Complete genome sequence and characterisation of a novel flexivirus infecting the necrotrophic conifer pathogen Diplodia sapinea

Abstract

Using total RNA sequencing, we detected a novel RNA virus infecting a Finnish isolate of the fungal conifer pathogen Diplodia sapinea, and its genome sequence was verified by Sanger sequencing. This virus, which we have named "Diplodia sapinea flexi-like virus 1" (DsFLV1), has a 7,494-nucleotide-long positive-sense single-stranded RNA genome, excluding the poly(A) tail. The largest open reading frame (ORF1) encodes a replication-associated polyprotein (RP) that contains conserved domains characteristic of members of the order Tymovirales, including viral methyltransferase (Mtr), viral helicase (Hel), and RNA-dependent RNA polymerase (RdRP) domains. Four smaller ORFs were also identified. ORF2 potentially encodes a capsid protein, while the other three ORFs, initiated by alternative start codons, encode putative proteins with unknown functions. Phylogenetic analysis placed DsFLV1 in a distinct clade within the family Deltaflexiviridae, close to Erysiphe necator-associated flexivirus 2. This is the first report of a positive-sense single-stranded RNA virus infecting D. sapinea.

Fig. 1

(A) Schematic representation of the genome of Diplodia sapinea flexi-like virus 1 (DsFLV1). The RP ORF is shown in green, and the smaller putative ORFs are shown in blue. Yellow rectangular boxes indicate conserved domains (Mtr, viral methyltransferase; Hel, viral RNA helicase; RdRP, catalytic core domain of RNA-dependent RNA polymerase conserved in + ssRNA viruses and closely related viruses). (B) Symptomatic Scots pine shoot with Diplodia tip blight from which D. sapinea was isolated. (C) D. sapinea strain 138 after cultivation at 20°C for 14 days. (D) ML (RAxML) phylogenetic tree based on amino acid sequences of the DsFLV1 RP (in red with an asterisk) and those of other members of the order Tymovirales (Supplementary Table S3). Bootstrap values ≥ 50% are shown at the nodes. Branch lengths represent the number of aa substitutions per site. Scale bar, 2.0 substitutions per site per branch. The tree is rooted at the midpoint