الفهرس 
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Abstract
Viral diseases on plants are still until now a great puzzle because it is very difficult to managing it. For example potato viruses cause economically important diseases in potatoes and their effect greatly induces serious reduction of the yield quantity and quality for almost cultivated potato varieties. The present work was conducted to study the control of certain viruses Alfalfa mosaic virus ( AMV) and Potato virus Y (PVY) which greatly affected Burna potato variety through using certain beneficial microorganisms formulations as well as vaccination of potato plants with mild isolate of (PVY ) was used to control (PVY) virus in order to limits its damage on Burna potato plants in the greenhouse or field experiments.
The results could be summarized as follows:
In the present study, Burna potato variety which showing naturally viral infection with (AMV) that has pronounced calico pattern (yellow blotching) and rugose mosaic caused by (PVY) were collected from EL-Minia and Assiut Governorates localities.
1-The causal viruses were identified by using differential plants, serological methods ”Precipitation DROP method and Indirect Enzyme-Linked Immunosorbent Assay (ELISA)”and molecular detection methods reverse-transcription polymerase chain reaction (RT-PCR).
2-Some of the differential plants were inoculated mechanically to prove its sensitivity to detect Potato virus Y including false acacia locust tree (Pseudo-acacia L.) which produced minute faint pink to light brown local lesions (L.L.) were started in appearance after 10-14 days from mechanically inoculation of its young leaves .These results demonstrated that the aforesaid indicator plants are considered as new differential hosts for (PVY) detection.
3-In case of Nabak (Zizphusspina -csisti L.) yellows necrotic lesions appeared after 5-10 and it was gradually changed to violet or pale brown in color. In addition, after about 20-22 days vein clearing was noticed on the new leaves of the same inoculated leaves with (AMV) , it means that this virus could be able to spread across the plant cells from local lesions areas to form a systematically case in the whole plant. This plant might be a new plant indicator for AMV.
4-When detached leaves of Nasturtium (Tropaeo lummajus L.) plant was inoculated by dusting this leaves with 405 mesh Carborundum, then inoculated mechanically with ( AMV) , the inoculated leaves greatly reacted with the viral infection of the aforesaid virus by producing yellowing areas due to the effect of the viral infection after 5 days from leaves inoculation . However, detached leaves of Nasturtium T. majus L. did not react strongly with the infection with (PVY) and just produced very light yellowing appearance on inoculated leaves after 7 days from inoculation. Data also indicated that this plant may be considered a new differential plant to distinguish (AMV) and (PVY). However, it may be very beneficial for both viruses detection.
5-The electron micrograph showing single flexible filamentous particle of (PVY) under electron microscope in Burna potato variety sap and viral particles (723nm) in length and (9.25) in width meanwhile, the electron micrograph showing (AMV) particles are composed of icosahedralcapsids under electron microscope in Burna potato variety sap and viral particles (30-57nm) in length and (18) in diameter were found separately or in mixed infection with (AMV) under electron microscope.
6-RNA extracted from leaves of infected Burna potato variety with either AMV or PVY (based on ELISA results) was tested in RT-PCR, by using specific primers to amplify the CP genes of AMV or PVY separately. RT-PCR result confirmed the presence of AMV and PYV in symptomatic potato plants. The tested potato plants were either infected with one virus (single infection), while some plants were infected with the two viruses simultaneously. Mixed Infection with AMV & PVY usually resulted in severer symptoms compared with symptoms occurred in case of single infection. May this severity due to the suppression of post-transcriptional gene silencing by one virus, and this is will allow increase in replication rate of the another virus and severity of the symptoms. These results proved that AMV and PVY are the main viruses infecting potato in Assiut governorate.
7-Comparison between nucleotide sequences of AMV isolates from Assiut and worldwide isolates had been done and Neighbor- Joining tree generated from 20 nucleotide (nt) sequences of Assiut-AMV and worldwide AMV isolates.
8-Data indicated that AMV isolates clustered showed that the two main groups (group I and II), and Egyptian isolates fall in both of these groups.Results indicated that Assiut-AMV isolate fall in group II with another Egyptian isolate from WadyElnatron (Accession Number: HG315522), while another Egyptian isolate from Elmonyfeya governorate fall into group I.
9-The majority of AMV isolates in group I originated from countries in North, South America, Europe, and Australia including USA, France, Italy, Canada, Chile, Mexico and Brazil, beside AMV isolate from Elmonyfeya-Egypt.
10-Results also indicated that the majority of isolates in group II originated from Asian countries including China, Korea and Japan, beside isolates form Egypt (Assiut and WadyElnatron).
11-Results revealed that AMV has a wide host range, and to study whether, host and geographical origin of each isolate has any effect on variation, a neighbor Joining tree was constructed from CP gene AMV worldwide isolates.
12-Phylogenetic analysis showed AMV–Assiut showed tendency to cluster according to geographic and host origin, as AMV-Assiut which was closely related to an isolate from Egypt (WadyElnatron), and both isolates were originated from potato plants,
13-Comparison of CP gene of PVY isolate from Upper Egypt and worldwide isolates had been done and analysis revealed that coat protein gene of PVY isolate from Upper Egypt (PVY-Assiut) shared identity ranged from 88.7 -99.6 %.
