Background and rationale

1. Detection and molecular characterisation of potyviruses infecting potato and vegetables in Iraq • NAWRES SADEQ, MN MARUTHI and SUSAN SEAL • Natural Resources Institute, University of Greenwich, Chatham Maritime, Kent ME4 4TB, UK Background and rationale Plant viruses are a major limiting factor for potato and vegetable production in Iraq. Local reports described a range of viruses in Iraq on potato, tomato, cucurbits and broad bean, identified mainly on the basis of biological and serological properties, with limited electron microscopy, which lack accurate identification and characterisation of viruses. This study was initiated with the following objectives: 1) to generate basic information on the type of potyviruses infecting potato and vegetables in Iraq, 2) to study their molecular diversity and phylogenetic relationships, and 3) to develop reliable RT-PCR diagnostic tests. Diseased leaf samples from potato, tomato, broad bean and cucurbits were collected from Baghdad and Al-Anbar provinces in 2008. Total nucleic acids were extracted using CTAB protocol and virus genomes amplified by RT-PCR using various sets of degenerative potyvirus primers (Fig 1). RT-PCR amplicons of the expected size were cloned and screened by restriction digestion with AluΙ and EcoRI (Fig 2 A&B) to assist the selection of clones for sequencing. Phylogenetic analyses of the sequences were carried out using MEGA4. All the crops tested (potato, tomato, broad bean and cucurbits) by RT-PCR were infected with potyviruses. Use of primers Pot1/Pot2 and S primer/M4T gave rise to the highest number of positive samples (Table 1). PCR products of size 1.3 Kb with Pot1/Pot2, and 1.6-1.8 Kb with S primer/M4T sets were observed in 52 and 57 samples out of 138 samples tested, respectively (Fig 2 C&D). Methodology: RT-PCR tests to amplify Iraqi potyviruses Fig 2. RT-PCR products from broad bean, tomato and potato using A) S primer/M4T, and B) pot1/pot2 primers. Restriction digestion pattern using C) AluΙ and D) EcoRI. BB1: broad bean, PC & PM: potato, CuMMo: zucchini, To23 & To24: tomato. Sequencing of the PCR products revealed the occurrence of three potyviruses in Iraqi samples. Bean yellow mosaic virus (BYMV) in broad bean (97% similar to isolates from Japan), Zucchini yellow mosaic virus (ZYMV) in squash (95% similar to isolates from Pakistan), and two strains of Potato virusY in potato (PVY-NTN 92% similar to strains from Syria, USA and UK; and PVY N:O 95-99% similar to isolates from USA, Canada and Germany), and PVY N:O in tomato only. Phylogenetic analysis of the sequences clustered these viruses separately according to their nucleotide similarities (Fig 3). At 92% similarity Three potyviruses, BYMV, ZYMV and PVY were found to infect potato, tomato, broad bean and cucurbits in Iraq. All three are isolates of previously described viruses. PVY-NTN was the most divergent poty- virus found in Iraq, the deduced amino acid sequences of which is presented together with the selected vir-uses from the data-base (Fig 4). Fig 1. Genome map of a potyvirus indicating conserved motifs and degenerate primer sites Results: Three potyviruses were detected in potato and vegetables of Iraq Fig 3. Neighbour joining tree with 70% bootstrap scores based on NIb (GNNS) motif /CP/ 3`UTR nucleotide sequences of four Iraqi potyvirus isol-ates (highlighted in red) along with related sequences from the database. Conclusions Table 1. Degenerate potyvirus primers used to detect potyviruses in Iraqi plants by RT-PCR. NT: not tested, NS: non specific bands, +: positive, -: negative, +/-: weak positive.*: expected product size. Fig 4. PVY-NTN coat protein amino acid sequences isolated from potato in Iraq aligned with closely related sequences from GenBank.1:Iraqi,2:Syrian,3:USA isolates. 1 ALKKLYVDRTVDEEELQGFRGMVPPYNNEIEGISYKMQRRAQDTIVSGPKIKKDGEKGKRGPHVNVGNLLEEVVHIVARGIKTIFRSSKIGAITQRSKKGCRTVLNLEPLLECSPKQIDISNTRATQSQFDTWYEAVQLAFDIGEMEMPTVMNGLMVWCIENEPSPNINGVWVMM 1-175 2 ALKKLYMDRTVDEEELKAFTEMMVALDDELECDTYEVHHQGNDTIDAGGSTKKDAKQEQGSIQPNLNKGKEKDVNVGTSGTHTVPRIKAITSKMRMPKSKGATVLNLEHLLEYAPQQIDISNTRATQSQFDTWYEAVQLAYDIGETEMPTVMNGLMVWCIENGTSPNINGVWVMM 3 ALKKLYMNRTVDEEELKAFTEMMVALDDELECDTYEVHHQGNDTIDAGGSTKKDAKQEQGSIQPNLNKEKEKDVNVGTSGTHTVPRIKAITSKMRMPKSKGATVLNLEHLLEYAPQQIDISNTRATQSQFDTWYEAVQLAYDIGETEMPTVMNGLMVWCIENGTSPNINGVWVMM