An insight into curcumin-based photosensitization as a promising and green food preservation technology

Consumer awareness on the side effects of chemical preservatives has increased the demand for natural preservation technologies. An efficient and sustainable alternative to current conventional preservation techniques should guarantee food safety and retain its quality with minimal side effects. Photosensitization, utilizing light and a natural photosensitizer, has been postulated as a viable and green alternative to the current conventional preservation techniques. The potential of curcumin as a natural photosensitizer is reviewed in this paper as a practical guide to develop a safe and effective decontamination tool for industrial use. The fundamentals of the photosensitization mechanism are discussed, with the main emphasis on the natural photosensitizer, curcumin, and its application to inactivate microorganisms as well as to enhance the shelf life of foods. Photosensitization has shown promising results in inactivating a wide spectrum of microorganisms with no reported microbial resistance due to its particular lethal mode of targeting nucleic acids. Curcumin as a natural photosensitizer has recently been investigated and demonstrated efficacy in decontamination and delaying spoilage. Moreover, studies have shown the beneficial impact of an appropriate encapsulation technique to enhance the cellular uptake of photosensitizers, and therefore, the phototoxicity. Further studies relating to improved delivery of natural photosensitizers with inherent poor solubility should be conducted. Also, detailed studies on various food products are warranted to better understand the impact of encapsulation on curcumin photophysical properties, photo-driven release mechanism, and nutritional and organoleptic properties of treated foods

Evaluation of growth performance, oxidative stress and immune response in gilthead sea bream fed with novel feed formulations

Trabajo presentado en Aquaculture Europe 2020, celebrado en modalidad virtual del 12 al 15 de abril de 2021.[Introduction]: As the aquaculture sector continues to expand while being more environmentally conscious, the development of sustainable aquafeeds is becoming increasingly important (FAO, 2020). Tolerance to the replacement of fishmeal and fish oil in feeds has been largely studied in gilthead seabream (Sparus aurata) (Gasco et al., 2018; Karapanagiotidis, Psofakis, Mente, Malandrakis, & Golomazou, 2019), and many products emerge now as potential alternatives to ingredients used in conventional formulations. A main goal of GAIN EU project is to evaluate emerging ingredients, already commercially available, using different formulation concepts that consider all fish nutritional requirements. GAIN diets are based on circularity principles, maximizing resource efficiency, while contributing towards zero waste in the agro-food value chain, feed cost-effectiveness, and having good social acceptance. The present study aims to understand the real impacts of these novel feed formulations on growth performance, nutritional condition, immunity, and oxidative status using biomarkers.[Methods]: Quadruplicate groups of gilthead seabream (Sparus aurata) were fed ad libitum with four different diets. Three of them have been designed to facilitate aquaculture eco-intensification through increased circularity and resource utilization: NOPAP - formula without terrestrial animal by-products processed animal protein; PAP - formula with terrestrial animal by-products processed animal protein; and MIX - a mixture of NOPAP and PAP. The fourth feed followed a standard commercial formulation and was used as a control diet. After a 77-day feeding trial, plasma samples were collected to evaluate humoral parameters (protease, anti-protease, bactericidal activity and IgM). Liver and head kidney tissues were collected for the simultaneous profiling of a panel of 42 (liver) or 29 (head kidney) genes, as markers of growth performance, lipid and energy metabolism, and immune and antioxidant activities by qPCR. Liver samples were also used to analyse oxidative biomarker (Lipid peroxidation and catalase).[Results]: Tested feed formulations did not affect growth performance or feed intake. However, fish fed PAP and MIX diets had a higher feed conversion ratio (FCR) and protein efficiency ratio than control and NOPAP groups. This impairment was accompanied by a decreased hepatic expression of igf-i and ghr1. NOPAP diet slightly increased innate immunity parameters, showing better results on bactericidal, IgM, and anti-protease activity, as well as a significant up-regulation of il-8 in head kidney. Fish fed with PAP diet displayed an up-regulation of pro-inflammatory genes, namely il-8 and other cytokines (il-1β, tnf-α), chemokines (ck8), and chemokine receptors (ccr3). The same pattern was found for the T-cell markers cd3x, cd4, and cd8a. The activity of the antioxidant enzyme catalase was significantly lower in fish fed with PAP and MIX diet, being a possible indication of decreased antioxidant defences. This is supported by the observed regulation of antioxidant genes (mn-sod/sod2, gpr-170, gpr-94, and gpr-75), although not statistically significant.[Discussion]: The similar performance of novel formulations and the control diet indicates that they can be considered as viable options for seabream feeds. Differences in FCR suggest that NOPAP can promote a better bioavailability and/or increased absorption of key nutrients than PAP and MIX diets. Indeed, this impairment was also evidenced by their hepatic expression pattern of markers of growth performance. In general, PAP exhibited an opposite response to the NOPAP group. NOPAP was closer to the control diet, and MIX showed intermediate values between PAP and NOPAP in almost all parameters. The markedly pro-inflammatory head kidney expression profile in PAP fish may be also indicative of an impaired response at the mucosal level. In any case, the low proportion of differentially expressed genes between the experimental diets and control (18 out of 71) constitutes an additional and indirect confirmation of their suitability.[Conclusions]: Novel feed formulations for gilthead seabream seem to be viable options for a near future. In any case, all results are related to the formulation itself and cannot be attributed to a specific ingredient alteration. More studies are necessary to understand the cost-benefit of these new formulations and their market acceptability to optimize sustainability within the current/predictable European regulatory framework.“This project was financed from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 773330 (GAIN), with additional support from Nord university (Norway) and Sparos SA (Portugal)”

