S Felsenstein, J. (1985). CONFIDENCE LIMITS ON PHYLOGENIES: AN APPROACH USING THE BOOTSTRAP. Evolution, 39(4), 783-791. doi:10.1111/j.1558-5646.1985.tb00420.x

[EN] The diversity of Phytophthora species in declining Fagaceae forests in Europe is increasing in the last years. The genus Quercus is one of the most extended Fagaceae genera in Europe, and Q. ilex is the dominant tree in Spain. The introduction of soil-borne pathogens, such as Phytophthora in Fagaceae forests modifies the microbial community present in the rhizosphere, and has relevant environmental and economic consequences. A better understanding of the diversity of Phytophthora spp. associated with Q. ilex is proposed in this study by using Next Generation Sequencing (NGS) in six Q. ilex stands located in three regions in Spain. Thirty-seven Phytophthora phylotypes belonging to clades 1 to 12, except for clades 4, 5 and 11, are detected in this study, which represents a high diversity of Phytophthora species in holm oak Spanish forests. Phytophthora chlamydospora, P. citrophthora, P. gonapodyides, P. lacustris, P. meadii, P. plurivora, P. pseudocryptogea, P. psychrophila and P. quercina were present in the three regions. Seven phylotypes could not be associated with known Phytophthora species, so they were putatively named as Phytophthora sp. Most of the detected phylotypes corresponded to terrestrial Phytophthora species but aquatic species from clades 6 and 9 were also present in all regions. We would like to thank M. Leon from the Instituto Agroforestal Mediterraneo-UPV (Spain) for its technical assistance. This research was supported by funding from the project AGL2011-30438-C02-01 (Ministerio de Economia y Competitividad, Spain) and Euphresco [Instituto Nacional de Investigacion y Tecnologia Agraria y Agroalimentaria (EUPHESCO-CEP: "Current and Emerging Phytophthoras: Research Supporting Risk Assesssment and Risk Management")]. Mora-Sala, B.; Gramaje Pérez, D.; Abad Campos, P.; Berbegal Martinez, M. (2019). Diversity of Phytophthora Species Associated with Quercus ilex L. in Three Spanish Regions Evaluated by NGS. Forests. 10(11):1-16. https://doi.org/10.3390/f10110979 Mideros, M. F., Turissini, D. A., Guayazán, N., Ibarra-Avila, H., Danies, G., Cárdenas, M., … Restrepo, S. (2018). Phytophthora betacei, a new species within Phytophthora clade 1c causing late blight on Solanum betaceum in Colombia. Persoonia - Molecular Phylogeny and Evolution of Fungi, 41(1), 39-55. doi:10.3767/persoonia.2018.41.03 Tremblay, É. D., Duceppe, M.-O., Bérubé, J. A., Kimoto, T., Lemieux, C., & Bilodeau, G. J. (2018). Screening for Exotic Forest Pathogens to Increase Survey Capacity Using Metagenomics. Phytopathology®, 108(12), 1509-1521. doi:10.1094/phyto-02-18-0028-r Brasier, C. M. (1992). Oak tree mortality in Iberia. Nature, 360(6404), 539-539. doi:10.1038/360539a0 Jung, T., Blaschke, H., & Neumann, P. (1996). Isolation, identification and pathogenicity of Phytophthora species from declining oak stands. Forest Pathology, 26(5), 253-272. doi:10.1111/j.1439-0329.1996.tb00846.x Jung, T., Cooke, D. E. L., Blaschke, H., Duncan, J. M., & Oßwald, W. (1999). Phytophthora quercina sp. nov., causing root rot of European oaks. Mycological Research, 103(7), 785-798. doi:10.1017/s0953756298007734 Jung, T., Blaschke, H., & Osswald, W. (2000). Involvement of soilborne Phytophthora species in Central European oak decline and the effect of site factors on the disease. Plant Pathology, 49(6), 706-718. doi:10.1046/j.1365-3059.2000.00521.x