Detección molecular de Potato yellow vein virus en el vector natural Trialeurodes vaporariorum, Westwood (mosca blanca)

Abstract

ABSTRACT: Potato yellow vein virus (PYVV) expresses yellowing symptoms on plants and it is transmitted by the whitefly vector Trialeurodes vaporariorum Westwood. No molecular viral detection from the vector extracts has been reported to date. The objectives of this study were I) amplification of the PYVV major coat protein (CP) gene from viruliferous vector extracts by RT-PCR using specific primers, II) cloning and III) sequencing and sequences analysis. Some Colombian Solanum phureja (Jus et Buk) field potato plants expressing yellowing symptoms and confirmed PYVV positive by RT-PCR were used as virus donor plants for feeding whiteflies. Viruliferous vectors were obtained by depositing aviruliferous whiteflies on symptomatic leaves and kept in a muslin cloth-covered glass flask for 48 h. A kit SV for total RNA Isolation System (Promega®) was used for extracting the viral RNA from one viruliferous whitefly and from pools of viruliferous vectors (2, 5, 10, 15 and 20 individuals); CP gene amplicons of about 765bp were cloned and sequenced to confirm the PYVV genome. Blast alignment established more than 99% similarity between CP gene amplified from vector extracts and several GenBank reported PYVV CP genes from Colombian and Peruvian isolates.This study presents information concerning the molecular detection of PYVV in its natural vector for the first time by using a very simple viral acquisition technique for obtaining viruliferous vectors that could be of interest for future studies concerning virus and vector gene characterization, plant-virus and vector interaction and/or epidemiological studies.

Key words: PYVV, RNA, whitefly, detection, vector.

DETECCIÓN MOLECULAR DE POTATO YELLOW VEIN VIRUS EN EL VECTOR NATURAL TRIALEURODES VAPORARIORUM,  WESTWOOD (MOSCA BLANCA)

RESUMEN: El Potato yellow vein virus (PYVV) produce síntomas de amarillamiento en las nervaduras de la hoja de papa, y es transmitido por el vector natural Trialeurodes vaporariorum Westwood (moscablanca). Hasta la fecha no se ha detectado molecularmente PYVV en extractos de moscas virulíferas. Los objetivos de este estudio fueron I) amplificar el gen de la proteína mayor de la cápside (CP) de PYVV a partir de extractos del vector, utilizando RT-PCR con cebadores específicos, II) clonar los amplicones y III)secuenciar y analizar las secuencias. Se utilizaron como donadoras de virus, las plantas de papa Solanum phureja (Jus et Buk), colectadas en campo con síntomas de amarillamiento y confirmadas positivas para PYVV por RT-PCR. Se depositaron moscas aviruliferas en hojas sintomáticas cubiertas con tela de muselina, para la adquisición de PYVV durante 48 h. Para la extracción de RNA viral se empleó el kit SV total RNA Isolation System (Promega®), utilizando una mosca o grupos (2, 5, 10, 15 y 20 individuos). Los amplicones obtenidos de CP, aproximadamente 765 pb se clonaron y secuenciaron. Las secuencias obtenidas se alinearon con la herramienta BLAST; se establecieron similitudes del 99% entre el amplicón derivado del vector y secuencias del gen CP de aislados de PYVV colombianos y peruanos.

Palabras clave: PYVV, RNA, mosca blanca, detección, vector.