Identification of Cotton leaf curl Gezira virus in Papaya in Oman.

Papaya is an important fruit crop in Oman covering some 130 ha with an annual production of 20 tonnes. In 2011, during surveys of farms in the Quriyat region of Oman, papaya plants were found severely affected by leaf curl disease. Leaves with severe curling, vein darkening, and vein thickening were collected for study. Disease incidence ranged from 30 to 50%, particularly in fields with young papaya. A begomovirus (family Geminiviridae) was suspected as the causal agent based on symptoms (1) and the presence of whiteflies in the field. Samples (four to five) were collected from three farms. Total nucleic acids extracted from symptomatic leaves using the CTAB method were used as templates to amplify circular DNAs using Φ29 DNA polymerase and products were digested with restriction enzymes to identify fragments of 2.6 to 2.8 kb typical of geminiviruses. PstI yielded a fragment of ~1.8 kb when the digested product was analyzed by electrophoresis on a 1% agarose gel. The fragment was cloned and sequenced using primer walking strategy in both directions. The sequencing confirmed the exact size (1,764 bp) and the sequence was deposited in GenBank (HE800524). The viral sequence from Oman (isolate Pap-2) showed four open reading frames (ORFs) in the complementary sense (replication associated protein [Rep] gene, the C2 gene, the replication enhancer protein [REn] gene, and the C4 gene) and the virion-sense ORFs (V1 and V2) were missing in the sequence. An initial comparison to NCBI database sequences using BLAST showed the clone from Oman had the highest level of sequence identity to Cotton leaf curl Gezira virus (CLCuGeV) (FJ868828) cloned from okra in Sudan. Subsequent pair wise sequence comparison was done using ClustalV algorithm. Full length sequences of CLCuGeV from database were trimmed according to the size and genomic coordinates of Pap-2 isolate. The Pap-2 isolate sequence was found to have 83.3 to 95.1% sequence identity to CLCuGeV sequences with maximum value to the Sudan isolate. Amino acid sequence comparison showed that the four predicted proteins (Rep, C2, REn, and C4) encoded by the Pap-2 isolate shared 95.3%, 97.8%, 97.7%, and 87.6% sequence identity, respectively, with the homologous proteins of CLCuGeV-SD (FJ868828). The absence of virion-sense protein sequences indicated it to be a subgenomic molecule of CLCuGeV. According to the recommendations of International Committee on Taxonomy of Viruses, these results indicate that the virus identified in association with papaya leaf curl disease in Oman is a variant of CLCuGeV. CLCuGeV is a begomovirus of African origin which is distinct from the begomoviruses of the Middle East and Asia. To our knowledge, this is the first report of CLCuGeV, or any other cotton infecting begomovirus, from papaya in Oman. The presence of a recombinant fragment of CLCuGeV in a Tomato yellow leaf curl virus isolate from Iran (2), and the association of CLCuGeV with cotton in Pakistan (3) and hollyhock in Jordan (GU945265) suggests this virus has moved into the Middle East and Asia from Africa. The identification of CLCuGeV in Oman shows the widespread occurrence of this virus species. This discovery is important since Oman, and other countries in the area, are a hub of international trade and travel, particularly by air and sea, meaning that the virus could spread further. References: (1) R. W. Briddon and P. G. Markham. Virus Res. 71:151, 2000. (2) P. Lefeuvre et al. PLoS Pathog. 6:e1001164, 2010. (3) M. N. Tahir et al. PLoS ONE 6:e20366, 2011.