الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
 |  | from | 166 |  |  |
| | | from | 166 | | |
Abstract
It is well known that ZYMV is considered as the most series potyviruses affected cucurbit crops worldwide including Egypt. This is due to it causes destructive diseases to a large number of cucurbitaceous plants including squash. Therefore, this study was designed to identify an Egyptian isolate of ZYMV obtained from infecting squash leaves or fruits, whatever, grown under open field and/or greenhouse based on its biological and molecular characteristics. Also, three molecular tools, i.e., SDS-PAGE, ISSR-PCR and SCoT were used to determine the genetic variation between ZYMV-infected and healthy cucurbits.
To achieve such goal the following experiments were carried out:
1. Collecting of squash plant samples (leaves or fruits) showing virus-like symptoms from greenhouse and open field area(s).
2. Detection of the presence of ZYMV in collected samples based on DAS-ELISA technique.
3. Molecular detection using RT-PCR tool for ZYMV in collected samples.
4. Biological characterization of an isolate of ZYMV based on: differential hosts, aphid transmission, amorphous inclusion via light microscopy, cylindrical inclusions via examination of ultrathin sections of ZYMV-infected squash leaves among transmission electron microscopy and morphology of ZYMV particles in partial purified virus preparation based on polyethylene glycol via transmission electron microscopy.
5. Molecular characterization of the selected ZYMV isolate based on isolation of ZYMV-cp gene via RT-PCR using the purified virus particles in the presence of two specific primers, extraction of PCR product from agarose gel, purification of PCR product using PCR Clean-Up kit, determining the nucleotide sequences of purified PCR product, and bioinformatics analysis of obtained sequences based on nucleotides and deduced amino acids (Open reading frame-ORF).
6. DNA fingerprinting for some ZYMV-infected cucurbits using SDS-PAGE analysis.
7. Assessment of genetic variation of six ZYMV-infected cucurbit species as follows: extraction of total DNA from some ZYMV-infected and healthy cucurbits, DNA polymorphisms generated using some ISSR and SCoT primers, determining the similarities between DNA profiles, and finally phylogenetic relationship between the tested cucurbits (ZYMV-infected and healthy).
The results of this study could be summarized as follows:
1. A number of 27 squash samples (22 leaves and 5 fruits) exhibited virus-like symptoms (mosaic, severe mosaic, vein banding, yellows, leaf deformation, blisters, rolling, and filiform shapes) were collected from two different environments, i.e., greenhouse (16 samples) and open field (11 samples) conditions.
2. The presence of ZYMV in the collected squash samples was detected via DAS-ELISA using specific antiserum.
3. Twelve (54.55%) out of 22 leaf samples showed ELISA positive values indicating its infection with ZYMV.
4. All positive-ELISA samples were appeared positive results when subjected to RT-PCR detection and two negative-ELISA samples were also appeared PCR products, indicated the high sensitivity of RT-PCR for virus detection.
5. ZYMV isolate under investigation was biologically characterized based on mechanically inoculation of some cucurbits, i.e., squash cv. Eskandarani, cantaloupe, watermelon, cucumber, luffa, in addition to Chenopodium amaranticolor as an indicator host. These hosts showed different severe symptoms in case of systemic infection, while Ch. Amaranticolor appeared necrotic local lesions.
6. ZYMV was successfully transmitted in a non-persistent manner to squash via cotton aphid (Aphis gossypii).
7. Light microscopy of ZYMV-infected squash stripes revealed that the presence of proteinous crystalline and amorphous inclusions in the cytoplasm of infected squash leaves.
8. Transmission electron microscopy of ultrathin sections of ZYMV-infected squash leaves showed the presence of cylindrical inclusions (pinwheels and scroll) belonging to subdivision I. Results also showed that ZYMV particles were found near to mitochondria and scroll inclusions.
9. The virus was successfully purified among polyethylene glycol method from infected squash leaves 10 days post virus-inoculation, and flexuous filaments virus particles were occurred with a titer of 11-12 mg/100 g of ZYMV.
10. A size of 831 nucleotides were sequenced from the PCR product of ZYMV-cp gene after cleaning from agarose gel containing an open reading frame in sense orientation direction which deduced to 236 amino acid of ZYMV cp gene of which documented in GenBank as a ZYMV Shrouk-23 strain (LC778450.1), and analyzed against 25 isolates or strains of ZYMV documented in GenBank, with a similarly ranged from 91.94 to 99.16%, and 96.19 to 100.00% for nucleotides and deduced amino acids, respectively.
11. When SDS-PAGE analysis was used to determine the genetic variability of four cucurbits (Qethaa, luffa, squash and cantaloupe) infected with ZYMV compared to healthy, a total of 15 storable bands was found.
12. The experimental results showed that DNA polymorphisms include 38 polymorphic DNA fragments were generated using the five ISSR-PCR used primers. Samples of squash, cantaloupe, qethaa, watermelon, cucumber and luffa appeared an average of 12.5, 20, 15.5, 13.5, 13.5 and 10.5 DNA fragments.
13. The maximum identity percent between the ZYMV-infected and healthy samples was 59% between samples of watermelon followed by 56% for squash samples, 45% for cantaloupe samples, 40% for qethaa samples, 30% for cucumber samples and 29% for luffa samples.
14. Phylogenetic tree of DNA polymorphisms confirmed the genetic relationship among each of the healthy samples of watermelon and cucumber, and the healthy samples of qethaa and luffa; ZYMV-infected samples of watermelon and squash, and ZYMV-infected samples of kantaloupe and qethaa.
15. A number of nine SCoT primers were used to determine the DNA fingerprinting of six ZYM-infected cucurbit species compared with healthy of the same species. A total of 88 polymorphisms DNA fragments distributed as follows: 13, 7, 10, 6, 9, 10, 15, 8 and 10 were generated using SCoT-02, SCoT-03, SCoT-04, SCoT-06, SCoT-08, SCoT-11, SCoT-12, SCoT-13 and SCoT-14, respectively.
16. DNA polymorphisms of the six cucurbit species infected with ZYMV showed identities ranged from 32 to 79% when compared to healthy using among SCoT assessment using nine SCoT primers.