Detection and identification of Phytophthora spp. in woody plant nurseries and holm oak forests

Tesis por compendio[ES] Phytophthora es uno de los géneros fitopatógenos más relevantes y agresivos en la agricultura y silvicultura. Muestreos realizados en la última década han revelado una gran cantidad de interacciones entre especies de Phytophthora y plantas, desconocidas con anterioridad. La introducción de nuevos patógenos de suelo, como Phytophthora en los bosques de Fagaceae, modifica la comunidad microbiana presente en la rizosfera, con importantes consecuencias ambientales y económicas. El género Quercus es uno de los géneros de Fagaceae más extendidos en Europa, y Quercus ilex es la especie dominante en España. El vínculo entre la dispersión de Phytophthora en los ecosistemas naturales y las actividades del ser humano, ha sido previamente estudiado. Numerosos muestreos en viveros y espacios públicos, mostraron la presencia de gran diversidad de especies de Phytophthora que podían suponer una amenaza para la producción y los ecosistemas naturales. En este contexto, se realizó un muestreo de viveros ornamentales y forestales en cuatro comunidades autónomas españolas, centrándose en los posibles síntomas asociados a Phytophthora en diferentes hospedantes e incluyendo muestras de agua de los viveros. Los resultados mostraron 17 filotipos de Phytophthora que afectan a 22 especies vegetales incluidas en 19 géneros. Algunas de estas interacciones se citaron por primera vez en España. Entre los patógenos de suelo aislados en los viveros, se identificó una gran cantidad de formas asexuales tipo Cylindrocarpon en las raíces de hospedantes leñosos. Se caracterizó una colección de aislados mediante estudios morfológicos y moleculares. Se identificaron 12 especies pertenecientes a los géneros Cylindrodendrum, Dactylonectria e Ilyonectria en hospedantes pertenecientes a 15 géneros y otras cuatro nuevas especies se describieron. El estudio demostró la prevalencia de este grupo fúngico asociado con plántulas de diversos hospedantes que muestran síntomas de decaimiento en viveros forestales. Se evaluó la susceptibilidad de Q. ilex a la inoculación con ocho especies de Phytophthora obtenidas de muestreos en viveros. Las especies más agresivas fueron Phytophthora cinnamomi, Phytophthora cryptogea, Phytophthora gonapodyides, Phytophthora plurivora y Phytophthora psychrophila, seguidas de Phytophthora megasperma, mientras que Phytophthora quercina y Phytophthora nicotianae fueron las especies menos agresivas. Los resultados obtenidos en el ensayo de patogenicidad confirmaron que todas las especies de Phytophthora evaluadas podrían representar una amenaza para los encinares. En este contexto, se realizó un estudio para verificar la presencia y / o detección de especies de Phytophthora en dos áreas de España (dehesas del sudoeste y bosque del noreste) utilizando diferentes métodos de aislamiento y detección. El aislamiento directo y el método de trampeo vegetal en muestras obtenidas a partir de encinas con y sin decaimiento, identificaron Phytophthora cambivora, P. cinnamomi, P. gonapodyides, P. megasperma y Phytophthora pseudocryptogea en las dehesas, mientras que, en el bosque del noreste, no se aisló Phytophthora spp. Los análisis estadísticos indicaron que no había una relación significativa entre la frecuencia de aislamiento de las especies de Phytophthora y la expresión de los síntomas de la enfermedad en las encinas de las dehesas. Además, P. quercina se detectó con mayor frecuencia que P. cinnamomi en las dos áreas estudiadas utilizando sondas TaqMan de PCR a tiempo real. Se evaluaron seis masas de Q. ilex ubicadas en tres comunidades autónomas de España mediante “Next Generation Sequencing” (NGS) para tener un mayor conocimiento sobre la diversidad de Phytophthora spp. en los bosques de encinas. Se detectaron 37 filotipos de Phytophthora pertenecientes a los clados 1 al 12, excepto los clados 4, 5 y 11, lo que demuestra una gran diversidad de Phytophthora en los encinares estudiados. Los filotipos más abundantes fueron P. quercina, P. psychrophila, P. cinnamomi y P. plurivora.[EN] Phytophthora is one of the most relevant and aggressive plant pathogenic genus in agriculture and forestry. Due to the increasing environmental threat of invasive plant pathogens, monitoring new areas in the last decade has revealed a large number of new Phytophthora species-plant host interactions. The introduction of soilborne pathogens, such as Phytophthora in Fagaceae forests modifies the microbial community present in the rhizosphere with relevant environmental and economic consequences. The genus Quercus is one of the most extended Fagaceae genera in Europe, and Q. ilex is the dominant tree in Spain. The link between Phytophthora dispersion in natural ecosystems and human derived activities has been previously studied. Numerous sampling in nurseries and public spaces revealed a great diversity of Phytophthora species that compromised production and threated natural ecosystems. In this context, sampling ornamental and forest nurseries in four Spanish regions was carried out focusing on possible symptoms associated to Phytophthora on different hosts and including water samples from the nurseries. The results showed 17 Phytophthora phylotypes affecting 22 plant species included in 19 plant genera and some of them reported for the first time in Spain. Among the soilborne pathogens isolated in the nurseries, a large number of Cylindrocarpon-like asexual morphs were identified from the roots of woody hosts. A collection of Cylindrocarpon-like isolates recovered from Spanish nurseries was characterised by morphological and molecular studies. Twelve species belonging to the genera Cylindrodendrum, Dactylonectria and Ilyonectria were identified from damaged roots of 15 different host genera and other four species were newly described. The study demonstrated the prevalence of this fungal group associated with seedlings of diverse hosts showing decline symptoms in forest nurseries in Spain. The susceptibility of Quercus ilex to the