Characterization of the partial RNA1 and RNA2 3′ untranslated region of Tomato ringspot virus isolates from North America

Abstract

The 3 untranslated regions (UTRs) of RNA1 and RNA2 of Tomato ringspot virus (ToRSV) are 1.3 kb in size and virtually identical. In this study, sequences containing most of the 3 UTRs (1168-1265 bp) were determined from 18 ToRSV isolates collected from fruit trees, small fruits and grapevines in North America. Pairwise nucleotide sequence comparisons with sequences of two isolates available in GenBank showed identities ranging from 95.1 to 100% for the majority of ToRSV isolates. Most genetic variation occurs as point mutations and single insertions and/or deletions. Phylogenetic analysis showed that 17 of the 20 isolates grouped together into a major cluster and three isolates grouped into two minor clusters, with genetic divergence rates among the three clusters of 11.8% to 23.8%. No correlation was found between the 3 UTR sequences and symptom expression on Nicotiana benthamiana, host plant or geographic origin of ToRSV isolates. RT-PCR using primers designed within the highly conserved 3 UTR regions detected all 18 ToRSV isolates as well as two isolates from a vineyard. This assay can serve as a practical methodology for the sensitive detection of varied ToRSV isolates as it is more sensitive than a RT-PCR assay based on previously reported U1/D1 primers. Résumé:Les séquences non traduites (3 UTR) de l'ARN1 et de l'ARN2 du virus des taches annulaires de la tomate (ToRSV) mesurent 1,3 kb et sont essentiellement identiques. Dans cette étude, les séquences contenant la majorité des 3 UTR (1168-1265 bp) ont été déterminées á partir de 18 isolats de ToRSV collectés en Amérique du Nord sur des arbres fruitiers, des petits fruits et des vignes. Les comparaisons binaires de séquences de nucléotides avec les séquences de deux isolats de la GenBank ont montré des similarités variant de 95.1% á 100% en ce qui a trait á la majorité des isolats de ToRSV. La plupart des variations génétiques se produisent en tant que mutations ponctuelles et qu'insertions/délétions uniques. L'analyse phylogénétique a montré que 17 des 20 isolats étaient groupés en super-famille et que trois isolats l'étaient en deux familles mineures, dont les taux de divergence génétique, chez les trois familles, variaient de 11.8% á 23.8%. Nous n'avons trouvé aucune corrélation entre les séquences 3 UTR et l'expression des symptômes sur Nicotiana benthamiana, plante hôte ou origine géographique des isolats de ToRSV. Des amorces RT-PCR conçues dans les régions 3 UTR hautement conservées ont permis de détecter les 18 isolats de ToRSV ainsi que deux isolats provenant d'un vignoble. Ce test peut être utilisé comme méthode pratique pour la détection précise d'isolats de ToRSV puisqu'il est plus sensible qu'un test RT-PCR basé sur les amorces U1/D1 utilisées précédemment.

An Investigation of Apple Chlorotic Leaf Spot (ACLSV) and Tomato Ring Spot (ToRSV) in Some Iranian Pear Gardens

“…Inoculation assays of different ToRSV isolates collected from various sources, showed four biological groups using N. benthamian (17). Inoculation of Iranian ToRSV-B2 isolate on N. bethamiana induced necrotic local lesions on the inoculated leaves fallowed by mottle and mosaic, and it seems that ToRSV-B2 has different biological reaction from those reported by Li et al (17). Four weeks post inoculation all symptomatic and asymptomatic plants were assayed by ELISA (data not shown).…”

“…The herbaceous host range of ACLSV and ToRSV isolates from pear were similar to those previously reported for corresponding isolates from other crops (15,16). Inoculation assays of different ToRSV isolates collected from various sources, showed four biological groups using N. benthamian (17). Inoculation of Iranian ToRSV-B2 isolate on N. bethamiana induced necrotic local lesions on the inoculated leaves fallowed by mottle and mosaic, and it seems that ToRSV-B2 has different biological reaction from those reported by Li et al (17).…”

“…ToRSV infects a broad range of herbaceous and perennial plants, and causes serious diseases like peach stem pitting and yellow bud mosaic, prune brown line, and grapevine decline. Multiple sequence alignment indicated a high variability in coat protein of ToRSV isolates; however, the 3'UTR region is highly conserved (17). In addition, phylogenetic analysis using 3'UTR sequences showed two main groups that most of the isolates except two of them (PV-100 and RIB9001 from grapevine and redcurrant, respectively) clustered in group I.…”

Complete genome sequence of three tomato ringspot virus isolates: evidence for reassortment and recombination

“…1A). The Rasp1-2014 RNA2 3 0 UTR branched with the majority of the ToRSV isolates, but the Rasp1-2014 RNA1 3 0 UTR grouped separately with two isolates from grapevine (PV-100) and from red currant (RIB9001) [10]. Similarly, the Rasp1-2014 RNA2 5 0 UTR branched with the reference raspberry and 13C280 5 0 UTRs, while the Rasp1-2014 RNA1 5 0 UTR separated in a distinct branch (Fig.…”

“…Available 3 0 UTR sequences from other isolates were included, although these sequences were obtained using internal primers within the 3 0 UTRs and were not assigned to either RNA1 or RNA2 [10]. While the 3 0 UTRs of RNA1 and of RNA2 branched together for the 13C280, GYV-2014 and reference raspberry isolates, this was not the case for the Rasp1-2014 isolate (Fig.…”