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 verifies the presence and/or detection of Phytophthora species in two holm oak areas of Spain (southwestern "dehesas" and northeastern woodland) using different isolation and detection approaches. Direct isolation and baiting methods in declining and non-declining holm oak trees revealed Phytophthora cambivora, Phytophthora cinnamomi, Phytophthora gonapodyides, Phytophthora 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 both P. cinnamomi and P. quercina are involved 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 first report of P. pseudocryptogea in Europe and its role in the holm oak decline should be further studied.

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.

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, β-tubulin (tub2), and translation elongation factor 1-α (tef1) 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.

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