Fish meal-free diets supplemented with health promoters support optimal growth in gilthead sea bream, with benefitial changes in gene expression, intestinal microbiota and improved intestinal disease recovery

Trabajo presentado en la International Conference & Exposition Aquaculture Europe, celebrada en Funchal, Maderia (Portugal) del 04 al 07 de octubre de 2021.[Introduction]: The exponential growth of the aquaculture sector requires the development of sustainable aquafeeds with less dependence on marine products. Tolerance to fish meal (FM) and fish oil replacement in the economically important gilthead sea bream (Sparus aurata) is being extensively studied with many products emerging as alternative feed ingredients. It has been demonstrated that alternative diets influence the composition of intestinal adherent microbial populations, which have a key role on host metabolism, health and disease resistance. In addition, low fish meal diets showed an increased susceptibility to enteric parasites (Piazzon et al., 2017). Clearly, differences in diet have an impact on the overall health and metabolism of the fish and many parameters have to be taken into account when studying alternative diets for their use in aquaculture. In this study we evaluated the effect of a novel feed formulation (NoPAP SANA) with total replacement of FM by insect meal and bacterial fermentation biomass, and supplemented with the health-promoter additive SANACORE®GM (Palenzuela et al., 2020), on growth performance, gene expression, intestinal microbiota and disease resistance in gilthead sea bream.[Methods]: Tagged gilthead sea bream of mean weight 21.3 g were distributed in two open-flow tanks (160 fish/tank) and fed ad libitum during 34 days with control or NoPAP SANA diets. Twelve fish/diet were sacrificed and head kidney (HK), liver (L) and posterior intestine (PI) were taken for RNA extraction. From the same fish, the adherent bacteria of PI were collected and immediately used for DNA extraction. RNA from HK, L and PI was used to run three customized PCR-arrays including genes of interest for each tissue, with markers of performance and metabolism (L), immune system (HK and PI), epithelial integrity, nutrient transport and mucins (PI). Using the bacterial DNA, the V3-V4 region of the 16S rRNA of each individual sample was amplified and sequenced by Illumina MiSeq. After quality filtering, taxonomic assignment was performed with a custom-made pipeline using the RDP database. Alpha diversity was calculated using Phyloseq and beta diversity using PERMANOVA and PLS-DA models. Metagenome prediction and pathway analysis were performed using Piphillin. Differential gene expression and OTU presence and abundance correlations were studied using the corrplot R package. From the remaining fish, 70 fish/group were challenged with the intestinal parasite Enteromyxum leei by effluent exposure and the remaining fish were used as controls. The challenge lasted 78 days, including a non-lethal diagnosis sampling at day 40. At the end of the challenge all fish were sampled for histological and molecular diagnosis. Biometric values from all fish were taken in all sampling points.[Results]: A slight decrease in condition factor and specific growth rate was detected in the NoPAP SANA group. However, all fish grew efficiently considering gilthead sea bream standards. NoPAP SANA group showed differential expression of 17 out of 44 genes in L, two out of 29 in HK, and 4 out of 44 in PI. The bacterial composition at the PI showed no major differences in diversity or at the phylum level. However, 29 abundant (>1%) OTUs significantly changed with the diet. From these, 10 OTUs were significantly correlated with differential expression of genes in the different tissues, highlighting Pseudoxanthomonas which was positively correlated with the expression of seven L genes, or Actinomyces, significantly correlated with the expression of L and HK genes (Fig. 1). Inferred metagenome analyses revealed that the altered microbiota with NoPAP SANA diet could account for changes in 15 metabolic pathways. The intensity and prevalence of infection after the parasite challenge was not significantly different between diets. In fact, infected fish from both groups showed similar recovery rates.[Conclusions]: NoPAP SANA promoted good growth parameters and efficient conversions arising as a good alternative for a FMbased diet in gilthead sea bream diets. This diet modulated the expression of several genes in L showing the capacity to reduce lipogenesis, mitochondrial activity and the risk of oxidative stress and, at the same time, promoting an antiinflammatory gene expression profile in HK and PI. Changes were also detected in the adherent bacterial populations of PI, with significant changes of OTUs that could potentially account for significant metabolic alterations. The correlations between presence and abundance of intestinal bacteria with changes in gene expression of different tissues, together with the pathway analysis results, show that microbiota changes can have an impact on host metabolism at a systemic level, and vice versa. Clearly, the changes induced by this novel FM-free diet supported an accelerated growth with an overall feed conversion ratio close to 1 and no increased susceptibility against this intestinal parasite, as often observed in studies when replacing a FM-based diet.GAIN (EU-H2020 #773330); RYC2018-024049-I/AEI/10.13039/50110001103

