High Diversity of Novel Viruses in the Tree Pathogen Phytophthora castaneae Revealed by High-Throughput Sequencing of Total and Small RNA

Abstract

Phytophthora castaneae, an oomycete pathogen causing root and trunk rot of different tree species in Asia, was shown to harbor a rich diversity of novel viruses from different families. Four P. castaneae isolates collected from Chamaecyparis hodginsii in a semi-natural montane forest site in Vietnam were investigated for viral presence by traditional and next-generation sequencing (NGS) techniques, i.e., double-stranded RNA (dsRNA) extraction and high-throughput sequencing (HTS) of small RNAs (sRNAs) and total RNA. Genome organization, sequence similarity, and phylogenetic analyses indicated that the viruses were related to members of the order Bunyavirales and families Endornaviridae, Megabirnaviridae, Narnaviridae, Totiviridae, and the proposed family "Fusagraviridae." The study describes six novel viruses: Phytophthora castaneae RNA virus 1-5 (PcaRV1-5) and Phytophthora castaneae negative-stranded RNA virus 1 (PcaNSRV1). All six viruses were detected by sRNA sequencing, which demonstrates an active RNA interference (RNAi) system targeting viruses in P. castaneae. To our knowledge, this is the first report of viruses in P. castaneae and the whole Phytophthora major Clade 5, as well as of the activity of an RNAi mechanism targeting viral genomes among Clade 5 species. PcaRV1 is the first megabirnavirus described in oomycetes and the genus Phytophthora.

Keywords: RNA interference; RdRp; dsRNA; forest pathogen; multiple viral infections; mycovirus; oomycetes; ssRNA.

Figure 1

sRNA size distribution of raw VN999 ,VN1004 , VN1008 , and VN1012 reads, respectively, after mapping to the final genomes of viruses PcaNSRV1 and PcaRV1-5, respectively. Mapping was done in Geneious Prime^® 2020.2.3 using Bowtie mapper. The final graphs were constructed in R Core Team (2020). Sizes of sRNA profiles starting from 28 nt that were represented with less than 1.5% of total reads per virus were omitted during graph preparation for a better visualization.

Figure 2

Genome organization of viruses PcaRV1-4 and PcaNSRV1. Genome sequence of PcaRV5 is not graphically represented as it is partial and requires further characterization.

Figure 3

Phylogenetic RAxML tree including the three dsRNA viruses described in this study, PcaRV1 (megabirna-like virus), PcaRV3 (toti-like virus), and PcaRV4 (fusagra-like virus; all three indicated by a yellow star ), and related members belonging to the families Megabirnaviridae, Toriviridae proposed family “Fusagraviridae.” The viruses described from oomycetes are indicated by a green hexagone . Nodes are labeled with bootstrap percentages ≥50% only. Family classification and the corresponding pBLAST accession numbers are shown next to the virus names. The tree is rooted in the midpoint. Branch lengths are scaled to the expected underlying number of amino acid substitutions per site. The scale bar is indicatig 2.0 aa substitutions per site, per branch.

Figure 4

Maximum likelihood tree (RAxML) showing the phylogenetic relationships of PcaRV2 (indicated by the yellow star ) with other (+)ssRNA viruses belonging to families Narnaviridae, Mitoviridae, Leiviviridae, and Botourmiaviridae. The viruses described from oomycetes are indicated by a green hexagone . Nodes are labeled with bootstrap percentages ≥50%. Branch lengths are scaled to the expected underlying number of amino acid substitutions per site. Family classification and the corresponding pBLAST accession numbers are shown next to the virus names. The tree is rooted in the family Leviviridae.

Figure 5

Maximum likelihood tree (RAxML) depicting the phylogenetic relationship of the predicted RdRp of PcaNSRV1 (indicated by the yellow star ) with other complete RdRp belonging to related (−)ssRNA viruses from the orders Bunyavirales and Mononegavirales. The viruses described from oomycetes are indicated by a green hexagone . Nodes are labeled with bootstrap support values. Branch lengths present calculated evolutionary distannce and are scaled to the expected underlying number of amino acid substitutions per site. Nodes are marked with bootstrap percentages ≥50% only. The tree is rooted in Mononegaviales viruses, which are classified within the family Mymonaviridae. Family classification and the corresponding pBLAST accession numbers are shown next to the virus names. The scale bar is indicatig 0.7 aa substitutions per site, per branch.