Marine oomycetes have recently been shown to be concurrently infected by (-)ssRNA viruses of the order Bunyavirales. In this work, even higher virus variability was found in a single isolate of Phytophthora condilina, a recently described member of Phytophthora phylogenetic Clade 6a, which was isolated from brackish estuarine waters in southern Portugal. Using total and small RNA-seq the full RdRp of 13 different potential novel bunya-like viruses and two complete toti-like viruses were detected. All these viruses were successfully confirmed by reverse transcription polymerase chain reaction (RT-PCR) using total RNA as template, but complementarily one of the toti-like and five of the bunya-like viruses were confirmed when dsRNA was purified for RT-PCR. In our study, total RNA-seq was by far more efficient for de novo assembling of the virus sequencing but small RNA-seq showed higher read numbers for most viruses. Two main populations of small RNAs (21 nts and 25 nts-long) were identified, which were in accordance with other Phytophthora species. To the best of our knowledge, this is the first study using small RNA sequencing to identify viruses in Phytophthora spp.

Biotechnological Centre Faculty of Agriculture University of South Bohemia Na Sadkach 1780 37005 Ceske Budejovice Czech Republic

Phytophthora Research Centre Department of Forest Protection and Wildlife Management Faculty of Forestry and Wood Technology Mendel University in Brno Zemědělská 1 61300 Brno Czech Republic

Zobrazit více v PubMed

Burgess T.I., Simamora A.V., White D., Wiliams B., Schwager M., Stukely M.J.C., Hardy G.E.S.J. New species from Phytophthora clade 6a: Evidence for recent radiation. Persoonia Mol. Phylogeny Evol. Fungi. 2018;41:1–17. doi: 10.3767/persoonia.2018.41.01. PubMed DOI PMC

Reddell P., Bowen G.D., Robson A.D. Nodulation of Casuarinaceae in relation to host species and soil properties. Aust. J. Bot. 1986 doi: 10.1071/BT9860435. DOI

Jung T., Stukely M.J.C., Hardy G.E.S.J., White D., Paap T., Dunstan W.A., Burgess T.I. Multiple new Phytophthora species from ITS clade 6 associated with natural ecosystems in Australia: Evolutionary and ecological implications. Persoonia Mol. Phylogeny Evol. Fungi. 2011 doi: 10.3767/003158511X557577. PubMed DOI PMC

Hacker C.V., Brasier C.M., Buck K.W. A double-stranded RNA from a Phytophthora species is related to the plant endornaviruses and contains a putative UDP glycosyltransferase gene. J. Gen. Virol. 2005;86:1561–1570. doi: 10.1099/vir.0.80808-0. PubMed DOI

Kozlakidis Z., Brown N.A., Jamal A., Phoon X., Coutts R.H.A. Incidence of endornaviruses in Phytophthora taxon douglasfir and Phytophthora ramorum. Virus Genes. 2010;40:130–134. doi: 10.1007/s11262-009-0421-7. PubMed DOI

Cai G., Myers K., Hillman B.I., Fry W.E. A novel virus of the late blight pathogen, Phytophthora infestans, with two RNA segments and a supergroup 1 RNA-dependent RNA polymerase. Virology. 2009;392:52–61. doi: 10.1016/j.virol.2009.06.040. PubMed DOI

Cai G., Hillman B.I. Phytophthora Viruses. 1st ed. Volume 86. Elsevier Inc.; Amsterdam, The Netherlands: 2013.

Cai G., Krychiw J.F., Myers K., Fry W.E., Hillman B.I. A new virus from the plant pathogenic oomycete Phytophthora infestans with an 8 kb dsRNA genome: The sixth member of a proposed new virus genus. Virology. 2013;435:341–349. doi: 10.1016/j.virol.2012.10.012. PubMed DOI

Cai G., Myers K., Fry W.E., Hillman B.I. A member of the virus family Narnaviridae from the plant pathogenic oomycete Phytophthora infestans. Arch. Virol. 2012;157:165–169. doi: 10.1007/s00705-011-1126-5. PubMed DOI