A novel fish meal-free diet formulation supports proper growth and does not impair intestinal parasite susceptibility in gilthead sea bream (Sparus aurata) with a reshape of gut microbiota and tissue-specific gene expression patterns

The exponential growth of the aquaculture sector requires the development of sustainable aquafeeds with less dependence on marine products. The maximized replacement of fish meal (FM) and fish oil (FO) with plant ingredients has been extensively studied in the economically important species gilthead sea bream (Sparus aurata). Recently, major progress has been done with other alternative raw materials, though some non-pathological inflammatory response persisted with feed formulations that increased the circularity of resource utilization. In the present study, we evaluated the effects on growth performance, gene expression, intestinal microbiota and disease resistance of a FM-free diet (NoPAP SANA), based on plant ingredients, aquaculture by-products, algae oil, insect meal and bacterial fermentation biomasses as main dietary oil and protein sources, and supplemented with a commercially available health-promoting feed additive (SANACORE®GM). Juveniles of 21 g initial body weight were fed control or NoPAP SANA diets for 34 days, and head kidney, liver and posterior intestine were collected for gene expression analyses using customized PCR-arrays. Each tissue-specific PCR covered 96 genes in total and included markers of growth performance, lipid and energy metabolism, antioxidant defence, immune system, and intestinal function and integrity. From the same fish, the adherent bacteria of the posterior intestine were studied by Illumina sequencing of the V3-V4 region of the 16S rRNA. The remaining fish were challenged with the intestinal parasite Enteromyxum leei for 78 days and sampled for parasite diagnosis. Both control and NoPAP SANA fish grew efficiently considering gilthead sea bream standards. Before parasite challenge, the NoPAP SANA group showed differential expression of 17, 2 and 4 genes in liver, head kidney and posterior intestine, respectively. The intestinal bacterial composition showed no major differences in diversity or at the phylum level. However, 29 abundant OTUs significantly changed with the diet. From these, 10 OTUs were significantly correlated with differentially expressed genes in the different target tissues. Inferred metagenome analyses revealed that the altered microbiota with NoPAP SANA diet could account for changes in 15 metabolic pathways. The intensity and prevalence of infection after the parasite challenge did not significantly vary between dietary treatments, and infected fish from both groups showed similar disease outcome. Altogether, these results indicate that the NoPAP SANA diet promoted optimal growth and a healthy condition in gilthead sea bream without affecting susceptibility against the tested intestinal parasite, as often observed with alternative diets following current industry formulations.This project has received funding from the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No 773330 (GAIN, Green Aquaculture Intensification). This publication reflects the views only of the authors, and the European Commission cannot be held responsible for any use which may be made of the information contained therein. Additional funding was obtained by a Spanish MICINN project (Bream-AquaINTECH, RTI2018–094128-B-I00, AEI/FEDER, UE). M.C·P was funded by a Ramón y Cajal Postdoctoral Research Fellowship (RYC2018-024049-I, co-funded by the European Social Fund & ACOND/2020 Generalitat Valenciana)

How do novel feed formulations affect growth performance, oxidative stress and immune response of atlantic salmon?

