Genetic diversity and a heterogeneous population of Citrus mosaic virus within a single citrus tree

Abstract

Plural variants of Citrus mosaic virus (CiMV), a Satsuma dwarf virus (SDV) strain, were detected from a single citrus tree. Reverse transcription-polymerase chain reaction amplifi cation of 1139 nucleotides (nt) of 3′-terminal RNA1 and analyses of nucleotide sequences revealed three CiMV variants that differed by up to 5.4%. The analyses of 1403-1493 nt of 3′-terminal RNA2 showed four CiMV variants, two of which differed in the presence of an 89-nt insert related to a 29-to 30-nt repeat in the 3′-noncoding regions. This is the fi rst report to show multiple infections by SDV variants that can be generated by genetic mutations.

A broad spectrum, one-step RT-PCR to detect Satsuma dwarf virus variants using universal primers targeting both segmented RNAs 1 and 2

“…Except for reactions using SDV-CP/SDV-CM primers, that did not detect HV, reactions with the other primer sets could detect all the SDV strains (Ito et al 2007;Iwanami 2010). However, they might not detect all SDV variants because the primers target the region for which fewer nucleotide sequences have been reported.…”

“…SDV RNAs 1 or 2 (Ito et al 2007;Iwanami 2010). Except for reactions using SDV-CP/SDV-CM primers, that did not detect HV, reactions with the other primer sets could detect all the SDV strains (Ito et al 2007;Iwanami 2010).…”

“…GenBank accessions for the reported sequences of SDV isolates are given after the colon except for undeposited sequence of Ci-968 RNA 1 (Iwanami and Ieki 1996). Primers reported by Ito et al (2007) and Iwanami (2010) are underlined. In primers,…”

“…1. SRNA1P/ SRNA1M primers (Ito et al 2007) and FW146/RV488 primers (Iwanami 2010) were designed to target RdRp to 3 0 -NCR of RNA 1 and 5 0 -conserved terminal region of RNA 2 of SDV, respectively. Of these primers, primer SRNA1M could anneal to both RNAs 1 and 2 (Fig.…”

“…Second, RNA extracted using Isogen (Nippon Gene, Tokyo, Japan) from new leaves of 28 laboratory-collection citrus trees infected with variants of four SDV strains was used for the one-step RT-PCR detection. SDV strains in the samples were identified in advance by partial nucleotide sequence analysis of fragments amplified in a two-step RT-PCR using SRNA1P/SRNA1M primers or SDV-CP/SDV-CM primers following the methods described by Ito et al (2007), except for those already reported and listed in Fig. 1.…”