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International audience; Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. Previously (Geiser et al. 2013; Phytopathology 103:400-408. 2013), the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani Species Complex (FSSC). Subsequently, this concept was challenged by one research group (Lombard et al. 2015 Studies in Mycology 80: 189-245) who proposed dividing Fusarium into seven genera, including the FSSC as the genus Neocosmospora, with subsequent justification based on claims that the Geiser et al. (2013) concept of Fusarium is polyphyletic (Sandoval-Denis et al. 2018; Persoonia 41:109-129). Here we test this claim, and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species recently described as Neocosmospora were recombined in Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural and practical taxonomic option available.

Degradation of natural ecosystems increases the risk of infections in wildlife due to microbiota dysbiosis. However, little is known about its influence on the development of fungal communities in predators and facultative avian scavengers. We evaluated the incidence of oral disease in wild nestling black kites (Milvus migrans) under contrasting environmental degradation conditions, and explored their oral fungal patterns using molecular methods and multivariate analysis. Oral lesions were found in 36.8% of the 38 nestlings examined in an anthropogenically altered habitat (southeastern Madrid, Spain), but in none of the 105 nestlings examined in a well-conserved natural area (Doñana National Park, Spain). In a subsample of 48 black kites, the composition of the oral fungal community differed among symptomatic nestlings from Madrid (SM) and asymptomatic nestlings from Madrid (AM) and Doñana (AD). Opportunistic fungal pathogens (e.g., Fusarium incarnatum-equiseti species complex, Mucor spp., Rhizopus oryzae) were more prevalent in SM and AM than in AD. Hierarchical clustering and principal component analyses revealed that fungal patterns were distinct between both study areas, and that anthropogenic and natural environmental factors had a greater impact on them than oral disease. Fungal signatures associated with anthropogenic and natural stresses harbored some taxa that could be used to flag oral infection (F. incarnatum-equiseti species complex and Alternaria), indicate environmental degradation (Alternaria) or provide protective benefits in degraded environments (Trichoderma, Epicoccum nigrum and Sordaria). Co-occurrence associations between potentially beneficial and pathogenic fungi were typical of AM and AD, hinting at a possible role in host health. This study shows that early-life exposure to highly degraded environments induces a shift towards a higher prevalence of pathogenic species in the oral cavity of black kites, favoring oral disease. Furthermore, our findings suggest potential ecological applications of the monitoring of oral mycobiome as a bioindication of oral disease and environmental degradation., This work was supported by projects CGL2009-12753-C02-01/BOS, CGL2010-15726 and PGC2018-095860-B-I00 of the Spanish Ministry of Science, Innovation and Universities, and FEDER funds and P18-FR-4239 of the Junta de Andalucía (Consejería de Conocimiento, Investigación y Universidad).

The oomycete Aphanomyces astaci is an emerging infectious pathogen affecting freshwater crayfish worldwide and is responsible for one of the most severe wildlife pandemics ever reported. The pathogen has caused mass mortalities of freshwater crayfish species in Europe and Asia, and threatens other susceptible species in Madagascar, Oceania and South America. The pathogen naturally coexists with some North American crayfish species that are its chronic carriers. Presumptions that A. astaci originated in North America are based on disease outbreaks that followed translocations of North American crayfish and on the identification of the pathogen mainly in Europe. We studied A. astaci in the southeastern US, a center of freshwater crayfish diversity. In order to decipher the origin of the pathogen, we investigated (1) the distribution and haplotype diversity of A. astaci, and (2) whether there are crayfish species-specificities and/or geographical restrictions for A. astaci haplotypes. A total of 132 individuals, corresponding to 19 crayfish species and one shrimp species from 23 locations, tested positive for A. astaci. Mitochondrial rnnS and rnnL sequences indicated that A. astaci from the southeastern US exhibited the highest genetic diversity so far described for the pathogen (eight haplotypes, six of which we newly describe). Our findings that A. astaci is widely distributed and genetically diverse in the region supports the hypothesis that the pathogen originated in the southeastern US. In contrast to previous assumptions, however, the pathogen exhibited no clear species-specificity or geographical patterns.

