<p>El presente estudio se adelantó dentro de un proyecto de investigación para obtención de plantas resistentes al virus del mosaico del pepino (CMV), mediante la transformación y expresión del gen de la proteína de la cápside de CMV en plantas transgénicas de variedades comerciales de plátano y banano (Musa spp). Se trata de una estrategia reciente que ha sido utilizada con éxito en varios cultivos comerciales. El CMV fue detectado serológicamente (DAS-EUSA) en hojas de plátano cv. Dominico Hartón y de banano cv. Gros Michel que presentaban síntomas de mosaico. El virus se purificó parcialmente a partir de hojas infectadas de <em>Nicotiana tabacum</em> y con la ayuda del microscopio electrónico de transmisión, se identificaron partículas isométricas de aproximadamente 30 nm dc diámetro. El peso molecular de la proteína de la cápside en geles desnaturalizados de poliacrilamida (SDS-PAGE) fué de 28 kDa. El patrón electroforético del RNA viral de doble cadena (dcRNA) fué similar para los dos aislamientos de CMV obtenidos de plátano y banano. Utilizando la dcRNA como patrón, se sintetizó cADN por transcripción reversa y, a partir de éste, se amplificaron por PCR los genes de la proteína de Ia cápside (GPC) de ambos aislamientos. Los productos de la amplificación de aproximadamente 890 pares de bases (pb), que contenían el GPC, se donaron en el plásmido vector Bluescript KS (+/-). El análisis de restricción con endonucleasas y la secuenciación del GPC de ambos aislamientos, indicó que éstos pertenecen al subgrupo I (tipificado por el aislamiento DTL). El análisis de la secuencia nucleotídica y de aminoácidos de ambos genes, indicó una homología del 99% y un alto grado de conservación con otros miembros del subgrupo I de CMV. El GPC se está donando en plásmidos vectores para su transformación en variedades comerciales de plátano y banano en Colombia.</p><p> </p><p><strong>Characterization of the Coat Protein Gene of two Cucumber MosaicVirus (CMC) Isolates from Plantain and Banana (Musa spp)</strong></p><p>The final objective of this study is to genetically engineer cucumber mosaic virus (CMV) resistance in edible <em>Musa</em> spp.in Colombia, by means of expressing the CMV coat protein gene in transgenic plants. This strategy has been successfully employed in other crops. CMV was serologically detected in leaves of commercially grown bananas (cv. Gros Michel) and plantains (cv. Dominico-Harton) showing mosaic symptoms. CMV was partially purified from infected tissue of Nicotiana tabacum. Transmission electron microscopy examination showed the presence of viral isometric particles of approximately 30 nm in diameter. The molecular weight of the coat protein subunit was approximately 28 kDa as determined by SDS-PAGE. Analysis of double-stranded RNA (dsRNA) indicated that the isolations present in both varieties had similar dsRNA profiles, characteristic of CMV. cDNA was reverse transcribed using viral dsRNA as the template, and the coat protein genes (CPG) of the two CMV isolates were amplified by the polymerase chain reaction (PCR). The amplified products of approximately 890 bp, containing the CPG, were cloned into the plasmid vector Bluescript KS (+/-). Based on the pattern obtained from the digestion of the amplified CP genes with Msp 1 and DNA sequencing data, the two CMV isolates were classified into the serotype subgroup I (typified by isolate DTL). Computer analysis of DNA sequences of the CP genes derived from both CMV isolates showed a 99% homology between them at the nucleotide and amino acid level as well as a high level of conservation with other members of the CMV subgroup I. The CPG of CMV are now being cloned into plant transformation vectors for their genetic engineering into banana and plantain commercial cultivars.</p>
Plantains (Musa AAB) are important sources of food and income for millions of people in Colombia and other developing countries. Colombia is the largest producer of plantains (2) and the third largest exporter of bananas in the world. In 2001, plants of ‘Dominico-Hartón’ plantain showing mild chlorotic streak symptoms were observed in northwestern Colombia. Electron microscopy of symptomatic tissue extracts revealed the presence of filamentous virus-like particles approximately 800 nm long. Immunocapture reverse-transcription polymerase chain reaction was performed to test for the presence of Banana mild mosaic virus (BanMMV) as described by J. E. Thomas (unpublished, Queensland Department of Primary Industries, Australia) and Sharman et al. (3). For polymerase chain reaction (PCR), the upstream primer No. 193 (5′-CAC TTA GGT TTG TGT GAT GT-3′) (designed in this study by using the computer Program DNAMAN Version 4.13) and the downstream primer Poty1 (5′-GGA TCC CGG GTT TTT TTT TTT TTT TTT V-3′) (1,3; J. E. Thomas, unpublished, Queensland Department of Primary Industries, Australia) were used. Amplification products of the expected size (approximately 900 bp) were obtained and sequenced after cloning in a pCR2.1 plasmid vector. Analyses of nucleic acid sequences using the international sequence databases and the BLAST program yielded nucleotide and amino acid sequence similarities of 80 to 83% and 90 to 92%, respectively, with an Australian isolate of BanMMV (GenBank Accession No. AF314662). The coat protein (CP) gene of the Colombian BanMMV isolate consists of 717 nucleotides. When the CP of the Colombian BanMMV isolates (GenBank Accession Nos. AY319331, AY319332, and AY319333) was compared with the CP of the Australian isolate, a highly variable region was observed in the N-terminus region. To our knowledge, this is the first report of BanMMV isolated from plantains in Colombia and the presence of molecular variability in the CP of BanMMV isolates. BanMMV has been found in Colombia associated with Banana streak virus and Cucumber mosaic virus in plantain. References: (1) A. Gibbs and A. Mackenzie. J. Virol.Methods 63:9, 1997 (2) N. S. Price. Infomusa 8(2):26, 1999. (3) M. Sharman et al. J. Virol. Methods 89:75, 2000.
We have recently reported on the presence of banana streak virus (BSV) affecting plantains (Musa spp.) in Colombia (2). BSV is serologically related to sugarcane bacilliform virus and has been found to be transmitted by the pink mealybug (Saccharicoccus sacchari) from sugarcane to banana (1). In the vicinity of affected plantain crops in the localities of Andes (Antioquia) and Montenegro (Quindio), we observed sugarcane (Saccharum officinarum L.) plants with chlorotic streaks on their leaves, as well as arrowroot (Canna edulis Ker-Gawl.) plants with mild mosaic symptoms. The foliar tissue of symptomatic plants of these two species was tested for BSV and cucumber mosaic virus (CMV) by double antibody sandwich-enzyme-linked immunosorbent assay with commercial polyclonal antisera (Agdia Inc., Elkhart, IN). BSV was detected in samples of both plant species, whereas CMV was not detected in either one. Immunosorbent electron microscopy analysis of BSV-infected, symptomatic, foliar tissue of sugarcane showed the presence of viral-like bacilliform particles measuring approximately 150 × 30 nm, typical of BSV. This is the first report of BSV infecting Saccharum officinarum in Colombia and the first report of Canna edulis as a host for this virus. References: (1) B. E. L. Lockhart and L. J. C. Autrey. Plant Dis. 72:230, 1988. (2) H. Reichel et al. Plant Dis. 80:463, 1996.
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