الفهرس | Only 14 pages are availabe for public view |
Abstract The present study aimed to achieve the following objectives: 1. Confirmation of viruses. 2. Preparation and characterization of Chitosan Nanoparticles. 3. Application of nanoparticles in vitro 4. Application of nanoparticles in vivo The results might be summarized as follows: PART I 1. Confirmation of viruses 1.1 Potato virus Y (PVY) 1.1.1 Confirmation of the Virus Isolate Biologically Symptoms developed on the five host plants (Datura metel, Datura stramonium, Nicotiana tabacum cv.White Burley, N. tabacum cv. Samsun, Nicotiana glutinosa, Solanum lycopersicum and Solanum tuberosum) were used for biological confirmation of potato virus Y. Datura metel showed mottling, vein clearing and crinkle while Datura stramonium showed no symptomes. N. tabacum cv.White Burley showed mosaic, veinal necrosis while N. tabacum cv. Samsun showed severe mosaic, vein clearing and Nicotiana glutinosa showed severe mosaic. Solanum lycopersicum showed mosaic and Solanum tuberosum showed mosaic, necrosis, deformation and leaf narrow 1.1.2. Confirmation of the Virus Isolate Serologically PVY has detected by DAS-ELISA. The positive result was record by ELISA reader and compared with negative control. 111 SUMMARY Nesma I. Helmy (2017), M.Sc., Fac. Agric., Ain Shams Univ. 1.1.3. Confirmation of the Virus Isolate Molecularly 1.1.3.1 One Step RT-PCR RT-PCR was carry out using Plant Total RNA Mini Kit from RBC and primer specific sites for the viral coat protein gene of PVY with an expected size of 800 bp on agarose gel electrophoresis. 1.1.4. Virus morphology The morphology of viral particle of PVY showed difference in the morphological shape by electron micrograph using negative staining method. The Examination showed viral particles with a clear modal length of 650 nm and 11nm wide. 1.2. Tobacco mosaic virus (TMV) 1.2.1. Confirmation of the Virus Isolate Biologically Symptoms developed on the five host plants (Datura metel, Datura stramonium, Chenopodium quinoa, Chenopodium amaranticolor, Nicotiana glutinosa, N. tabacum cv. Samsun and Solanum lycopersicum) were used for biological confirmation of potato virus Y. Datura metel, Datura stramonium, Chenopodium quinoa, Chenopodium amaranticolor, Nicotiana glutinosa showed local lesion symptoms after 4 day from mechanically inoculation while N. tabacum cv. Samsun and Solanum lycopersicum showed severe mosaic after 15 day from mechanically inoculation. 1.2.2. Confirmation of the Virus Isolate Serologically TMV has detected by DAS-ELISA. The positive result 0.973 O. D was record by ELISA reader and compared with negative control 0.084 O.D. 112 SUMMARY Nesma I. Helmy (2017), M.Sc., Fac. Agric., Ain Shams Univ. 1.2.3. Confirmation of the Virus Isolate Molecularly 1.2.3.1 One Step RT-PCR RT-PCR was carry out using Plant Total RNA Mini Kit from RBC and primer specific sites for the viral movement protein gene of TMV with an expected size of 422bp on agarose gel electrophoresis. 1.2.4. Virus morphology The morphology of viral particle of TMV showed difference in the morphological shape by electron micrograph using negative staining method. The Examination showed rod shaped of viral particles. viral particles with a clear modal length of 300 x 15 nm. PART II Preparation and characterization of Chitosan Nanoparticles 1. Size, Zeta Potential, and Morphology of Chitosan Nanoparticles The chitosan nanoparticles prepared in the experiment exhibit a white powdered shape, and are insoluble in water. The mean size and size distribution of each batch of nanoparticle suspension was analyzed using the Zeta-sizer analysis. The size distribution profile, represents a typical batch of nanoparticles with a mean diameter of 29 nm and a polydispersity index < 0.371. Zeta potential that is surface charge. It was noticed that the surfaces of chitosan nanoparticles have a positive charge about 33.5mV. Chitosan nanoparticles were observed by the transmission electron microscope and it was less than 20 nm in diameter with smooth and spherical in shape. 