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.

Biotechnological Centre Faculty of Agriculture University of South Bohemia Ceske Budejovice Czechia

Centre for Marine Sciences University of Algarve Faro Portugal

Phytophthora Research Centre Department of Forest Protection and Wildlife Management Faculty of Forestry and Wood Technology Mendel University in Brno Brno Czechia

Zobrazit více v PubMed

Abudurexiti A., Adkins S., Alioto D., Alkhovsky S. V., Avšič-Županc T., Ballinger M. J., et al. (2019). Taxonomy of the order bunyavirales: update 2019. Arch. Virol. 164 1949–1965. PubMed PMC

Barr J. N., Fearns R. (2010). How RNA viruses maintain their genome integrity. J. Gen. Virol. 91 1373–1387. 10.1099/vir.0.020818-20810 PubMed DOI

Bennett R. M., de Cock A. W. A. M., Lévesque C. A., Thines M. (2017). Calycofera gen. nov., an estuarine sister taxon to Phytopythium. Peronosporaceae. Mycol. Prog. 16 947–954. 10.1007/s11557-017-1326-1329 DOI

Bennett R. M., Thines M. (2019). Revisiting salisapiliaceae. FUSE 3 171–184. 10.3114/fuse.2019.03.10 PubMed DOI PMC

Breitbart M. (2012). Marine viruses: truth or dare. Ann. Rev. Mar. Sci. 4 425–448. 10.1146/annurev-marine-120709-142805 PubMed DOI

Briese T., Calisher C. H., Higgs S. (2013). Viruses of the family Bunyaviridae : are all available isolates reassortants? Virology 446 207–216. 10.1016/j.virol.2013.07.030 PubMed DOI

Cai G., Fry W. E., Hillman B. I. (2019). PiRV-2 stimulates sporulation in Phytophthora infestans. Virus Res 271:197674. 10.1016/J.VIRUSRES.2019.197674 PubMed DOI

Cai G., Krychiw J. F., Myers K., Fry W. E., Hillman B. I. (2013). 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 435 341–349. PubMed

Cai G., Myers K., Fry W. E., Hillman B. I. (2012). A member of the virus family Narnaviridae from the plant pathogenic oomycete Phytophthora infestans. Arch. Virol. 157 165–169. PubMed

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

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

Coupeau D., Bayrou C., Baillieux P., Marichal A., Lenaerts A. C., Caty C., et al. (2019). Host-dependence of in vitro reassortment dynamics among the sathuperi and shamonda simbuviruses. Emerg. Microbes Infect. 8 381–395. 10.1080/22221751.2019.1586410 PubMed DOI PMC

Coy S., Gann E., Pound H., Short S., Wilhelm S. (2018). Viruses of eukaryotic algae: diversity, methods for detection, and future directions. Viruses 10:487. 10.3390/v10090487 PubMed DOI PMC

Desnues C., Rodriguez-Brito B., Rayhawk S., Kelley S., Tran T., Haynes M., et al. (2008). Biodiversity and biogeography of phages in modern stromatolites and thrombolites. Nature 452 340–343. 10.1038/nature06735 PubMed DOI

Donaire L., Pagán I., Ayllón M. A. (2016). 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 499 212–218. 10.1016/J.VIROL.2016.09.017 PubMed DOI

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

Fahlgren N., Bollmann S. R., Kasschau K. D., Cuperus J. T., Press C. M., Sullivan C. M., et al. (2013). Phytophthora have distinct endogenous small RNA populations that include short interfering and microRNAs. PLoS One 8:e77181. 10.1371/journal.pone.0077181 PubMed DOI PMC

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

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

Govers L. L., Man In ‘T Veld W. A., Meffert J. P., Bouma T. J., van Rijswick P. C. J., Heusinkveld J. H. T., et al. (2016). Marine Phytophthora species can hamper conservation and restoration of vegetated coastal ecosystems. Proc. R. Soc. B Biol. Sci. 283:20160812. 10.1098/rspb.2016.0812 PubMed DOI PMC

Grasse W., Spring O. (2017). ssRNA viruses from biotrophic Oomycetes form a new phylogenetic group between Nodaviridae and Tombusviridae. Arch. Virol. 162 1319–1324. 10.1007/s00705-017-3243-3242 PubMed DOI

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

Hacker C. V., Brasier C. M., Buck K. W. (2005). A double-stranded RNA from a Phytophthora species is related to the plant endornaviruses and contains a putative UDP glycosyltransferase gene. J. Gen. Virol. 86 1561–1570. 10.1099/vir.0.80808-80800 PubMed DOI