14-Results indicated that the nucleotide level (nt) with 69 worldwide PVY isolates used in this study. PVY-Assiut shared the highest identity at nt level with PVY-NTN strain from South Africa, while shared the least identity with PVY-O strain from Poland.
15-Data indicated that there was no clear clustering depending on the host origin, but in case of aa trees, isolates clustered partially according to their host origin to form different sub-groups. Interestingly, when a neighbor joining tree was constructed from nt sequences of recombinant PVY isolates (PVY-NTN), these isolates clustered into three main groups A & B & C according to their host origin, as group A contained PVY isolates infecting potato, group B contained PVY isolates infecting pepper, tomato, and group C contained PVY isolates infecting tobacco.
16-Results also were indicated that analysis of PVY-Assiut isolates and other Egyptian isolates available in gene bank showed that these isolates clustered in two different groups in both cases of nt and aa nucleotide sequences. PVY-Assiut clustered with PVY isolates infecting potato in Giza governorate.
17-Data showed that PVY-Assiut was the ancestor these two isolate, these isolate from Giza were previously described as PVY-NTN strain, while the group two contained isolate from North of Egypt.
18-For induction of acquired systemic resistance in Burna potato variety against the tested viruses formulation were prepared from five of microorganisms as (biotic agents) Bio-I , (Pseudomonas flurescens L. ), Bio-II (Bacillus subtilisL.), Bio-III (Rhizobium leguminosarum L.), Bio-IV (TichodermaharizianumRifi) and Bio-V (Saccharomyces exiguous L.) to treat the infected potato tubers with (AMV) and (PVY).
19- Data showed that biotic formulations which used in treating infected potato Burna variety with ( AMV) and ( PVY) greatly affected on the concentration of the aforesaid virus within potato plant tissues which indicted as local lesions induced on the mechanically inoculated leaves of indicator plant broad bean (Viciafaba L.) Compared with untreated potato tubers (Control). Data also indicated that significant increase in tuber weight was occurred after 24 h from treating infected potato tubers with all biotic formulations tested compared with control. However, highly tuber weight was occurred after 24h in case of biotic formulation (Bio- V).
20- In addition to results of the count of viable bacterial and fungal propagules CFU/ml ofall formulations tested for two years preservation it was notices that the counts of its propagules were increased by the of preservation even under the room temperature or the refrigerator.
21- In this respect, it had been noticed that the high count of propagules of all formulations was recorded under preservation under room temperature except Bio-II and Bio-IV which gave high count of propagules under preservation on the refrigerator. Meanwhile, results indicated that there were no significant differences between preservation of the rest tested formulations under room temperature or preserved it in the refrigerator.
22- It is clear that, formulations of microorganisms, which used for induced resistances of potato against certain viruses were formed from corn starch supplemented with water mushroom compost and preserved in a refrigerator at 4°C or at room temperature. These formulations showed long period preservation with a tangible viability of its microorganisms.
23-Vaccination is the phenomenon through the activity of a mild or weak virus isolate in a plant could prevent the expression of a subsequent challenge virus. Vaccination has been used successfully to control potato against Potato virus Y (PVY) which causing a serious diseases on Burna potato variety in the green house as well as in the field experiments.
24- Results of the greenhouse and field experiments indicated that vaccinated potato plants ”Burna” variety with the mild isolate of (PVY) was capable to protect these plants against severe isolate of PVY, when potato plants were inoculated with this virus. However, mild isolate gives a tangible vaccination, when these plants were inoculated with severe isolate of PVY.
25- The best results of vaccination of potato plants against the aforesaid severe isolate was obtained when potato plants were inoculated firstly with mild isolate and then re-inoculated with the severe isolate after two or three days later.
26- It clear that the results ofBurna potato variety which vaccinated with mild isolate induced great reduction in local lesions of (PVY) produced on the indicator plant leaves when the potato sap was taken from vaccinated, non-vaccinated plants and inoculated mechanically on the indicator plant. However, the mild isolate virus affected significantly on the challenging virus concentration which indicated as the average number of local lesions within potato tissues in all periods of the treatment of vaccination compared with control (non-vaccinated potato plants). Furthermore, the mild isolate virus was highly significantly reduced the average of local lesions after 3 days period from vaccination.
27- These results which indicated that the mild isolate of (PVY) protected potato plants from homologous severe isolate of (PVY).
28- Data of vaccinated potato plants Burna variety in the greenhouse and field experiments with mild isolate had been found its capabilities to protect potato plants Burna variety against severe strain of PVY. This protection was indicated as reduction in viral concentration within plant tissues and also viral syndrome.
29- The results wereindicated that vaccination of potato plants with mild strain against the severe challenging virus after 0,1 , 2 and 3 days prior the falling of severe challenging virus highly protected plants from the great damage induced by tested virus .
30-Data also showed that vaccination of potato which used in treating infected potato Burna variety withPVYgreatly affected significantly on the tuber yield in the field experiments compared with unvaccinated potato tubers (Control). Data also indicated that significant increase in tuber weight was occurred after 3 days period.
31- Furthermore, the average of highly tuber weight in the greenhouse was 946.8 gm/plant in case of Mild isolate+ PVY after (3) days period and 232.48 gm/plant in case of PVY after (3) days period. Meanwhile, it was 3205.6 gm/plant in case of Mild isolate +PVY after (3) day period in the field experiments.