Jung, T., Hansen, E. M., Winton, L., Oswald, W., & Delatour, C. (2002). Three new species of Phytophthora from European oak forests. Mycological Research, 106(4), 397-411. doi:10.1017/s0953756202005622 Jung, T., Nechwatal, J., Cooke, D. E. L., Hartmann, G., Blaschke, M., Oßwald, W. F., … Delatour, C. (2003). Phytophthora pseudosyringae sp. nov., a new species causing root and collar rot of deciduous tree species in Europe. Mycological Research, 107(7), 772-789. doi:10.1017/s0953756203008074 JUNG, T., HUDLER, G. W., JENSEN-TRACY, S. L., GRIFFITHS, H. M., FLEISCHMANN, F., & OSSWALD, W. (2006). Involvement of Phytophthora species in the decline of European beech in Europe and the USA. Mycologist, 19(04), 159. doi:10.1017/s0269915x05004052 Jung, T., Jung, M. H., Cacciola, S. O., Cech, T., Bakonyi, J., Seress, D., … Scanu, B. (2017). Multiple new cryptic pathogenic Phytophthora species from Fagaceae forests in Austria, Italy and Portugal. IMA Fungus, 8(2), 219-244. doi:10.5598/imafungus.2017.08.02.02 Robin, C., Desprez-Loustau, M.-L., Capron, G., & Delatour, C. (1998). First record of Phytophthora cinnamomi on cork and holm oaks in France and evidence of pathogenicity. Annales des Sciences Forestières, 55(8), 869-883. doi:10.1051/forest:19980801 Hansen, E., & Delatour, C. (1999). Phytophthora species in oak forests of north-east France. ANNALS OF FOREST SCIENCE, 56(7), 539-547. doi:10.1051/forest:19990702 VETTRAINO, A. M., BARZANTI, G. P., BIANCO, M. C., RAGAZZI, A., CAPRETTI, P., PAOLETTI, E., … VANNINI, A. (2002). Occurrence of Phytophthora species in oak stands in Italy and their association with declining oak trees. Forest Pathology, 32(1), 19-28. doi:10.1046/j.1439-0329.2002.00264.x Vettraino, A. M., Morel, O., Perlerou, C., Robin, C., Diamandis, S., & Vannini, A. (2005). Occurrence and distribution of Phytophthora species in European chestnut stands, and their association with Ink Disease and crown decline. European Journal of Plant Pathology, 111(2), 169-180. doi:10.1007/s10658-004-1882-0 Rizzo, D. M., Garbelotto, M., Davidson, J. M., Slaughter, G. W., & Koike, S. T. (2002). Phytophthora ramorum as the Cause of Extensive Mortality of Quercus spp. and Lithocarpus densiflorus in California. Plant Disease, 86(3), 205-214. doi:10.1094/pdis.2002.86.3.205 Rizzo, D. M., & Garbelotto, M. (2003). Sudden oak death: endangering California and Oregon forest ecosystems. Frontiers in Ecology and the Environment, 1(4), 197-204. doi:10.1890/1540-9295(2003)001[0197:sodeca]2.0.co;2 Balci, Y., & Halmschlager, E. (2003). Incidence of Phytophthora species in oak forests in Austria and their possible involvement in oak decline. Forest Pathology, 33(3), 157-174. doi:10.1046/j.1439-0329.2003.00318.x Balci, Y., & Halmschlager, E. (2003). Phytophthora species in oak ecosystems in Turkey and their association with declining oak trees. Plant Pathology, 52(6), 694-702. doi:10.1111/j.1365-3059.2003.00919.x Balci, Y., Balci, S., Eggers, J., MacDonald, W. L., Juzwik, J., Long, R. P., & Gottschalk, K. W. (2007). Phytophthora spp. Associated with Forest Soils in Eastern and North-Central U.S. Oak Ecosystems. Plant Disease, 91(6), 705-710. doi:10.1094/pdis-91-6-0705 Balci, Y., Balci, S., MacDonald, W. L., & Gottschalk, K. W. (2008). Relative susceptibility of oaks to seven species ofPhytophthoraisolated from oak forest soils. Forest Pathology, 38(6), 394-409. doi:10.1111/j.1439-0329.2008.00559.x Balci, Y., Balci, S., Blair, J. E., Park, S.