Molecular characterization of plant viruses infecting potato and vegetables in Iraq

1. Molecular characterization of plant viruses infecting potato and vegetables in Iraq NAWRES AL_KUWAITY, SUSAN SEAL and M. N. MARUTHINatural Resources Institute, University of Greenwich at Medway, Chatham, Maritime, Kent, ME4 4TB, UK

2. Introduction: • Potato and vegetables are grown in Iraq and have significant economic importance as they are used for daily consumption and a source of income. • Plant viruses are one of the factors that limit the production of potato and vegetables worldwide. • A number of viruses have been studied in Iraq using conventional methods. However there is no molecular information about plant viruses from Iraq.

3. Potato and vegetable production (in metric tonnes) in Iraq according to FAO statistics in 2010

4. Objectives: • To identify plant viruses occurring in potato and vegetable samples collected from Iraqi fields by PCR/RT-PCR using genus/family specific degenerate primers. • To study the relatedness of viruses isolated from Iraq to other viruses from different geographical regions by phylogenetic analyses.

5. The following virus groups/families were selected to screen for, in potato and vegetables based on their economic importance: • 1. Potyviridae • 2. Geminiviridae • 3. Carlaviruses • 4. Potexviruses • 5. Alfamoviruses • 6. Cucumoviruses • 7. Tobamoviruses • 8. Tombusviruses About 60% of known plant viruses are members of these families Previously reported in Iraq Symptoms on certain samples

6. Potato and vegetables Potato Cow pea

7. Gel recovery and extraction RT-PCR using genus/family specific primer sets Dried samples Cloning into pGEMT-vector DNA fragments RNA CTAB extraction Ethidium bromide gel electrophoresis Blue/white colony and T7/SP6 screening DNA Sequencing and sequence analysis Cloning into pGEM-3zf vector PCR using genus specific primer sets RCA Full length DNA fragments Restriction digestion Screening and detection scheme for plant virus groups in potato and vegetable samples collected from Iraq.

8. Genus or family specific primer sets used to screen potato and vegetable samples

9. CI NIaVPg HC-Pro NIa Pro P1 P3 CP NIb ~1.8 kb ~1.6 kb • Results: 5`UTR Detection of potyviruses in Iraqi samples 3`UTR Poly A Tail M p To P Tomato Broad bean Sample screening for potyviruses by Sprimer/M4T primer set. P: potato, To:tomato. Zucchini leaf showing mosaic symptoms from naturally infected plant

10. Potato virus Y-O:N IRQ 99 PM2 IRQ 99 PM6 IRQ PM9 IRQ PM8 IRQ 98 PVY-Wi 99 PVYO:N 86 PB209 USA N:O-Mb112 Canada OR-1 USA PVY P55 IRQ 92 99% P8-2 IRQ P8-6 IRQ 96 SYR-II-DrH PVYNW P54A-3 IRQ 100 87 PVY-To IRQ 78 P46-1 IRQ 98 96 P54A-5 IRQ P54A-1 IRQ PVYNTN PVY-NTN IRQ 100 PVY-12 96 HR1 Potato virus A 93 2002 Japan Kunchyna Slovakia 98% ZYMV 100 ZYMV IRQ B strain Isael 100 AG Israel NAT Israel 96 Gladiolus BYMV-S 90-2 Japan 100 BYMV-IRQ1 BB5 IRQ 88 BYMV BB3-IRQ 97% BB17 IRQ BB1-1 IRQ 100 BYMV-IRQ2 100 BB6 IRQ BB15-IRQ BB2 IRQ BB3-1 IRQ BB3-2 IRQ Yam chlorotic necrotic mosaic virus Blackberry virus Y Squash vein yellowing virus Brome streak mosaic virus 100 Oat necrotic mottle virus 100 Neighbor-Joining phylogenetic tree of potyviruses.

11. Detection of begomoviruses in Iraqi samples ori CR AV2 AC4 DNA-A V1 or CP C2 or TrAP C2 or REP Naturally infected tomato plant showing yellowing and leaf curling symptoms C3 or REn ~540 bp ~2.8 kb L Tomato Eggplant + L Tomato Gel electrophoresis of DNA fragments amplified by PCR using Deng primers RLFP of RCA products

12. New Caledonia Moroccan Almeria 97 RE4 89 95 Mauritius Netherland TYLCV IRQ Jordan 100 Cuba 100 Dominican Japan-Misumi 100 TYLCV-GJ 100 99 Bos Korea 100 Iran Kuwait 94 TLCOV TLCV-AU TYLCV-China TCTV 99% TYLCV Evolutionary relationships of Tomato yellow leaf curl virus isolated from tomato in Iraq to GenBank isolates/viruses.

13. ORF4 (TGB3) 25K 223K 7K 11K ORF2 (TGB1) ORF6 (NBP) ORF1 (RRP) ORF4(CP) ORF3 (TGB2) Poly A Tail 12K 34K Detection of carlaviruses in Iraqi samples ~950 bp Cowpea P M RT-PCR using Car-F1/M4T primer set to screen samples for carlaviruses. P:potato

14. P3-2 P3-4 82 P3-11 P3-11 P3-1 P3-1 P3-3 P3-12 P3-12 PMO2-cp PMO2-cp PVS3-5 Syria Syria P19-18-cp P19-18-cp P19-23-cp P19-23-cp P19-24-cp P19-24-cp 89 P18-33 P18-33 99 P18-34 P18-34 KER.SA.28 Iran AZA.TA.6 Iran P4-23 P4-23 PC11-cp PC11-cp Karla Hangzhou China China IdDef-US Id4106-US 90 PC6 Maranhao Brazil 91 Arachis Brazil 92 CpMMV-PR Puerto Rico Barreiras Brazil 83 CPMMV-M USA CPMMV-H USA Ghana Cp2-8 92 96 Cp2-9 99% PVS 80% CoMMV Neighbor-Joining phylogenetic tree of carlaviruses.

15. 33K 92K 22KD ORF3 ORF4 41K 19K ~ 1.2Kb M Tomato E Tombusviruses RT-PCR using CIR1/CIR2 primers. E: eggplant sample Detection of tombusviruses in Iraqi samples 3`OH 5` ORF1 ORF1 (RT) ORF2 (CP) Naturally infected eggplant showing leaf malformation symptoms

16. Tom9 Tom13 Tom10 Tom1 77 Tom5 Tom8 93 Tom11 Tom12 98 Tom6 GALV Tom7 Japan 98 92 Limo 08 GYP2 92 Lim 4 99 WaterDoss Lim 3 99 Italy 72 SchunterRiver TBSV 94 CIRSV CNV CPMoV 100 LWSV 98% Phylogenetic tree of Grapevine Algerian latent virus nucleotide sequences

17. Viruses detected in samples tested PVY PVS GALV PVY TYLCV GALV ZYMV BYMV CpMMV

18. Conclusions This study confirmed the occurrence of three potyviruses (PVY, ZYMV and BYMV), one begomovirus (TYLCV), two carlaviruses (PVS and CpMMV) and one tombusvirus (GALV) in potato and vegetables grown in Iraq. Provided the first molecular information about plant viruses from Iraq and distinguished three recombinant strains of PVY; namely PVYNTN, PVYO:N and PVYNW occurring in potato and tomato samples. The data presents a new report of CpMMV in cowpea and GALV in eggplant and tomato from Iraq. Nucleotide sequence analysis revealed that all detected isolates showed high identity to GenBank isolates . As Iraq imports most of its planting material, the high identity of sequences obtained to isolates from Genebank suggests that most viruses detected may have been introduced into Iraq in imported planting material.

19. Thank you