Cai G., Fry W.E., Hillman B.I. PiRV-2 stimulates sporulation in Phytophthora infestans. Virus Res. 2019;271:197674. doi: 10.1016/j.virusres.2019.197674. PubMed DOI

Poimala A., Vainio E.J. Complete genome sequence of a novel toti-like virus from the plant—Pathogenic oomycete Phytophthora cactorum. Arch. Virol. 2020;165:1679–1682. doi: 10.1007/s00705-020-04642-2. PubMed DOI PMC

Uchida K., Sakuta K., Ito A., Takahashi Y., Katayama Y., Fukuhara T., Uematsu S., Okada R., Moriyama H. Two novel endornaviruses co-infecting a Phytophthora pathogen of Asparagus officinalis modulate the developmental stages and fungicide sensitivities of the host oomycete. Front. Microbiol. 2021;12:122. doi: 10.3389/fmicb.2021.633502. PubMed DOI PMC

Yokoi T., Yamashita S., Hibi T. The nucleotide sequence and genome organization of Sclerophthora macrospora virus A. Virology. 2003;311:394–399. doi: 10.1016/S0042-6822(03)00183-1. PubMed DOI

Grasse W., Zipper R., Totska M., Spring O. Plasmopara halstedii virus causes hypovirulence in Plasmopara halstedii, the downy mildew pathogen of the sunflower. Fungal Genet. Biol. 2013;57:42–47. doi: 10.1016/j.fgb.2013.05.009. PubMed DOI

Grasse W., Spring O. ssRNA viruses from biotrophic Oomycetes form a new phylogenetic group between Nodaviridae and Tombusviridae. Arch. Virol. 2017;162:1319–1324. doi: 10.1007/s00705-017-3243-2. PubMed DOI

Chiapello M., Rodríguez-Romero J., Nerva L., Forgia M., Chitarra W., Ayllón M.A., Turina M. Putative new plant viruses associated with Plasmopara viticola-infected grapevine samples. Ann. Appl. Biol. 2020;176:180–191. doi: 10.1111/aab.12563. DOI

Chiapello M., Rodríguez-Romero J., Ayllón M.A., Turina M. Analysis of the virome associated to grapevine downy mildew lesions reveals new mycovirus lineages. Virus Evol. 2020;6:veaa058. doi: 10.1093/ve/veaa058. PubMed DOI PMC

Gillings M.R., Tesoriero L.A., Gunn L.V. Detection of double-stranded RNA and virus-like particles in Australian isolates of Pythium irregulare. Plant Pathol. 1993;42:6–15. doi: 10.1111/j.1365-3059.1993.tb01466.x. DOI

Shiba K., Hatta C., Sasai S., Tojo M., Ohki S.T., Mochizuki T. Genome sequence of a novel partitivirus isolated from the oomycete Pythium nunn. Arch. Virol. 2018;163:2561–2563. doi: 10.1007/s00705-018-3880-0. PubMed DOI

Sasai S., Tamura K., Tojo M., Herrero M.L., Hoshino T., Ohki S.T., Mochizuki T. A novel non-segmented double-stranded RNA virus from an Arctic isolate of Pythium polare. Virology. 2018;522:234–243. doi: 10.1016/j.virol.2018.07.012. PubMed DOI

Botella L., Janoušek J., Maia C., Jung M.H., Raco M., Jung T. Marine oomycetes of the genus Halophytophthora harbor viruses related to Bunyaviruses. Front. Microbiol. 2020;11:1467. doi: 10.3389/fmicb.2020.01467. PubMed DOI PMC

Kreuze J.F., Perez A., Untiveros M., Quispe D., Fuentes S., Barker I., Simon R. Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: A generic method for diagnosis, discovery and sequencing of viruses. Virology. 2009;388:1–7. doi: 10.1016/j.virol.2009.03.024. PubMed DOI

Segers G.C., Zhang X., Deng F., Sun Q., Nuss D.L. Evidence that RNA silencing functions as an antiviral defense mechanism in fungi. Proc. Natl. Acad. Sci. USA. 2007;104:12902–12906. doi: 10.1073/pnas.0702500104. PubMed DOI PMC