Oomycetes are fungal-like microorganisms parasitic towards a large number of plant and animal species. Genera from order Saprolegniales, such as Saprolegnia and Aphanomyces, cause devastating infections of freshwater animals. Saprolegnia parasitica is a widely distributed oomycete pathogen that causes saprolegniosis, a disease responsible for significant economic losses in aquaculture, as well as declines of natural populations of fish and other freshwater organisms. Despite its negative impact, no monitoring protocol for S. parasitica has been established to date. Thus, we aimed to develop a droplet digital PCR (ddPCR) assay for the detection and quantification of S. parasitica in environmental DNA samples. Saprolegnia parasitica-specific primers were designed to target internal transcribed spacer region 2 (ITS 2), based on the alignment of ITS sequences of S. parasitica and a range of Saprolegnia spp., as well as other oomycetes. The specificity of primers was tested using genomic DNA of S. parasitica (as positive control) and DNA of non–S. parasitica oomycete isolates, as well as trout/crayfish DNA (as negative control). The primers specifically amplified a segment of the ITS region of oomycete pathogen S. parasitica, while no amplification (i.e. no positive droplets) was obtained for closely related Saprolegnia spp. (e.g. Saprolegnia sp. 1 and S. ferax) and other more distantly related oomycetes. Next, the limit of detection (LOD) of the assay was established by using serial dilutions of the S. parasitica genomic DNA. The determined sensitivity of the assay was high: LOD was 15 fg of pathogen’s genomic DNA per µL of the reaction mixture. Assay performance was further assessed with environmental DNA samples isolated from water from the trout farms and natural environments, as well as (ii) biofilm from the host surface (swab samples). Water samples were collected from 21 different locations in Croatia, while swab samples were collected from S. parasitica host/carrier species: (i) skin and eggs of the rainbow trout (Oncorhynchus mykiss Walbaum, 1792) and brown trout (Salmo trutta Linnaeus, 1758), and (ii) cuticle of signal crayfish (Pacifastacus leniusculus Dana, 1852) and narrow clawed crayfish (Pontastacus leptodactylus Eschscholtz, 1823). Samples were classified into agent levels A0 to A6, depending of the number of S. parasitica ITS copies per ng of total DNA. Saprolegnia parasitica was detected in 76 % of water samples (16/21) and the range of pathogen’s ITS copies in positive samples was between 0.02 and 14 copies/ng of total DNA (agent levels A1 to A3). Regarding the swab samples, S. parasitica load was significantly higher in diseased trout than in those with healthy appearance: 9375 vs 3.28 S. parasitica copies/ng of total swab DNA (average agent level A6 vs. A2, respectively). Despite the fact that none of the sampled crayfish had signs of infection, the pathogen was detected in all tested cuticle swabs. Swabs of P. leniusculus, a known S. parasitica host, had significantly higher S. parasitica load than swabs of P. leptodactylus, S. parasitica carrier: 390 vs 83 S. parasitica copies/ng (agent level A5 vs. A4, respectively). In conclusion, our results demonstrate the applicability of the newly developed ddPCR assay in monitoring and early detection of S. parasitica in aquaculture facilities and natural freshwater environments. This would help in a better understanding of S. parasitica ecology and its effects on the host populations.

The endangered yellow-spotted river turtle (Podocnemis unifilis) has experienced a dramatic population decline in the Ecuadorian Amazonia, mainly due to overexploitation of its eggs. To reverse this trend, the Wildlife Conservation Society has developed a head-start program in Yasuní National Park since 2008, but the potential risk that microbes associated with its eggs might represent for hatching success has not been evaluated yet. Members of the Fusarium solani species complex (FSSC) are involved in egg failure in sea turtles under natural and hatchery conditions, but their role in infecting the eggs of P. unifilis is unknown. In this study, we collected eggshells of P. unifilis and obtained 50 fungal and bacterial isolates. Some potentially pathogenic fungi of the genera Fusarium, Penicillium and Rhizopus were identified based on molecular data. Most importantly, the sea turtle pathogenic species F. keratoplasticum not only was present, but it was the most frequently found. Conversely, we have also isolated other microorganisms, such as Pseudomonas or Phoma-like species, producing a wide spectrum of antifungal compounds that may have a protective role against fungal diseases. Our survey provides useful information on potential pathogens found in P. unifilis eggshells, upon which the success of conservation programs may depend., This research was funded by the Real Jardín Botánico (Spanish National Research Council, CSIC), and the MS program “Biodiversity and its Conservation in Tropical Areas” of the Menéndez Pelayo International University-CSIC.

