Trude Vrålstad

High diversity of root associated fungi in both alpine and arctic Dryas octopetala.

BackgroundDryas octopetala is a widespread dwarf shrub in alpine and arctic regions that forms ectomycorrhizal (ECM) symbiotic relationships with fungi. In this study we investigated the fungal communities associated with roots of D. octopetala in alpine sites in Norway and in the High Arctic on Svalbard, where we aimed to reveal whether the fungal diversity and species composition varied across the Alpine and Arctic regions. The internal transcribed spacer (ITS) region of nuclear ribosomal DNA was used to identify the fungal communities from bulk root samples obtained from 24 plants.ResultsA total of 137 operational taxonomic units (OTUs) were detected (using 97% similarity cut off during sequence clustering) and well-known ECM genera such as Cenococcum, Cortinarius, Hebeloma, Inocybe and Tomentella occurred frequently. There was no decrease in fungal diversity with increasing latitude. The overall spatial heterogeneity was high, but a weak geographical structuring of the composition of OTUs in the root systems was observed. Calculated species accumulation curves did not level off.ConclusionsThis study indicates that the diversity of fungi associated with D. octopetala does not decrease in high latitude arctic regions, which contrasts observations made in a wide spectrum of other organism groups. A high degree of patchiness was observed across root systems, but the fungal communities were nevertheless weakly spatially structured. Non-asymptotical species accumulation curves and the occurrence of a high number of singletons indicated that only a small fraction of the fungal diversity was detected.

New insights into the mycorrhizal Rhizoscyphus ericae aggregate: spatial structure and co‐colonization of ectomycorrhizal and ericoid roots

• Fungi in the Rhizoscyphus ericae aggregate have been recovered from the roots of co-occurring ericaceous shrubs and ectomycorrhizal trees. However, to date, there is no evidence that the same individual genotypes colonize both hosts, and no information on the extent of the mycelial networks that might form. • Using spatially explicit core sampling, we isolated fungi from neighbouring Pinus sylvestris (ectomycorrhizal) and Vaccinium vitis-idaea (ericoid mycorrhizal) roots and applied intersimple sequence repeat (ISSR) typing to assess the occurrence and extent of shared genets. • Most isolates were identified as Meliniomyces variabilis, and isolates with identical ISSR profiles were obtained from neighbouring ericoid and ectomycorrhizal roots on a number of occasions. However, genet sizes were small (< 13  cm), and several genets were found in a single soil core. Genetic relatedness was independent of spatial separation at the scales investigated (< 43  m) and M. variabilis populations from sites 20  km apart were genetically indistinguishable. • We conclude that individual genets of M. variabilis can simultaneously colonize Scots pine and Vaccinium roots, but there is no evidence for the formation of large mycelial networks. Our data also suggest significant genotypic overlap between widely separated populations of this ubiquitous root-associated fungus.

The postfire discomycete Geopyxis carbonaria (Ascomycota) is a biotrophic root associate with Norway spruce (Picea abies) in nature

The hypothesis that the postfire discomycete Geopyxis carbonaria (Ascomycota, Pezizales, Pyronemataceae) has a biotrophic association with roots of Norway spruce (Picea abies) in nature was tested by isolation of fungal strains from fresh, brown, smooth mycorrhiza‐like root tips of Norway spruce collected from below the depth of detrimental heat penetration in a postfire site. The morphology of seven culture isolates originating from the smooth mycorrhiza‐like root tips of two different spruce trees was congruent with the morphology of axenic culture isolates obtained from ascospores of G. carbonaria. DNA sequences of the nuclear ribosomal internal transcribed spacers ITS1 and ITS2 from these root‐derived cultures and the ascosporic G. carbonaria culture isolates were found to be identical, further supporting the conclusion that the isolates were conspecific. The extensive ascocarp and ascospore formation of G. carbonaria which succeeds a forest fire may be explained in terms of a fungal escape from a moribund tree associate. Possible ecological adaptations of G. carbonaria to the pre‐ and postfire community are discussed.

Detection and quantification of the crayfish plague agent in natural waters: direct monitoring approach for aquatic environments.

