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العنوان
SEROLOGICAL STUDIES ON SOME PLANT VIRUSES \
المؤلف
HEGAZY, AHMED HAMDY ABDELMOAMEN.
هيئة الاعداد
باحث / أحمد حمدي عبد المؤمن حجازي
مشرف / خالد عبد الفتاح الدجدج
مناقش / هاشم محمد ابراهيم البنا
مشرف / عاطف شكري صادق
تاريخ النشر
2018.
عدد الصفحات
174 p. :
اللغة
الإنجليزية
الدرجة
ماجستير
التخصص
الزراعية والعلوم البيولوجية (المتنوعة)
تاريخ الإجازة
10/5/2018
مكان الإجازة
جامعة عين شمس - كلية الزراعة - الميكروبيولوجيا الزراعية
الفهرس
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Abstract

Forty samples of naturally infected potato plants exhibiting symptoms that is suspected to be viral infection including severe and mild mosaic, vein clearing, leafroll, deformation, veinal necrosis, rugosity and chlorosis were collected from the field. According to DAS-ELISA values, the PVY was found to be the most naturally existed virus alone 27.5% and with the other two viruses 42.5%, followed by PLRV which existed in 20% of the tested 40 plants as single infection and 22.5% as mixed infection. characterization of PVY, PVX and PLRV was performed biologically and serologically.
Tomato mosaic virus (ToMV) isolate used in this work was maintained on N. tabacum cv. Samson and provided by virology Labs., Microbiology Dept., Ain shams University.
The obtained results can be summarized as follow:
1- PVY isolate was tested on thirteen plant species and cultivars which belonged to five families for host range studies and the results were confirmed by DAS-ELISA. Some hosts had no reaction and showed no visible symptoms, other hosts exhibited leaf Necrosis and some developed mild and severe mosaic.
2- Reaction and symptoms developed on five differential host plants species belonging to two families Chenopodiaceae (Chenopodium amaranticolor, C. quinoa) and Solanaceae (Datura metel, D. stramonium, and N. tabacum cv. White burley), were used for biological differentiation between virus isolates. D. stramonium, C. amaranticolor and C. quinoa were immune to the PVY isolate, however, D. stramonium showed mild mosaic and veinal chlorosis with PVX. On the other hand, PVY caused severe mosaic symptoms on Datura metel. N. tabacum cv. White Burley showed mosaic, veinal necrosis and leaf deformation with the PVY isolate, however, systemic veinal chlorosis noticed in its reaction with PVX. PLRV showed no symptoms on the previous hosts.
3- Polyclonal antibodies from commercial ELISA kit provided by (LOEWE Biochemica GmbH Germany) were used to identify the PVY isolate in differential hosts and host range.
4- Clarified PVY preparation from tobacco infected leaf was negatively stained by two % Uranyl acetate and was investigated using transmission electron microscope. Filamentous viral particles with length of 720 nm and 11 nm width were detected in the clarified sap preparation.
5- Crude sap of ToMV infected N. tabacum cv. Samson was mechanically inoculated on N. glutinosa. Morphologically identical local lesions were developed after three days of inoculation.
6- ToMV isolate was tested on fifteen plant species and cultivars for host range studies and the results were confirmed by DAS-ELISA. Systemic symptoms appeared on Nicotiana tabacum cvs. White Burley and Samsun 10 days post inoculation. Inoculated N. rustica leaves showed local lesions 3 days post inoculation, followed by systemic symptoms 10 days of inoculation. The L. esculentum cv. Castle Rock plants produced systemic symptomes 12 days after inoculation. Local symptoms were observed after 4-5 days of inoculation on N. glutinosa, Datura metel, C. amaranticolor, C. quinoa, D. stramonium and Gomphrena globosa. After one month post inoculation no symptoms were appeared on Vicia faba plants.
7- Polyclonal antibodies from commercial ELISA kit provided by (LOEWE Biochemica GmbH Germany) were used to identify the ToMV isolate in differential hosts and host range.
8- Clarified ToMV preparation from tobacco infected leaf was negatively stained by two % Uranyl acetate and was investigated using transmission electron microscope. Rod shaped viral particles with length of 300 nm and 18 nm width were detected in the clarified sap preparation.