Global climate patterns are shifting due to anthropogenic causes, putting pressure on many species that are unable to adapt to such rapid changes. Sea turtle clutches, for example, normally develop between 26 and 33°C, but tolerate higher temperatures at the end of development. High temperatures, protracted rainfall, and droughts have been linked to fewer hatchlings. Likewise, climatic conditions can affect the size of sea turtle hatchlings, which could affect their probability of survival. We analyzed the effects of a biotic factor (egg mass) and environmental factors (precipitation and incubation temperature) on East Pacific leatherback turtle Dermochelys coriacea hatchling sizes in Costa Rica. Hatchling size (mass, straight carapace length, straight carapace width, and head width) was affected by both biotic and environmental factors. While egg mass was the strongest predictor for hatchling mass, temperature was the strongest for carapace length and head width. Carapace width was most strongly predicted by precipitation. Hatchling carapace length and width decreased with increases in temperature and increased with higher levels of precipitation. Hatchling mass also decreased with increasing temperature, although the relationship was not statistically significant. Head width decreased with greater precipitation during development. These findings suggest that increases in temperature and decreases in precipitation may not only decrease the number of hatchlings produced, but may also produce smaller leatherback hatchlings in Pacific Costa Rica, which may impair hatchling survival in the first hours after emergence.

The causative agent of crayfish plague, Aphanomyces astaci (Saprolegniales, Oomycota), is one of the 100 world’s worst invasive alien species and represents a major threat to freshwater crayfish species worldwide. A better understanding of the biology and epidemiology of A. astaci relies on the application of efficient tools to detect the pathogen and assess its genetic diversity. In this study, we validated the specificity of two recently developed PCR-based approaches used to detect A. astaci groups. The first relies on the analysis of mitochondrial ribosomal rnnS (small) and rnnL (large) subunit sequences and the second, of sequences obtained by using genotype-specific primers designed from A. astaci whole genome sequencing. For this purpose, we tested the specificity against 76 selected isolates, including other oomycete species and the recently described species Aphanomyces fennicus, which, when used in nrITS-based specific tests for A. astaci, is known to result in a false positive. Under both approaches, we were able to efficiently and accurately identify A. astaci and its genetic groups in both pure cultures and clinical samples. We report that sequence analysis of the rnnS region alone is sufficient for the identification of A. astaci and a partial characterization of haplogroups. In contrast, the rnnL region alone is not sufficiently informative for A. astaci identification as other oomycete species present sequences identical to those of A. astaci.

The crayfish plague pathogenAphanomyces astaciis one of the main factors responsible for the decline in European and Asian native crayfish species. This pathogen was transported to these regions through its natural carriers, North American crayfish species, which were introduced during the last century. Since then, the carrier species and the pathogen have spread worldwide due to globalization and the highly invasive nature of these species. In Europe, five carrier species have been categorized as high-risk as they are responsible for the loss of provisioning services, which endangers freshwater ecosystems. The red swamp crayfishProcambarus clarkii, in particular, is currently one of the most concerning species as its spread threatens crayfish biodiversity and freshwater ecosystems worldwide. In this study, we describe the first detection ofA. astaciin an introduced population ofP. clarkiiin Central America, specifically in Costa Rica. Using molecular approaches, we analyzed 48 crayfish samples collected from Reservoir Cachí and detected the presence ofA. astaciin four of these samples. The introduction ofP. clarkiiand the incorrect management of the species (related to its fishery and the commercialization of live specimens) over the past decades in Europe are mistakes that should not be repeated elsewhere. The detection of the pathogen is a warning sign about the dangerous impact that the introduction of this invasive crayfish may have, not only as a carrier of an emerging disease but also as a direct risk to the invaded ecosystems. Our results may serve to (1) assess current and future consequences, and (2) direct future research activities, such as determining the potential impacts ofA. astacion native decapod species, or on other introduced crayfish species that are used for aquaculture purposes, such asCherax quadricarinatus.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the

