Identificación y caracterización molecular de virus transmitidos por mosca blanca bemisia tabaci que infectan tomate en la región andina de Colombia

Abstract

Los Begomovirus son virus que pertenecen a la familia Geminiviridae, se caracterizan por infectar plantas dicotiledóneas y ser transmitidos por mosca blanca (Bemisia tabaci, biotipo B). El genoma está constituido por una o dos moléculas circulares de ADN de cadena sencilla, cuyos genes están asociados con procesos de replicación, transcripción, encapsidación y movimiento. Los Begomovirus son considerados virus emergentes debido al aumento en la incidencia y severidad en las enfermedades causadas en diferentes cultivos en áreas tropicales y subtropicales del mundo. En América Latina se ha reportado el surgimiento de nuevos Begomovirus que afectan diferentes cultivos, generando pérdidas económicas para los agricultores. Con base en lo anterior, la presente investigación trazo los siguientes objetivos: identificar y caracterizar Begomovirus que afectan tomate Solanum lycopersicum L. en zonas productoras de la región Andina de Colombia por métodos moleculares; conocer la diversidad genética de los Begomovirus identificados en las diferentes muestras a través del análisis bioinformático; adquirir la secuencia de nucleótidos del componente A y el componente B de los principales Begomovirus identificados, y construir clones infectivos con begomovirus identificados en el Valle del Cauca y calcular la eficiencia de los clones infectivos por medio de inoculación por biobalística en plantas de tabaco Nicotiana tabacum y tomate comercial Solanum lycopersicum L.

//Abstract: Begomovirus (family Geminiviridae), are characterized by infecting dicotyledonous plants and are transmitted by the whitefly (Bemisia tabaci biotype B). It´s genome consists of one or two circular molecules of single-stranded DNA, whose genes are associated with replication, transcription, encapsidation and movement processes. Begomovirus are considered emerging viruses due to increased incidence and severity in diseases reported in different cultures in tropical and subtropical areas of the world. In Latin America there has been reported the emergence of new Begomovirus, affecting different cultures, and causing economic losses for farmers. Based on the above, the current study aims to identify and analyze the genetic diversity of endemic Begomovirus in Colombia by molecular methods; to know genetic diversity of Begomovirus in the different samples identified through bioinformatic analysis; to acquire the nucleotide sequence of component A and component B of the main Begomovirus, and to construct infectious clones with begomoviruses identified in Valle del Cauca and to calculate the efficiency of infectious clones by biolistic inoculation in tobacco and tomato plants. To carry out these objectives, samples of plant tissue (leaves) of tomato plants were collected in the open field and greenhouse, exhibiting typical symptoms of Begomovirus disease in the departments of Boyacá, Cundinamarca, Antioquia, Valle del Cauca and Santander. Following the collection, these materials were used for the extraction of total DNA, this one was used to determine the presence of Begomovirus through nucleic acid hybridization technique Dot blot and PCR. The PCR products associated with the DNA A and B, they were cloned and sequenced, in total 10 clones were obtained associated with the component A and 9 clones associated with the component B, these clones allowed to determine the presence of three viral entities associated to Potato yellow mosaic virus-PYMV. These results allowed the attention of the complete viral genome in tomato samples collected in the departments of Valle del Cauca, Cundinamarca and Santander. Using the model rolling circle amplification (RCA), both viral components (A and B) were cloned and partially sequenced, in total 30 complete genomes were obtained, 12 associated with the DNA A and 18 associated with the DNA B. Two of this clones (component A and B) isolated in the city of Tuluá were sequenced completely; the total sequences were deposited in the Genbank data base NCBI as PYMV [Colombia] Component A and as PYMV [Colombia] Component B. These sequences were the basis for the construction of infectious clones which were used in trial for biolistic inoculation in tomato and tobacco plants; Begomovirus detection in the inoculated material was performed by PCR. When we observed characteristic symptoms of viral disease; the biolistic inoculation trials allowed provided efficiencies between 44 and 87.5%. The results obtained in this research are a valuable contribution in the search for sources of resistance in Colombian endemics Begomovirus in tomato germplasm in the National University of Colombia, Palmira