Nuss D.L. Mycoviruses, RNA silencing, and viral RNA recombination. Adv. Virus Res. 2011;80:25–48. doi: 10.1016/B978-0-12-385987-7.00002-6. PubMed DOI PMC

Chiba S., Lin Y.H., Kondo H., Kanematsu S., Suzuki N. A novel betapartitivirus RnPV6 from Rosellinia necatrix tolerates host RNA silencing but is interfered by its defective RNAs. Virus Res. 2016;219:62–72. doi: 10.1016/j.virusres.2015.10.017. PubMed DOI

Ozkan S., Mohorianu I., Xu P., Dalmay T., Coutts R.H.A. Profile and functional analysis of small RNAs derived from Aspergillus fumigatus infected with double-stranded RNA mycoviruses. BMC Genom. 2017;18:416. doi: 10.1186/s12864-017-3773-8. PubMed DOI PMC

Silvestri A., Turina M., Fiorilli V., Miozzi L., Venice F., Bonfante P., Lanfranco L. Different genetic sources contribute to the small RNA population in the arbuscular mycorrhizal fungus Gigaspora margarita. Front. Microbiol. 2020 doi: 10.3389/fmicb.2020.00395. PubMed DOI PMC

Vainio E.J., Jurvansuu J., Streng J., Rajama M., Hantula J., Valkonen J.P.T. Diagnosis and discovery of fungal viruses using deep sequencing of small RNAs. J. Gen. Virol. 2015;96:714–725. doi: 10.1099/jgv.0.000003. PubMed DOI

Donaire L., Pagán I., Ayllón M.A. Characterization of Botrytis cinerea negative-stranded RNA virus 1, a new mycovirus related to plant viruses, and a reconstruction of host pattern evolution in negative-sense ssRNA viruses. Virology. 2016;499:212–218. doi: 10.1016/j.virol.2016.09.017. PubMed DOI

Nerva L., Ciuffo M., Vallino M., Margaria P., Varese G.C., Gnavi G., Turina M. Multiple approaches for the detection and characterization of viral and plasmid symbionts from a collection of marine fungi. Virus Res. 2016;219:22–38. doi: 10.1016/j.virusres.2015.10.028. PubMed DOI

Vetukuri R.R., Åsman A.K.M., Tellgren-Roth C., Jahan S.N., Reimegård J., Fogelqvist J., Savenkov E., Söderbom F., Avrova A.O., Whisson S.C., et al. Evidence for small RNAs homologous to effector-encoding genes and transposable elements in the oomycete Phytophthora infestans. PLoS ONE. 2012;7:e51399. doi: 10.1371/journal.pone.0051399. PubMed DOI PMC

Fahlgren N., Bollmann S.R., Kasschau K.D., Cuperus J.T., Press C.M., Sullivan C.M., Chapman E.J., Hoyer J.S., Gilbert K.B., Grünwald N.J., et al. Phytophthora have distinct endogenous small RNA populations that include short interfering and microRNAs. PLoS ONE. 2013;8:e77181. doi: 10.1371/journal.pone.0077181. PubMed DOI PMC

Jia J., Lu W., Zhong C., Zhou R., Xu J., Liu W., Gou X., Wang Q., Yin J., Xu C., et al. The 25-26 nt small RNAs in Phytophthora parasitica are associated with efficient silencing of homologous endogenous genes. Front. Microbiol. 2017;8:773. doi: 10.3389/fmicb.2017.00773. PubMed DOI PMC

Dunker F., Trutzenberg A., Rothenpieler J.S., Kuhn S., Pröls R., Schreiber T., Tissier A., Kemen A., Kemen E., Hückelhoven R., et al. Oomycete small RNAs bind to the plant RNA-induced silencing complex for virulence. Elife. 2020;9:e56096. doi: 10.7554/eLife.56096. PubMed DOI PMC

Suttle C.A. Marine viruses—Major players in the global ecosystem. Nat. Rev. Microbiol. 2007;5:801–812. doi: 10.1038/nrmicro1750. PubMed DOI