Halldorsson S., Li S., Li M., Harlos K., Bowden T. A., Huiskonen J. T. (2018). Shielding and activation of a viral membrane fusion protein. Nat. Commun. 9 1–9. 10.1038/s41467-017-02789-2782 PubMed DOI PMC

Huiskonen J. T., Hepojoki J., Laurinmaki P., Vaheri A., Lankinen H., Butcher S. J., et al. (2010). Electron cryotomography of tula hantavirus suggests a unique assembly paradigm for enveloped viruses. J. Virol. 84 4889–4897. 10.1128/jvi.00057-10 PubMed DOI PMC

Jung T., Chang T. T., Bakonyi J., Seress D., Pérez-Sierra A., Yang X., et al. (2017a). Diversity of Phytophthora species in natural ecosystems of Taiwan and association with disease symptoms. Plant Pathol. 66 194–211. 10.1111/ppa.12564 DOI

Jung T., Scanu B., Bakonyi J., Seress D., Kovács G. M., Durán A., et al. (2017b). Nothophytophthora gen. nov., a new sister genus of Phytophthora from natural and semi-natural ecosystems. Persoonia 39 143–174. 10.3767/persoonia.2017.39.07 PubMed DOI PMC

Kormelink R., Garcia M. L., Goodin M., Sasaya T., Haenni A.-L. (2011). Negative-strand RNA viruses: the plant-infecting counterparts. Virus Res. 162 184–202. 10.1016/j.virusres.2011.09.028 PubMed DOI

Kozlakidis Z., Brown N. A., Jamal A., Phoon X., Coutts R. H. A. (2010). Incidence of endornaviruses in Phytophthora taxon douglasfir and Phytophthora ramorum. Virus Genes 40 130–134. 10.1007/s11262-009-0421-427 PubMed DOI

Lee S. Y. (1998). Ecological role of grapsid crabs in mangrove ecosystems: a review. Mar. Freshw. Res. 49 335–343. 10.1071/mf97179 DOI

Leslie J. F., Zeller K. A. (1996). Heterokaryon incompatibility in fungi—more than just another way to die. J. Genet. 75 415–424. 10.1007/BF02966319 DOI

Li C.-X., Shi M., Tian J.-H., Lin X.-D., Kang Y.-J., Chen L.-J., et al. (2015). Unprecedented genomic diversity of RNA viruses in arthropods reveals the ancestry of negative-sense RNA viruses. Elife 4:e05378. 10.7554/eLife.05378 PubMed DOI PMC

Li H., Roossinck M. J. (2004). Genetic bottlenecks reduce population variation in an experimental rna virus population. Society 78 10582–10587. 10.1128/JVI.78.19.10582 PubMed DOI PMC

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

Lu S., Wang J., Chitsaz F., Derbyshire M. K., Geer R. C., Gonzales N. R., et al. (2020). CDD/SPARCLE: the conserved domain database in 2020. Nucleic Acids Res. 48 D265–D268. 10.1093/nar/gkz991 PubMed DOI PMC

Man in ’t Veld W. A., Rosendahl K. C. H. M., van Rijswick P. C. J., Meffert J. P., Boer E., Westenberg M., et al. (2019). Multiple Halophytophthora spp. and Phytophthora spp. including P. gemini, P. inundata and P. chesapeakensis sp. nov. isolated from the seagrass Zostera marina in the Northern hemisphere. Eur. J. Plant Pathol. 153 341–357. 10.1007/s10658-018-1561-1561 DOI

Marano A. V., Jesus A. L., de Souza J. I., Leaño E. M., James T. Y., Jerônimo G. H., et al. (2014). A new combination in Phytopythium: P. kandeliae (Oomycetes. Straminipila). Mycosphere 5 510–522. 10.5943/mycosphere/5/4/3 DOI

Margaria P., Bosco L., Vallino M., Ciuffo M., Mautino G. C., Tavella L., et al. (2014). The NSs Protein of tomato spotted wilt virus is required for persistent infection and transmission by Frankliniella occidentalis. J. Virol. 88 5788–5802. 10.1128/jvi.00079-14 PubMed DOI PMC

Marklewitz M., Zirkel F., Kurth A., Drosten C., Junglena S. (2015). Evolutionary and phenotypic analysis of live virus isolates suggests arthropod origin of a pathogenic RNA virus family. Proc. Natl. Acad. Sci. U.S.A. 112 7536–7541. 10.1073/pnas.1502036112 PubMed DOI PMC

