RESUMO
The Rioverde Valley is an important farming area of the San Luis Potosi State in the north-central region of Mexico, where a variety of horticultural crops (i.e., tomato, pepper, cucumber, and watermelon) are annually cultivated. In the summer of 2005, a number of plants exhibiting a variety of symptoms, including leaf yellowing, curling, and stunted growth, were observed in several tomato (Lycopersicon esculentum L.) fields. The presence of whiteflies (Bemisia tabaci Genn.) and symptoms seemed to suggest a begomoviral etiology. Leaves of 12 symptomatic tomato plants and seven plants of the weed Solanum rostratum (Dunal) growing into the same area were collected in July and September from several fields throughout the Rioverde area and assessed for the presence of begomoviruses (genus Begomovirus, family Geminiviridae) by PCR using the degenerate primers prRepDGR (CCTCCTCTAGCASWTCTNCCGTC), SL2050 (2), and prC889 (3). Amplicons of 1.4 kb were derived from viral DNA-A present in all examined S. rostratum and tomato samples, which were cloned into pGEM-T Easy Vector (Promega, Madison, WI) and subsequently analyzed by restriction fragment length polymorphism (RFLP) using MspI and HinfI. Several restriction fragment patterns were observed among the cloned PCR products, hence indicating the occurrence of different begomoviruses in the sampled fields. Sequencing of amplicons derived from one S. rostratum plant revealed the concurrent presence of Tomato severe leaf curl virus (ToSLCV; GenBank Accession No. DQ347946; [2]) and a distinct virus (GenBank Accession No. EF501978) displaying a high sequence identity with Tomato golden mottle virus from Guatemala (ToGMoV-GT94-R2; GenBank Accession No. AF32852). Restriction fragment patterns identical to that of the ToGMoV-like isolate were found in PCR clones from three additional S. rostratum plants and five tomato samples. A set of partially overlapping PCR products of 1.8 and 1.4 kb encompassing the complete DNA-A component of ToGMoV were obtained from one tomato sample by using two pairs of degenerate primers, prRepQGR-rev and prCP70 (1) and prRepDGR and prC889. Amplicons were cloned, sequenced, and compared with viral sequences available in the GenBank database using BlastN and Clustal V alignments (MegAlign, DNASTAR, Madison, WI). The 2,614-bp DNA-A sequence of the Rioverde isolate (GenBank Accession No. DQ520943) displays 93% sequence identity with the Guatemalan isolate of ToGMoV. In addition, a number of B. tabaci specimens of unidentified biotype were collected in one tomato field and total DNA was isolated from them by a modified Dellaporta method. Amplification of viral DNA present in the whiteflies was carried out and the PCR products were cloned and sequenced. One of the begomoviral DNA-A genomes isolated from the whiteflies (GenBank Accession No. EF501976) displayed 99% sequence identity with the virus isolated from plants. Previously, ToGMoV had been found only in Central America ( http://gemini.biosci.arizona.edu/viruses ), but this report considerably expands its known geographical distribution. References: (1) R. De La Torre-Almaraz et al. Plant Dis. 90:378, 2006. (2) J. A. Mauricio-Castillo et al. Plant Dis. 90:1116, 2006. (3) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.
RESUMO
Okra (Abelmoschus esculentus L. Moench), an annual vegetable of African origin, has been cultivated in Mexico for 3 decades. Since 2000, the most important okra-producing areas in the states of Guerrero and Morelos have been affected by a disease causing yellow streak and severe distortion of fruits, a bright yellow mottle, and curling and distortion of leaves. These symptoms and the presence of whiteflies (Bemisia tabaci Gennadius) suggest a viral etiology. Samples of symptomatic plants from three localities, Iguala (Guerrero), Mazatepec, and Xochitepec (Morelos) were collected in November 2004 and tested for the presence of viruses. Single whitefly transmissions, grafting experiments, and experimental inoculation of healthy plants by biolistic delivery of DNA extracts from symptomatic plants consistently induced yellow mottle in okra plants and suggest the presence of a DNA virus. Total DNA extracts from symptomatic plants from field and greenhouse conditions were analyzed by Southern blot hybridization using the coat protein gene of Pepper yellow vein huasteco virus as a probe at low stringency. More than 20 positive samples were subsequently used as templates for polymerase chain reaction (PCR) amplification with the degenerate primers pRepMot and pCPMot (1). PCR products of approximately 600 bp were obtained and directly sequenced. Eight isolates from the three localities (GenBank Accession Nos. AY624016 to AY624023) shared 97 to 100% nucleotide identity but were significantly different from other known begomoviruses. The complete genome A sequence of one isolate from Mazatepec (Ok-M3) was determined using PCR amplification of viral DNA with the degenerate primers PAL1v1978 and PAL1c1960 (3) and four new universal primers, pRepQGR (5'-TCCCTGWATGTTYGGATGGAAATG-3'), pRepQGR-rev (5'-CATTTCCATCCRAACATWCAGGGA-3'), pCp70-MAC (5'-GTC TAGACCTTRCANGGNCCTTCACA-3'), and pCp70-MAC-rev (5'-GAA GGSCCNTGYAAGGTNCAGTC-3'). Partially overlapping PCR products of 0.9, 1.3, and 1.7 kb were cloned into pGEM-T easy vector (Promega, Madison, WI) and sequenced. The 2612-bp DNA-A sequence of Ok-M3 (GenBank Accession No. DQ022611) was compared with sequences available from GenBank using the Clustal alignment method (MegAlign, DNASTAR software, London). The highest sequence identity was obtained with Sida yellow vein virus (SiYVV; Accession No. Y11099), Sida golden mosaic Honduras virus (SiGMHV; Accession No. Y11097), and Chino del tomate virus (CdTV; Accession No. AF101478) that had 85.4, 85.4, and 84.4% nucleotide sequence identity with the Ok-M3 isolate, respectively. Comparative analysis of the intergenic region of the Ok-M3 isolate and its closest relatives revealed that these viruses display different putative Rep-binding sites (iterons): Ok-M3 (GGTACACA), SiYVV (GGAGTA), and SiGMHV (GGKGTA). Current taxonomic criteria for the classification of begomoviruses establishes that less than 89% DNA-A nucleotide sequence identity with the closest relative of a virus is indicative of a separate species (2). Our results indicate that the okra-infecting virus identified in this study is a new begomovirus species, and the provisional name of Okra yellow mottle Mexico virus is proposed. References: (1) J. T. Ascencio-Ibañez et al. Plant Dis. 86:692, 2002. (2) C. Fauquet et al. Arch. Virol. 148:405, 2003. (3) M. Rojas et al. Plant Dis. 77:340, 1993.