Wommack K.E., Colwell R.R. Virioplankton: Viruses in aquatic ecosystems. Microbiol. Mol. Biol. Rev. 2000;64:69–114. doi: 10.1128/MMBR.64.1.69-114.2000. PubMed DOI PMC

Weinbauer M.G., Rassoulzadegan F. Are viruses driving microbial diversification and diversity? Environ. Microbiol. 2004;6:1–11. doi: 10.1046/j.1462-2920.2003.00539.x. PubMed DOI

Breitbart M. Marine viruses: Truth or Dare. Ann. Rev. Mar. Sci. 2012;4:425–448. doi: 10.1146/annurev-marine-120709-142805. PubMed DOI

Chen X., Wei W., Wang J., Li H., Sun J., Ma R., Jiao N., Zhang R. Tide driven microbial dynamics through virus-host interactions in the estuarine ecosystem. Water Res. 2019;160:118–129. doi: 10.1016/j.watres.2019.05.051. PubMed DOI

Hingamp P., Grimsley N., Acinas S.G., Clerissi C., Subirana L., Poulain J., Ferrera I., Sarmento H., Villar E., Lima-Mendez G., et al. Exploring nucleo-cytoplasmic large DNA viruses in Tara Oceans microbial metagenomes. ISME J. 2013;7:1678–1695. doi: 10.1038/ismej.2013.59. PubMed DOI PMC

Jung T., Scanu B., Brasier C., Webber J., Milenković I., Corcobado T., Tomšovský M., Pánek M., Bakonyi J., Maia C., et al. A survey in natural forest ecosystems of Vietnam reveals high diversity of both new and described Phytophthora taxa including P. ramorum. Forests. 2020;11:93. doi: 10.3390/f11010093. DOI

Grabherr M.G., Haas B.J., Yassour M., Levin J.Z., Thompson D.A., Amit I., Adiconis X., Fan L., Raychowdhury R., Zeng Q., et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 2011;29:644–652. doi: 10.1038/nbt.1883. PubMed DOI PMC

Edgar R.C. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32:1792–1797. doi: 10.1093/nar/gkh340. PubMed DOI PMC

Miller M.A., Pfeiffer W., Schwartz T. Creating the CIPRES Science Gateway for Inference of Large Phylogenetic Trees; Proceedings of the Gateway Computing Environments Workshop (GCE); New Orleans, LA, USA. 14 November 2010; pp. 1–8.

Wickner R.B., Ghabrial S.A., Nibert M.L., Patterson J.L., Wang C. Virus Taxonomy. Elsevier; Amsterdam, The Netherlands: 2012. Totiviridae; pp. 639–650.

Doherty M., Sanganee K., Kozlakidis Z., Coutts R.H.A., Brasier C.M., Buck K.W. Molecular Characterization of a totivirus and a partitivirus from the Genus Ophiostoma. For. Res. 2007;192:188–192. doi: 10.1111/j.1439-0434.2007.01207.x. DOI

Sasaki A., Kanematsu S., Onoue M., Oyama Y., Yoshida K. Infection of Rosellinia necatrix with purified viral particles of a member of Partitiviridae (RnPV1-W8) Arch. Virol. 2006;151:697–707. doi: 10.1007/s00705-005-0662-2. PubMed DOI

Deakin G., Dobbs E., Bennett J.M., Jones I.M., Grogan H.M., Burton K.S. Multiple viral infections in Agaricus bisporus—Characterisation of 18 unique RNA viruses and 8 ORFans identified by deep sequencing. Sci. Rep. 2017;7:2469. doi: 10.1038/s41598-017-01592-9. PubMed DOI PMC

Zhu J.Z., Zhu H.J., Gao B.D., Zhou Q., Zhong J. Diverse, Novel mycoviruses from the virome of a hypovirulent Sclerotium rolfsii strain. Front. Plant Sci. 2018;9:1738. doi: 10.3389/fpls.2018.01738. PubMed DOI PMC

Hillman B.I., Annisa A., Suzuki N. Advances in Virus Research. Volume 100. Academic Press Inc.; New York, NY, USA: 2018. Viruses of plant-interacting fungi; pp. 99–116. PubMed

