First Report of Tomato spotted wilt virus Infecting African Clover (Trifolium tembense) in Georgia

Tomato spotted wilt virus (TSWV; family Bunyaviridae, genus Tospovirus), which is vectored by several species of thrips (order Thysanoptera, family Thripidae), causes a destructive disease that affects many economically important host plants such as tomatoes, peppers, and peanuts. Controlling the spread of this disease is challenging, and currently, only limited strategies are available to prevent and/or control its dissemination, including early diagnosis, destruction of infected material, and elimination of the vector. TSWV has been previously reported in subterranean clover (Trifolium subterraneum), white clover (T. repens), and various unidentified wild clovers (Trifolium spp.) in North America and Australia (1,3), but never before in an African species. T. tembense (Fresen.), an herbaceous annual African clover that is mainly used for grazing, is part of the national germplasm collection housed at the Plant Genetic Resources Conservation Unit in Griffin, GA. TSWV was found naturally infecting several accessions of this species being grown for regeneration in a greenhouse during 2008. Initial putative identification of the virus was done by visual inspection of host symptoms that included ringspots, necrotic and chlorotic local lesions, sometimes mild systemic wilting, and eventually an overall decline of healthy tissue in the infected plants. This was subsequently confirmed by double-antibody sandwich (DAS)-ELISA and reverse transcription (RT)-PCR. Primers (5'-ATGTCTAAGGTTAAGCTC-3' forward and 5'-TTAAGCAAGTTCTGTGAG-3' reverse) targeted the nucleocapsid gene of TSWV and amplified an expected product of approximately 800 bp (2). No product was amplified in any of the negative controls. Twenty-six individuals representing twelve plant accessions (PI 517788, 517790, 517792, 517793, 517809, 517832, 517842, 517845, 517851, 517871, 517876, and 517889) were screened for TSWV. Two to three individuals were targeted from each accession. Samples were chosen on the basis of the availability of leaf tissue to perform two diagnostic assays, ELISA and RT-PCR. Samples chosen for this study were all naturally infected by thrips. All but four individuals representing two plant accessions tested positive for the virus. The RT-PCR data substantiated the DAS-ELISA results and confirmed the suspected infection. More than 26% of the positive samples naturally infected by TSWV were further characterized by purifying and sequencing (bidirectionally) the RT-PCR product on an automated CEQ 8000 sequencer (Beckman Coulter, Fullerton, CA). The resulting sequences were aligned and edited using AlignIR (LI-COR, Lincoln, NE). More than 700 bp of sequence data (GenBank Accession No. FJ183743-FJ183746) was compiled and they displayed 98% identity with deposited TSWV nucleocapsid gene sequences in GenBank, with no similarity to any other targets. To our knowledge, this is the first report of TSWV infection in T. tembense. Accessions potentially resistant to TSWV within this species were identified and need to be further substantiated. T. tembense is a wild, native clover in Africa and could serve as a weed host for infection of nearby agronomically important crops. References: (1) I. Bitterlich and L. S. MacDonald. Can. Plant Dis. Surv. 73:137, 1993. (2) R. J. Holguin-Pena and E. O. Rueda-Puente. Plant Dis. 91:1682, 2007. (3) C. R. Wilson. Plant Pathol. 47:171, 1998.