Marzano S.-Y. L., Nelson B. D., Ajayi-Oyetunde O., Bradley C. A., Hughes T. J., Hartman G. L., et al. (2016). Identification of diverse mycoviruses through metatranscriptomics characterization of the viromes of five major fungal plant pathogens. J. Virol. 90 6846–6863. 10.1128/jvi.00357-316 PubMed DOI PMC

Melzer M. S., Ikeda S. S., Boland G. J. (2002). Interspecific Transmission of Double-Stranded RNA and Hypovirulence from Sclerotinia sclerotiorum to S. minor. Phytopathology 92 780–784. 10.1094/PHYTO.2002.92.7.780 PubMed DOI

Miller M. A., Pfeiffer W., Schwartz T. (2010). “Creating the CIPRES Science Gateway for inference of large phylogenetic trees,” in 2010 Gateway Computing Environments Workshop, GCE 2010. 10.1109/GCE.2010.5676129 DOI

Morris T. J., Dodds J. A. (1979). Isolation and analysis of double-stranded RNA from virus-infected plant and fungal tissue. Phytopathology 69 854 10.1094/Phyto-69-854 DOI

Muller R., Poch O., Delarue M., Bishop D. H. L., Bouloy M. (1994). 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 75(Pt 6), 1345–1352. 10.1099/0022-1317-75-6-1345 PubMed DOI

Nagasaki K., Tomaru Y., Takao Y., Nishida K., Shirai Y., Suzuki H., et al. (2005). Previously unknown virus infects marine diatom. Appl. Environ. Microbiol 71 3528–3535. 10.1128/AEM.71.7.3528-3535.2005 PubMed DOI PMC

Nerva L., Ciuffo M., Vallino M., Margaria P., Varese G. C., Gnavi G., et al. (2015). Multiple approaches for the detection and characterization of viral and plasmid symbionts from a collection of marine fungi. Virus Res 219 22–38. 10.1016/j.virusres.2015.10.028 PubMed DOI

Nerva L., Forgia M., Ciuffo M., Chitarra W., Chiapello M., Vallino M., et al. (2019a). The mycovirome of a fungal collection from the sea cucumber Holothuria polii. Virus Res. 273:197737. 10.1016/j.virusres.2019.197737 PubMed DOI

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

Newell S. Y., Fell J. W. (1992). Distribution and experimental responses to substrate of marine oomycetes (Halophytophthora spp.) in mangrove ecosystems. Mycol. Res. 96 851–856. 10.1016/S0953-7562(09)81030-81037 DOI

Newell S. Y., Fell J. W. (1997). Competition among mangrove oomycotes, and between oomycotes and other microbes. Aquat. Microb. Ecol. 12 21–28. 10.3354/ame012021 DOI

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

Obijeski J. F., Bishop D. H., Murphy F. A., Palmer E. L. (1976). Structural proteins of La Crosse virus. J. Virol. 19 985–997. 10.1128/jvi.19.3.985-997.1976 PubMed DOI PMC

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

Poimala A., Vainio E. J. (2020). Complete genome sequence of a novel toti - like virus from the plant - pathogenic oomycete Phytophthora cactorum. Arch. Virol. 165, 1679–1682. 10.1007/s00705-020-04642-4642 PubMed DOI PMC

Rozas J., Ferrer-Mata A., Sanchez-Del Barrio J. C., Guirao-Rico S., Librado P., Ramos-Onsins S. E., et al. (2017). DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol. Biol. Evol. 34 3299–3302. 10.1093/molbev/msx248 PubMed DOI

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

Schardl C. L., Craven K. D. (2003). Interspecific hybridization in plant-associated fungi and oomycetes: a review. Mol. Ecol. 12 2861–2873. 10.1046/j.1365-294X.2003.01965.x PubMed DOI

Schneider W. L., Roossinck M. J. (2001). Genetic diversity in RNA virus quasispecies is controlled by host-virus interactions. Society 75 6566–6571. 10.1128/JVI.75.14.6566 PubMed DOI PMC

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

Shi M., Lin X. D., Tian J. H., Chen L. J., Chen X., Li C. X., et al. (2016). Redefining the invertebrate RNA virosphere. Nature 540 539–543. 10.1038/nature20167 PubMed DOI

Shi M., Zhang Y. Z., Holmes E. C. (2018). Meta-transcriptomics and the evolutionary biology of RNA viruses. Virus Res. 243 83–90. 10.1016/j.virusres.2017.10.016 PubMed DOI PMC