inoculation with eight Phytophthora species obtained from nurseries was evaluated. The most aggressive species were Phytophthora cinnamomi, Phytophthora cryptogea, Phytophthora gonapodyides, Phytophthora plurivora and Phytophthora psychrophila followed by Phytophthora megasperma, while Phytophthora quercina and Phytophthora nicotianae were the least aggressive species. Results obtained in the pathogenicity test confirmed that all Phytophthora species tested could represent a threat to holm oak ecosystems. In this context, a study to verify the presence and/or detection of Phytophthora species was conducted in two holm oak areas of Spain (southwestern dehesas and northeastern woodland) using different isolation and detection methods. Direct isolation and baiting methods in declining and non-declining holm oak trees revealed Phytophthora cambivora, P. cinnamomi, P. gonapodyides, P. megasperma, and Phytophthora pseudocryptogea in the dehesas, while in the northeastern woodland, no Phytophthora spp. were recovered. Statistical analyses indicated that there was not a significant relationship between the Phytophthora spp. isolation frequency and the disease expression of the holm oak stands in the dehesas. Phytophthora quercina and P. cinnamomi TaqMan real-time PCR probes showed that P. quercina was detected in a higher frequency than P. cinnamomi in both studied areas. A better understanding of the Phytophthora spp. diversity in holm oak forests was assessed 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, were detected in this study, demonstrating a high diversity of Phytophthora species in holm oak Spanish forests. The most abundant phylotypes were P. quercina, P. psychrophila, P. cinnamomi and P. plurivora. In summary, this Thesis demonstrated a high diversity of Phytophthora and Cylindrocarpon-like species in Spanish nurseries, reporting new pathogen-plant host in[CA] Phytophthora és un dels gèneres fitopatògens més rellevants i agressius en l'agricultura i la silvicultura. Mostrejos realitzats en l'última dècada han revelat una gran quantitat d'interaccions desconegudes fins ara entre espècies de Phytophthora i plantes. La introducció de nous patògens del sòl, com Phytophthora en els boscos de Fagaceae, modifica la comunitat microbiana present en la rizosfera, amb importants conseqüències ambientals i econòmiques. El gènere Quercus és un dels gèneres de Fagaceae més estesos a Europa, i Quercus ilex és l'espècie dominant a Espanya. El vincle entre la dispersió de Phytophthora en els ecosistemes naturals i les activitats de l'ésser humà, ja ha sigut estudiat prèviament. Nombrosos mostrejos en vivers i espais públics, van mostrar la presència d'una gran diversitat d'espècies de Phytophthora que podien suposar una amenaça per a la producció i els ecosistemes naturals. En aquest context, es va realitzar un mostreig de vivers ornamentals i forestals en quatre comunitats autònomes espanyoles, centrant-se en els possibles símptomes associats a Phytophthora en diferents hostes i incloent mostres d'aigua dels vivers. Els resultats van mostrar 17 filotipus de Phytophthora que afecten 22 espècies vegetals incloses en 19 gèneres. Algunes d'aquestes 'interaccions es van citar per primera vegada a Espanya. Entre els patògens del sòl aïllats en els vivers, es va identificar una gran quantitat de formes asexuals tipus Cylindrocarpon en les arrels de plantes llenyoses. Es va caracteritzar una col·lecció d'aïllats mitjançant estudis morfològics i moleculars. Es van identificar 12 espècies pertanyents als gèneres Cylindrodendrum, Dactylonectria i Ilyonectria en hostes pertanyents a 15 gèneres i altres quatre noves espècies es van descriure. L'estudi va demostrar la prevalença d'aquest grup fúngic associat amb plàntules de diversos hostes que mostren símptomes de decaïment en vivers forestals espanyols. Es va avaluar la susceptibilitat de Q. ilex a la inoculació amb huit espècies de Phytophthora obtingudes de mostrejos en vivers. Les espècies més agressives van ser Phytophthora cinnamomi, Phytophthora cryptogea, Phytophthora gonapodyides, Phytophthora plurivora i Phytophthora psychrophila, seguides de Phytophthora megasperma, mentre que Phytophthora quercina i Phytophthora nicotianae van ser les espècies menys agressives. Els resultats obtinguts en l'assaig de patogenicitat van confirmar que totes les espècies de Phytophthora avaluades podrien representar una amenaça per als ecosistemes d'alzines. En aquest context, es va realitzar un estudi per a verificar la presència i / o detecció d'espècies de Phytophthora en dues àrees d'Espanya (deveses del sud-oest i bosc del nord-est) utilitzant diferents mètodes d'aïllament i detecció. L'aïllament directe i el mètode de parany vegetal en mostres obtingudes a partir d'alzines, amb i sense decaïment, van identificar Phytophthora cambivora, P. cinnamomi, P. gonapodyides, P. megasperma i Phytophthora pseudocryptogea en les deveses, mentre que, en el bosc del nord-est, no es va aïllar Phytophthora spp. Les anàlisis estadístiques van indicar que no hi havia una relació significativa entre la freqüència d'aïllament de les espècies de Phytophthora i l'expressió dels símptomes de la malaltia en les alzines en les deveses. A més, P. quercina es va detectar amb major freqüència que P. cinnamomi en les dues àrees estudiades utilitzant sondes TaqMan de PCR a temps real. Es van avaluar sis masses de Q. ilex situades en tres comunitats autònomes d'Espanya mitjançant "Next Generation Sequencing" (NGS) per a tindre un major coneixement sobre la diversitat de Phytophthora spp. en els boscos d'alzines. Es van detectar 37 filotipus de Phytophthora pertanyents als clades 1 al 12, excepte els clades 4, 5 i 11, la qual cosa demostra una gran diversitat d'espècies de Phytophthora en els alzinars estudiats. Els filotipus més abundants van ser P. quercina, P. psychrophila, P. cinnamomi i P. plurivora.Mora Sala, B. (2020). Detection and identification of Phytophthora spp. in woody plant nurseries and holm oak forests [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/153808TESISCompendi

