الفهرس 
Chapter I : IntroductionOnly 14 pages are availabe for public view
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Abstract
• Name of Candidate: Mohamed Atef Abdel-Rahman Nasr-Eldin
B.Sc. (Microb.&Chem.), Zagazig Univ., Benha Branch, 2003
M.Sc. (Microbiology), Benha Univ., 2007
• Degree: Ph.D.
• Title of thesis: Molecular Studies on Some Viroids Infecting Grapevine
in Egypt
• Department: Botany Branch: Microbiology, Virology
Faculty of Science, University of Benha, Egypt, 2013
Hop stunt viroid (HSVd), Citrus exocortis viroid (CEVd), Potato
spindle tuber viroid (PSTVd) and Grapevine yellow speckle viroid-1
(GYSVd-1) were characterized from naturally infected grapevine Vitis
vinifera cv. Balady/banaty during two summer seasons 2009/2010,
2010/2011 in Egypt. One hundred sticks of grapevine trees were collected out
of three thousand grapevine trees showing symptoms such as stem cracking,
torsion, leaf deformation, mosaic vein-banding, yellow batches, Also some
leaves showing distinctive viroid and virus like symptoms involved ”yellow
speckle, vein clearing and wavy leaflets margin”.
Nucleic acid hybridization using digoxygenin-labelled riboprobes for
HSVd, CEVd and PSTVd was used to detect the viroid-infected samples. The
total percentage of positive infection was 88.88%. Viroid-infected samples
gave negative results in DAS-ELISA using specific polyclonal antibodies
against ToRSV and GFLV.
The frequency of HSVd, CEVd and PSTVd naturally infected
grapevine trees recorded different percentages as in single (8.33, 8.33 and
11.11%) respectively, double (11.11, 11.11 and 19.44%) for CEVd+HSVd,
CEVd+PSTVd and HSVd+PSTVd respectively and mixed infection
(19.44%) with different disease symptoms.
Grapevine viroids (HSVd, CEVd, PSTVd, and may be GYSVd) gave
different disease symptoms on some host range plants (indicator plants).
HSVd, gave characteristic symptoms as mosaic with chlorotic spots, vein
clearing, rugosity and stunting on Cucumis sativus L. cv. alpha, CEVd gave
mosaic and vein banding on Gynura aurantiaca, and PSTVd gave leaf
deformation and epinasity on Lycopersicon esculantum L. cv. Castle rock).
GYSVd did not give any symptoms on indicator hosts but gave yellow
speckle and yellowing spots on the main host V. vinifera cv. Balady/banaty.
Grapevine viroids isolates were transmitted through grafting by eye
buds from infected grapevine cv. Balady/ banaty, to healthy one and the
symptoms appeared after 6-8 weeks.CF-11 cellulose column chromatography was utilized to remove
contaminating host RNAs from viroid preparations as well as to separate
individual viroids with selective elusion by different ethanol concentrations.
Serial elusion with 25 % ethanol-STE, 15 % ethanol-STE and 0%
ethanol-STE buffer eluant by CF-11 cellulose column chromatography was
done, and grapevine viroids-RNA yield was determined by UV
Spectrophotometer.
Sequential PAGE confirmed the presence of GYSVd-1, CEVd and
HSVd. In the 15 % ethanol-STE buffer eluant was highly enriched in
grapevine viroids. This manipulation greatly improved the recovery of
viroids, the four fractionated infected samples appears different band patterns
as mixed and single infection whereas, the mixed infection with HSVd,
GYSVd, and CEVd, appeared in two samples, while another two samples
revealed single infection with single band for HSVd. In the 0% ethanol-STE
buffer eluant did not appears any bands.
Total RNA was extracted from four group infected grapevine plant
leaves. The integrity and quantity of the purified RNA were confirmed by
UV Spectrophotometer and gel electrophoresis. About 368 bp DNA fragment
was amplified from the samples by RT-PCR using specific GYSVd-1 (+) and
GYSVd-1 (-) primers.
The nucleotide sequence of the PCR-amplified fragment for the
GYSVd-1 genome (Egyptian isolate) was done to determine the relationship
with other recommended GYSVd-1 registered in Gen Bank.
Comparison between bases composition of complete genome sequence
for GYSVd-1 (Egyptian isolate) and seven GYSVd-1–isolates was done to
determine the relationship with other recommended GYSVd-1. A Similarity
precentage index of GYSVd-1(EG) revealed 99.55% a high degree of
similarity to the other isolates sequences of GYSVd-1 in Genbank
The minimum free energy of a secondary structure for RNA-GYSVd-
1(EG) isolate was determined from its primary sequence by summing the
energy contribution of all base pairs, interior loops, hairpin loop, bugle loops
and external loop at 37 °C using MFOLD program was -173.33 kcal mol-1. It
is rod-shaped structure composed of alternating single- and double-stranded
regions.
Grapevine viroids reduce vegetative growth, fruit yield quality and
quantity. The reduction rate in total pigment content nearly to 50 % for
viroids-infected grapevine plants comparing with healthy ones.
The rate of reduction in viroids-infected grapevine berries juice total
soluble solids comparing with healthy ones was 27.77%. While the rate of
increase in acidity of the berries juice of viroids infected grapevine was
33.33%. Total soluble sugar content differs greatly in viroids infectedgrapevine yielding than healthy ones. The reduction percentage of total
soluble sugar content in viroids infected grapevine yielding was 11.11%. and
the rate of reduction in viroids-infected grapevine berries total insoluble
sugars was 22.99%. The rate of reduction in total carbohydrate content of
viroids-infected grapevine yielding was 14.26%. The rate of reduction in
Sugar:acid ratio of viroids-infected grapevine yielding comparing with
healthy ones was 35.68%.
Shoot tip cultures coupled with cold-therapy were successfully used
to eliminate grapevine viroids from infected mother plants, cold therapy at 4
°C for 1, 2 and 3 months, treated plantlets on M.S. media showing the
percentage of survival was 73, 64, 45 % and viroids-free plants were 18, 27
and 40 %, and thus reduces the risk of introducing these viroids to Egypt.
Key words: Hop stunt viroid (HSVd), Citrus exocortis viroid (CEVd),
Potato spindle tuber viroid (PSTVd) and Grapevine yellow speckle viroid-1
(GYSVd-1), Nucleic acid hybridization, DAS-ELISA, CF-11 cellulose
column chromatography, S-PAGE, RT-PCR, Sequencing, Tissue culture,
Cold therapy.