9- The total RNA was extracted from infected N. tabacum cv. Xanthi and N. tabacum cv. Samson leaves with PVY and ToMV, respectively. It was evaluated before reverse transcription using Qubit 3.0 fluorometer. The concentration of total RNA was 200 ng per µl. Then the overall RNA quality and yield was assessed by electrophoresis on 1% denaturing agarose gel. RNA fragments appeared intact on the gel and had sharp 28S and 18S rRNA bands. The 28S rRNA band was approximately twice as intense as the 18S rRNA band. This 2:1 ratio (28S:18S) indicated the success of total RNA extraction with high density.
10- The designed primer for PVY coat protein gene matched with more than 1000 results on the gene bank when the organism is limited to potato virus Y (taxid:12216). The designed primer pair have a perfect melting temperature of 59.46 and 60.25 that aids in preventing the tendency for secondary annealing. In case of ToMV, the used primer pair showed a perfect amplification capacity at its melting temperature (Tm) of 50.18 and 55.02.
11- The total RNA from PVY and ToMV infected N. tabacum cv. Xanthi and N. tabacum cv. Samson leaves, respectively, were reverse transcribed by RT-PCR using the one oligonucleotide reverse primer of each PVY-CP and ToMV-CP primer pair. The amplified DNA for PVY-CP and ToMV-CP was in the expected size calculated of approximately (400 bp) and (500 bp), respectively.
12- The PCR products were purified by QIAquick PCR Purification Kit. The concentration of the purified PCR product was 0.729 µg/µl, a total of 48 µl were obtained for each virus.
13- The partial nucleotide sequences of the PCR-amplified fragments of the PVY and ToMV coat protein (CP) genes were generated at Macrogen, Korea. The sequencing was done on both strands. The cDNA sequence was performed using the produced PCR products when the specific (downstream and upstream) primers for CP gene of PVY and ToMV were used. The total nucleotide count of amplified PVY coat protein gene was found to be 381 bp, and 482 bp in the case of ToMV CP gene.
14- The CP gene of PVY revealed the highest content for Adenine (A) 142 (37.3%) followed by Thymine (T) 79 (20.7%), then Guanine (G) 86 (22.6%) and finally Cytosine (C) 74 (19.4%) while %G-C content was found to be 42%. While, the CP gene of ToMV revealed the highest content for Thymine (T) 143 (29.6%) followed by Adenine (A) 142 (29.4%), then Guanine (G) 102 (21.1%) and finally Cytosine (C) 96 (19.8%), while the %G-C content was found to be 41%.
15- The partial nucleotide sequence of PVY and ToMV coat protein genes were aligned with ten isolates in the Gene bank according to the E value. The studied PVY isolate tented to cluster with group N and sub group NTN and had 100% identity with the other ten isolates from the gene bank. For ToMV the partial coat protein gene was aligned with ten isolates of ToMV in the Gene bank according to the E value. The isolated ToMV had 100% identity with camellia strain and 99% identity with the other nine.
16- The nucleotide sequence was translated into 126 and 159 amino acids for PVY and ToMV, respectively, then aligned with ten amino acid sequences using Blastx tool from the gene bank sorted by E value.
17- For the prediction of the linear B-cell epitopes in the primary amino acids sequence of PVY and ToMV coat proteins, BCPREDS server was used. Three epitopes, whose sequence length was 14 residues, were retrieved from the PVY amino acid sequence and five epitopes were retrieved from ToMV sequence.
18- Potato virus Y was purified from systemically infected leaves of N. tabacum cv. Xanthi. Two molar concentrations of potassium phosphate buffer were used in the extraction step. Two clarifying agents were used in the clarification step. For the purpose of obtaining a sufficient quantity of highly purified virus to be used as the inject antigen for antiserum production, the use of high molar extraction buffer alongside Triton X-100 for clarification was found the most satisfactory of several methods investigated. The ratio A260/A280 varied from 1.19 to 1.30 suggesting the presence of low host impurities. Attempts were made to assess virus losses (by DAS-ELISA and by inoculation on D. metel) which occurred during the various stages of the purification procedure. Additional indication of the purity of purified PVY suspensions was provided by electron micrographs. No obvious contaminants were observed in photographs of negatively stained preparations of the virus, which showed the presence of numerous flexuous rod-shaped particles.
