الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Viroids are the smallest known agents of infectious disease— small
(246–399 nt), highly structured, circular RNAs that lack the protective capsid and mRNA activity characteristic of viral RNAs yet are able to replicate
autonomously in susceptible plant species.
Grapevine viroids diseases often have an economic impact on
grapevine growth, yield and fruit quality and also increase plant mortality.
Full range virus, viroid testing combined with a comprehensive grapevine
certification program are needed to reduce or eliminate these detrimental
effects.
Grapevine ( Vitis vinifera ) is one of the most important crops in
Egypt. Viroids are widespread throughout all grapevine growing areas of the
world. According to sequence analysis, five distinct viroids have been
recognized on grapevine (Szychowski et al., 1988b). A total of 100 samples
of the major grapevine variety balady/banaty were collected from three
thousand grapevine trees. Vein banding, yellow vein, leaf rolling, yellowing,
chrome yellow spots, mosaic, yellow speckle, vein clearing, yellowing
blotches spotting and small leaf symptoms were detected in all areas
surveyed. In this study grapevine trees leaves samples were examined for the
presence of grapevine viroids depending on the external symptoms and
molecular hybridization, tissue print and dot blot hybridization, sPAGE and
RT-PCR. The viroids were detected by sPAGE essentially as described by
(Semancik and Harper 1984; Szychowski et al., 1988b) following nucleic
acid extraction, RT-PCR detection.
5.1. Detection of grapevine viroids:
5.1.1. Nucleic acid hybridization
The samples were tested for the presence of grapevine viroids by
Nucleic acid hybridization using digoxygenin-labelled riboprobes for Hop
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stunt viroid (HSVd), Citrus exocortis viroid (CEVd), and Potato spindle
tuber viroid (PSTVd). By tissue printing hybridization, HSVd was detected
in 10 samples, CEVd in 12 and PSTVd in 10. while by dot blot hybridization
HSVd gave 11 positive results from 100, CEVd gave 7 positive results and
PSTVd gave 12 positive results.
5.2. Detection of viruses in naturally infected grapevine
samples
Serological diagnosis by DAS-ELISA using specific polyclonal
antibodies against ToRSV and GFLV were done for ten grapevine trees
samples which gave positive results with grapevine viroids out of 100 trees
gave negative results comparing with the positive and negative controls.
5.3. Frequency of viroids naturally infected grapevine trees
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.
5.4. Biological indexing of grapevine viroids:
5.4.1. Host range and symptomatology
Viroids usually have relatively narrow host ranges, with each species
infecting one or a few plant species in the field.
In biological identification, grapevine viroids (HSVd, CEVd, PSTVd,
and may be GYSVd) gave different disease symptoms on some host range
plants (indicator plants). HSVd, gave vein clearing on Gynura aurantiaca,
mosaic with chlorotic spots, vein clearing, rugosity and stunting on Cucumis
sativus L. cv. alpha, mild mosaic on Lycopersicon esculantum L. cv. Castle
rock, but HSVd did not give any symptoms on Chenopodium amaranticolor
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L, Nicotiana glutinosa L., Datura metel L. and Vitis vinifera cv.
Balady/banaty.
CEVd gave mosaic and vein banding on G. aurantiaca, mild mosaic
on Cucumis sativus L. cv. alpha, but CEVd did not give any symptoms also
on Ch. amaranticolor L, L. esculantum L. cv. Castle rock, N. glutinosa L. D.
metel L. and, V. vinifera cv. Balady/banaty.
PSTVd gave mild mosaic on G. aurantiaca, severe mosaic, leaf
deformation and epinasity on Lycopersicon esculantum L. cv. Castle rock).
PSTVd did not give any symptoms on Ch. amaranticolor L, C. sativus L. cv.
alpha, N. glutinosa L., D. metel L. and V. vinifera cv. Balady/banaty
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.
5.4.2. Mode of transmission of grapevine viroids
5.4.2. A. Mechanical transmission
HSVd, PSTVd and CEVd were mechanically inoculated on herbaceous
plants with infectious sap using cotton swap and producing different viroid
disease symptoms.
5.4.2. B. Grafting transmission:
The 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.
HSVd gave yellowing, leaf curl and mosaic, CEVd gave vein
chlorosis, mosaic while PSTVd gave mosaic, leaf deformation, and
yellowing, GYSVd gave severe mosaic, leaf deformation and yellowing
spots.
