الفهرس | Only 14 pages are availabe for public view |
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 Summary 173 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 Summary 174 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. Summary 175 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. Summary 176 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 Summary 177 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 Summary 178 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%. Summary 179 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. Summary 180 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. |