Natural Populations from the Phytophthora palustris Complex Show a High Diversity and Abundance of ssRNA and dsRNA Viruses

Abstract

We explored the virome of the "Phytophthora palustris complex", a group of aquatic specialists geographically limited to Southeast and East Asia, the native origin of many destructive invasive forest Phytophthora spp. Based on high-throughput sequencing (RNAseq) of 112 isolates of "P. palustris" collected from rivers, mangroves, and ponds, and natural forests in subtropical and tropical areas in Indonesia, Taiwan, and Japan, 52 putative viruses were identified, which, to varying degrees, were phylogenetically related to the families Botybirnaviridae, Narnaviridae, Tombusviridae, and Totiviridae, and the order Bunyavirales. The prevalence of all viruses in their hosts was investigated and confirmed by RT-PCR. The rich virus composition, high abundance, and distribution discovered in our study indicate that viruses are naturally infecting taxa from the "P. palustris complex" in their natural niche, and that they are predominant members of the host cellular environment. Certain Indonesian localities are the viruses' hotspots and particular "P. palustris" isolates show complex multiviral infections. This study defines the first bi-segmented bunya-like virus together with the first tombus-like and botybirna-like viruses in the genus Phytophthora and provides insights into the spread and evolution of RNA viruses in the natural populations of an oomycete species.

Keywords: Phytophthora; RNA-sequencing; multiple viral infections; mycovirus; natural habitat; oomycetes; virus ecology; virus evolution; virus reservoirs.

Figure 8

Plot representing the virus occurrence and diversity for each “P. palustris” isolate.

Figure 9

Plot representing the virus occurrence and diversity for each sampling site.

Figure 1

(a). Graphical representation of the L segment (RNA 1) and S segment (RNA 2) of PpaBLV11. (b). Alignment of 5′ and 3′ terminal sequences of PpaBLV11 RNA1 and RNA 2 with RNA1 or L segment sequences from the following viruses: Rift Valley fever virus (RVFV, DDBJ/EMBL/GenBank accession number: X56464), tulip streak virus (TuSV, LC571987), Uukuniemi virus (UUKV, D10759), Gouleako virus (GOLV, HQ541738), Lentinula edodes negative-strand RNA virus 2-HG3 (LeNSRV2, LC466007). Asterisks indicate that the nucleotides are 100% identical in all the viruses. (c). Complementary structure between the 3′ and 5′ termini in the putative PpaBLV11 genome.

Figure 2

(a) Maximum likelihood tree (RAxML) depicting the phylogenic relationship of the predicted RdRP of Phytophthora palustris bunya-like viruses with other complete RdRP belonging to related viruses from the order Bunyavirales. (b) RAxML tree with the NC of PpaBLV11 and other bunyaviruses of the family Phenuiviridae. Nodes are labeled with bootstrap support values ≥50%. Branch lengths are scaled to the expected underlying number of amino acid substitutions per site. Phytophthora palustris bunya-like viruses 1–13 (and variants) are represented by their abbreviated names (PpaBLV1-13) and indicated with a red asterisk (*). Family classification and the corresponding GenBank accession numbers are shown next to the virus names. Colorful squares represent the virus host kingdom or phylum: Fungi, Nematoda, Oomycota, Arthropoda, Plants, Mammalia, (Fishes) Chordata, Ochrophyta (Heterokonta), Excavata. Scale bars represent expected changes per site per branch.

Figure 2

(a) Maximum likelihood tree (RAxML) depicting the phylogenic relationship of the predicted RdRP of Phytophthora palustris bunya-like viruses with other complete RdRP belonging to related viruses from the order Bunyavirales. (b) RAxML tree with the NC of PpaBLV11 and other bunyaviruses of the family Phenuiviridae. Nodes are labeled with bootstrap support values ≥50%. Branch lengths are scaled to the expected underlying number of amino acid substitutions per site. Phytophthora palustris bunya-like viruses 1–13 (and variants) are represented by their abbreviated names (PpaBLV1-13) and indicated with a red asterisk (*). Family classification and the corresponding GenBank accession numbers are shown next to the virus names. Colorful squares represent the virus host kingdom or phylum: Fungi, Nematoda, Oomycota, Arthropoda, Plants, Mammalia, (Fishes) Chordata, Ochrophyta (Heterokonta), Excavata. Scale bars represent expected changes per site per branch.