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5.5. RNA analysis of grapevine viroids:
5.5.1. Extraction and purification of grapevine viroids-RNA:
The purified grapevine viroids-RNA were obtained from 10g infected
grapevine leaves according to CF-11 cellulose column chromatography. This
procedure can be utilized to remove contaminating host RNAs from viroid
preparations as well as to separate individual viroids with selective elusion by
different ethanol concentrations. And now known to comprise a mixture of
four viroids, were analysed by sequential elution from CF-11 cellulose.
Serial elusion with an ethanol gradient and STE by CF-11 cellulose
column chromatography was done.
In the 25 % ethanol-STE buffer eluant was moderate enriched in
grapevine viroids. The values of A260/A280 were 1.53, 1.3, 1.08 and 1.34
respectively for four groups. And the values of A280/A260 were 0.65, 0.76,
0.92 and 0.74 respectively for four groups 1, 2, 3 and 4. Grapevine viroids
yield were 0.36, 1.7, 0.88 and 0.64 μg/μl infected grapevine leaves.
In the 15 % ethanol-STE buffer eluant was highly enriched in
grapevine viroids. This manipulation greatly improved the recovery of
viroids. The values of A260/A280 were 1.95, 1.38, 1.8 and 1.92 respectively
for four groups. And the values of A280/A260 were 0.51, 0.72, 0.56 and
0.52 respectively for four groups 1, 2, 3 and 4. Grapevine viroids yield were
0.47, 1.8, 0.72 and 0.96 μg/μl infected grapevine leaves.
In the 0% ethanol-STE buffer eluant was enriched in grapevine
viroids. The values of A260/A280 were 1.23, 1.43, 1.51 and 1.6 respectively
for four groups. And the values of A280/A260 were 0.80, 0.69, 0.66 and 0.63
respectively for four groups 1, 2, 3 and 4. Grapevine viroids yield were 0.58,
1.82, 0.60 and 0.44 μg/μl infected grapevine leaves.
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5.5.2. Electrophoretic forms of grapevine viroids-RNA:
Sequential PAGE confirmed the presence of GYSVd-1, CEVd and
HSVd. The solution of RNA (four group represent 12 positive sample)
obtained after the cellulose chromatography (15% E.OH-STE and 0% E.OHSTE)
was subjected to one cycle of 5 % polyacrylamide gel electrophoresis
under non- denaturing conditions. The final purified viroid RNA species
migrates as a single electrophoretic component under standard conditions of
PAGE. where sequential polyacrylamide gel electrophoresis (s-PAGE) is
very efficient method for circular RNA viroid detection in partially purified
RNA viroid with two steps procedure.
In the 15 % ethanol-STE buffer eluant was highly enriched in grapevine
viroids. This manipulation greatly improved the recovery of viroid, the four
fractionated infected samples appears different band patterns as mixed and
single infection whereas, group 1 appears mixed infection with HSVd,
GYSVd, and CEVd, group 2 appears mixed infection also with HSVd,
GYSVd, and CEVd, and group 3, 4 revealed to single infection with single
band for HSVd. In the 0% ethanol-STE buffer eluant did not appears any
bands.
5.6. Molecular characterization of GYSVd-1
5.6.1. Reverse transcription polymerase chain reaction (RTPCR)
5.6.1. A. RNA yield
Total RNA was extracted from four group infected grapevine plant
leaves according to Astruc et al., (1996) and Pallás et al., (1998) as
described under materials and methods. The integrity and quantity of the
purified RNA were confirmed by UV Spectrophotometer and gel
electrophoresis. The protocol described under materials and methods was
used successfully to isolate a high yield of total RNA (1.9, 1.76, 1.23, 1.09
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μg/0.5 g leaf tissues) from naturally infected grapevine tissues for group 1, 2,
3 and 4 respectively.
5.6.1. B. RT-PCR amplification
RT-PCR of GYSVd-1 Egyptian-isolate was used to amplify a
fragments of about (368 bp). The size of the amplified PCR product from
grapevine viroid infected leaves was estimated by comparing its
electrophoretic mobility with those standard DNA marker. The amplified
DNAs was in the expected sizes calculated (368 bp) from the position of the
primers.
5.6.2. Nucleotide sequence analysis:
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 GenBank. The cDNA
sequence was performed using PCR product when the specific (downstream
and upstream) primers for GYSVd-1 were used. Nucleotide were found to
be 368 bp.
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
registered in GenBank. 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.