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

[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/f10110979S1161011Mideros, 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.2018.41.03Tremblay, 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-rBrasier, C. M. (1992). Oak tree mortality in Iberia. Nature, 360(6404), 539-539. doi:10.1038/360539a0Jung, 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.xJung, 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/s0953756298007734Jung, 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.xJung, 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/s0953756202005622Jung, 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/s0953756203008074JUNG, 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/s0269915x05004052Jung, 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.2017.08.02.02Robin, 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:19980801Hansen, 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:19990702VETTRAINO, 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.xVettraino, 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/s10658-004-1882-0Rizzo, 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.2002.86.3.205Rizzo, 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;2Balci, 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.xBalci, 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.xBalci, 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-91-6-0705Balci, 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.xBalci, 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.2008.02.008Vannini, A., & Vettraino, A. (2011). Phytophthora cambivora. Forest Phytophthoras, 1(1). doi:10.5399/osu/fp.1.1.1811Pérez-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.12037Brasier, 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:19960217Jung, 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.12239Vannini, 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:10.1111/1574-6941.12132Jankowiak, 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/s12223-014-0331-5Scanu, 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.0143234Corcobado, 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.12627Corcobado, 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.2044-0588.2010.022.033Hansen, E. M., Reeser, P. W., & Sutton, W. (2012). PhytophthoraBeyond Agriculture. Annual Review of Phytopathology, 50(1), 359-378. doi:10.1146/annurev-phyto-081211-172946Català, 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.0119311Jung, 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/f10030259Jung, 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.2018.40.08Brasier, 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.xO’Brien, P. A., Williams, N., & Hardy, G. E. S. (2009). DetectingPhytophthora. Critical Reviews in Microbiology, 35(3), 169-181. doi:10.1080/10408410902831518Berlanas, 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.2019.01142TABERLET, 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.xOulas, 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.s12462Vettraino, 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.xCatalà, 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.12541Prigigallo, 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-rScibetta, 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.2011.12.012Burgess, 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.03904Mora-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/f9110697Altschul, 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/25.17.3389Park, 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-92-6-0966Edgar, R. C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32(5), 1792-1797. doi:10.1093/nar/gkh340Tamura, 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/mst197Felsenstein, J. (1985). CONFIDENCE LIMITS ON PHYLOGENIES: AN APPROACH USING THE BOOTSTRAP. Evolution, 39(4), 783-791. doi:10.1111/j.1558-5646.1985.tb00420.xGlynou, 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.14873Dhariwal, 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/gkx295Ruiz 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-yRedondo, 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/s41396-018-0229-3BRASIER, 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.xGallego, 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.xSANCHEZ, 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.xDe 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-zSerrano, 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/s10658-011-9871-6Mora-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/s10658-018-01650-6Jung, 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:10.3767/003158509x442612Ioos, 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.xZhao, 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-91-12-1669Alaei, 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.2008.10.001Mrázková, 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-pp

The Use of qPCR Reveals a High Frequency of Phytophthora quercina in Two Spanish Holm Oak Areas

[EN] The struggling Spanish holm oak woodland situation associated with Phytophthora root rot has been studied for a long time. Phytophthora cinnamomi is considered the main, but not the only species responsible for the decline scenario. This study veri¿es the presence and/or detection of Phytophthora species in two holm oak areas of Spain (southwestern ¿dehesas¿ and northeastern woodland)usingdifferentisolationanddetectionapproaches. Directisolationandbaitingmethodsin declining and non-declining holm oak trees revealed Phytophthoracambivora, Phytophthoracinnamomi, Phytophthoragonapodyides, Phytophthoramegasperma, and Phytophthorapseudocryptogea in the dehesas, while in the northeastern woodland, no Phytophthora spp. were recovered. Statistical analyses indicated that there was not a signi¿cant relationship between the Phytophthora spp. isolation frequencyandthediseaseexpressionoftheholmoakstandsinthedehesas. Phytophthoraquercinaand P.cinnamomiTaqManreal-timePCRprobesshowedthatbothP.cinnamomiandP.quercinaareinvolved in the holm oak decline in Spain, but P. quercina was detected in a higher frequency than P. cinnamomi in both studied areas. Thus, this study demonstrates that molecular approaches complement direct isolation techniques in natural and seminatural ecosystem surveys to determine the presence and distribution of Phytophthora spp. This is the ¿rst report of P. pseudocryptogea in Europe and its role in the holm oak decline should be further studied.This research was supported by funding from the project AGL2011-30438-C02-01 (Ministerio de Economia y Competitividad, Spain).Mora-Sala, B.; Berbegal Martinez, 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):1-14. https://doi.org/10.3390/f9110697S11491

Survey, identification, and characterization of Cylindrocarpon-like asexual morphs in Spanish forest nurseries

[EN] Cylindrocarpon-like asexual morphs infect herbaceous and woody plants, mainly in agricultural scenarios, but also in forestry systems. The aim of the present study was to characterize a collection of Cylindrocarpon-like isolates recovered from the roots of a broad range of forest hosts from nurseries showing decline by morphological and molecular studies. Between 2009 and 2012, 17 forest nurseries in Spain were surveyed and a total of 103 Cylindrocarpon-like isolates were obtained. Isolates were identified based on DNA sequences of the partial gene regions histone H3 (his3). For the new species, the internal transcribed spacer and intervening 5.8S nrRNA gene (ITS) region, beta-tubulin (tub2), and translation elongation factor 1-alpha (tefl) were also used to determine their phylogenetic position. Twelve species belonging to the genera Cylindrodendrum, Dactylonectria, and Ilyonectria were identified from damaged roots of 15 different host genera. The species C. alicantinum, D. macrodidyma, D. novozelandica, D. pauciseptata, D. pinicola, D. torresensis, I. capensis, I. cyclaminicola, I. liriodendri, I. pseudodestructans, I. robusta, and I. rufa were identified. In addition, two Dactylonectria species (D. hispanica sp. nov. and D. valentina sp. nov.), one Ilyonectria species (I. ilicicola sp. nov.), and one Neonectria species (N. quercicola sp. nov.) are newly described. The present study demonstrates the prevalence of this fungal group associated with seedlings of diverse hosts showing decline symptoms in forest nurseries in Spain.This research was supported by funding from the Spanish project AGL2011-30438-C02-01 (Ministerio de Economia y Competitividad, Spain). It was also funded by Portuguese national funds through Fundacao para a Ciencia e a Tecnologia grant SFRH/BPD/84508/2012 for Ana Cabral and FCT Unit funding UID/AGR/04129/2013). C. Agusti-Brisach is the holder of a 'Juan de la Cierva-Formacion' fellowship from MINECO (Spain).Mora-Sala, B.; Cabral, A.; León Santana, M.; Agusti Brisach, C.; Armengol Fortí, J.; Abad Campos, P. (2018). Survey, identification, and characterization of Cylindrocarpon-like asexual morphs in Spanish forest nurseries. Plant Disease. 102(11):2083-2100. https://doi.org/10.1094/PDIS-01-18-0171-RES208321001021