Figure 3

(a). Graphical representation of the tombus-like virus contig (PpaTbLV1). (b). Phylogenetic analysis (RAxML) based on the predicted RdRP of Phytophthora palustris tombus-like virus 1, abbreviated and indicated by a red asterisk (*), with other complete unclassified tombus-like viruses and members of the family Tombusviridae. Nodes are labeled with bootstrap support values ≥50%. Branch lengths are scaled to the expected underlying number of amino acid substitutions per site. Tree is rooted in the midpoint. Family classification and the corresponding pBLAST accession numbers are shown next to the virus names. Colorful squares represent the virus host kingdom or phylum: Fungi, Oomycota, Arthropoda, Animalia, Plants. Scale bar = 0.4 expected changes per site per branch.

Figure 4

(a). Graphical representation of a narna-like (PpaNLV7) virus contig. (b). Phylogenetic analysis (RAxML) based on the predicted RdRP of Phytophthora palustris (+)ssRNA viruses with other complete classified and unclassified members of the families Mitoviridae, Narnaviridae, Botourmiaviridae, Leviviridae. Nodes are labeled with bootstrap support values ≥50%. Branch lengths are scaled to the expected underlying number of amino acid substitutions per site. Tree is rooted in the midpoint. Phytophthora palustris narna-like viruses are abbreviated PpaNLV1-8, and indicated with a red asterisk (*). Family classification and the corresponding pBLAST accession numbers are shown next to the virus names. Colorful squares represent the virus host kingdom or phylum: Fungi, Oomycota, Arthropoda, Animalia, Plants, Ochrophyta (Heterokonta), Apicomplexa (Protists), Bacteria. Scale bar = 0.6 expected changes per site per branch.

Figure 5

(a). Graphical representation of the genomes of one botybirna-like virus (PpaBbLV2) and one toti-like virus (PpaTLV1-1). (b). Phylogenetic analysis (RAxML) based on the predicted RdRP of the dsRNA viruses discovered in Phytophthora palustris with other classified and unclassified members of the families Totiviridae and Botybirnaviridae. Nodes are labeled with bootstrap support values ≥50%. Branch lengths are scaled to the expected underlying number of amino acid substitutions per site. Tree is unrooted and branches are shown in decreasing order. Variants of Phytophthora palustris toti-like and botybirna-like viruses are abbreviated PpaTLV1-12 and PpaBbLV1 and 2, respectively, and indicated with a red asterisk (*). Family classification and the corresponding pBLAST accession numbers are shown next to the virus names. Colorful squares represent the virus host kingdom or phylum: Fungi, Oomycota, Arthropoda, Chordata, Ochrophyta (Heterokonta), Excavata. Scale bar = 0.6 expected changes per site per branch.

Figure 5

(a). Graphical representation of the genomes of one botybirna-like virus (PpaBbLV2) and one toti-like virus (PpaTLV1-1). (b). Phylogenetic analysis (RAxML) based on the predicted RdRP of the dsRNA viruses discovered in Phytophthora palustris with other classified and unclassified members of the families Totiviridae and Botybirnaviridae. Nodes are labeled with bootstrap support values ≥50%. Branch lengths are scaled to the expected underlying number of amino acid substitutions per site. Tree is unrooted and branches are shown in decreasing order. Variants of Phytophthora palustris toti-like and botybirna-like viruses are abbreviated PpaTLV1-12 and PpaBbLV1 and 2, respectively, and indicated with a red asterisk (*). Family classification and the corresponding pBLAST accession numbers are shown next to the virus names. Colorful squares represent the virus host kingdom or phylum: Fungi, Oomycota, Arthropoda, Chordata, Ochrophyta (Heterokonta), Excavata. Scale bar = 0.6 expected changes per site per branch.

Figure 6

Map of Southeast and East Asia showing the sampling locations in Indonesia, Taiwan, and the Japanese Okinawa and Amami islands (indicated by red dots and arrows) where “P. palustris” isolates were collected.

Figure 7

(a). Map showing the sites of the virus-hosting “P. palustris” isolates in Sumatra. (b). Map showing the sites of the virus-hosting “P. palustris” isolates in Kalimantan. The pie charts illustrate the relative frequency of each virus family/order in Sumatra and Kalimantan.