Saprolegnia infections are among the main parasitic diseases affecting farmed salmonids. The distribution and potential transfer of Saprolegnia spp. between farms and the natural environment has been scarcely investigated. Therefore, this work aimed to study the diversity and abundance of oomycete species in salmonid farms, tributary water, and effluent water systems. Four trout farms in Italy and two Atlantic salmon farms in Scotland were considered. In Italian farms, 532 isolates of oomycetes were obtained from fish and water, at upstream, inside, and downstream the farms. In Scottish farms, 201 oomycetes isolates were obtained from water outside the farm and from fish and water inside the farming units. Isolates were identified to the species level through amplification and sequencing of the ITS rDNA region. In Italy, S. parasitica was significantly more present in farmed than in wild fish, while in water it was more frequently isolated from the wild, particularly in effluent systems, not associated with more frequent isolation of S. parasitica in wild fish downstream the farm. In Scotland, S. parasitica was the most prevalent species isolated from fish, while isolates from water were mostly Pythium spp. with few S. parasitica isolates from upstream and downstream the farms., European Union’s Horizon 2020 research and innovation programme under grant agreement No. 634429

Crayfish plague, caused by the pathogen Aphanomyces astaci, is one of the main factors responsible for the decimation of the native European crayfish species Austropotamobius pallipes. In Spain, two North American freshwater crayfish species, Procambarus clarkii and Pacifastacus leniusculus, were intentionally introduced during the 1970s for aquaculture and fishery purposes. Since then, incidences of crayfish plague have been continually reported. In this work, we evaluated more than 50 diagnosed cases of crayfish plague that have occurred in the Iberian Peninsula since 2004 by performing a microscopic examination of infected specimens and by molecularly identifying and haplotyping the pathogen. Our results showed that (i) the pathogen A. astaci has been active 45 years since the first introductions of the invasive North American crayfish species in the Iberian Peninsula, and (ii) P. clarkii and P. leniusculus are chronic reservoirs of the crayfish plague pathogen. Moreover, our data confirmed a correspondence between pathogen origin and spread and the specific haplotypes carried by the North American invasive crayfish located in the vicinity of each outbreak. We generated a crayfish plague incidence map of the Iberian Peninsula that shows (i) a northern area, mainly inhabited by alien P. leniusculus, where crayfish plague cases are associated with the b-haplotype specific to P. leniusculus, and (ii) southern, central and eastern areas that are basically inhabited by alien P. clarkii, where crayfish plague cases are associated with the d1- and d2-haplotypes specific to P. clarkii. The results presented here are evidence of the long standing and negative impact of the two invasive crayfish species on the native species, indicating the need for more extensive control measures.

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Global introductions of aquatic species and their associated pathogens are threatening worldwide biodiversity. The introduction of two North American crayfish species, Procambarus clarkii and Pacifastacus leniusculus, into Japan in 1927 seems to have negatively affected native Japanese crayfish populations of Cambaroides japonicus. Several studies have shown the decline of these native populations due to competition, predation and habitat colonization by the two invasive North American crayfish species. Here, we identify an additional factor contributing to this decline. We report the first crayfish plague outbreaks in C. japonicus populations in Japan, which were diagnosed using both histological and molecular approaches (analyses of the internal transcribed spacer region). Subsequent analyses of the mitochondrial ribosomal rnnS and rnnL regions of diseased specimens indicate that these outbreaks originated from a P. clarkii population and identify a novel haplotype of Aphanomyces astaci, d3-haplotype, hosted by P. clarkii. Overall, our findings demonstrate the first two cases of crayfish plague in Japan, and the first case in a non-European native crayfish species, which originated from the red swamp crayfish P. clarkii. This finding is a matter of concern for the conservation of the native freshwater species of Japan and also highlights the risk of introducing crayfish carrier species into biogeographic regions harboring species susceptible to the crayfish plague., This research was partially supported by a grant from the Spanish Ministry of Economy and Competitiveness (CGL2016-80526-R).

