Sequence and phylogenetic analysis of a novel alphaendornavirus, the first virus described from the oomycete plant pathogen Phytophthora heveae.

Here, we report the discovery and complete genome sequence of a novel virus, designated as "Phytophthora heveae alphaendornavirus 1" (PhAEV1), from a single isolate of the plant pathogenic oomycete Phytophthora heveae (kingdom Stramenipila) isolated from a tropical evergreen lowland rainforest in northern Vietnam. PhAEV1 was detected by both cellulose affinity chromatography of dsRNA and high-throughput sequencing of total RNA, and its presence and sequence were confirmed by RT-PCR and Sanger sequencing. The PhAEV1 genome, 12,820 nucleotides (nt) in length, was predicted to encode a single large polyprotein with the catalytic core domain of viral (superfamily 1) RNA helicase (HEL, amino acid [aa] positions 1,287-1,531), glycosyltransferase (GT, aa positions ca. 2,800-3,125), and RNA-directed RNA polymerase (RdRp, aa positions 3,875-4,112). PhAEV1 is the most similar to Phytophthora cactorum alphaendornavirus 3, sharing 39.4% and 39.1% nt and aa sequence identity, respectively. In addition to the first 5'-terminal AUG codon, three additional in-frame methionine codons were found in close proximity (nt 14-16, 96-98, and 176-178), suggesting potential additional translation initiation sites. Conserved RdRp motifs (A-E) similar to those detected in related endornaviruses were identified in PhAEV1, as well as in several previously described alphaendornaviruses from other Phytophthora species in which these motifs had not been identified previously. Phylogenetic analysis showed that PhAEV1 clusters with members of the genus Alphaendornavirus in the family Endornaviridae and is basal to two other alphaendornaviruses described from another oomycete, Phytophthora cactorum. PhAEV1 is the first virus reported in P. heveae.

Bunyaviruses Affect Growth, Sporulation, and Elicitin Production in Phytophthora cactorum.

Phytophthora cactorum is an important oomycetous plant pathogen with numerous host plant species, including garden strawberry (Fragaria × ananassa) and silver birch (Betula pendula). P. cactorum also hosts mycoviruses, but their phenotypic effects on the host oomycete have not been studied earlier. In the present study, we tested polyethylene glycol (PEG)-induced water stress for virus curing and created an isogenic virus-free isolate for testing viral effects in pair with the original isolate. Phytophthora cactorum bunya-like viruses 1 and 2 (PcBV1 & 2) significantly reduced hyphal growth of the P. cactorum host isolate, as well as sporangia production and size. Transcriptomic and proteomic analyses revealed an increase in the production of elicitins due to bunyavirus infection. However, the presence of bunyaviruses did not seem to alter the pathogenicity of P. cactorum. Virus transmission through anastomosis was unsuccessful in vitro.

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

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.

Marine Oomycetes of the Genus Halophytophthora Harbor Viruses Related to Bunyaviruses.

We investigated the incidence of RNA viruses in a collection of Halophytophthora spp. from estuarine ecosystems in southern Portugal. The first approach to detect the presence of viruses was based on the occurrence of dsRNA, typically considered as a viral molecule in plants and fungi. Two dsRNA-banding patterns (∼7 and 9 kb) were observed in seven of 73 Halophytophthora isolates tested (9.6%). Consequently, two dsRNA-hosting isolates were chosen to perform stranded RNA sequencing for de novo virus sequence assembly. A total of eight putative novel virus species with genomic affinities to members of the order Bunyavirales were detected and their full-length RdRp gene characterized by RACE. Based on the direct partial amplification of their RdRp gene by RT-PCR multiple viral infections occur in both isolates selected. Likewise, the screening of those viruses in the whole collection of Halophytophthora isolates showed that their occurrence is limited to one single Halophytophthora species. To our knowledge, this is the first report demonstrating the presence of negative (-) ssRNA viruses in marine oomycetes.