Response of Quercus ilex seedlings to Phytophthora spp. root infection in a soil infestation test

[EN] Phytophthora species are the main agents associated with oak (Quercus spp.) decline, together with the changing environmental conditions and the intensive land use. The aim of this study was to evaluate the susceptibility of Quercus ilex to the inoculation with eight Phytophthora species. Seven to eight month old Q. ilex seedlings grown from acorns, obtained from two Spanish origins, were inoculated with P. cinnamomi, P. cryptogea, P. gonapodyides, P. megasperma, P. nicotianae, P. plurivora, P. psychrophila and P. quercina. All Phytophthora inoculated seedlings showed decline and symptoms including small dark necrotic root lesions, root cankers, and loss of fine roots and tap root. The most aggressive species were P. cinnamomi, P. cryptogea, P. gonapodyides, P. plurivora and P. psychrophila followed by P. megasperma., while Phytophthora quercina and P. nicotianae were the less aggressive species. Results obtained confirm that these Phytophthora species could constituted a threat to Q. ilex ecosystems and the implications are further discussed.The authors are grateful to A. Solla and his team from the Centro Universitario de Plasencia-Universidad de Extremadura (Spain) for helping in the acorns collection and to the CIEF (Centro para la Investigación y Experimentación Forestal, Generalitat Valenciana, Valencia, Spain) for providing the acorns. This research was supported by funding from the project AGL2011- 30438-C02-01 (Ministerio de Economía y Competitividad, Spain).Mora-Sala, B.; Abad Campos, P.; Berbegal Martinez, M. (2018). Response of Quercus ilex seedlings to Phytophthora spp. root infection in a soil infestation test. European Journal of Plant Pathology. https://doi.org/10.1007/s10658-018-01650-6SÁlvarez, L. A., Pérez-Sierra, A., Armengol, J., & García-Jiménez, J. (2007). Characterization of Phytophthora nicotianae isolates causing collar and root rot of lavender and rosemary in Spain. Journal of Plant Pathology, 89, 261–264.Balci, Y., & Halmschlager, E. (2003a). Incidence of Phytophthora species in oak forests in Austria and their possible involvement in oak decline. Forest Pathology, 33, 157–174.Balci, Y., & Halmschlager, E. (2003b). Phytophthora species in oak ecosystems in Turkey and their association with declining oak trees. Plant Pathology, 52, 694–702.Brasier, C. M. (1992a). Oak tree mortality in Iberia. Nature, 360, 539.Brasier, C. M. ((1992b)). Phytophthora cinnamomi as a contributory factor on European oak declines. In N. by Luisi, P. Lerario, & A. B. Vannini (Eds.), Recent Advances in Studies on Oak Decline. Proc. Int. Congress, Brindisi, Italy, September 13-18, 1992 (pp. 49–58). Italy: Università degli Studi.Brasier, C. M. (1996). Phytophthora cinnamomi and oak decline in southern Europe. Environmental constraints including climate change. Annales des Sciences Forestieres, 53, 347–358.Brasier, C. M. (2008). The biosecurity threat to the UK and global environment from international trade in plants. Plant Pathology, 57, 792–808.Brasier, C. M., Hamm, P. B., & Hansen, E. M. (1993a). Cultural characters, protein patterns and unusual mating behaviour of P. gonapodyides isolates from Britain and North America. Mycological Research, 97, 1287–1298.Brasier, C. M., Robredo, F., & Ferraz, J. F. P. (1993b). Evidence for Phytophthora cinnamomi involvement in Iberian oak decline. Plant Pathology, 42, 140–145.Camilo-Alves, C. S. P., Clara, M. I. E., & Ribeiro, N. M. C. A. (2013). Decline of Mediterranean oak trees and its association with Phytophthora cinnamomi: a review. European Journal of Forest Research, 132, 411–432.Català, S., Berbegal, M., Pérez-Sierra, A., & Abad-Campos, P. (2017). Metabarcoding and development of new real-time specific assays reveal Phytophthora species diversity in holm oak forests in eastern Spain. Plant Pathology, 66, 115–123.Collett, D. (2003). Modelling survival data in medical research (2nd ed.). Boca Raton: Chapman & Hall/CRC, 410 pp.Corcobado, T., Cubera, E., Pérez-Sierra, A., Jung, T., & Solla, A. (2010). First report of Phytophthora gonapodyides involved in the decline of Quercus ilex in xeric conditions in Spain. New Disease Reports, 22, 33.Corcobado, T., Cubera, E., Moreno, G., & Solla, A. (2013). Quercus ilex forests are influenced by annual variations in water table, soil water deficit and fine root loss caused by Phytophthora cinnamomi. Agricultural and Forest Meteorology, 169, 92–99.Corcobado, T., Vivas, M., Moreno, G., & Solla, A. (2014). Ectomycorrhizal symbiosis in declining and non-declining Quercus ilex trees infected with or free of Phytophthora cinnamomi. Forest Ecology and Management, 324, 72–80.Corcobado, T., Miranda-Torres, J. J., Martín-García, J., Jung, T., & Solla, A. (2017). Early survival of Quercus ilex subspecies from different populations after infections and co-infections by multiple Phytophthora species. Plant Pathology, 66, 792–804.Erwin, D. C., & Ribeiro, O. K. (1996). Phytophthora diseases worldwide. St. Paul, Minnesota,USA: APS Press, American Phytopathological. Society 562pp.Gallego, F. J., Perez de Algaba, A., & Fernandez-Escobar, R. (1999). Etiology of oak decline in Spain. European Journal of Forest Pathology, 29, 17–27.Hansen, E., & Delatour, C. (1999). Phytophthora species in oak forests of north-east France. Annals of Forest Science, 56, 539–547.Hardham, A. R., & Blackman, L. M. (2010). Molecular cytology of Phytophthora plant interactions. Australasian Plant Pathology, 39, 29.Hernández-Lambraño, R. E., González-Moreno, P., & Sánchez-Agudo, J. Á. (2018). Environmental factors associated with the spatial distribution of invasive plant pathogens in the Iberian Peninsula: The case of Phytophthora cinnamomi Rands. Forest Ecology and Management, 419, 101–109.