Molecular Characterization of Watermelon Chlorotic Stunt Virus (WmCSV) from Palestine
Abstract
:1. Introduction
2. Results and Discussion
2.1. Field Survey
Season/Year | Area | Crop | Fields | +ve by deg. primers * | +ve for WmCSV (PCR) | +ve for WmCSV (RCA) | Total no. of samples +ve for WmCSV (%) | No. of Samples mixed Infected with SLCV (% ϕ) |
---|---|---|---|---|---|---|---|---|
Summer/2010 | Tulkarm | Watermelon | 3 | 58/62 | 0 | 0 | 0 (0) | 50 (80.6) |
Jenin | Watermelon | 4 | 65/110 | 43 | 37 | 43 (39.1) | 27 (62.7) | |
Summer/2011 | Jenin | Watermelon | 4 | ND (120) | 0 | 0 | 0 (0) | 100 (83.3) |
Arifa S. Khan | Jericho | Watermelon | 7 | 59/84 | 0 | 0 | 0 (0) | 54 (64.3) |
Total | 23 | 288/501 | 53 | 73 | 79 (15.8) | 261 (52.1) |
2.2. Cloning and Sequencing of WmCSV-[PAL]
Primer Name | 5'–3' Sequence | Target | Fragment Size |
---|---|---|---|
WmA150F 1 | GTCAGTATGTGGGATCCATTGC | DNA-A | 1201 bp |
WmA 1350R 1 | GCAAATACGATTCAACCACAACC | ||
WmA170R 1 | GCAATGGATCCCACATACTGAC | DNA-A | 1597 bp |
WmA1325F 1 | GGTTGTGGTTGAATCGTATTTGC | ||
WmB672F | CGCCGTTGCCTGGAGGATGTTCAC | DNA-B | 1329 bp |
WmB2000R | GCAGCACAGGCTGCCTTCACCTTC | ||
WmB1977F | GAAGGTGAAGGCAGCCTGTGCTGC | DNA-B | 1479 bp |
WmB695R | GTGAACATCCTCCAGGCAACGGCG |
DNA-A | DNA-B | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Position of Genes (Coordinates of Start/Stop Codons [Predicted Coding Capacity in kDa] | Position of Genes (Coordinates of Start/Stop Codons [Predicted Coding Capacity in kDa] | ||||||||||||
AV2 | AV1 | AC1 | AC2 | AC3 | AC4 | AC5 | BC1 | BV1 | |||||
Isolate | Accession Number | Size (nt) | nt | nt | Nt | nt | Nt | nt | nt | Accession Number | Size (nt) | nt | nt |
PAL-1-J40 | KC462552 | 2751 | 154–513 (13.2) | 314–1090 (28.7) | 1539–2624 (40.2) | 1234–1641 (15.1) | 1087–1491 (14.9) | 2327–2470 (5.3) | 218–985 (28.4) | KC462553 | 2760 | 1326–2321 (36.5) | 522–1286 (28.3) |
PAL-2-J44 | KJ854912 | 2753 | 156–515 (13.2) | 316–1092 (28.7) | 1541–2626 (40.2) | 1234–1641 (15.1) | 1089–1493 (14.9) | 2329–2472 (5.3) | 220–987 (28.4) | - | - | ||
PAL-3-J45 | KJ854913 | 2752 | 155–514 (13.2) | 315–1091 (28.7) | 1540–2625 (40.2) | 1233–1640 (15.1) | 1088–1492 (14.9) | 2328–2471 (5.3) | 219–986 (28.4) | - | - | ||
PAL-4-J56 | KJ854914 | 2752 | 155–514 (13.2) | 315–1091 (28.7) | 1540–2625 (40.2) | 1233–1640 (15.1) | 1088–1492 (14.9) | 2328–2471 (5.3) | 219–986 (28.4) | - | - | ||
PAL-5-J67 | KJ854915 | 2752 | 155–514 (13.2) | 315–1091 (28.7) | 1540–2625 (40.2) | 1233–1640 (15.1) | 1088–1492 (14.9) | 2328–2471 (5.3) | 219–986 (28.4) | - | - | ||
PAL-6-Q13 | KJ854916 | 2753 | 156–515 (13.2) | 316–1092 (28.7) | 1541–2626 (40.2) | 1234–1641 (15.1) | 1089–1493 (14.9) | 2329–2472 (5.3) | 220–987 (28.4) | - | - | ||
PAL-7-Q17 | KJ854917 | 2752 | 156–515 (13.2) | 316–1092 (28.7) | 1541–2626 (40.2) | 1233–1640 (15.1) | 1088–1492 (14.9) | 2328–1471 (5.3) | 219–986 (28.4) | - | - | ||
