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Genetic Variability of Papaya Ringspot Virus Isolates In Genetic variability of Papaya ringspot virus isolates in Norte de Santander - Colombia Variabilidad genética del virus de la mancha anular de la papaya en Norte de Santander - Colombia Giovanni Chaves-Bedoya1 and Luz Yineth Ortiz-Rojas1 ABSTRACT RESUMEN The Papaya ringspot virus (PRSV), a member of the potyvirus El virus de la mancha anular de la papaya (PRSV), un virus that is transmitted by aphids within the Potyviridae family, miembro de los potyvirus transmitidos por áfidos dentro de is the main limiting factor for papaya (Carica papaya L.) and la familia Potyviridae, es el principal factor limitante para los Cucurbits worldwide and causes losses of up to 100%. In this cultivos de papaya (Carica papaya L.) y cucurbitáceas en todo study, we conducted research on the genetic diversity of PRSV el mundo, llegando a ocasionar pérdidas hasta del 100% de la isolates collected from two locations in the department of producción. En este trabajo se realizó el estudio de la diversi- Norte de Santander, Colombia. The analysis was performed by dad genética de diferentes aislados de PRSV colectados en dos comparing the nucleotide sequences of the region that encode localidades del departamento de Norte de Santander, Colombia. the coat protein (CP) of nine PRSV isolates from the Villa del El análisis se realizó comparando las secuencias nucleotídicas Rosario location and 12 isolates from the Campo Hermoso loca- de la región que codifica para la proteína de la cubierta (CP) tion. The analysis included three sequences of the CP of PRSV de nueve aislados de la localidad de Villa del Rosario y 12 ais- isolates reported in the Colombian departments of Arauca and lados de la localidad de Campo Hermoso. Para comparación Valle del Cauca. The bioinformatic analysis suggested that the el análisis incluyó tres secuencias de la CP de PRSV de aislados PRSV isolates from the locations in Norte de Santander were reportados en los departamentos colombianos de Arauca y different from each other, grouping into different phylogenetic Valle del Cauca y de otras secuencias informadas en el Gen- groups. Anexistence of recombination events in the coat protein Bank. Los análisis bioinformáticos sugieren que los aislados de was observed. This is the first study of PRSV genetic variability PRSV de las localidades en Norte de Santander son distintos that has been conducted at the local level in Colombia. entre sí agrupándose en grupos filogenéticos diferentes, y se demostró la existencia de eventos de recombinación. Este es el primer estudio de variabilidad genética de PRSV a nivel local en Colombia. Key words: PRSV, coat protein, recombination, papaya, Palabras clave: PRSV, proteína de la cubierta, recombinación, sequencing. papaya, secuenciación. Introduction cultivated in land rich with organic matter and abundant moisture. The papaya was first described in 1,526 by the The papaya (Carica papaya L.) is a species whose origin is Spanish chronicler Oviedo, who found it on the coasts of registered as Tropical America, especially Central America Panama and Colombia (Cabrera et al., 2011). and the west coast of South America. Currently, it is grown in all of the tropical and subtropical areas of the world, A limiting factor for papaya yield worldwide is the dis- between 32 degrees north latitude and south of the Equa- ease caused by PRSV. Discovered in 1945 (Jensen, 1949), tor (Arango et al., 2010). It is an herbaceous plant with PRSV is the most widely distributed and harmful virus large leaves and rapid growth. Botanically, Carica papaya in papaya. Plants infected at the seedling stage or within (L.) cannot be clearly described as a tree, plant or shrub. It two months after planting do not produce normal, mature reaches a height of 10 m and an age of 15 to 30 years. The fruits. The virus is transmitted by many species of aphids size of the fruit can weigh between 100 g and 10 kg. Its in a non-persistent manner (Suzuki et al., 2007). PRSV shape is round-oval to oblong and the color of its flesh is strains are divided into two biotypes: the papaya infecting yellowish-white, deep yellow, orange or reddish yellow. It is type (PRSV-P), which affects both papaya and cucurbits, Received for publication: 10 April, 2015. Accepted for publication: 30 June, 2015. Doi: 10.15446/agron.colomb.v33n2.50095 1 Plantae Research Group, Faculty of Basic Sciences, Universidad Francisco de Paula Santander. Cucuta (Colombia). [email protected] Agronomía Colombiana 33(2), 184-193, 2015 and the cucurbit infecting type (PRSV-W) from Taiwan, diversity of crops. The papaya crop occupies the ninth which affects cucurbits but not papaya (Gonsalves et al., position of importance for fruit crops (Tafur et al., 2006). 