[EN] Amphibian populations are declining due to a myriad of abiotic and biotic factors, including invasive species and pathogens. In temperate freshwater ecosystems, for example, amphibian populations are threatened by the predation of introduced salmonids. Salmonids not only directly predate upon amphibian eggs and larvae but may also transport deadly pathogens into freshwater systems. Though most research has focused on temperate systems, much less is known about the effects of introduced species in Neotropical streams. We conducted two experiments to investigate the impacts of rainbow trout (Oncorhynchus mykiss) in two Neotropical anurans. First, we assessed the effect of the rainbow trout on tadpole survivorship and morphology in Nymphargus grandisonae, a glassfrog species endemic of the Andes. Tadpoles of N. grandisonae were subjected to three treatments involving the absence of rainbow trout (control) and its presence with different types of chemical cues – kairomones (from rainbow trout) and cue alarms (from tadpole prey); the results show that the presence of rainbow trout affects the larval morphology of this glassfrog. In the second experiment, to test whether rainbow trout is a vector of the pathogenic freshwater mold Saprolegnia diclina (Oomycetes), eggs of Engystomops petersi were placed with infected and uninfected rainbow trout. There was a high mortality rate in the embryos of E. petersi exposed to trout infected with S. diclina. This represents the first evidence that rainbow trout may have a direct negative effect on Neotropical amphibian populations, and thus should be considered a threat. Management programs should be implemented to eradicate trout from Andean rivers, especially in areas with high number of endangered amphibians., [ES] Las poblaciones de anfibios están disminuyendo debido a una serie de factores, incluyendo las especies invasoras y patógenos. En los ecosistemas templados, por ejemplo, los anfibios están amenazados por salmónidos introducidos. Los salmónidos no solo depredan directamente los huevos y larvas, sino que también son vectores de patógenos en los ecosistemas acuáticos. A pesar de que existe una serie de investigaciones en zonas templadas, el efecto de las especies introducidas en sistemas neotropicales es prácticamente desconocido. En este trabajo estudiamos experimentalmente el efecto de la trucha arcoíris (Onchorhynchus mykiss) en dos especies de anuros neotropicales. Primero, determinamos el efecto de la trucha en la supervivencia y morfología de renacuajos de Nymphargus grandisonae, una rana de cristal endémica a los Andes. Los renacuajos fueron sometidos a tres tratamientos, los cuales combinan la ausencia de la trucha (control) y su presencia con diferentes señales químicas: cairomonas (de las truchas) y de alarma (producidas por los renacuajos depredados). Los resultados demuestran que la presencia de la trucha afecta la morfología de los renacuajos. En el segundo experimento, para determinar si la trucha actúa como vector del pátogeno Saprolegnia diclina (Oomycetes), los huevos del sapo Engystomops petersi fueron expuestos a la presencia truchas infectadas o no infectadas con S. diclina. El experimento muestra que la trucha actúa como vector de S. diclina, la cual produce una alta mortalidad en los huevos de E. petersi. Este es el primer estudio que claramente indica el efecto negativo de la trucha arcoíris en anfibios neotropicales. Recomendamos implementar programas de manejo dirigidos a erradicar a este pez introducido de los ríos andinos, especialmente en áreas con una alta presencia de especies amenazadas de anfibios.

The red swamp crayfish (Procambarus clarkii) is considered one of the most invasive species for aquatic ecosystems. Since its introduction in Spain in 1973, it has spread over different wetlands throughout Western Eu- rope, having rapidly adapted to lakes, ponds and coastal marshes. Among the invaded ecosystems, rice-growing areas have been largely affected by crayfish habits, mainly due to their digging behaviour. In pursuit of this, we at- tempted to draw some patterns about the dynamics of the red swamp crayfish population inhabiting the Ebro Delta Natural Park (NE Spain) - one of the main rice-producing areas within the Mediterranean region - in relation to the structural damage caused to draining structures. More specifically, we aimed at establishing links between the local densities of crayfish, the abundance of burrows they use to shelter, and the intensity of damage involved by crayfish digging activity. For this purpose, two approaches relating crayfish density data with abundances of burrows, com- plemented with in-situ observations of structural damage, were designed. Preliminary results indicated that crayfish density and the number of active burrows were positively related, with higher values implying severe damage to draining structures. However, intense digging activity periods did not directly relate to crayfish density, since other factors were also involved. The information obtained could be useful in planning future surveys in which sound details on digging behaviour and, consequently, the structural damage entailed, could be profitably assessed.