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, 531–542.Jeffers, S. N., & Aldwinckle, H. S. (1987). Enhancing detection of Phytophthora cactorum in naturally infested soil. Phytopathology, 77, 1475–1482.Jiménez, A. J., Sánchez, E. J., Romero, M. A., Belbahri, L., Trapero, A., Lefort, F., & Sánchez, M. E. (2008). Pathogenicity of Pythium spiculum and P. sterilum on feeder roots of Quercus rotundifolia. Plant Pathology, 57, 369.Jönsson, U. (2006). A conceptual model for the development of Phytophthora disease in Quercus robur. New Phytologist, 171, 55–68.Jönsson, U., Jung, T., Rosengren, U., Nihlgard, B., & Sonesson, K. (2003). Pathogenicity of Swedish isolates of Phytophthora quercina to Quercus robur in two different soils. New Phytologist, 158, 355–364.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, 22, 95–110.Jung, T., Blaschke, H., & Neumann, P. (1996). Isolation, identification and pathogenicity of Phytophthora species from declining oak stands. European Journal of Forest Pathology, 26, 253–272.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, 785–798.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, 706–718.Jung, T., Hansen, E. M., Winton, L., Oßwald, W., & Delatour, C. (2002). Three new species of Phytophthora from European oak forests. Mycological Research, 106, 397–411.Jung, T., Orlikowski, L., Henricot, B., Abad-Campos, P., Aday, A. G., Aguín Casal, O., Bakonyi, J., Cacciola, S. O., Cech, T., Chavarriaga, D., Corcobado, T., Cravador, A., Decourcelle, T., Denton, G., Diamandis, S., Dogmus-Lehtijärvi, H. T., Franceschini, A., Ginetti, B., Glavendekic, M., Hantula, J., Hartmann, G., Herrero, M., Ivic, D., Horta Jung, M., Lilja, A., Keca, N., Kramarets, V., Lyubenova, A., Machado, H., Magnano di San Lio, G., Mansilla Vázquez, P. J., Marçais, B., Matsiakh, I., Milenkovic, I., Moricca, S., Nagy, Z. Á., Nechwatal, J., Olsson, C., Oszako, T., Pane, A., Paplomatas, E. J., Pintos Varela, C., Prospero, S., Rial Martínez, C., Rigling, D., Robin, C., Rytkönen, A., Sánchez, M. E., Scanu, B., Schlenzig, A., Schumacher, J., Slavov, S., Solla, A., Sousa, E., Stenlid, J., Talgø, V., Tomic, Z., Tsopelas, P., Vannini, A., Vettraino, A. M., Wenneker, M., Woodward, S., & Peréz-Sierra, A. (2016). Widespread Phytophthora infestations in European nurseries put forest, semi-natural and horticultural ecosystems at high risk of Phytophthora diseases. Forest Pathology, 46, 134–163.Kroon, L. P., Brouwer, H., de Cock, A. W., & Govers, F. (2012). The genus Phytophthora anno 2012. Phytopathology, 102, 348–364.Linaldeddu, B. T., Scanu, B., Maddau, L., & Franceschini, A. (2014). Diplodia corticola and Phytophthora cinnamomi: the main pathogens involved in holm oak decline on Caprera Island (Italy). Forest Pathology, 44, 191–200.Luque, J., Parladé, J., & Pera, J. (2000). Pathogenicity of fungi isolated from Quercus suber in Catalonia (NE Spain). Forest Pathology, 30, 247–263.Luque, J., Parladé, J., & Pera, J. (2002). Seasonal changes in susceptibility of Quercus suber to Botryosphaeria stevensii and Phytophthora cinnamomi. Plant Pathology, 51, 338–345.MAGRAMA. (2014). Diagnóstico del Sector Forestal Español. Análisis y Prospectiva - Serie Agrinfo/Medioambiente n° 8. Ed. Ministerio de Agricultura, Alimentación y Medio Ambiente. In NIPO: 280-14-081-9.Martín-García, J., Solla, A., Corcobado, T., Siasou, E., & Woodward, S. (2015). Influence of temperature on germination of Quercus ilex in Phytophthora cinnamomi, P. gonapodyides, P. quercina and P. psychrophila infested soils. Forest Pathology, 45, 215–223.Maurel, M., Robin, C., Capron, G., & Desprez-Loustau, M. L. (2001). Effects of root damage associated with Phytophthora cinnamomi on water elations, biomass accumulation, mineral nutrition and vulnerability to water deficit of five oak and chestnut species. Forest Pathology, 31, 353–369.McKinney, H. H. (1923). Influence of soil temperature and moisture on infection of wheat seedlings by Helminthosporium sativum. Journal of Agricultural Research, 26, 195–217.Moralejo, E., Pérez-Sierra, A., Álvarez, L. A., Belbahri, L., Lefort, F., & Descals, E. (2009). Multiple alien Phytophthora taxa discovered on diseased ornamental plants in Spain. Plant Pathology, 58, 100–110.