5.6.3. GYSVd-1RNA structure analysis:
The minimum free energy of a secondary structure for RNAGYSVd-
1(EG) isolate is 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
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mol-1. It is rod-shaped structure composed of alternating single- and doublestranded
regions
5.7. Vegetative growth and yield quality parameters of viroidsinfected
grapevine trees.
Grapevine viroids infection affected on the quality and quantity of grape
yield, whereas, the reduction rate in viroids-infected grapevine shoot length
was 32.91 %. comparing with healthy ones, and the reduction percentage of
leaf area, leaf fresh, leaf dry weights and leaf dry matter content in viroidsinfected
grapevine plants was 21.83, 13.12, 7.03 and 15.77 % respectively.
And also the reduction rate in total pigment content nearly to 50 % for
viroids-infected grapevine plants comparing with healthy ones. The reduction
rate percentage of bunch weight, no. of berries/ bunch, bunch length and
bunch width in viroids-infected grapevine plants was 40.48, 8.29, 26.84 and
16.99% respectively
Data obtained in the present study showed the reduction rate in
viroid-infected grapevine berries weight comparing with healthy ones was
51.80%, and berries size was 54.83%.
The rate of reduction in viroid-infected grapevine berries total soluble
solids comparing with healthy ones was 27.77%. While the rate of increase in
acidity of the berries of viroid infected grapevine was 33.33%.
Total soluble sugar content differs greatly in viroid infected grapevine
yielding than healthy ones. The reduction percentage of total soluble sugar
content in viroid infected grapevine yielding was 11.11%. and the rate of
reduction in viroid-infected grapevine berries total insoluble sugars was
22.99%.
The rate of reduction in total carbohydrate content of viroid-infected
grapevine yielding was 14.26%. And the rate of reduction in Sugar:acid ratio
of viroid-infected grapevine yielding comparing with healthy ones was
35.68%.
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5.8. Grapevine viroids-elimination
from infected grapevine plants, molecular hybridization and s-PAGE
were done to detect HSVd, CEVd, PSTVd, and GYSVd in stem nodle of
mother grapevine cv. balady/banaty, as well as micro-propagated shoot. Stem
nodle culture has been frequently used to eliminate grapevine viroids from
infected plants.
The results indicated that 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 viroid-free plants were 18, 27 and 40 %.
In conclusion, from the obtained pervious study results clearly
demonstrated that the relationship between viroid infection and some
phytopathological phenomena etiology in grapevine in Egypt. Grapevine
plants act a reservoir of viroids which infected with mixed, double and single
infections showing some disease symptoms in Egyptian fields. The modified
method developed in this study allowed the isolation of intact, high yield and
quality RNA from grapevine leaves and may be other plant tissues with high
phenolic compounds and polysaccharides. S-PAGE is an efficient
biochemical method for separation and identification of viroids. The
minimum free energy of a secondary structure for RNA-GYSVd-1(EG)
isolate is determined from its primary sequence by summing the energy
contribution of all base pairs, loops at 37°C using MFOLD program was -
173.33 kcal mol-1. It is rod-shaped structure composed of alternating singleand
double-stranded regions. Grapevine viroids reduce vegetative growth,
fruit yield quality and quantity. Shoot tip cultures coupled with cold-therapy
were successfully used to eliminate grapevine viroids, with high eradication
rate after three months of cold-therapy treatment.
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Recommendations:
our results summarizes the economic effects, distribution and
measures currently used to control the viroid diseases affecting of
economically important crop (Grapevine). Under most conditions,; the
primary means of viroid spread are vegetative propagation and mechanical
transmission and the best means for control of viroid diseases are extensions
of those used for virus diseases and include: (i) elimination of viroids from
planting material (certified stock programmes); (ii) control of viroid spread in
the field (eradication); and (iii) quarantine exclusion of new infection. Proper
sanitation, including sterilization of tools and equipment between each plant
(for certified stock) or between each bed, row or section (to prevent spread in
the field), is critical. Many quarantine and certification programmes are
currently in place to prevent viroid introduction from germplasm collections.
Results are uneven, but in those cases where control has been
achieved. Three factors have proven critical: first, the availability of rapid
and sensitive diagnostic test(s) second, adoption of either a one-pass
production system or a clean stock programme where mother plants
maintained under protected conditions are repeatedly tested for their infection
status; and third, strict adherence to a zero-tolerance policy. To date, no
useful sources of genetic resistance to viroid infection have been identified in
any plant species.