1.2. Infrared Spectroscopy Chitosan nanoparticles were prepared between chitosan and tripolyphosphate. FTIR studies of chitosan and chitosan nanoparticles 113 SUMMARY Nesma I. Helmy (2017), M.Sc., Fac. Agric., Ain Shams Univ. were performed to characterize the chemical structure of nanoparticles. A band at 3429cm 1 corresponds to the combined peaks of the NH2 and OH group stretching vibration in chitosan. The band at 1633cm 1 is attributed to the CONH2 group. A shift from 3429 to 3431cm 1 is shown, and the peak is sharper in the chitosan nanoparticles, which appear the hydrogen bonding is enhanced. The intensities of (NH2) band at 1550cm and (CONH2) band at 1637cm, which can be observed clearly in pure chitosan, decrease dramatically, and two new sorption bands at 1633 and 1428cm 1 appear which shows that the ammonium groups are crosslinked with tripolyphosphate molecules. Part III Application of nanoparticles in vitro 1. Effect of the nanoparticles on virus morphology The morphology of viral particles of PVY and TMV showed difference in morphological shape by electron micrograph using negative staining method. TEM examination was done on PVY and PVY treated with silver nanoparticles, PVY and PVY treated with chitosan nanoparticles, TMV and TMV treated with silver nanoparticles, TMV and TMV treated with chitosan nanoparticles. The results observed that silver and chitosan nanoparticles bind at a limit site on virus particles. 2. Effect of nanoparticles on virus infectivity 2.1. Effect of nanoparticles on PVY infectivity After 21 and 28 days of inoculation on D. metel plants. The chitosan nanoparticles, silver nanoparticles mixed with virus crude sap due to reduction of virus infectivity. The results of D.metel plants which mechanically inoculated with virus only give infection rate 100% whereas 114 SUMMARY Nesma I. Helmy (2017), M.Sc., Fac. Agric., Ain Shams Univ. all plants were reaction with ELISA. The symptoms were Mosaic, vein clearing, crinkle and leaf cup shape. The results of D. metel plants which mechanically inoculated with mixture of 2 g/L of chitosan nanoparticles and PVY suspension after 21 and 28 day were no symptoms and not reaction with ELISA test. The results of D. metel plants which mechanically inoculated with mixture of 50 ppm of silver nanoparticles and PVY suspension due to reduction of symptoms with ratio 60%. The symptoms were vein clearing and leaf cup shape. It is reaction with ELISA test. 2.2. Effect of nanoparticles on TMV infectivity After 6 days of inoculation on N.glutinosa plants. The chitosan nanoparticles, silver nanoparticles mixed with virus crude sap due to reduction of virus infectivity. The half leaf treated with TMV only showed 47.5 local lesions while the half leaf treated with mixture of chitosan nanoparticle and TMV suspension showed 16.5 local lesions. The reduction of viral activity expressed by local lesions was 65.3% on N. glutinosa. The half leaf treated with TMV only showed 65.75 local lesions while the half leaf treated with mixture of silver nanoparticle and TMV suspension showed 39.5 local lesions. The reduction of viral activity expressed by local lesions was 40% on N. glutinosa. This finding suggests that the chitosan nanoparticles are effective antiviral agent PART IV Application of nanoparticles in vivo 1. Application of nanoparticles as an antiviral agent A) Silver nanoparticles 1.1. Virus infectivity All concentrations were able to reduce virus infectivity after 25 days of inoculation in potato plants treated with silver nanoparticles preinoculation. Data indicate that the potato plants treated with concentration of 50 ppm silver nanoparticles display a higher significant reduction in the PVY virus infectivity than other concentrations. The highest percentage reduction of virus infectivity with concentration of 50 ppm was 39.5 %. This is followed by concentration of 55 ppm. While the less reduction percentage achieved with concentration of 40 ppm 18.7% and 45 ppm 24.3 compared to the infected control. 1.2. Disease severity Data indicate that all concentrations were able to reduce disease severity after 25 days of inoculation in potato plants treated with silver nanoparticles pre-inoculation. Data indicate that concentration of 50 ppm showed the highest significant reduction percentage of disease severity 51.2% followed by concentration of 55 ppm 47.5% and 60 ppm 42.6%. However, concentration of 40 ppm had the lowest significant percentage of disease severity 31.1% compared to the infected control. 1.3. Virus concentration At 21 days of inoculation, concentration of 60 ppm for silver nanoparticles showed the lowest significant concentration of virus replication followed by concentration of 55 ppm, 50 ppm. 116 SUMMARY Nesma I. Helmy (2017), M.Sc., Fac. Agric., Ain Shams Univ. 1.4. Reverse transcriptase - Polymerase chain reaction (RT-PCR) The resulet showed that the virus found in all plants treated with concentrations of silver nanoparticles and inoculated with PVY. RNA amplification produced band with expected size 800 bp on agarose gel. However, there were differences in the density of each amplicon but the ampilicon produced from the 50 mg /l treatment was less dense. 