Trabajo presentado en la International Conference & Exposition Aquaculture Europe, celebrada en Funchal, Maderia (Portugal) del 04 al 07 de octubre de 2021.[Introduction]: The aquaculture industry continues to grow faster than any other sector of food production. The need to make aquaculture as sustainable and more environmentally conscious as possible is becoming clearer everyday (FAO, 2020). With this in mind, the replacement of fishmeal and fish oil in aquafeeds has been studied in Atlantic salmon (Salmo salar) (e.g., Bendiksen et al., 2011) with many products emerging as potential alternatives to conventional ones (e.g., Hodar et al., 2020). One of the main objectives of the EU project GAIN is to evaluate new ingredients that are already commercially available using different formulation concepts that consider all the fish nutritional needs. GAIN diets are based on circular economy principles and maximize resource efficiency, while contributing to zero waste in the agri-food value chain, being cost-effective feeds, and having good social acceptability. The present study aims to understand the actual effects of these novel feed formulations on growth performance, nutritional status, immunity and oxidative status.[Methods]: Quadruplicate groups of Atlantic salmon were fed ad libitum with three different diets. Two diets were developed to facilitate the eco-intensification of aquaculture through increased circularity and resource utilization (NOPAP - formula without processed animal protein - and PAP - formula with processed animal protein). The third diet was a commercial-like formulation that was used as a control. After a 96-day feeding trial, plasma samples were collected to evaluate humoral parameters (protease, anti-protease, bactericidal activity, and IgM). Liver and head kidney tissues (collected at day 45 and 96) were used for the simultaneous profiling by PCR array of a panel of 38 or 28 genes, respectively, as markers of growth performance, lipid and energy metabolism, and immune and antioxidant activities. Liver samples were also used to analyse lipid peroxidation. In addition, after 45 and 96 days, the lice count and fish welfare were also assessed by standard methods. The dorsal skin and foregut were collected at days 45 and 96 for mucosal mapping (mucous cell area, density, and barrier status).[Results]: Growth performance was adequate and comparable to commercial standards for the novel diets tested. Other parameters analysed, including those related to key performance indicators, intestinal and skin dorsal mucosal mapping, plasma innate immune defences, and lipid peroxidation in the liver did not significantly differ across diets. Regarding head kidney gene expression, at Day 45, 2 out of 28 genes in the array were differentially expressed (p<0.05). Gene expression of fish fed with novel feed formulations showed a pro-inflammatory profile evidenced by the up regulation of il-8, and a down regulation of il-10.At Day 96, the same genes continued to be differentially expressed, but gene clec1b (membrane protein) was also up-regulated. However, the rest of the analyses do not support this pro-inflammatory profile. A longer trial may bring light to some of the current results. In turn, the liver had a differential gene expression only at the second sampling point (Day96), where 4 out of 38 genes in the array were affected, including growth performance (igf2), lipid metabolism, elongases (elovl4), and energy metabolism (ucp2l and sirt1). These transcriptomic changes may be attributed to an initial response to the experimental diets. Cross-analysis of gene expression by time points and dietary treatment (two-way ANOVA) yielded only 2 out of 38 genes that had significantly different expression across treatments. The differentially expressed genes were related to growth performance (igf2) and lipid metabolism (elovl4).[Conclusions]: The novel feed formulations of the GAIN project for Atlantic Salmon seem to be viable options for the near future. In any case, all results are related to the formulation itself and cannot be attributed to a specific ingredient alteration. More studies are necessary to understand the cost-benefit of these new formulations and their market acceptability to optimize sustainability within the current/predictable European regulatory framework.This project was financed by the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 773330 (GAIN), with additional support from Nord University (Norway) and SPAROS Lda (Portugal)

Increased pain intensity is associated with greater verbal communication difficulty and increased production of speech and co-speech gestures

