in the infected
one, moreover, they were separated and decreased in the length of the
infected than in the healthy one.. The chloroplast alterations were studied
and compared with healthy ones. The number and organization of
chloroplasts were different in cells of infected tissues, and exhibited
several degrees of deformation and lyses. It showed slightly rounded
with irregular rows of grana, plastoglobules in unusual numbers within
grana and stroma of chloroplast and destructed regions in chloroplast
which does not organized into grana and thylakoid system.
28. The nucleus shows with several dark-stained bodies and sometimes
nucleolus appeared as kidney shaped. The infected cells revealed the
presence of structures consisting of multiple membranes, frequently
associated with an internal core either simple or compound and
usually appeared along plasma-membrane protruded into vacuoles
and tubules in their longitudinal section.
29. ApMV and ASGV infection, affected on the quality and quantity of
yield. Represented in the reduction of the height of the infected trees,
reduction of the trunk diameter and the yield compared with the
healthy trees
30. Changes in some biochemical components associated with ApMV
and ASGV infection were studied. ApMV and ASGV were able to
reduce the photosynthetic pigments (Chlorophyll A and B) content of
infected apple and pear trees, compared with the healthy ones. It was
observed that the infected apple leaves with ApMV showed marked
biochemical changes represented in relative reduction in Chl a, Chl. b
and carotenoid contents. It was observed that the highest reduction in
Chlorophyll b, being 9.35% followed by chlorophyll a, 1.95% and
Carotenoids 7.59% respectively, when compared with the healthy
control. Also the infection with ASGV of apple plants showed the
highest reduction in chlorophyll b 0.97%, followed by chlorophyll a,
SUMMARY 201
1.01% and Carotenoids 5.72%, when compared with the healthy
control.
31. Moreover, ApMV infected pear leaves show the highest reduction in
chlorophyll a, followed by chlorophyll b and carotenoids 50.12, 32.84
and 40.67, respectively when compared with the healthy plants. While
the infected pear leaves with ASGV show the highest reduction in
chlorophyll a, chlorophyll b and carotenoids 29.84, 0.48, and 23.73,
respectively when compared with the healthy plants.
32. The infected apple and pear seedlings with ApMV and ASGV showed
biochemical changes represented in marked increasing in total amino
acids 0.450 and 0.856 in infected apple trees respectively compared
with the healthy control 0.370. While in infected pear trees with
ApMV and ASGV 1.94 and 1.671, respectively compared with the
healthy control 0.502
33. Also marked increasing in total indol in the infected apple and pear
leaves was observed 1.206 and 1.684 in infected apple trees
respectively compared with the healthy control 1.129. While in
infected pear trees with ApMV and ASGV 1.764 and 1.179
respectively compared with the healthy control 1.114
34. Also marked increasing in total phenols in the infected apple and pear
leaves was observed 2.797 and 2.664 in infected apple trees
respectively compared with the healthy control 2.617. While in
infected pear trees with ApMV and ASGV 2.748 and 2.723
respectively compared with the healthy control 2.419.
35. Also marked increasing in total sugars in the infected apple and pear
leaves was observed 2.269 and 2.269 in infected apple trees,
respectively compared with the healthy control 1.966. While in
infected pear trees with ApMV and ASGV 2.334 and 2.322,
respectively compared with the healthy control 1.283
36. A significant reduction of some chemical characters of apple fruits.
The obtained results showed clear significant decreasing in infected
fruit weights with ApMV and ASGV 190.77 and 203.5, compared
with the healthy fruit 320.54. Marked inceasing in Ascorbic acid (VC
SUMMARY 202
mg/100g) 5.717 and 4.922 in ApMV and ASGV respectively when
compared with the healthy fruits 4.878. Also the results showed clear
decreasing in contents of titrable acidity 0.750 and 0.438 in ApMV
and ASGV respectively when compared with the healthy fruits
0.933. Also the results showed clear decreasing in contents of .total
sugar 8.294 and 8.416 in ApMV and ASGV respectively when
compared with the healthy fruits 11.165. While marked increasing in
total soluble solids (TSS) was observed in infected ApMV (14) and
ASGV (16) fruits when compared with the healthy control (12).
37. from infected ApMV and ASGV of apple (Malus) and pear
(Communis) plants, production of virus free plants was done using
thermotherapy with meristem tip culture.
38. The results indicated that High virus eradication efficiency was
achieved by thermotherapy at 36°C for 60 days combined with the
culturing of 0.5 mm long meristem-tips, the results were confirmed
by DAS-ELISA and PCR.
39. The changes in the activity of peroxidase and polyphenoloxidase were
estimated in healthy and infected apple trees with ApMV and ASGV, as
well as infected plants treated with some compounds in attempt for virus
elimination. The infected plants showed higher enzymatic activity (1.15
for ApMV, and 1.70 for ASGV) compared with healthy ones (0.864).
The infected plants treated with different compounds were found to be
unable to induce the highest polyphenoloxidase activity in an the
infected plants except in case of Virocide which gave the highest activity
(1.6 for ApMV and 1.78 for ASGV) followed by kombucha compared
with the healthy and inoculated control (0.864, 1.15 and 1.70).
Concerning peroxidase activity, its rates were increased in infected apple
trees when compared with the healthy ones.
40. The infected apple and pear seedlings with ApMV and ASGV
showed biochemical changes represented in marked increasing in
proline compared with the healthy control