A Metagenomic Investigation of the Viruses Associated with Shiraz Disease in Australia

Abstract

Shiraz disease (SD) is an economically important virus-associated disease that can significantly reduce yield in sensitive grapevine varieties and has so far only been reported in South Africa and Australia. In this study, RT-PCR and metagenomic high-throughput sequencing was used to study the virome of symptomatic and asymptomatic grapevines within vineyards affected by SD and located in South Australia. Results showed that grapevine virus A (GVA) phylogroup II variants were strongly associated with SD symptoms in Shiraz grapevines that also had mixed infections of viruses including combinations of grapevine leafroll-associated virus 3 (GLRaV-3) and grapevine leafroll-associated virus 4 strains 5, 6 and 9 (GLRaV-4/5, GLRaV-4/6, GLRaV-4/9). GVA phylogroup III variants, on the other hand, were present in both symptomatic and asymptomatic grapevines, suggesting no or decreased virulence of these strains. Similarly, only GVA phylogroup I variants were found in heritage Shiraz grapevines affected by mild leafroll disease, along with GLRaV-1, suggesting this phylogroup may not be associated with SD.

Keywords: Australia; Shiraz disease; dsRNA; grapevine leafroll-associated virus 3; grapevine leafroll-associated virus 4; grapevine virus A; metagenomic sequencing; phylogenetic groups.

Figure 1

Symptoms of grapevine virus A (GVA)-infected Shiraz from the Willunga, Langhorne Creek and Barossa Valley sites. Symptoms of Shiraz-disease-affected Shiraz grapevines at (a) Willunga and (b) Langhorne Creek, and infected with GVA^II variants (c) mild grapevine leafroll disease symptoms on an unknown clone of Shiraz that tested positive to GVA^I variant and grapevine leafroll-associated virus 1 at Barossa Valley. (d) An asymptomatic Shiraz, clone BVRC12 at Willunga.

Figure 2

Neighbour-joining phylogenetic analysis of grapevine virus A (GVA) using 35 metagenomic sequences and all available sequences from GenBank. A neighbour-joining tree using (a) complete genome sequences alignment above 6991 nts, (GVA isolates associated with SD-affected grapevines are labelled with red triangles and isolates associated with asymptomatic grapevines are labelled with green rhombus), (b) full-length nucleotide (nt) sequences of RNA-dependent RNA polymerase (RdRp), (c) full-length amino acid (aa) sequences of RdRp, (d) nt sequences of complete coat protein (CP) gene, (e) aa sequence of CP gene, (f) nt sequence of complete movement protein (MP) gene, (g) nt sequences of RNA-binding (RB) gene. All phylogenetic trees were constructed using MEGA (7.0.26) software and the neighbour-joining method with 1000 bootstrap replicates. Bootstrap values below 50% were not shown. Australian sequences generated by a previous study by Meta-HTS [1] are labelled using open circles and sequences generated by NovaSeq are labelled by black dots. Isolates for which the phylogroup assigned differed depending on the gene used is marked by * in figure (g). The red squares indicate the primary branches which show ≥ 99% bootstrap values. The colour-coded I, II and III labelled in figure (g) is based on the phylogroup assigned using the CP gene, for comparison. Grapevine virus F (GVF) isolate AUD46129 (accession no. NC018458) was used as an outgroup.

Figure 3

Amino acid alignment of the N-terminal (90–94 aa) of the RNA-binding gene of grapevine virus A (GVA) of Australian and international isolates. The phylogenetic group (I, II or III) of each isolate is on the left. Red * indicates isolates associated with Shiraz disease (SD) and green # indicates isolates not associated with SD. The blue question mark indicates a SD-negative isolate in phylogenetic group II [11]. The two black arrows point to positions 31 and 61 of the alignment.

Figure 4

Phylogenetic analysis of 21 near-complete genome sequences (above 17,027 nts) of Australian grapevine leafroll-associated virus 3 (GLRaV-3) isolates and 77 publicly available full genome GLRaV-3 sequences from GenBank. A neighbour-joining tree of (a) complete genome sequences, (b) nucleotide sequence of the coat protein gene, was constructed with 1000 bootstrap replicates by MEGA software. Black dots denote sequences generated by Meta-HTS of this study. The bootstrap values below 50% are not shown. Sequences generated by this study are labelled by black dots. Roman numerals I–V represent the five distinct GLRaV-3 phylogroups observed in this study.

Figure 5

Neighbour-joining phylogenetic trees of grapevine leafroll-associated virus 4 (GLRaV-4) detected by metagenomic high-throughput sequencing and isolates from the GenBank database. Phylogenetic trees constructed by alignments of (a) complete genome sequences (above 12600 nts), (b) amino acid (aa) sequences of the RNA-dependent RNA polymerase (RdRp), (c) aa sequences of the heat shock protein 70 homologue (HSP70h) and (d) aa sequences of the coat protein (CP) gene. Bootstrap values less than 50% are not shown. * Indicates GLRaV-4 isolates with similarities to the exemplar isolate LR106 (accession no. FJ467503) below the demarcation of this species. Open circles indicate sequences generated by a previous study [1] and black dots display the sequences by this study.