1.5. Plant growth parameters Result showed that treatment with 50 ppm increased the fresh weight of inoculated-treated potato plants significantly as compared with infected ones. PVY infection reduced the fresh weight of control plants at 25-day 32.6 g compared to healthy plants 67.5 g. Effect of virus infection on dry weight was clear of control plants 3.6 g/ plant, while 50 ppm treatment increased the dry weight under infection condition significantly to 5.7 g. treatment without inoculation increased the dry weight to 7.5 g for 50 ppm treatment. This increasing was significant higher than healthy plants 6.5 g. Data showed that treatment with 50 ppm increased the leaf area of inoculated-treated potato plants significantly as compared with infected plants. PVY infection reduced the leaf area of control plants at 25-day 7.1 cm compared to healthy plants 13.7 cm. while 50 ppm treatment increased the leaf area under infection condition to 11.7 cm. treatment without inoculation increased the leaf area to 17.6 for 50 ppm treatment. Effect of virus infection on tuber weight was clear of control plants 9.1 g/ plant, while 50 ppm treatment increased the tuber weight under infection condition significantly to 17.3 g. treatment without 117 SUMMARY Nesma I. Helmy (2017), M.Sc., Fac. Agric., Ain Shams Univ. inoculation increased the tuber weight to 30.3 g for 50 ppm treatment. This increasing was significant higher than healthy plants 27.5 g. 1.6. Antioxidant enzyme activites 1.6.1. Peroxidase (PO) activity Data indicted that the activites of PO at 14 days of inoculation was non-significant higher with concentration of 60 ppm of silver nanoparticles. While, the lowest PO activity was at concentration of 40 ppm compared to control. 1.6.2. Polyphenoloxidase (PPO) activity Data indicted that the activites of PO at 14 days of inoculation was significant higher with concentration of 60 ppm of silver nanoparticles. While, the significant lowest PO activity was at concentration of 40 ppm compared to control. 1.7. Total Phenol contents Treatment (60 ppm) was the most effective on accumulation of phenol content compared to the other treatments. This is followed by Treatment (45 ppm). All concentrations resulted in the significant increase of phenol content at 14 days of inoculation. 1.8. Total amino acid contents In this analysis, amino acids content in potato leaves treated with silver nanoparticles and inoculated with PVY was increased comparing with healthy and inoculated controls significantly. After 14 days of applying AgNPs, treatment (50 ppm) was the most effective on significant increase of amino acids content compared to the other treatments. This is followed by treatment (45 ppm). All concentrations resulted in the increase of amino acids content at 14 days of inoculation. 118 SUMMARY Nesma I. Helmy (2017), M.Sc., Fac. Agric., Ain Shams Univ. 1.9. Photosynthetic pigments Chlorophyll A, B and carotenoids contents were determined afar 14 days from inoculation to detect the effect of treatments with silver nanoparticles on those pigments in potato plants. In general, the results showed that PVY infection significantly reduced chlorophyll A, B, carotenoids compared to those of healthy plants. Treatment of plants with AgNPs increased chlorophyll A, B, carotenoids contents. Data showed that treatment (50 ppm) was the most effective on increase of Chlorophyll A, B and carotenoids contents compared to the other treatments. This is followed by treatment (45 ppm). 1.10. Total silver in potato tuber In this analysis, total silver in potato tuber treated with silver nanoparticles and inoculated with PVY was determined. After 14 days of applying AgNPs, all concentrations was Less than the limit (10 ppm). The differences in treatment are significant. B) chitosan nanoparticles 1. Virus infectivity All concentrations of ChNPs were able to reduce virus infectivity after 25 days of each pre and post virus inoculation in potato plants treated with ChNPs compared with virus inoculated plant without ChNPs treated. Potato plants treated with ChNPs pre and post PVY inoculation not appeared symptoms while virus inoculated plants without ChNPs treatment showed mild symptoms (vein clearing and mild mosaic). This result was confirmed by DAS-ELISA test using PVY specific poly clonal antibody and PCR test using PVY coat protein gene. 119 SUMMARY Nesma I. Helmy (2017), M.Sc., Fac. Agric., Ain Shams Univ. 2. Virus concentration Result of ELISA test demonstrated that all treatments reduced the virus concenration signficantly compared to the control. At 21 days of inoculation, all concentration of chitosan nanoparticles to pre-inoculation and post-inoculation showed the lowest concentration of virus replication followed by concentration of control. 