Detection of Two Bipartite Geminiviruses Infecting Dicotyledonous Weeds in Trinidad.

Severe symptoms of suspected geminivirus etiology were manifested as intense yellow or golden mottling or mosaic of the lamina accompanied by mild leaf margin distortion on dicotyledonous weed species, Sida rhombifolia (L.) and Rhynchosia minima (L.), collected from 1999 to 2002 from the northeastern and central regions of Trinidad. S. rhombifolia is a common roadside weed while R. minima may have been introduced through restricted cultivation as a forage legume for livestock. Potato yellow mosaic virus-Trinidad isolate (PYMV-TT) has been implicated as the primary causal agent of begomoviral disease in large-scale tomato cultivation in Trinidad (2). It has been suggested that these weeds may be alternative hosts to PYMV-TT. However, all samples tested negative for PYMV-TT in dot blot hybridization assays using a PYMV-TT-specific DNA-A probe under high stringency. These results excluded the presence of PYMV-TT in these weeds. Polymerase chain reaction (PCR) amplification using clarified leaf extracts with degenerate primers for DNA-A (MP16 and MP82, PAL1v1978 and PAR1c715, and prV324 and prC889) and for DNA-B (PBL1v2040 and PCRc1) was performed on the weed samples (S. N. Rampersad and P. Umaharan, unpublished). Degenerate primers MP16 and MP82 target the 5' terminal region of the coat protein (cp) (2); PAL1v1978 and PAR1c715 direct amplification of the replication-associated protein gene (rep) and part of the cp gene (1); prV324 and prC889 amplify the core cp sequence (3). Primers PBL1v2040 and PCRc1 target the intergenic region and the 5' terminal of the BL1 ORF (1). PCR fragments obtained through amplification using this primer pair confirmed the presence of a DNA-B component for the unknown viruses. PCR fragments were sequenced and alignments were performed using DNASTAR (DNASTAR Inc., Madison, WI) and BLASTN ( www.ncbi.nlm.nih.gov/blast/ ) programs. None of the partial nucleotide sequences obtained for the viruses produced significant alignments with each other (5' terminal cp: 74% identity; core cp sequence: 78% identity), suggesting the detection of two distinct viruses. In addition, the partial sequences obtained were aligned to sequences of homologous regions of 11 New World begomoviruses (from the major representative clusters). The nearest match for R. minima, using alignments with 5' terminal cp (GenBank Accession No. AY221124), core cp (GenBank Accession No. AY217344), and 5' terminal BL1 region (GenBank Accession No. AY220490) was obtained for Rhynchosia golden mosaic virus (RhGMV, GenBank Accession Nos. AF408199 and AF442117) with 84 and 88% identity. There were no significant similarities found for sequence comparisons of the BL1 ORF. For S. rhombifolia, the highest homology using the 5' terminal cp (GenBank Accession No. AY220489), core cp (GenBank Accession No. AY217345), rep/cp region (GenBank Accession No. AY220488), and the 5' terminal BL1 region (GenBank Accession No. AY221125) was obtained for Sida golden mosaic virus (SiGMV, GenBank Accession Nos. AF049336, AF070923, and Y11100), with 82, 89, 84, and 87% identity. To our knowledge, this is the first report of geminivirus infection in these weed species in Trinidad. This may have substantial implications to future geminivirus disease outbreaks especially if there is expansion of the host range of these viruses to include economically important crops. References: (1) M. R. Rojas et al. Plant Dis. 77:340, 1993. (2) P. Umaharan et al. Phytopathology 88:1262, 1998. (3) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.