Aphanomyces astaci, a specialised parasite of North American freshwater crayfish, is the disease agent of crayfish plague that is lethal to European freshwater crayfish. The life cycle of A. astaci has been inferred from experimental laboratory studies, but less is known about its natural sustainability and ecology. To address such questions, tools for monitoring of A. astaci directly in aquatic environments are needed. Here, we present an approach for detecting and quantifying A. astaci directly from water samples using species-specific TaqMan minor groove binder real-time PCR. Samples of a 10-fold dilution series from approximately 10(4) to approximately 1 spore of A. astaci were repeatedly tested, and reliable detection down to 1 spore was demonstrated. Further, to simulate real-life samples from natural water bodies, water samples from lakes of various water qualities were spiked with spores. The results demonstrated that co-extracted humic acids inhibit detection significantly. However, use of bovine serum albumin or the TaqMan Environmental Master Mix largely removes this problem. The practical application of the approach was successfully demonstrated on real-life water samples from crayfish farms in Finland hosting infected North American signal crayfish Pacifastacus leniusculus. Direct monitoring of A. astaci from aquatic environments may find application in the management of wild noble crayfish Astacus astacus stocks, improved aquaculture practices and more targeted conservation actions. The approach will further facilitate studies of A. astaci spore dynamics during plague outbreaks and in carrier crayfish populations, which will broaden our knowledge of the biology of this devastating crayfish pathogen.

Invasive crayfish and crayfish plague on the move: first detection of the plague agent Aphanomyces astaci in the Romanian Danube.

Native European crayfish, such as Astacus leptodactylus, are threatened, among other factors, by the crayfish plague agent Aphanomyces astaci, dispersed by invasive North American crayfish. Two of these invaders, Pacifastacus leniusculus and Orconectes limosus, have extended their distribution in the River Danube catchment; the latter was detected for the first time in Romania in 2008. We monitored, at monthly intervals for over 2 yr, occurrence of native A. leptodactylus and invasive O. limosus at 6 sites on the Romanian Danube and checked for the invasive species in 4 of its tributaries. Between January 2009 and March 2011, the relative abundances of O. limosus steadily increased with time, while the native A. leptodactylus dramatically decreased in abundance. O. limosus expanded downstream at a rate of ca. 15 km yr-1; in August 2011, it was already present in the upper 105 km of the Romanian Danube. An agent-specific real-time PCR analyses demonstrated the presence of A. astaci DNA in at least 32% of the analysed invasive (n = 71) and 41% of the native (n = 49) crayfish coexisting in the Danube. Furthermore, A. astaci was also detected in A. leptodactylus captured about 70 km downstream of the O. limosus invasion front (at the time of sampling). Assuming a steady rate of expansion, O. limosus may invade the sensitive Danube delta area in the mid-2060s, even without long-distance dispersal. The crayfish plague agent, however, may reach the delta substantially earlier, through dispersal downstream among populations of native crayfish.

Re-examination of the prevalence of Aphanomyces astaci in North American crayfish populations in Central Europe by TaqMan MGB real-time PCR

We applied quantitative TaqMan minor groove binder real-time polymerase chain reaction (PCR) on DNA isolates from soft abdominal cuticle of 460 North American crayfish Orconectes limosus and Pacifastacus leniusculus, previously tested for Aphanomyces astaci presence by conventional semi-nested PCR. Both approaches target the internal transcribed spacers of the pathogen nuclear ribosomal DNA, but apply different specific sequence motifs and technologies. The real-time PCR approach seems to provide higher sensitivity; the number of crayfish that tested positive increased from 23 to 32%, and 10 additional crayfish populations were indicated as hosting the disease agent. However, the vast majority of newly recorded positives contained very low agent levels, from 5 to 50 PCR-forming units. An isolate producing a false positive result by the semi-nested PCR (apparently undescribed Aphanomyces related to A. astaci) remained negative using the real-time PCR. The present study shows that previous results based on the semi-nested PCR were not substantially influenced by false positives but might have suffered from some false negatives at low agent levels. Combining alternative methods may therefore provide more reliable conclusions on the pathogens presence. Further, we found positive correlation between the prevalence of infection carriers in American crayfish populations and the average amounts of A. astaci DNA detected in infected local crayfish individuals.

Monitoring the spore dynamics of Aphanomyces astaci in the ambient water of latent carrier crayfish.