Hantula J., Mäkelä S., Xu P., Brusila V., Nuorteva H., Kashif M., Hyder R., Vainio E.J. Multiple virus infections on Heterobasidion sp. Fungal Biol. 2020;124:102–109. doi: 10.1016/j.funbio.2019.12.004. PubMed DOI

Botella L., Hantula J. Description, distribution, and relevance of viruses of the forest pathogen Gremmeniella abietina. Viruses. 2018;10:654. doi: 10.3390/v10110654. PubMed DOI PMC

Vainio E.J., Müller M.M., Korhonen K., Piri T., Hantula J. Viruses accumulate in aging infection centers of a fungal forest pathogen. ISME J. 2015;9:497–507. doi: 10.1038/ismej.2014.145. PubMed DOI PMC

Ferron F., Weber F., de la Torre J.C., Reguera J. Transcription and replication mechanisms of Bunyaviridae and Arenaviridae L proteins. Virus Res. 2017;234:118–134. doi: 10.1016/j.virusres.2017.01.018. PubMed DOI PMC

Nerva L., Forgia M., Ciuffo M., Chitarra W., Chiapello M., Vallino M., Varesec G.C., Turinaa M. The mycovirome of a fungal collection from the sea cucumber Holothuria polii. Virus Res. 2019;273:197737. doi: 10.1016/j.virusres.2019.197737. PubMed DOI

Lin Y.-H., Fujita M., Chiba S., Hyodo K., Andika I.B., Suzuki N., Kondo H. Two novel fungal negative-strand RNA viruses related to mymonaviruses and phenuiviruses in the shiitake mushroom (Lentinula edodes) Virology. 2019;533:125–136. doi: 10.1016/j.virol.2019.05.008. PubMed DOI

Muller R., Poch O., Delarue M., Bishop D.H.L., Bouloy M. Rift valley fever virus L segment: Correction of the sequence and possible functional role of newly identified regions conserved in RNA-dependent polymerases. J. Gen. Virol. 1994;75:1345–1352. doi: 10.1099/0022-1317-75-6-1345. PubMed DOI

Makarova K.S., Aravind L., Grishin N.V., Rogozin I.B., Koonin E.V. A DNA repair system specific for thermophilic Archaea and bacteria predicted by genomic context analysis. Nucleic Acids Res. 2002;30:482–496. doi: 10.1093/nar/30.2.482. PubMed DOI PMC

Terns M.P., Terns R.M. CRISPR-based adaptive immune systems. Curr. Opin. Microbiol. 2011;14:321–327. doi: 10.1016/j.mib.2011.03.005. PubMed DOI PMC

Touriol C., Bornes S., Bonnal S., Audigier S., Prats H., Prats A.C., Vagner S. Generation of protein isoform diversity by alternative initiation of translation at non-AUG codons. Biol. Cell. 2003;95:169–178. doi: 10.1016/S0248-4900(03)00033-9. PubMed DOI

Bekaert M., Richard H., Prum B., Rousset J.P. Identification of programmed translational -1 frameshifting sites in the genome of Saccharomyces cerevisiae. Genome Res. 2005;15:1411–1420. doi: 10.1101/gr.4258005. PubMed DOI PMC

Firth A.E., Brierley I. Non-canonical translation in RNA viruses. J. Gen. Virol. 2012;93:1385. doi: 10.1099/vir.0.042499-0. PubMed DOI PMC

Weber F., Wagner V., Rasmussen S.B., Hartmann R., Paludan S.R. Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses. J. Virol. 2006;80:5059–5064. doi: 10.1128/JVI.80.10.5059-5064.2006. PubMed DOI PMC

O’Brien C.A., Hobson-Peters J., Yam A.W.Y., Colmant A.M.G., McLean B.J., Prow N.A., Watterson D., Hall-Mendelin S., Warrilow D., Ng M.L., et al. Viral RNA intermediates as targets for detection and discovery of novel and emerging mosquito-borne viruses. PLoS Negl. Trop. Dis. 2015;9:e0003629. doi: 10.1371/journal.pntd.0003629. PubMed DOI PMC