The oomyceteAphanomyces astaci, the causative agent of crayfish plague, is listed as one of the 100 worst invasive species in the world, destroying the native crayfish populations throughout Eurasia. The aim of this study was to examine the potential of selected mitochondrial (mt) genes to track the diversity of the crayfish plague pathogenA. astaci. Two sets of primers were developed to amplify the mtDNA of ribosomal rnnS and rnnL subunits. We confirmed two main lineages, with four different haplogroups and five haplotypes among 27 studiedA. astacistrains. The haplogroups detected were (1) the A-haplogroup with the a-haplotype strains originating fromOrconectessp.,Pacifastacus leniusculusandAstacus astacus; (2) the B-haplogroup with the b-haplotype strains originating from theP. leniusculus; (3) the D-haplogroup with the d1 and d2-haplotypes strains originating fromProcambarus clarkii; and (4) the E-haplogroup with the e-haplotype strains originating from theOrconectes limosus. The described markers are stable and reliable and the results are easily repeatable in different laboratories. The present method has high applicability as it allows the detection and characterization of theA. astacihaplotype in acute disease outbreaks in the wild, directly from the infected crayfish tissue samples.

Worldwide biodiversity is suffering a “biogeographical homogenization” due to biological invasions. The translocation of freshwater crayfish around the world for aquaculture and the pet trade is also spreading their symbionts, so that all of the association's members have the potential to form invasive populations. In Europe, the signal crayfish, Pacifastacus leniusculus, is often concomitant with the branchiobdellidan Xironogiton victoriensis and through repeated translocations their ranges are expanding. In this study, we report the first observation of X. victoriensis on a native European crayfish population of Austropotamobius pallipes. This work is an alert to the authorities and decision makers on the risks to native species when aquaculture projects introduce alien crayfish species and their symbionts into an area. Future studies are needed to evaluate the impact of this non-native ectosymbiont on native crayfish populations.

The crayfish plague agent Aphanomyces astaci is one of the world's most threatening invasive species. Originally from North America, the pathogen is being imported alongside American crayfish species, which are used for various purposes. In this study, we investigated the marginal, currently known distribution area of the pathogen in Eastern Europe by sampling narrow-clawed crayfish (Astacus leptodactylus) and spiny-cheek crayfish (Orconectes limosus) populations. In addition, using specific real-time PCR, we tested several marine decapod species, which also occur in brackish waters of the Danube at the West coast of the Black Sea and the Dniester River basin. By sequencing the nuclear chitinase gene, mitochondrial rnnS/rnnL DNA and by genotyping using microsatellite markers, we identified the A. astaci haplogroups of highly infected specimens. The A. astaci DNA was detected in 9% of the investigated A. leptodactylus samples, both in invaded and non-invaded sectors, and in 8% of the studied O. limosus samples. None of the marine decapods tested positive for A. astaci. The results revealed that narrow-clawed crayfish from the Dniester River carried the A. astaci B-haplogroup, while A. astaci from the Danube Delta belonged to the A- and B-haplogroups. In the invaded sector of the Danube, we also identified the A-haplogroup. Microsatellite analysis revealed a genotype identical to the genotype Up. It might be that some of the detected A. astaci haplogroups are relics from older outbreaks in the late 19th century, which may have persisted as a chronic infection for several decades in crayfish populations.