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. https://doi.org/10.3390/f9110697 .Moreira, A. C., & Martins, J. M. S. (2005). Influence of site factors on the impact of Phytophthora cinnamomi in cork oak stands in Portugal. Forest Pathology, 35, 145–162.Mrázková, M., Černý, K., Tomosovsky, M., Strnadová, V., Gregorová, B., Holub, V., Panek, M., Havrdová, L., & Hejná, M. (2013). Occurrence of Phytophthora multivora and Phytophthora plurivora in the Czech Republic. Plant Protection Science, 49, 155–164.Navarro, R. M., Gallo, L., Sánchez, M. E., Fernández, P., & Trapero, A. (2004). Efecto de distintas fertilizaciones de fósforo en la resistencia de brinzales de encina y alcornoque a Phytophthora cinnamomi Rands. Investigación Agraria. Sistemas y Recursos Forestales, 13, 550–558.Panabières, F., Ali, G., Allagui, M., Dalio, R., Gudmestad, N., Kuhn, M., Guha Roy, S., Schena, L., & Zampounis, A. (2016). Phytophthora nicotianae diseases worldwide: new knowledge of a long-recognised pathogen. Phytopathologia Mediterranea, 55, 20–40.Pérez-Sierra, A., & Jung, T. (2013). Phytophthora in woody ornamental nurseries. In: Phytophthora: A global perspective (pp. 166-177). Ed. by Lamour, K. Wallingford: CABI.Pérez-Sierra, A., Mora-Sala, B., León, M., García-Jiménez, J., & Abad-Campos, P. (2012). Enfermedades causadas por Phytophthora en viveros de plantas ornamentales. Boletín de Sanidad Vegetal-Plagas, 38, 143–156.Pérez-Sierra, A., López-García, C., León, M., García-Jiménez, J., Abad-Campos, P., & Jung, T. (2013). Previously unrecorded low-temperature Phytophthora species associated with Quercus decline in a Mediterranean forest in eastern Spain. Forest Pathology, 43, 331–339.Redondo, M. A., Pérez-Sierra, A., & Abad-Campos, P. (2015). Histology of Quercus ilex roots during infection by Phytophthora cinnamomi. Trees - Structure and Function, 29, 1943–5197.Ríos, P., Obregón, S., de Haro, A., Fernández-Rebollo, P., Serrano, M. S., & Sánchez, M. E. (2016). Effect of Brassica Biofumigant Amendments on Different Stages of the Life Cycle of Phytophthora cinnamomi. Journal of Phytopathology, 164, 582–594.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, 205–214.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 Forestieres, 55, 869–883.Robin, C., Capron, G., & Desprez-Loustau, M. L. (2001). Root infection by Phytophthora cinnamomi in seedlings of three oak species. Plant Pathology, 50, 708–716.Rodríguez-Molina, M. C., Torres-Vila, L. M., Blanco-Santos, A., Núñez, E. J. P., & Torres-Álvarez, E. (2002). Viability of holm and cork oak seedlings from acorns sown in soils naturally infected with Phytophthora cinnamomi. Forest Pathology, 32, 365–372.Romero, M. A., Sánchez, J. E., Jiménez, J. J., Belbahri, L., Trapero, A., Lefort, F., & Sánchez, M. E. (2007). New Pythium taxa causing root rot in Mediterranean Quercus species in southwest Spain and Portugal. Journal of Phytopathology, 115, 289–295.Sánchez de Lorenzo-Cáceres J. M. (2001). Guía de las plantas ornamentales. S.A. Mundi-Prensa Libros. ISBN 9788471149374. 688 pp.Sánchez, M. E., Caetano, P., Ferraz, J., & Trapero, A. (2002). Phytophtora disease of Quercus ilex in south-western Spain. Forest Pathology, 32, 5–18.Sánchez, M. E., Sánchez, J. E., Navarro, R. M., Fernández, P., & Trapero, A. (2003). Incidencia de la podredumbre radical causada por Phytophthora cinnamomi en masas de Quercus en Andalucía. Boletín de Sanidad Vegetal-Plagas, 29, 87–108.Sánchez, M. E., Andicoberry, S., & Trapero, A. (2005). Pathogenicity of three Phytophthora spp. causing late seedling rot of Quercus ilex ssp. ballota. Forest Pathology, 35, 115–125.Sánchez, M. E., Caetano, P., Romero, M. A., Navarro, R. M., & Trapero, A. (2006). Phytophthora root rot as the main factor of oak decline in southern Spain. In: Progress in Research on Phytophthora Diseases of Forest Trees. Proceedings of the Third International IUFRO Working Party S07.02.09. Meeting at Freising. Germany 11-18 September 2004. Brasier C. M., Jung T., Oßwald W. (Eds). Forest Research. Farnham, UK. pp. 149-154.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. https://doi.org/10.1371/journal.pone.0143234 .Schmitthenner, A. F., & Canaday, C. H. (1983). Role of chemical factors in the development of Phytophthora diseases. In: Phytophthora. Its biology, taxonomy, ecology, and pathology (pp.189-196). Ed. by Erwin D. C., Bartnicki-Garcia S., Tsao P. H. St. Paul, : The American Phytopathological Society.Scibetta, S., Schena, L., Chimento, A., Cacciola, S. A., & Cooke, D. E. L. (2012). A molecular method to assess Phytophthora diversity in environmental samples. Journal of Microbiological Methods, 88, 356–368.Sena, K., Crocker, E., Vincelli, P., & Barton, C. (2018). Phytophthora cinnamomi as a driver of forest change: Implications for conservation and management. Forest Ecology and Management, 409, 799–807.Thines, M. (2013). Taxonomy and phylogeny of Phytophthora and related oomycetes In: Phytophthora: A global perspective (pp. 11-18). Ed. by Lamour, K. Wallingford: CABI.Tsao, P. H. (1990). Why many Phytophthora root rots and crown rots of tree and horticultural crops remain undetected. EPPO Bulletin, 20, 11–17.Tuset, J. J., Hinarejos, C., Mira, J. L., & Cobos, M. (1996). Implicación de Phytophthora cinnamomi Rands en la enfermedad de la seca de encinas y alcornoques. Boletín de Sanidad Vegetal-Plagas, 22, 491–499.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, 19–28.Xia, K., Hill, L. M., Li, D. Z., & Walters, C. (2014). Factors affecting stress tolerance in recalcitrant embryonic axes from seeds of four Quercus (Fagaceae) species native to the USA or China. Annals of Botany, 114, 1747–1759