3. Reverse transcriptase - Polymerase chain reaction (RT-PCR) Detection of potato virus Y (PVY) in plants treated with different concentration of ChNPs pre and post inoculation by RT-PCR and 1% agarose gel electrophoresis analysis and hundred ladder marker showed that postive plants in all treatment. RNA amplification produced band with expected size 800 bp on agarose gel. However, there were differences in the density of each amplicon but the ampilicon produced from the 2 g /l treatment was less in density. 4. Plant growth parameters Data showed that treatment with 2 g/l increased the fresh weight of inoculated-treated potato plants significantly to 65 g pre-inoculation and 75.3 g post-inoculation as compared with infected ones. This increasing was a little than healthy plants. However, PVY infection reduced the fresh weight of control plants significantly at 25 days 48 g compared to healthy plants 86 g. Treatment of post-inoculation showed an increasing of fresh weight than that of pre-inoculation. Effect of virus infection on dry weight was clear of control plants 6.2 g/ plant, while 2 g/l treatment increased the dry weight under infection condition significantly to 9.6 g pre-inoculation and 10.9 g postinoculation. Treatment of post-inoculation showed an increasing of dry 120 SUMMARY Nesma I. Helmy (2017), M.Sc., Fac. Agric., Ain Shams Univ. weight than that of pre-inoculation significantly at 25 days of inoculation. This increasing was higher than healthy plants 13 g. Data showed that treatment with 2 g/l increased the leaf area of inoculated-treated potato plants significantly to 14.4 cm pre-inoculation and 17.2 cm post-inoculation as compared with infected ones. This increasing was a little than healthy plants. However, PVY infection reduced the leaf area of control plants significantly at 25 days 12.7 cm compared to healthy plants 18.3 cm. Treatment of post-inoculation showed an increasing of leaf area than that of pre-inoculation. Effect of virus infection on tuber weight was clear of control plants 8.7 g/ plant, while 2 g/l treatment increased the tuber weight under infection condition significantly to 14 g pre-inoculation and 15.1 g postinoculation. Treatment of post-inoculation showed an increasing significantly of tuber weight than that of pre-inoculation at 25 days of inoculation. This increasing was higher than healthy plants 17.3 g. 3. Antioxidant enzyme activites 3.1. Peroxidase (PO) activity Data indicted that the activites of PO at 14 days of inoculation was significant higher with concentration of 2 g/l of chitosan nanoparticles compared to control. Treatment of post-inoculation showed an increasing of PO activity than that of pre-inoculation. 3.2. Polyphenoloxidase (PPO) activity Data indicted that the activites of PO at 14 days of inoculation was significant higher with concentration of 2 g/l of chitosan nanoparticles compared to control. Treatment of post-inoculation showed an increasing of PPO activity than that of pre-inoculation. 121 SUMMARY Nesma I. Helmy (2017), M.Sc., Fac. Agric., Ain Shams Univ. 4. Total Phenol contents Our results indicated that PVY increased levels of phenol content significantly in inoculated plants compared with healthy ones. High acculmulation of phenol content was observed at 14 days with silver nanoparticles. concentration of chitosan nanoparticles (2 g/l) was the most effective on accumulation of phenol content compared to the other concentration. Treatment of post-inoculation showed an increasing of phenol content than that of pre-inoculation. 5. Total amino acid contants In this analysis, amino acids content in potato leaves treated with chitosan nanoparticles and inoculated with PVY was increased significantly comparing with control. After 14 days of applying chitosan nanoparticles, concentration of 2 g/l was the most effective on increase of amino acids content compared to the other concentration. Treatment of post-inoculation showed an increasing of amino acid content than that of pre-inoculation. All concentrations resulted in the increase of amino acids content at 14 days of inoculation. 6. Photosynthetic pigments Chlorophyll A, B and carotenoids contents were determined afar 14 days from inoculation to detect the effect of treatments with chitosan nanoparticles on those pigments in potato plants. In general, the results showed that PVY infection significantly reduced chlorophyll A, B and non-significantly carotenoids compared to those of healthy plants. Treatment of plants with chitosan nanoparticles increased chlorophyll A, B, carotenoids contents. Result showed that concentrations of 2 g/l was the most effective on increase of Chlorophyll 122 SUMMARY Nesma I. Helmy (2017), M.Sc., Fac. Agric., Ain Shams Univ. A, B and carotenoids contents compared to the other treatments. Treatment of post-inoculation showed an increasing of plant pigments content than that of pre-inoculation |