PAL-8-Q20 | KJ854918 | 2753 | 156–515 (13.2) | 316–1092 (28.7) | 1541–2626 (40.2) | 1234–1641 (15.1) | 1089–1493 (14.9) | 2329–2472 (5.3) | 220–987 (28.4) | - | - | ||
PAL-9-Q23 | KJ854919 | 2753 | 156–515 (13.2) | 316–1092 (28.7) | 1541–2626 (40.2) | 1234–1641 (15.1) | 1089–1493 (14.9) | 2329–2472 (5.3) | 220–987 (28.4) | - | - |
2.3. Sequence Analysis of DNA-A and DNA-B of WmCSV-[PA:Pal:10]
Virus | Total nt | DNA-A | DNA-B | |||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
AV2 | AV1 | AC1 | AC2 | AC3 | AC4 | AC5 | BC1 | BV1 | ||||||||||||
DNA-A | DNA-B | Nt | aa | nt | Aa | Nt | Aa | nt | Aa | Nt | Aa | nt | aa | nt | aa | nt | aa | nt | aa | |
WmCSV [YE] | 97.60 | 95.80 | 99.44 | 98.32 | 97.68 | 95.35 | 97.88 | 97.50 | 97.55 | 95.00 | 97.28 | 94.78 | 98.61 | 95.74 | 98.57 | 96.86 | 89.86 | 91.56 | 96.60 | 93.65 |
WmCSV [SD] | 98.22 | 95.36 | 98.89 | 98.32 | 98.97 | 100.00 | 98.25 | 98.61 | 97.30 | 94.17 | 97.78 | 95.52 | 100.00 | 100.00 | 99.09 | 97.65 | 89.96 | 91.87 | 95.03 | 92.11 |
WmCSV [JO] | 99.20 | 95.80 | 99.72 | 100.00 | 99.61 | 99.61 | 99.45 | 99.17 | 98.28 | 95.83 | 98.27 | 96.27 | 100.00 | 100.00 | 99.61 | 99.22 | 90.16 | 91.46 | 96.60 | 92.59 |
WmCSV [IL] | 99.42 | 98.26 | 100.00 | 100.00 | 99.49 | 99.61 | 99.63 | 99.17 | 98.77 | 96.67 | 98.77 | 97.76 | 100.00 | 100.00 | 99.61 | 99.22 | 91.57 | 99.59 | 97.78 | 94.71 |
WmCSV [IR] | 98.26 | 94.46 | 99.44 | 99.16 | 98.97 | 100.00 | 98.16 | 98.06 | 96.57 | 92.50 | 97.04 | 95.52 | 100.00 | 100.00 | 99.22 | 97.65 | 89.66 | 91.06 | 96.56 | 92.67 |
WmCSV [LB] | 99.06 | 98.26 | 100.00 | 100.00 | 98.58 | 97.29 | 99.45 | 99.17 | 98.77 | 96.67 | 99.01 | 98.51 | 100.00 | 100.00 | 98.83 | 97.25 | 98.69 | 99.59 | 98.04 | 94.74 |
WmCSV [OM:1] | 97.86 | 93.16 | 99.44 | 99.16 | 98.46 | 98.84 | 97.61 | 97.50 | 96.57 | 92.50 | 97.78 | 95.52 | 99.31 | 97.87 | 98.96 | 96.86 | 98.16 | 89.84 | 94.77 | 93.72 |
WmCSV [OM:2] | 97.79 | 93.77 | 99.44 | 99.16 | 98.46 | 98.84 | 97.43 | 97.23 | 96.53 | 93.33 | 97.78 | 95.52 | 99.31 | 97.87 | 98.96 | 96.86 | 88.96 | 89.02 | 92.96 | 92.15 |
Virus Name | Abbreviation | DNA-A | DNA-B |
---|---|---|---|
Watermelon chlorotic stunt virus-[Yemen] | WmCSV-[YE] | AJ012081 | AJ012082 |
Watermelon chlorotic stunt virus-[Sudan] | WmCSV-[SD] | AJ245650 | AJ245651 |
Watermelon chlorotic stunt virus-[Oman]-Als-2 | WmCSV-[Als-2] | JN618982 | HE800539 |
Watermelon chlorotic stunt virus-[Oman]-Als-1 | WmCSV-[Als-1] | JN618981 | JN618980 |
Watermelon chlorotic stunt virus-[Lebanon] | WmCSV-[LB] | HM368371 | HM368372 |
Watermelon chlorotic stunt virus-[Jordan] | WmCSV-[JO] | EU561237 | EU561236 |
Watermelon chlorotic stunt virus-[Israel] | WmCSV-[IL] | EF201809.1 | EF201810.1 |
Watermelon chlorotic stunt virus-[Iran] | WmCSV-[IR] | AJ245652 | AJ245653 |