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Survey and identification of Phytophthora spp. in ornamental nurseries

[EN] Phytophthora is one of the most destructive groups of plant pathogens, which has broad host range. It is globally responsible for large losses in nurseries. The problem increases when Phytophthora spp. escape from the nursery and spread to natural ecosystems, altering them and diminishing biodiversity thereof. Due to the increasing threat of invasive species of Phytophthora, it was conducted a survey in ten nurseries for the production of woody ornamental plant, located in Catalonia. Plants with symptoms of possible infection by Phytophthora (including substrate or rhizosphere soil) and water samples were processed. The species were identified by direct sequencing of the ITS region of ribosomal DNA. The isolated species were P. cactorum, P.cambivora, P. cinnamomi, P. citricola, P. citrophthora, P. cryptogea, P. gonapodyides, P. lacustris, P. nicotianae, P. palmivora, P. plurivora, P. tropicalis, Phytophthora taxon Pgchlamydo, and Phytophthora taxon oaksoil. The presence of these pathogens has been detected in a third of the collected plants and all water samples. Other oomycetes and fungi were also isolated, highlighting the genera Pythium, Fusarium and Cylindrocarpon (actualmente desglosado en tres géneros Cylindrocarpon, Ilyonectria y Campylocarpon). These results emphasize the need to improve health measures to ensure the production of nursery material free of pathogens.[ES] Phytophthora es uno de los grupos de patógenos vegetales más destructivos, que cuenta con amplia gama de hospedantes. Es responsable a nivel mundial de grandes pérdidas en viveros. El problema se agrava cuando Phytophthora spp. “escapan” de los límites de los viveros y se extienden a ecosistemas naturales, alterándolos y disminuyendo la biodiversidad de los mismos. Debido a la creciente amenaza de las especies invasoras de Phytophthora, se llevó a cabo una prospección en diez viveros dedicados a la producción de planta ornamental leñosa, situados en Cataluña. Se procesaron muestras de plantas con síntomas de posible infección por Phytophthora (incluyendo sustrato o suelo de la rizosfera) y muestras de agua. Las especies se identificaron por secuenciación directa de la región ITS del ADN ribosómico. Las especies aisladas fueron P. cactorum, P.cambivora, P. cinnamomi, P. citricola, P. citrophthora, P. cryptogea, P. gonapodyides, P. lacustris, P. nicotianae, P. palmivora, P. plurivora, P. tropicalis, Phytophthora taxon Pgchlamydo y Phytophthora taxon oaksoil. Se ha detectado la presencia de estos patógenos en un tercio de las plantas analizadas y en todas las muestras de agua. También se aislaron otros oomicetos y hongos, destacando los géneros Pythium, Fusarium y Cylindrocarpon (actualmente desglosado en Cylindrocarpon, Ilyonectria y Campylocarpon). Estos resultados remarcan la necesidad de mejorar las medidas sanitarias, que garanticen la producción de material de vivero libre de patógenos.Mora Sala, B. (2014). Survey and identification of Phytophthora spp. in ornamental nurseries. http://hdl.handle.net/10251/52334Archivo delegad

A nested-polymerase chain reaction protocol for the detection of Mycosphaerella nawae in persimmon