19- ToMV was purified from systemically infected leaves of N. tabacum cv. Samson. Two clarifying agents were used in the clarification step. Two concentrations of sodium chloride were used with PEG 6000 in the precipitation step. For the purpose of obtaining a sufficient quantity of highly purified virus to be used as the inject antigen for antiserum production, the use of Triton X-100 for clarification alongside with PEG6000 + 1% NaCl as a concentration step was found the most satisfactory of several methods investigated. The ratio A260/A280 varied from 1.22 to 1.23 suggesting the presence of low host impurities. Evaluation of purified ToMV was carried out as mentioned in PVY. No obvious contaminants were observed in photographs of negatively stained preparations of the virus, which showed the presence of numerous rod-shaped viral particles with length of 300 nm and 18 nm width.
20- Antiserum to intact PVY and ToMV was prepared in two white New Zealand rabbits each using two different immunization schedules depending on intravenous followed by intramuscular injections.
21- The produced PVY and ToMV antisera were evaluated and titred by slide agglutination, microprecipitin, indirect ELISA. The titer of PVY antiserum according to the microprecipitin test was 1:256 and the dilution end point of purified PVY was 1:16. Indirect ELISA titration curves of the produced PVY antiserum compared to IgGs from LOEWE’s commercial kit were generated. The sensitivity of antibodies was clearly at 1:256 dilution for produced PVY antiserum and 1:512 in LOEWE kit’s IgG.
22- The titer of ToMV antiserum according to the microprecipitin test was 1:1024 and the dilution end point of purified ToMV was 1:32. Indirect ELISA titration curves of the produced ToMV antiserum compared to IgGs from LOEWE’s commercial kit were generated. The sensitivity of antibodies was clearly at 1:1024 dilution for produced ToMV antiserum and 1:512 in LOEWE kit’s IgG.
23- For Antibody purification, The produced PVY and ToMV antisera were processed for lipoproteins removal by dextran sulfate (delipidation). Then they have undergone two purification procedures, firstly by Ammonium sulfate fractionation in which the antisera were precipitated sequentially first to an initial saturation of 25% and then to a final 50% ammonium sulfate saturation. Then, further purification was done by means of affinity chromatography using protein A magnetic beads. The antibodies were assessed spectrophotometrically. The concentration of ToMV-IgG purified by ammonium sulfate was 23 mg/ml, while, the conc. of that purified by protein A magnetic beads was 12.8 mg/ml.
24- For the purpose of lateral flow immunoassays (LFIAs) strips assembly colloidal gold nanoparticles were synthesized by citrate method from gold chloride and used in labeling the produced and purified PVY and ToMV IgGs. The produced nanogold were characterized by UV-VIS spectrophotometer. The prepared nanoparticles had absorbance peak at 530 nm representing the presence of 40 nm AuNPs.
25- The prepared nano-gold suspension in the present study, was measured on the Nano-ZS Malvern instrument for measuring dynamic light scattering (DLS) for particle size distribution. The results showed that the average size of the gold particles was 40 nm with acceptable polydispersity index (PDI) which is equal to 0.103 indicating monodispersity and excellent dispersion of AuNPs solution. Also, electrophoretic light scattering (ELS) for zeta potential analysis of the prepared GNPs (40 nm) by citrate method was -32 mV which confirms the formation of -ve charges on the surface of GNPs. This provides sufficiently stable dispersion of the nano particles.
26- High resolution TEM image of the synthesized AuNPs reveals a good performance of the applied citrate method in producing monodispersed spherical shape of nanoparticles averaged 40 nm in size. Finally, X-ray diffraction (XRD) confirmed that synthesized AuNPs were of crystalline nature with face-centered cubic gold structure.
27- The optimum concentration for the stable conjugation of gold nanoparticles with purified PVY and ToMV IgGs was estimated at 1:512 and 1:1024, respectively.
28- The immune-strips was assembled with different membranes materials (Nitrocellulose/Nilon) with different pore sizes (0.2 µm 0.45 µm 1.2 µm 5 µm). Nitrocellulose membranes with pore size of 5 µm was the best choice for ILFST assembly because it has a good capillary flow rate and perfect binding capacity. The produced immunostrips showed a very good sensitivity in the detection of PVY and ToMV in naturally infected plants.