The secondary cysts of the fish pathogen oomycete Saprolegnia parasitica possess bundles of long hooked hairs that are characteristic to this economically important pathogenic species. Few studies have been carried out on elucidating their specific role in the S. parasitica life cycle and the role they may have in the infection process. We show here their function by employing several strategies that focus on descriptive, developmental and predictive approaches. The strength of attachment of the secondary cysts of this pathogen was compared to other closely related species where bundles of long hooked hairs are absent. We found that the attachment of the S. parasitica cysts was around three times stronger than that of other species. The time sequence and influence of selected factors on morphology and the number of the bundles of long hooked hairs conducted by scanning electron microscopy study revealed that these are dynamic structures. They are deployed early after encystment, i.e., within 30 sec of zoospore encystment, and the length, but not the number, of the bundles steadily increased over the encystment period. We also observed that the number and length of the bundles was influenced by the type of substrate and encystment treatment applied, suggesting that these structures can adapt to different substrates (glass or fish scales) and can be modulated by different signals (i.e., protein media, 50 mM CaCl2 concentrations, carbon particles). Immunolocalization studies evidenced the presence of an adhesive extracellular matrix. The bioinformatic analyses of the S. parasitica secreted proteins showed that there is a high expression of genes encoding domains of putative proteins related to the attachment process and cell adhesion (fibronectin and thrombospondin) coinciding with the deployment stage of the bundles of long hooked hairs formation. This suggests that the bundles are structures that might contribute to the adhesion of the cysts to the host because they are composed of these adhesive proteins and/or by increasing the surface of attachment of this extracellular matrix.

The lack of a robust taxonomy in the genus Saprolegnia is leading to the presence of incorrectly named isolates in culture collections and of an increasing number of misassigned sequences in DNA databases. Accurate species delimitation is critical for most biological disciplines. A recently proposed approach to solve species delimitation (taxonomic diagnosis system) of difficult organisms is the definition of molecular operational taxonomic units (MOTUs). We have used 961 sequences of nrDNA ITS from culture collections (461 sequences) and GenBank (500 sequences), to perform phylogenetic and clustering optimization analyses. As result, we have identified 29 DNA-based MOTUs in agreement with phylogenetic studies. The resulting molecular clusters support the validity of 18 species of Saprolegnia and identify 11 potential new ones. We have also listed a number of incorrectly named isolates in culture collections, misassigned species names to GenBank sequences, and reference sequences for the species. We conclude that GenBank represents the main source of errors for identifying Saprolegnia species since it possesses sequences with misassigned names and also sequencing errors. The presented taxonomic diagnosis system might help setting the basis for a suitable identification of species in this economically important genus., This work was supported by grants from the European Union (ITN-SAPRO-238550), and Ministerio de Ciencia e Innovación, Spain (CGL2009-10032), and project, Fundación San Ignacio del Huinay, Endesa, Chile. José V. Sandoval-Sierra was supported by ITN-SAPRO-23855

International audience; Aphanomyces astaci is an invasive pathogenic oomycete responsible for the crayfish plague, a disease that has devastated European freshwater crayfish. So far, five genotype groups of this pathogen have been identified by applying random amplified polymorphic DNA analysis on axenic cultures. To allow genotyping of A. astaci in host tissue samples, we have developed co-dominant microsatellite markers for this pathogen, tested them on pure cultures of all genotype groups, and subsequently evaluated their use on tissues of (1) natural A. astaci carriers, i.e., North American crayfish species, and (2) A. astaci-infected indigenous European species from crayfish plague outbreaks. Out of over 200 potential loci containing simple sequence repeat (SSR) motifs identified by 454 pyrosequencing of SSR-enriched library, we tested 25 loci with highest number of repeats, and finally selected nine that allow unambiguous separation of all known RAPD-defined genotype groups of A. astaci from axenic cultures. Using these markers, we were able to characterize A. astaci strains from DNA isolates from infected crayfish tissues when crayfish had a moderate to high agent level according to quantitative PCR analyses. The results support the hypothesis that different North American crayfish hosts carry different genotype groups of the pathogen, and confirm that multiple genotype groups, including the one originally introduced to Europe in the 19th century, cause crayfish plague outbreaks in Central Europe. So far undocumented A. astaci genotype seems to have caused one of the analysed outbreaks from the Czech Republic. The newly developed culture-independent approach allowing direct genotyping of this pathogen in both axenic cultures and mixed genome samples opens new possibilities in studies of crayfish plague pathogen distribution, diversity and epidemiology.