Mycosphaerella nawae is the causal agent of circular leaf spot of persimmon. A polymerase chain reaction (PCR) based protocol was developed for M. nawae-specific identification from pure culture, or infected symptomatic and asymptomatic persimmon tissues. Variation among the internal transcribed spacer regions (ITS) of the ribosomal DNA (rDNA) sequences of potentially related fungal species in persimmon orchards was analyzed for a primer pair design. Specificity was confirmed using multiple isolates of these species, other fungal pathogens that cause foliar diseases in persimmon and contaminants commonly obtained in the isolation process. The detection threshold for M. nawae DNA was lowered from 50 pg to 500 fg when nested-PCR was evaluated instead of single PCR. The nested-PCR protocol developed in this study showed its suitability to be applied for the specific detection of M. nawae from three types of naturally infected persimmon tissues: from lesions in fresh leaves, from pseudothecia present in lesions in leaf litter, and from infected asymptomatic leaves. The protocol can be useful for routine diagnosis, disease monitoring programs and for epidemiological research.M. Berbegal was a contract holder of the "Campus de Excelencia Internacional" program of the Universitat Politecnica de Valencia. This research was financially supported by Fundacion Agroalimed (Conselleria de Agricultura, Pesca i Alimentacio, Generalitat Valenciana). We thank J. Armengol for critically reading the manuscript prior to submission.Berbegal Martinez, M.; Mora Sala, B.; García Jiménez, J. (2013). A nested-polymerase chain reaction protocol for the detection of Mycosphaerella nawae in persimmon. European Journal of Plant Pathology. 137(2):273-281. doi:10.1007/s10658-013-0237-0S2732811372Alaniz, S., Armengol, J., García-Jiménez, J., Abad-Campos, P., & León, M. (2009). A Multiplex PCR system for the specific detection of Cylindrocarpon liriodendri, C. macrodidymum, and C. pauciseptatum from grapevine. Plant Disease, 93, 821–825.Arnal, L., & del Río, M. A. (2003). Removing astringency by carbon dioxide and nitrogen-enriched atmospheres in persimmon fruit cv. Rojo Brillante. Journal of Food Science, 68, 1516–1518.Berbegal, M., Armengol, J., & García-Jiménez, J. (2011). Evaluation of fungicides to control circular leaf spot of persimmon caused by Mycosphaerella nawae. Crop Protection, 30, 1461–1468.Berbegal, M., Pérez-Sierra, A., Armengol, J., Park, C. S., & García-Jiménez, J. (2010). First report of circular leaf spot of persimmon caused by Mycosphaerella nawae in Spain. Plant Disease, 94, 374.Crous, P. W., Hong, J. W., Wingfield, B. D., & Wingfield, M. J. (2001). ITS rDNA phylogeny of selected Mycosphaerella species and their anamorphs occurring on Myrtaceae. Mycological Research, 105, 425–431.David, J. C. (2000). Pseudocercospora kaki. IMI descriptions of fungi and bacteria: 144, sheet 1439. Wallingford: CAB International.Food and Agriculture Organization of the United Nations (FAO) (2009). Crop production database FAOSTAT. http://faostat3.fao.org/home/index.html . Accessed 14 Sept 2012Gardes, M., & Bruns, T. D. (1993). ITS primers with enhanced specificity for basidiomycetes-applications to the identification of mycorrhizae and rusts. Molecular Ecology, 2, 113–118.Ikata, S., & Hitomi, T. (1929). Studies on circular leaf spot of persimmon caused by Mycosphaerella nawae. Special Bulletin of the Okayama Prefecture Agricultural Experiment Station, 33, 1–36 (In Japanese).Johanson, A., & Jeger, M. J. (1993). Use of PCR for detection of Mycosphaerella fijiensis and M. musicola, the causal agents of Sigatoka leaf spots in banana and plantain. Mycological Research, 97, 670–674.Kang, S. W., Kwon, J. H., & Kim, H. K. (1997). High sporulating medium for Cercospora kaki causing persimmon angular leaf spot. Korean Journal of Plant Pathology, 13, 69–71.Kang, S. W., Kwon, J. H., Lee, Y. S., & Park, C. S. (1993). Effects of meteorological factors on perithecial formation and release of ascospores of Mycosphaerella nawae from the overwintered persimmon. Rural Development Administration Journal of Agricultural Science, 35, 337–343 (In Korean).Kularatne, H. A. G., Lawrie, A. C., Barber, P. A., & Keane, P. J. (2004). A specific primer PCR and RFLP assay for the rapid detection and differentiation in planta of some Mycosphaerella species associated with foliar diseases of Eucalyptus globulus. Mycological Research, 108, 1476–1493.Kwon, J. H., Jeong, S. G., & Chung, B. K. (2007). Survey of overwintering potential of anthracnose of sweet persimmon caused by Colletotrichum gloeosporioides. Research in Plant Disease, 13, 204–206 (In Korean).Kwon, J. H., Kang, S. W., Park, C. S., & Kim, H. K. (1998). Identification of the imperfect stage of Mycosphaerella nawae causing circular leaf spot of persimmon in Korea. Korean Journal of Plant Pathology, 14, 397–401.Kwon, J. H., & Park, C. S. (2004). Ecology of disease outbreak of circular leaf spot of persimmon and inoculum dynamics of Mycosphaerella nawae. Research in Plant Disease, 10, 209–216 (In Korean).Nieto-Feliner, G., & Roselló, J. A. (2007). Better the devil you know? Guidelines for insightful utilization of nrDNA ITS in species-level evolutionary studies in plants. Molecular Phylogenetics and Evolution, 44, 911–919.Thompson, J. D., Higgins, D. G., & Gibson, T. J. (1994). CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22, 4673–4680.Truett, G. E., Heeger, P., Mynatt, R. L., Truett, A. A., Walker, J. A., & Warman, M. L. (2000). Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and Tris (HotSHOT). Biotechniques, 29, 52–54.Tuset, J. J., Hinarejos, C., & Mira, J. L. (1999). First report of leaf blight on sweet persimmon tree by Pestalotiopsis theae in Spain. Plant Disease, 83, 11.Vicent, A., Bassimba, D. D. M., Hinarejos, C., & Mira, J. L. (2012). Inoculum and disease dynamics of circular leaf spot of persimmon caused by Mycosphaerella nawae under semi-arid conditions. European Journal of Plant Pathology, 134, 289–299.Vicent, A., Bassimba, D. D. M., & Intrigliolo, D. S. (2011). Effects of temperature, water regime and irrigation on the release of ascospores of Mycosphaerella nawae, causal agent of circular leaf spot of persimmon. Plant Pathology, 60, 890–908.White, T. J., Burns, T., Lee, S., & Taylor, J. W. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetic. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, & T. J. White (Eds.), PCR protocols, a guide to methods and applications (pp. 315–322). San Diego: Academics Press

New Reports of Phytophthora Species in Plant Nurseries in Spain

The plant nursery industry has become an ideal reservoir for Phytophthora species and other soilborne pathogens. In this context, isolation from tissues and soil of ornamental and forest plants from nurseries in four regions of Spain was carried out. A high diversity of Phytophthora species was confirmed. Fourteen Phytophthora phylotypes (P. cactorum, P. cambivora, P. cinnamomi, P. citrophthora, P. crassamura, P. gonapodyides, P. hedraiandra, P. nicotianae, P. niederhauserii, P. palmivora, P. plurivora, P. pseudocryptogea, P. sansomeana, and Phytophthora sp. tropicalis-like 2) were isolated from over 500 plant samples of 22 species in 19 plant genera. Nine species were detected in water sources, two of them (P. bilorbang and P. lacustris) exclusively from water samples. P. crassamura was detected for the first time in Spain. This is the first time P. pseudocryptogea is isolated from Chamaecyparis lawsoniana and Yucca rostrata in Spain