Occurrence of Cucumber mosaic virus Subgroups IA and IB Isolates in Tomatoes in Nigeria

Tomato (Solanum lycopersicum L.) is a major economic crop consumed globally in fresh or processed forms. During a routine field survey of major tomato-producing areas of southwestern Nigeria in May/June 2013, tomato plants cv. Roma VF showing virus-like symptoms including stunting, chlorosis, and narrowing of leaf blades were observed in 10 farmers' fields with varying levels of incidence averaging ~27%. Moderate to high aphid infestations were also observed in affected fields, and fruit production was significantly impacted based on visual observations. Since symptoms observed on affected plants are similar to those described for Cucumber mosaic virus (CMV) infection in tomato (5), leaf tissue samples collected from a total of 92 tomato plants across 10 commercial farms were subjected to antigen coated plate (ACP)-ELISA essentially as described previously (2). In ACP-ELISA using a CMV polyclonal antibody, 24 of the 92 samples (26.1%) derived from 7 of the 10 survey locations spread across Oyo, Ogun, Ekiti, and Osun states of southwestern Nigeria tested positive for CMV. Based on the ACP-ELISA results, one randomly selected sample from each of the CMV-positive survey locations, seven samples in total, was subjected to total nucleic acid extraction (1) followed by one step-single tube RT-PCR using primers CMV1/CMV2 and conditions described previously (4) with appropriate virus-positive and -negative controls. A ~500 bp DNA band was amplified from these seven ACP-ELISA-positive samples, thus confirming the presence of CMV. To further confirm these results and to enable molecular typing of CMV isolates from southwest Nigeria, the amplified DNA fragments were precipitated with the addition of 70% ethanol and centrifugation and directly sequenced using the ABI 3130xL Genetic Analyzer (Applied Biosystems, California) at the Bioscience Center of the International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. Following the removal of primer- and 3'UTR-specific sequences, the remaining 366-bp partial CP-specific sequences (GenBank Accession Nos. KM091952 to 58) and corresponding sequences of global CMV isolates obtained from GenBank were subjected to multiple alignments using the MEGA 6.0 software. This analysis showed that tomato-infecting CMV isolates from southwest Nigeria shared 91.6 to 99.4% and 94.9 to 99.1% nucleotide (nt) and amino acid (aa) identities among themselves and 91.6 to 98.0% and 94.1 to 98.3%, 89.4 to 94.1% and 93.2 to 98.3%, and 75.2 to 78.8% and 84.0 to 87.3% with corresponding nt and aa sequences of representatives of CMV isolates belonging to subgroups IA (D10538), IB (AB008777), and II (M21464), respectively. Maximum likelihood phylogenetic analysis revealed the clustering of four and three CMV isolates obtained in this study into subgroups IA and IB, respectively, with >70% bootstrap support. CMV has been detected in tomato seeds (3) and its very wide host range includes cultivated crops and weed species (5). It is therefore plausible that contaminated seed lots and alternative weed and crop host plants serve as sources of CMV inoculum to cultivated tomato in affected farms. Although CMV has been reported from tomato from several countries worldwide, to our knowledge, this is the first empirical evidence for the occurrence of CMV subgroups IA and IB in cultivated tomato in Nigeria. References: (1) S. L. Dellaporta et al. Plant Mol. Biol. Rep. 1:19, 1983. (2) J. d'A. Hughes and S. A. Tarawali. Trop. Sci. 39:70, 1999. (3) K. H. Park and B. J. Cha. Res. Plant Dis. 8:101, 2002. (4) S. Wylie et al. Aus. J. Agric. Res. 44:41, 1993. (5) T. A. Zitter and J. F. Murphy. Plant Health Instructor. DOI: 10.1094/PHI-I-2009-0518-01, 2009.