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.

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).

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 virus Y 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.

Background and rationale

Methodology: RT-PCR tests to amplify Iraqi potyviruses

Conclusions

Results: Three potyviruses were detected in potato and vegetables of Iraq

Detection and molecular characterisation of potyviruses infecting potato and vegetables in

IraqNAWRES SADEQ, MN MARUTHI and SUSAN SEAL

Natural Resources Institute, University of Greenwich, Chatham Maritime, Kent ME4 4TB, UK

1 ALKKLYVDRTVDEEELQGFRGMVPPYNNEIEGISYKMQRRAQDTIVSGPKIKKDGEKGKRGPHVNVGNLLEEVVHIVARGIKTIFRSSKIGAITQRSKKGCRTVLNLEPLLECSPKQIDISNTRATQSQFDTWYEAVQLAFDIGEMEMPTVMNGLMVWCIENEPSPNINGVWVMM 1-1752 ALKKLYMDRTVDEEELKAFTEMMVALDDELECDTYEVHHQGNDTIDAGGSTKKDAKQEQGSIQPNLNKGKEKDVNVGTSGTHTVPRIKAITSKMRMPKSKGATVLNLEHLLEYAPQQIDISNTRATQSQFDTWYEAVQLAYDIGETEMPTVMNGLMVWCIENGTSPNINGVWVMM3 ALKKLYMNRTVDEEELKAFTEMMVALDDELECDTYEVHHQGNDTIDAGGSTKKDAKQEQGSIQPNLNKEKEKDVNVGTSGTHTVPRIKAITSKMRMPKSKGATVLNLEHLLEYAPQQIDISNTRATQSQFDTWYEAVQLAYDIGETEMPTVMNGLMVWCIENGTSPNINGVWVMM

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.

Fig 1. Genome map of a potyvirus indicating conserved motifs and degenerate primer sites

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.

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 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.