Shiba K., Hatta C., Sasai S., Tojo M., Ohki S. T., Mochizuki T. (2018). Genome sequence of a novel partitivirus identified from the oomycete Pythium nunn. Arch. Virol. 163 2561–2563. PubMed

Silvestri A., Turina M., Fiorilli V., Miozzi L., Venice F., Bonfante P., et al. (2020). Different genetic sources contribute to the small RNA Population in the arbuscular mycorrhizal fungus Gigaspora margarita. Front. Microbiol. 11:395. 10.3389/fmicb.2020.00395 PubMed DOI PMC

Stamatakis A., Hoover P., Rougemont J. (2008). A rapid bootstrap algorithm for the RAxML Web Servers. Syst. Biol. 57 758–771. 10.1080/10635150802429642 PubMed DOI

Sullivan B. K., Trevathan-Tackett S. M., Neuhauser S., Govers L. L. (2018). Review: host-pathogen dynamics of seagrass diseases under future global change. Mar. Pollut. Bull. 134 75–88. 10.1016/j.marpolbul.2017.09.030 PubMed DOI PMC

Sutela S., Poimala A., Vainio E. J. (2019). Viruses of fungi and oomycetes in the soil environment. FEMS Microbiol. Ecol. 95 1–18. 10.1093/femsec/fiz119 PubMed DOI

Suttle C. A. (2005). Viruses in the sea. Nature 437 356–361. 10.1038/nature04160 PubMed DOI

Thines M., Choi Y. J. (2016). Evolution, diversity, and taxonomy of the Peronosporaceae, with focus on the genus Peronospora. Phytopathology 106 6–18. 10.1094/PHYTO-05-15-0127-RVW PubMed DOI

Tuomivirta T. T., Uotila A., Hantula J. (2002). Two independent double-stranded RNA patterns occur in the Finnish Gremmeniella abietina var. abietina type A. For. Pathol. 32 197–205. 10.1046/j.1439-0329.2002.00285.x DOI

Vainio E. J., Hakanpää J., Dai Y.-C., Hansen E., Korhonen K., Hantula J. (2011). Species of Heterobasidion host a diverse pool of partitiviruses with global distribution and interspecies transmission. Fungal Biol. 115 1234–1243. 10.1016/j.funbio.2011.08.008 PubMed DOI

Van Diepeningen A. D., Debets A. J. M., Hoekstra R. F. (1998). Intra- and interspecies virus transfer in Aspergilli via protoplast fusion. Fungal Genet. Biol. 25 171–180. 10.1006/fgbi.1998.1096 PubMed DOI

Vega Thurber R. L., Barott K. L., Hall D., Liu H., Rodriguez-Mueller B., Desnues C., et al. (2008). Metagenomic analysis indicates that stressors induce production of herpes-like viruses in the coral Porites compressa. Proc. Natl. Acad. Sci. U.S.A. 105 18413–18418. 10.1073/pnas.0808985105 PubMed DOI PMC

Velasco L., Arjona-Girona I., Cretazzo E., López-Herrera C. (2019). Viromes in Xylariaceae fungi infecting avocado in Spain. Virology 532 11–21. 10.1016/j.virol.2019.03.021 PubMed DOI

Weiland J. J., Sundsbak J. L. (2000). Differentiation and detection of sugar beet fungal pathogens using pcr amplification of actin coding sequences and its region of the rRNA gene. Plant Dis. 84 475–482. 10.1094/PDIS.2000.84.4.475 PubMed DOI

Weinbauer M. G. (2004). Ecology of prokaryotic viruses. FEMS Microbiol. Rev. 28 127–181. 10.1016/j.femsre.2003.08.001 PubMed DOI

Weynberg K., Allen M., Wilson W. (2017). Marine prasinoviruses and their tiny plankton hosts: a review. Viruses 9:43. 10.3390/v9030043 PubMed DOI PMC

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

Yokoi T., Yamashita S., Hibi T. (2003). The nucleotide sequence and genome organization of Sclerophthora macrospora virus A. Virology 311 394–399. PubMed

Zhao G., Wu G., Lim E. S., Droit L., Krishnamurthy S., Barouch D. H., et al. (2017). VirusSeeker, a computational pipeline for virus discovery and virome composition analysis. Virology 503 21–30. 10.1016/j.virol.2017.01.005 PubMed DOI PMC

Diversity and impact of single-stranded RNA viruses in Czech Heterobasidion populations

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

Multiple Viral Infections Detected in Phytophthora condilina by Total and Small RNA Sequencing