-Y., Kang, S., & Macdonald, W. L. (2008). Phytophthora quercetorum sp. nov., a novel species isolated from eastern and north-central USA oak forest soils. Mycological Research, 112(8), 906-916. doi:10.1016/j.mycres.2008.02.008 Vannini, A., & Vettraino, A. (2011). Phytophthora cambivora. Forest Phytophthoras, 1(1). doi:10.5399/osu/fp.1.1.1811 Pérez-Sierra, A., López-García, C., León, M., García-Jiménez, J., Abad-Campos, P., & Jung, T. (2013). Previously unrecorded low-temperaturePhytophthoraspecies associated withQuercusdecline in a Mediterranean forest in eastern Spain. Forest Pathology, 43(4), 331-339. doi:10.1111/efp.12037 Brasier, C. (1996). Phytophthora cinnamomi and oak decline in southern Europe. Environmental constraints including climate change. Annales des Sciences Forestières, 53(2-3), 347-358. doi:10.1051/forest:19960217 Jung, T., Orlikowski, L., Henricot, B., Abad-Campos, P., Aday, A. G., Aguín Casal, O., … Chavarriaga, D. (2015). WidespreadPhytophthorainfestations in European nurseries put forest, semi-natural and horticultural ecosystems at high risk of Phytophthora diseases. Forest Pathology, 46(2), 134-163. doi:10.1111/efp.12239 Vannini, A., Bruni, N., Tomassini, A., Franceschini, S., & Vettraino, A. M. (2013). Pyrosequencing of environmental soil samples reveals biodiversity of thePhytophthoraresident community in chestnut forests. FEMS Microbiology Ecology, 85(3), 433-442. doi:10.1111/1574-6941.12132 Jankowiak, R., Stępniewska, H., Bilański, P., & Kolařík, M. (2014). Occurrence of Phytophthora plurivora and other Phytophthora species in oak forests of southern Poland and their association with site conditions and the health status of trees. Folia Microbiologica, 59(6), 531-542. doi:10.1007/s12223-014-0331-5 Scanu, B., Linaldeddu, B. T., Deidda, A., & Jung, T. (2015). Diversity of Phytophthora Species from Declining Mediterranean Maquis Vegetation, including Two New Species, Phytophthora crassamura and P. ornamentata sp. nov. PLOS ONE, 10(12), e0143234. doi:10.1371/journal.pone.0143234 Corcobado, T., Miranda-Torres, J. J., Martín-García, J., Jung, T., & Solla, A. (2016). Early survival of Quercus ilex subspecies from different populations after infections and co-infections by multiple Phytophthora species. Plant Pathology, 66(5), 792-804. doi:10.1111/ppa.12627 Corcobado, T., Cubera, E., Pérez-Sierra, A., Jung, T., & Solla, A. (2010). First report ofPhytophthora gonapodyidesinvolved in the decline ofQuercus ilexin xeric conditions in Spain. New Disease Reports, 22, 33. doi:10.5197/j.2044-0588.2010.022.033 Hansen, E. M., Reeser, P. W., & Sutton, W. (2012). PhytophthoraBeyond Agriculture. Annual Review of Phytopathology, 50(1), 359-378. doi:10.1146/annurev-phyto-081211-172946 Català, S., Pérez-Sierra, A., & Abad-Campos, P. (2015). The Use of Genus-Specific Amplicon Pyrosequencing to Assess Phytophthora Species Diversity Using eDNA from Soil and Water in Northern Spain. PLOS ONE, 10(3), e0119311. doi:10.1371/journal.pone.0119311 Jung, T., La Spada, F., Pane, A., Aloi, F., Evoli, M., Horta Jung, M., … Cacciola, S. O. (2019). Diversity and Distribution of Phytophthora Species in Protected Natural Areas in Sicily. Forests, 10(3), 259. doi:10.3390/f10030259 Jung, T., Pérez-Sierra, A., Durán, A., Jung, M. H., Balci, Y., & Scanu, B. (2018). Canker and decline diseases caused by soil- and airborne Phytophthora species in forests and woodlands. Persoonia - Molecular Phylogeny and Evolution of Fungi, 40(1), 182-220. doi:10.3767/persoonia.2018.40.08 Brasier, C. M. (2008). The biosecurity threat to the UK and global environment from international trade in plants. Plant Pathology, 57(5), 