The specialized crayfish parasite Aphanomyces astaci causes the devastating crayfish plague in European crayfish. Even though A. astaci sporulation has been thoroughly studied under pure culture conditions, little is known about the sporulation dynamic from its live host. Our purpose was to investigate the A. astaci spore dynamic in its native parasite-host relationship by monitoring the sporulation from carrier crayfish into the ambient water using agent specific qPCR. American signal crayfish (Pacifastacus leniusculus) with known positive carrier status were housed individually and communally in two experimental set-ups using multiple replicates and different temperatures. Water samples were collected weekly, and spore numbers were quantified. We demonstrate here that live latent carrier crayfish continuously released a moderate number of A. astaci spores (~2700 spores per crayfish/week) in the absence of death and moulting events. In contrast, a pronounced sporulation increase was seen already one week prior to death in moribund crayfish, suggesting a crayfish plague-like condition developing in weakened or stressed individuals. Significantly more spores were produced at 18°C compared to 4°C, while a negative correlation was detected between spore numbers and temperatures rising from 17 to 23°C. This study is the first attempt to quantify the spore release from carrier crayfish on the basis of qPCR applied on water samples, and demonstrate that the approach successfully unravel A. astaci sporulation patterns. The results emphasize that carrier crayfish pose a constant infection risk to highly susceptible crayfish species regardless of crayfish life cycle state.

Potent infection reservoir of crayfish plague now permanently established in Norway.

Noble crayfish Astacus astacus is threatened in Europe due to invasive crayfish carrying the crayfish plague agent Aphanomyces astaci. Norway is among the last countries in which the introduction of non-indigenous crayfish has been limited through strict legislation practices. However, North American signal crayfish Pacifastacus leniusculus were recently discovered in a water-course that has been repeatedly hit by the plague. We mapped the distribution and relative density (catch per unit effort) of signal crayfish within this lake, and performed agent-specific real-time PCR to estimate the prevalence of A. astaci in the population. The resulting length frequencies and relative density estimates clearly demonstrate a well-established signal crayfish population, in which 86.4% of the analysed individuals were confirmed carriers. The success of detection was significantly higher (84.1%) in the crayfish tailfan (i.e. uropods) than in the soft abdominal cuticle (38.4%), which is commonly used in prevalence studies. We therefore propose tailfan (uropods and telson) as the preferred tissue for studying A. astaci prevalence in signal crayfish populations. The likelihood of detecting an A. astaci-positive signal crayfish increased significantly with increasing crayfish length. Further, large female crayfish expressed significantly higher PCR-forming units values than large males. In surveys primarily exploring the presence of A. astaci-positive individuals in a population, large females should be selected for molecular analyses. Our study demonstrates that a potent crayfish plague infection reservoir, evidently originating from the illegal human introduction of signal crayfish, has permanently been established in Norway.

Microsatellite markers for direct genotyping of the crayfish plague pathogen Aphanomyces astaci (Oomycetes) from infected host tissues.

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.

ITS, OTUs and beyond—fungal hyperdiversity calls for supplementary solutions

Molecular species recognition of fungi emerged years before DNA barcoding ( Seifert 2009 ). While the ideal fungal DNA barcode seems Utopian, two research decades nevertheless highlight the internal transcribed spacer (ITS) as the best available choice ( Seifert 2009 ). Databases providing reliable ITS sequences of known fungi require enormous efforts, but are urgently needed ( Abarenkov et al. 2010a,b ; Begerow et al. 2010 ). Any criticism of such a commitment seems unjustified. However, exclusive focus on the development of ITS reference libraries will delay the progress towards a deeper ecological insight. It is widely acknowledged that ITS fails to recognize species, particularly in some ascomycete lineages ( Balajee et al. 2009 ; Seifert 2009 ). It also appears paradoxical to solely rely on ITS for ecological recognition of fungal species when modern fungal systematics rely on phylogenetic species recognition with concordance of multiple gene genealogies (see Blackwell 2011 ). Considering that at least 98% of the predicted ∼5 million fungal species remain undescribed ( Blackwell 2011 ), how will reliance on ITS alone influence the biodiversity estimates and ecological understanding? In this issue, Gazis et al. (2011) elegantly demonstrate through multi‐locus sequence phylogeny analyses that ITS largely underestimates the species diversity of tropical fungal endophytes and even more importantly obscures fundamental ecological and biogeographical patterns. This thorough reflection on species delimitation criteria and their implications for ecological and biogeographical inferences underline that ITS, particularly in hyperdiverse habitats, provides no shortcut to deeper knowledge of fungal ecology.