Wang Q., Li T., Xu K., Zhang W., Wang X., Quan J., Jin W., Zhang M., Fan G., Wang M.-B., et al. The tRNA-derived small rnas regulate gene expression through triggering sequence-specific degradation of target transcripts in the oomycete pathogen Phytophthora sojae. Front. Plant Sci. 2016;7 doi: 10.3389/fpls.2016.01938. PubMed DOI PMC

Mueller S., Gausson V., Vodovar N., Deddouche S., Troxler L., Perot J., Pfeffer S., Hoffmann J.A., Saleh M.-C., Imler J.-L. RNAi-mediated immunity provides strong protection against the negative-strand RNA vesicular stomatitis virus in Drosophila. Proc. Natl. Acad. Sci. USA. 2010;107:19390–19395. doi: 10.1073/pnas.1014378107. PubMed DOI PMC

Chiba S., Lin Y.-H., Kondo H., Kanematsu S., Suzuki N. A Novel victorivirus from a phytopathogenic fungus, Rosellinia necatrix, is infectious as particles and targeted by RNA silencing. J. Virol. 2013;87:6727–6738. doi: 10.1128/JVI.00557-13. PubMed DOI PMC

Nerva L., Turina M., Zanzotto A., Gardiman M., Gaiotti F., Gambino G., Chitarra W. Isolation, molecular characterization and virome analysis of culturable wood fungal endophytes in esca symptomatic and asymptomatic grapevine plants. Environ. Microbiol. 2019;21:2886–2904. doi: 10.1111/1462-2920.14651. PubMed DOI

Kuhn J.H., Adkins S., Alioto D., Alkhovsky S.V., Amarasinghe G.K., Anthony S.J., Avšiā-Županc T., Ayllón M.A., Bahl J. Taxonomic update for phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales. Arch. Virol. 2020;165:3023–3072. doi: 10.1007/s00705-020-04731-2. PubMed DOI PMC

Sutela S., Forgia M., Vainio E.J., Chiapello M., Daghino S., Vallino M., Martino E., Girlanda M., Perotto S., Turina M. The virome from a collection of endomycorrhizal fungi reveals new viral taxa with unprecedented genome organization. Virus Evol. 2020;6:veaa076. doi: 10.1093/ve/veaa076. PubMed DOI PMC

Shi M., Lin X.-D., Tian J.-H., Chen L.-J., Chen X., Li C.-X., Qin X.-C., Li J., Cao J.-P., Eden J.-S., et al. Redefining the invertebrate RNA virosphere. Nature. 2016;540:539–543. doi: 10.1038/nature20167. PubMed DOI

Charon J., Marcelino V.R., Wetherbee R., Verbruggen H., Holmes E.C. Metatranscriptomic identification of diverse and divergent RNA viruses in green and chlorarachniophyte algae cultures. Viruses. 2020;12:1180. doi: 10.3390/v12101180. PubMed DOI PMC

Beakes G.W., Sekimoto S. The evolutionary phylogeny of oomycetes—Insights gained from studies of holocarpic parasites of algae and invertebrates. In: Lamour K., Kamoun S., editors. Oomycete Genetics and Genomics: Diversity, Interactions, and Research Tools. John Wiley and Sons Inc.; Hoboken, NJ, USA: 2009. pp. 1–24.

O’Callaghan I., Harrison S., Fitzpatrick D., Sullivan T. The freshwater isopod Asellus aquaticus as a model biomonitor of environmental pollution: A review. Chemosphere. 2019;235:498–509. doi: 10.1016/j.chemosphere.2019.06.217. PubMed DOI

Thines M., Choi Y.-J. Evolution, Diversity, and Taxonomy of the Peronosporaceae, with Focus on the Genus Peronospora. Phytopathology. 2016;106:6–18. doi: 10.1094/PHYTO-05-15-0127-RVW. PubMed DOI

The virome of the panglobal, wide host-range plant pathogen Phytophthora cinnamomi: phylogeography and evolutionary insights

Characterization of Mycoviruses in Armillaria ostoyae and A. cepistipes in the Czech Republic

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

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

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

Eight new Halophytophthora species from marine and brackish-water ecosystems in Portugal and an updated phylogeny for the genus

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