792-808. doi:10.1111/j.1365-3059.2008.01886.x O’Brien, P. A., Williams, N., & Hardy, G. E. S. (2009). DetectingPhytophthora. Critical Reviews in Microbiology, 35(3), 169-181. doi:10.1080/10408410902831518 Berlanas, C., Berbegal, M., Elena, G., Laidani, M., Cibriain, J. F., Sagües, A., & Gramaje, D. (2019). The Fungal and Bacterial Rhizosphere Microbiome Associated With Grapevine Rootstock Genotypes in Mature and Young Vineyards. Frontiers in Microbiology, 10. doi:10.3389/fmicb.2019.01142 Oulas, A., Pavloudi, C., Polymenakou, P., Pavlopoulos, G. A., Papanikolaou, N., Kotoulas, G., … Iliopoulos, loannis. (2015). Metagenomics: Tools and Insights for Analyzing Next-Generation Sequencing Data Derived from Biodiversity Studies. Bioinformatics and Biology Insights, 9, BBI.S12462. doi:10.4137/bbi.s12462 Vettraino, A. M., Bonants, P., Tomassini, A., Bruni, N., & Vannini, A. (2012). Pyrosequencing as a tool for the detection ofPhytophthoraspecies: error rate and risk of false Molecular Operational Taxonomic Units. Letters in Applied Microbiology, 55(5), 390-396. doi:10.1111/j.1472-765x.2012.03310.x Català, S., Berbegal, M., Pérez-Sierra, A., & Abad-Campos, P. (2016). Metabarcoding and development of new real-time specific assays revealPhytophthoraspecies diversity in holm oak forests in eastern Spain. Plant Pathology, 66(1), 115-123. doi:10.1111/ppa.12541 Prigigallo, M. I., Abdelfattah, A., Cacciola, S. O., Faedda, R., Sanzani, S. M., Cooke, D. E. L., & Schena, L. (2016). Metabarcoding Analysis of Phytophthora Diversity Using Genus-Specific Primers and 454 Pyrosequencing. Phytopathology®, 106(3), 305-313. doi:10.1094/phyto-07-15-0167-r Scibetta, S., Schena, L., Chimento, A., Cacciola, S. O., & Cooke, D. E. L. (2012). A molecular method to assess Phytophthora diversity in environmental samples. Journal of Microbiological Methods, 88(3), 356-368. doi:10.1016/j.mimet.2011.12.012 Burgess, T. I., McDougall, K. L., Scott, P. M., Hardy, G. E. S., & Garnas, J. (2018). Predictors of Phytophthora diversity and community composition in natural areas across diverse Australian ecoregions. Ecography, 42(3), 565-577. doi:10.1111/ecog.03904 Mora-Sala, B., Berbegal, M., & Abad-Campos, P. (2018). The Use of qPCR Reveals a High Frequency of Phytophthora quercina in Two Spanish Holm Oak Areas. Forests, 9(11), 697. doi:10.3390/f9110697 Altschul, S. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25(17), 3389-3402. doi:10.1093/nar/25.17.3389 Park, J., Park, B., Veeraraghavan, N., Jung, K., Lee, Y.-H., Blair, J. E., … Kang, S. (2008). Phytophthora Database: A Forensic Database Supporting the Identification and Monitoring of Phytophthora. Plant Disease, 92(6), 966-972. doi:10.1094/pdis-92-6-0966 Edgar, R. C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32(5), 1792-1797. doi:10.1093/nar/gkh340 Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution, 30(12), 2725-2729. doi:10.1093/molbev/mst197 Glynou, K., Nam, B., Thines, M., & Maciá-Vicente, J. G. (2017). Facultative root-colonizing fungi dominate endophytic assemblages in roots of nonmycorrhizal Microthlaspi species. New Phytologist, 217(3), 1190-1202. doi:10.1111/nph.14873 Dhariwal, A., Chong, J., Habib, S., King, I. L., Agellon, L. B., & Xia, J. (2017). MicrobiomeAnalyst: a web-based tool for comprehensive statistical, visual and meta-analysis of microbiome data. Nucleic Acids Research, 45(W1), W180-W188. doi:10.1093/nar/gkx295 Ruiz Gómez, F. J., Navarro-Cerrillo, R. M., Pérez-de-Luque, A., Oβwald, W., Vannini, A., & Morales-Rodríguez, C. (2019). Assessment of