A novel weevil-transmitted tymovirus found in mixed infection on hollyhock

Abstract

Leaves of hollyhock (Alcea rosea) exhibiting vein chlorosis and yellow mosaic symptoms were collected at public sites in Lausanne and Nyon, two cities of western Switzerland. Diagnostic methods untangled in samples from both sites the mixed infections of a novel isometric virus, tentatively named "Alcea yellow mosaic virus" (AYMV) with the carlavirus Gaillardia latent virus. A new potyvirus was also identified in samples from Nyon. A combination of Illumina, Nanopore and Sanger sequencing was necessary to assemble the full-length genome of AYMV, revealing an exceptionally high cytidine content and other features typically associated with members of the genus Tymovirus. The host range of AYMV was found to be restricted to mallows, including ornamentals as well as economically important plants. Phylogenetic analyses further showed that AYMV belongs to a Tymovirus subclade that also gathers the other mallow-infecting members. The virus was readily transmitted by sap inoculation, and the weevil species Aspidapion radiolus was evidenced as a vector. Transmission assays using another weevil or other insect species did not succeed, and seed transmission was not observed.

Keywords: Carlavirus; Hollyhock; Mixed infection; Potyvirus; Tymoviridae; Tymovirus; Weevil.

Fig. 1

Identification of the novel tymovirus AYMV. a Hollyhock leaves collected in Lausanne showing vein clearing, chlorosis and mosaic symptoms. b Transmission electron micrograph of semi-purified virions negatively stained with 4% phosphotungstate. Note the staining penetration in empty particles. c Electrophoresis gel for RNA (left) and protein (right) associated with semi-purified particles. d Electrophoresis gel for the dsRNA extracted from symptomatic hollyhock leaves. e Healthy leaf of M. sylvestris (left) in comparison with an AYMV-infected leaf showing chlorotic spots (right), at 30 dpi

Fig. 2

Characterization of AYMV genome. a Base content for the gRNA of tymoviruses (box plots) and AYMV (black points). b Schematic representation of AYMV gRNA. ORFs are represented as colored boxes with associated positions and conserved domains/functions (see main text for details). The putative RP cleavage sites are indicated by gray arrows. The protonable hairpin (PH), tymobox and valine tRNA-like structure (^ValTLS) are indicated by vertical gray lines. c Alignment of the tymobox cDNA for AYMV and selected related species. d Alignment of the 3’-UTR cDNA for the same viruses described in Fig. 2c. The structural annotations are reproduced from a previous analysis of tymoviral tRNA (52). Colored background indicate ≥ 75% conservation. Full virus names and accession numbers are listed in Additional file 1: Table S1

Fig. 3

ML phylogenetic tree of the tymoviral proteins. a CP tree (model: rtREV + F + I + G4). b RP tree (model: rtREV + F + I + G4). c MP tree (model cpREV + F + I + G4). Full virus names and accession numbers are listed in Additional file 1: Table S1. The position of AYMV is highlighted by a black arrow. Selected members of the genera Marafivirus and Maculavirus were used to root the initial CP and RP trees. Black circles on branches indicate bootstrap support > 70%. The tree scales are given in substitution per site. Colored background are indicative of the family of the natural host

Fig. 4

Insect transmission assays. a Specimens of Coleoptera found on hollyhock and used for transmissions assay. Up: R. longirostre, middle: A. radiolus and low: P. fuscicornis. The bar represents 1 cm. b Transmission efficiencies for the different insect species. Pairs of insects were used for each transmission assay