Effective pain communication is essential if adequate treatment and support are to be provided. Pain communication is often multimodal, with sufferers utilising speech, nonverbal behaviours (such as facial expressions), and co-speech gestures (bodily movements, primarily of the hands and arms that accompany speech and can convey semantic information) to communicate their experience. Research suggests that the production of nonverbal pain behaviours is positively associated with pain intensity, but it is not known whether this is also the case for speech and co-speech gestures. The present study explored whether increased pain intensity is associated with greater speech and gesture production during face-to-face communication about acute, experimental pain. Participants (N = 26) were exposed to experimentally elicited pressure pain to the fingernail bed at high and low intensities and took part in video-recorded semi-structured interviews. Despite rating more intense pain as more difficult to communicate (t(25) = 2.21, p = .037), participants produced significantly longer verbal pain descriptions and more co-speech gestures in the high intensity pain condition (Words: t(25) = 3.57, p = .001; Gestures: t(25) = 3.66, p = .001). This suggests that spoken and gestural communication about pain is enhanced when pain is more intense. Thus, in addition to conveying detailed semantic information about pain, speech and co-speech gestures may provide a cue to pain intensity, with implications for the treatment and support received by pain sufferers. Future work should consider whether these findings are applicable within the context of clinical interactions about pain

Single-cell Atlas of common variable immunodeficiency shows germinal center-associated epigenetic dysregulation in B-cell responses

Common variable immunodeficiency (CVID), the most prevalent symptomatic primary immunodeficiency, displays impaired terminal B-cell differentiation and defective antibody responses. Incomplete genetic penetrance and ample phenotypic expressivity in CVID suggest the participation of additional pathogenic mechanisms. Monozygotic (MZ) twins discordant for CVID are uniquely valuable for studying the contribution of epigenetics to the disease. Here, we generate a single-cell epigenomics and transcriptomics census of naïve-to-memory B cell differentiation in a CVID-discordant MZ twin pair. Our analysis identifies DNA methylation, chromatin accessibility and transcriptional defects in memory B-cells mirroring defective cell-cell communication upon activation. These findings are validated in a cohort of CVID patients and healthy donors. Our findings provide a comprehensive multi-omics map of alterations in naïve-to-memory B-cell transition in CVID and indicate links between the epigenome and immune cell cross-talk. Our resource, publicly available at the Human Cell Atlas, gives insight into future diagnosis and treatments of CVID patients

Descriptive analysis of childbirth healthcare costs in an area with high levels of immigration in Spain

<p>Abstract</p> <p>Background</p> <p>The aim of this study was to estimate the cost of childbirth in a teaching hospital in Barcelona, Spain, including the costs of prenatal care, delivery and postnatal care (3 months). Costs were assessed by taking into account maternal origin and delivery type.</p> <p>Methods</p> <p>We performed a cross-sectional study of all deliveries in a teaching hospital to mothers living in its catchment area between October 2006 and September 2007. A process cost analysis based on a full cost accounting system was performed. The main information sources were the primary care program for sexual and reproductive health, and hospital care and costs records. Partial and total costs were compared according to maternal origin and delivery type. A regression model was fit to explain the total cost of the childbirth process as a function of maternal age and origin, prenatal care, delivery type, maternal and neonatal severity, and multiple delivery.</p> <p>Results</p> <p>The average cost of childbirth was 4,328€, with an average of 18.28 contacts between the mother or the newborn and the healthcare facilities. The delivery itself accounted for more than 75% of the overall cost: maternal admission accounted for 57% and neonatal admission for 20%. Prenatal care represented 18% of the overall cost and 75% of overall acts. The average overall cost was 5,815€ for cesarean sections, 4,064€ for vaginal instrumented deliveries and 3,682€ for vaginal non-instrumented deliveries (p < 0.001). The regression model explained 45.5% of the cost variability. The incremental cost of a delivery through cesarean section was 955€ (an increase of 31.9%) compared with an increase of 193€ (6.4%) for an instrumented vaginal delivery. The incremental cost of admitting the newborn to hospital ranged from 420€ (14.0%) to 1,951€ (65.2%) depending on the newborn's severity. Age, origin and prenatal care were not statistically significant or economically relevant.</p> <p>Conclusions</p> <p>Neither immigration nor prenatal care were associated with a substantial difference in costs. The most important predictors of cost were delivery type and neonatal severity. Given the impact of cesarean sections on the overall cost of childbirth, attempts should be made to take into account its higher cost in the decision of performing a cesarean section.</p

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