Tomato yellow spot virus[Brazil:Bicas 2:1999] | ToYSV-[BR:Bic2:99] | DQ336350 | DQ336351 |
Tomato yellow margin leaf curl virus-[Venezuela] | TYMLCV-[VE:Mer57] | AY508993 | AY508994 |
Tomato leaf curl New Delhi virus India-[India] | ToLCNDV-[INLuc] | Y16421 | X89653 |
Squash yellow mild mottle virus-[Costa Rica:1998] | SYMMV-[98:631] | AY064391 | AF440790 |
Squash mild leaf curl virus-[Imperial Valley] | SMLCV-[IV] | NC_004645 | NC_004646 |
Squash leaf curl virus-[Palestine] | SLCV-[PA: Pal:10] | KC441465 | KC441466 |
Squash leaf curl virus-[Jordan] | SLCV-[JO:Mal:08] | EF532620 | EF532621 |
Squash leaf curl China virus -[Pumpkin:Coimbatore] | SLCCV-[Pum:Coi] | AY184487 | AY184488 |
Potato yellow mosaic virus-[Puerto Rico] | PYMV | AY965897 | AY965898 |
Pepper yellow leaf curl virus-[Indonesia] | PepYLCIV-[IN] | AB267834 | AB267839 |
Mungbean yellow mosaic virus-[India] | MYMIV-[IN:Var:Dol] | AY547317 | DQ061273 |
Melon chlorotic leaf curl virus-[Guatemala] | MCLCV-[GUA] | AF325497 | AF325498 |
Loofa yellow mosaic virus-[Vietnam] | LYMV-[VI:02] | AF509739 | AF509740 |
Cucurbit leaf curl virus-[California] | CuLCV-[US:Cal:00] | AF224760 | AF224761 |
African cassava mosaic virus-[Cameroon] | ACMV-[CM:98] | AF112352 | AF112353 |
Spinach curly top virus-[USA] | SCTV-[USA] | NC_005860 | - |
Maize streak virus - [South Africa: Sasri_s:2007] | MSV-[SA:Sa:07] | - | EU152254 |
2.4. Discrimination between WmCSV-[PAL], WmCSV-[JO] and WmCSV-[IL] Isolates
2.5. Inoculation of Watermelon Plants by Particle Bombardment (Biolistic Inoculation)
3. Experimental
3.1. Samples Collection
3.2. Nucleic Acid Extraction
3.3. Detection of WmCSV-[PAL] by Polymerase Chain Reaction Using Degenerate Primers
3.3.1. Rolling Circle Amplification (RCA)
3.3.2. Restriction Fragment Length Polymorphism (RFLP)
3.3.3. Amplification of DNA-A and DNA-B of WmCSV-[PA]
3.4. Cloning and Sequence Analysis
3.5. Discrimination between WmCSV-[PAL] and WmCSV-[IL] and WmCSV-[JO] Isolates Using in Silico Enzymatic Digestion
3.6. Inoculation of Plants by Particle Bombardment (Biolistic Inoculation)
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References and Notes
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Ali-Shtayeh, M.S.; Jamous, R.M.; Mallah, O.B.; Abu-Zeitoun, S.Y. Molecular Characterization of Watermelon Chlorotic Stunt Virus (WmCSV) from Palestine. Viruses 2014, 6, 2444-2462. https://doi.org/10.3390/v6062444
Ali-Shtayeh MS, Jamous RM, Mallah OB, Abu-Zeitoun SY. Molecular Characterization of Watermelon Chlorotic Stunt Virus (WmCSV) from Palestine. Viruses. 2014; 6(6):2444-2462. https://doi.org/10.3390/v6062444
Chicago/Turabian StyleAli-Shtayeh, Mohammed S., Rana M. Jamous, Omar B. Mallah, and Salam Y. Abu-Zeitoun. 2014. "Molecular Characterization of Watermelon Chlorotic Stunt Virus (WmCSV) from Palestine" Viruses 6, no. 6: 2444-2462. https://doi.org/10.3390/v6062444