functional and structural changes of soil fungal and oomycete communities in holm oak declined dehesas through metabarcoding analysis. Scientific Reports, 9(1). doi:10.1038/s41598-019-41804-y Redondo, M. A., Boberg, J., Stenlid, J., & Oliva, J. (2018). Contrasting distribution patterns between aquatic and terrestrial Phytophthora species along a climatic gradient are linked to functional traits. The ISME Journal, 12(12), 2967-2980. doi:10.1038/s41396-018-0229-3 BRASIER, C. M., ROBREDO, F., & FERRAZ, J. F. P. (1993). Evidence forPhytophthora cinnamomiinvolvement in Iberian oak decline. Plant Pathology, 42(1), 140-145. doi:10.1111/j.1365-3059.1993.tb01482.x Gallego, B. F. J., de Algaba, A. P., & Fernandez-Escobar, R. (1999). Etiology of oak decline in Spain. Forest Pathology, 29(1), 17-27. doi:10.1046/j.1439-0329.1999.00128.x SANCHEZ, M. E., CAETANO, P., FERRAZ, J., & TRAPERO, A. (2002). Phytophthora disease of Quercus ilex in south-western Spain. Forest Pathology, 32(1), 5-18. doi:10.1046/j.1439-0329.2002.00261.x De Sampaio e Paiva Camilo-Alves, C., da Clara, M. I. E., & de Almeida Ribeiro, N. M. C. (2013). Decline of Mediterranean oak trees and its association with Phytophthora cinnamomi: a review. European Journal of Forest Research, 132(3), 411-432. doi:10.1007/s10342-013-0688-z Serrano, M. S., De Vita, P., Fernández-Rebollo, P., & Sánchez Hernández, M. E. (2011). Calcium fertilizers induce soil suppressiveness to Phytophthora cinnamomi root rot of Quercus ilex. European Journal of Plant Pathology, 132(2), 271-279. doi:10.1007/s10658-011-9871-6 Mora-Sala, B., Abad-Campos, P., & Berbegal, M. (2018). Response of Quercus ilex seedlings to Phytophthora spp. root infection in a soil infestation test. European Journal of Plant Pathology, 154(2), 215-225. doi:10.1007/s10658-018-01650-6 Jung, T., & Burgess, T. I. (2009). Re-evaluation of Phytophthora citricola isolates from multiple woody hosts in Europe and North America reveals a new species, Phytophthora plurivora sp. nov. Persoonia - Molecular Phylogeny and Evolution of Fungi, 22(1), 95-110. doi:10.3767/003158509x442612 Ioos, R., Laugustin, L., Rose, S., Tourvieille, J., & Tourvieille de Labrouhe, D. (2007). Development of a PCR test to detect the downy mildew causal agent Plasmopara halstedii in sunflower seeds. Plant Pathology, 56(2), 209-218. doi:10.1111/j.1365-3059.2006.01500.x Zhao, J., Wang, X. J., Chen, C. Q., Huang, L. L., & Kang, Z. S. (2007). A PCR-Based Assay for Detection of Puccinia striiformis f. sp. tritici in Wheat. Plant Disease, 91(12), 1669-1674. doi:10.1094/pdis-91-12-1669 Alaei, H., Baeyen, S., Maes, M., Höfte, M., & Heungens, K. (2009). Molecular detection of Puccinia horiana in Chrysanthemum x morifolium through conventional and real-time PCR. Journal of Microbiological Methods, 76(2), 136-145. doi:10.1016/j.mimet.2008.10.001 Mrázková, M., Černý, K., Tomšovský, M., Strnadová, V., Gregorová, B., Holub, V., … Hejná, M. (2013). Occurrence of Phytophthora multivora and Phytophthora plurivora in the Czech Republic. Plant Protection Science, 49(No. 4), 155-164. doi:10.17221/74/2012-pps

TABERLET, P., COISSAC, E., HAJIBABAEI, M., & RIESEBERG, L. H. (2012). Environmental DNA. Molecular Ecology, 21(8), 1789-1793. doi:10.1111/j.1365-294x.2012.05542.x

TABERLET, P., COISSAC, E., HAJIBABAEI, M., thereto RIESEBERG, L.H. (2012). Environmental DNA. Ecology molecular, 21 (8), 1789-1793. doi:10.1111/j.1365-294x.2012.05542.x

Diversity of Phytophthora Species Associated with Quercus ilex L. in Three Spanish Regions Evaluated by NGS

Abstract