Investigations of occurrence of Saprolegnia diclina from four limnocrenic springs of rivers within the BiaA‚ystok where eggs of Carassius carassius were the bait were done. Identification of isolates was accomplished on the basis of their vegetative, asexual reproduction, generative organs and by studying the sequencing of the internal transcribed spacer of nuclear ribosomal DNA (ITS1+5.8S+ITS2). S. diclina occurred in 29 (60.4%) [10(20.1%) in spring, 4 (8.3%) in summer, 11 (23.0%) in autumn and 4 (8.3%) in winter, 2005] of the 48 examined water samples. The results indicate the sequence comparisons of two ITS nuclear DNA for species identification: S. diclina. The results indicate that the sequence of our isolate correspond to S. diclina. It is very important that this study represents the first isolation (on the basis of molecular features) of S. diclina in fresh waters in Poland. Key words: Saprolegnia diclina, detection, nuclear DNA, eggs, Carassius carassius, springs, Poland.

A very large number of diseases in agriculture and aquaculture are caused by fungal-like micro-organisms, classified as Oomycetes or water moulds. Collectively, oomycetes represent a huge threat to global food security. In addition, several oomycetes can cause environmental disasters, by wiping out native species of trees, crustacians and amphibians. The group representing the aquatic oomycetes are relatively understudied in contrast to their terrestrial counterparts that predominantly infect plants (e.g. Phytophthora, Pythium and Bremia spp.). This review focuses on the unique characteristics of two aquatic Oomycetes, Saprolegnia parasitica and Saprolegnia diclina with respect to their impact on aquaculture, animal health and the surrounding environment. The species characteristics, ecology, biology, infectivity and identification methods are described and the latest research insights are discussed.

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricalesas examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM. Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells. The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics., This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 634429. This publication reflects the views only of the author, and the European Commission cannot be held responsible for any use which may be made of the information contained therein. We also acknowledge the financial contribution made by the Real Jardín Botánico, CSIC. SR is grateful to the European Union [ITN-SAPRO-238550] for the support of her research in Saprolegnia. We also want to thank Francisco Calonge for kindly provide the Phellorinia herculanea images and B. Pueyo for processing samples (Figure 5). All images were taken by the SEM service at the Real Jardín Botánico-CSIC in Madrid.

Fungal emerging pathogens are one of the main threats for global biodiversity. Sea turtles do not seem to be an exemption, and recent studies on important nesting areas worldwide have shown that two fungal pathogens, i.e. Fusarium falciforme and Fusarium keratoplasticum, are involved in low hatching success in nests of sea turtle species. Although the presence of these pathogens has been detected in Ascension Island, there are no investigations on the distribution of these two pathogens in main nesting beaches in the island. In this study, we analysed 109 eggshells of the species Chelonia mydas from four nesting areas in Ascension Island. We have isolated and identified a total of 46 fungal isolates. A phylogenetic analysis, of the ITS nrDNA region, with a number of reference sequences of the Fusarium solani species complex, showed that 23 of these isolates corresponded to the pathogen F. keratoplasticum. The analyses on isolation frequency, that included other previously obtained isolates, i.e. 11 F. keratoplasticum and one F. falciforme, showed that F. keratoplasticum was the species most frequently isolated in Ascension Island and it was found in all nesting beaches, while F. falciforme was only isolated from Pan Am beach. When compared with other nesting areas worldwide, the abundance of F. keratoplasticum over F. falciforme was higher than any other nesting region tested. These findings are important in order to evaluate the potential threat of this pathogen to nests of the sea turtle population of Ascension Island, and to develop future control strategies., This work was supported by grants of Ministerio de Ciencia e Innovacio´n, Spain (CGL2009-10032, CGL2012-32934), J.M.S.R. was supported by PhD fellowship of the CSIC (JAEPre 0901804). P.v.W. was supported by the University of Aberdeen and a Darwin award to Dr Paul Brickle.