2010). The virions of both biotypes cannot be distinguished in serological tests, but differ in their ability to infect pa- Given that there is not any information on PRSV in the paya (Bateson et al., 2002). PRSV-P infects papaya (Carica department of Norte de Santander, Colombia, this study papaya L.) naturally and is the main limiting factor in the was conducted for a preliminary determination of their production of papaya worldwide. PRSV-P was described for genetic variability, based on the sequence of the CP gene, the first time in Hawaii and, since then, has been reported using sequences of 21 PRSV isolates collected from symp- in several countries where it usually causes devastation tomatic papaya plantsin two different locations in the de- to the production of papaya in the early years of the first partment. Phylogenetic relationships between these isolates infection (Gonsalves, 1998). The epidemiology of PRSV-P and PRSV isolates reported in other parts of the world in papaya is similar to that of other non-persistent viruses were also evaluated. Studies on the molecular evolution and it is transmitted directly from papaya to papaya (Bate- of viruses are essential for understanding their biological son et al., 2002). characteristics, changes in virulence, geographical ranges and their emergence as new epidemics. This understand- PRSV has flexible, 760-780 x 12 nm filamentous, flexuous ing is crucial for the design of strategies for viral control rod particles and consists of a monopartite single-stranded (Moreno et al., 2004; Tan et al., 2004). positive sense RNA, about 10,326 nucleotides long, exclud- ing a poly-A tract found at its 3’end. PRSV encodes a single Materials and methods large protein which is subsequently cleaved into smaller proteins with several functions (Gonsalves et al., 2010). Field sampling. Symptomatic leaves of PRSV-infected pa- The central region of PRSV is highly conserved, while the paya plants were collected in the months of March and Sep- N-terminal region is more variable (Abdalla and Ali, 2012). tember of 2014 from different plots in the Campo Hermoso The symptoms on papaya are somewhat similar to those (12 isolates) and Villa del Rosario (nine isolates) locations, on cucurbits. In papaya, the leaves develop prominent for a total of 21 isolates. The harvested leaves were kept in mosaic and chlorosis on the leaf lamina and water-soaked, polystyrene coolers with ice to keep them fresh, taken to oily streaks on the petioles and upper part of the trunk the Plantae Research Group Laboratory at the Universidad (Gonsalves et al., 2010). Francisco de Paula Santander, and stored at -80°C until the extraction of total RNA from each sample. Genetic engineering (Kung et al., 2009; Mangrauthia et al., 2010; Yu et al., 2011), and cross protection (Gonsalves et RNA extraction and RT-PCR. The papaya leaves were al., 2010) are options for managing viral diseases such as used for total RNA extraction using the reagent TRIZOL PRSV. Knowledge of the nucleotide sequence and genetic (Invitrogen, Carlsbad, CA), according to the manufac- diversity is necessary to be able to create control strategies. turer’s instructions. The RNA extracted from each plant There have been different studies on the genetic diversity of was quantified in a spectrophotometer, Genesys 10S PRSV, particularly variant P, in different countries around UV-VIS (Thermo Scientific, Waltham, MA), reading at the world, including Australia (Bateson et al., 1994), Brazil a wavelength of 260 nm and 280 nm. The total RNA was (Lima et al., 2002), India (Jain et al., 2004), Jamaica and quantified and preserved at -80ºC in aliquots until use Venezuela (Chin et al., 2007), Mexico (Noa-Carrazana as a template for amplification by RT-PCR. The primers et al., 2007), Taiwan (Wang and Yeh, 1997), Thailand, for the RT-PCR reactions were designed based on the Vietnam and Philippines (Bateson et al., 2002), Venezuela consensus sequence of conserved regions reported in (Fernández-Rodríguez et al., 2008) and the USA (Abdalla the GenBank (Benson et al., 2014). The annealing sites and Ali, 2012). In Colombia, at the sequence level, data on were the 3’ region of the nuclear inclusion B (NIB) pro- PRSV are scarce, with the report of a few sequences cor- tein and the 5’ region of the untranslated region (3UTR). responding to the helper component proteinase (HC-Pro) The sequences of the primers were: forward 5’-GT- and the coat protein (CP) obtained in the departments of CATGGGGATATGGGGAGTTAACACA-3’ and reverse Valle del Cauca (four sequences) and Arauca (one sequence) 5’-TTTTTTTTCTCTCATTCTAAGAGGCTCG-3’. (Olarte Castillo et al., 2011). cDNA synthesis and cloning. Samples of the total RNA Norte de Santander is potentially an agricultural depart- were used as a template to make the RT-PCR reactions to ment due to its geographical conditions, presenting a amplify the CP of the PRSV. For the reverse transcription, Chaves-Bedoya and Ortiz-Rojas: Genetic variability of Papaya ringspot virus isolates in Norte de Santander - Colombia 185 we used a Tetro cDNA Synthesis Kit (Bioline, Tauton, MA).
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