Viruses Infecting Greenhood Orchids (Pterostylidinae) in Eastern Australia

Abstract

The Australasian biogeographic realm is a major centre of diversity for orchids, with every subfamily of the Orchidaceae represented and high levels of endemism at the species rank. It is hypothesised that there is a commensurate diversity of viruses infecting this group of plants. In this study, we have utilised high-throughput sequencing to survey for viruses infecting greenhood orchids (Pterostylidinae) in New South Wales and the Australian Capital Territory. The main aim of this study was to characterise Pterostylis blotch virus (PtBV), a previously reported but uncharacterised virus that had been tentatively classified in the genus Orthotospovirus. This classification was confirmed by genome sequencing, and phylogenetic analyses suggested that PtBV is representative of a new species that is possibly indigenous to Australia as it does not belong to either the American or Eurasian clades of orthotospoviruses. Apart from PtBV, putative new viruses in the genera Alphaendornavirus, Amalgavirus, Polerovirus and Totivirus were discovered, and complete genome sequences were obtained for each virus. It is concluded that the polerovirus is likely an example of an introduced virus infecting a native plant species in its natural habitat, as this virus is probably vectored by an aphid, and Australia has a depauperate native aphid fauna that does not include any species that are host-adapted to orchids.

Keywords: Solemoviridae; Tospoviridae; cryptic virus; disease; dsRNA virus; mycovirus; native vegetation; ssRNA virus; surveillance; thrips; virus discovery.

Figure 1

Pterostylis nutans infected with Pterostylis blotch virus (yellow frame) and healthy P. nutans (red frame), on Black Mountain, the Australian Capital Territory, in 2020.

Figure 2

Genome architectures of the viruses identified in this study. Abbreviations are as follows: PtAEV, Pterostylis alphaendornavirus; PtAV1, Pterostylis amalgavirus 1; PtAV2, Pterostylis amalgavirus 2; PtTV, Pterostylis totivirus; PtPV, Pterostylis polerovirus; RdRp, RNA-dependent RNA polymerase; NSm, non-structural protein, M segment; NSs, non-structural protein, S segment; N, nucleocapsid protein; CP, capsid protein; nt, nucleotide; aa, amino acid.

Figure 3

Maximum likelihood reconstruction of the phylogeny of orthotospoviruses based on concatenated sequence alignments of five genes (RNA-dependent RNA polymerase, non-structural protein M segment, glycoprotein precursor, non-structural protein S segment and nucleocapsid protein). The acronyms in red font correspond to the viruses discovered in this study (Table 1) and the other virus acronyms are defined in Table S3. The numbers to the left of the slash next to the branches are non-parametric bootstrap values based on 1000 replicates; the numbers to the right of the slash are the number of decisive gene trees (5 at maximum) supporting the branch. The nucleotide substitution models used are listed in Table S9.

Figure 4

Maximum likelihood reconstruction of the phylogeny of alphaendornaviruses based on the RNA-dependent RNA polymerase gene. The acronym in red font corresponds to the virus discovered in this study (Table 1) and the other virus acronyms are defined in Table S4. The numbers next to the branches are non-parametric bootstrap values based on 1000 replicates. The nucleotide substitution model used for the phylogenetic tree is listed in Table S9.

Figure 5

Maximum likelihood reconstruction of the phylogeny of amalgaviruses based on the RNA dependent RNA polymerase gene. The acronyms in red font correspond to the viruses discovered in this study (Table 1) and the other virus acronyms are defined in Table S5. The numbers next to the branches are non-parametric bootstrap values based on 1000 replicates. The nucleotide substitution model used for the phylogenetic tree is listed in Table S9.

Figure 6

Maximum likelihood reconstruction of the phylogeny of totiviruses based on the RNA-dependent RNA polymerase gene. The acronyms in red font correspond to the viruses s discovered in this study (Table 1) and the other virus acronyms are defined in Table S6. The numbers next to the branches are non-parametric bootstrap values based on 1000 replicates. The nucleotide substitution model used for the phylogenetic tree is listed in Table S9.

Figure 7

Maximum likelihood reconstruction of the phylogeny of poleroviruses based on concatenated codon sequence alignments of the conserved regions of four genes (serine proteinase, RNA-dependent RNA polymerase, capsid protein (P3) and the domain at the C-terminus of P3 in P3–P5 readthrough protein). The acronym in red font corresponds to the virus isolate discovered in this study (Table 1) and the other virus acronyms are defined in Table S7. The numbers to the left of the slash next to the branches are non-parametric bootstrap values based on 1000 replicates; the numbers to the right of the slash are the number of decisive gene trees (4 at maximum) supporting the branch. The nucleotide substitution models are listed in Table S9.