Ectomycorrhizal and non-mycorrhizal rhizosphere fungi increase root-derived C input to soil and modify enzyme activities: A 14 C pulse labelling of Picea abies seedlings.

Consequences of interactions between ectomycorrhizal fungi (EcMF) and non-mycorrhizal rhizosphere fungi (NMRF) for plant carbon (C) allocation belowground and nutrient cycling in soil remain unknown. To address this topic, we performed a mesocosm study with Norway spruce seedlings [Picea abies (L.) H. Karst] inoculated with EcMF, NMRF, or a mixture of both (MIX). 14 CO2 pulse labelling of spruce was applied to trace and visualize the 14 C incorporation into roots, rhizohyphosphere and hyphosphere. Activities and localization of enzymes involved in the C, nitrogen (N) and phosphorus (P) cycling were visualized using zymography. Spruce seedlings inoculated with EcMF and NMRF allocated more C to soils (EcMF: 10.7%; NMRF: 3.5% of total recovered C) compared to uninoculated control seedlings. The 14 C activity in the hyphosphere was highest for EcMF and lowest for NMRF. In the presence of both, NMRF and EcMF (MIX), the 14 C activity was 64% lower compared with EcMF inoculation alone. This suggests a suppressed C allocation via EcMF likely due to the competition between EcMF and NMRF for N and P. Furthermore, we observed 57% and 49% higher chitinase and leucine-aminopeptidase activities in the rhizohyphosphere of EcMF compared to the uninoculated control, respectively. In contrast, ß-glucosidase activity (14.3 nmol cm-2 h-1 ) was highest in NMRF likely because NMRF consumed rhizodeposits efficiently. This was further supported by that enzyme stoichiometry in soil with EcMF shifted to a higher investment of nutrient acquisition enzymes (e.g., chitinase, leucine-aminopeptidase, acid phosphatase) compared to NMRF inoculation, where investment in ß-glucosidase increased. In conclusion, the alleviation of EcMF from C limitation promotes higher activities of enzymes involved in the N and P cycle to cover the nutrient demand of EcMF and host seedlings. In contrast, C limitation of NMRF probably led to a shift in investment towards higher activities of enzymes involved in the C cycle.

Microbial functional changes mark irreversible course of Tibetan grassland degradation.

The Tibetan Plateau's Kobresia pastures store 2.5% of the world's soil organic carbon (SOC). Climate change and overgrazing render their topsoils vulnerable to degradation, with SOC stocks declining by 42% and nitrogen (N) by 33% at severely degraded sites. We resolved these losses into erosion accounting for two-thirds, and decreased carbon (C) input and increased SOC mineralization accounting for the other third, and confirmed these results by comparison with a meta-analysis of 594 observations. The microbial community responded to the degradation through altered taxonomic composition and enzymatic activities. Hydrolytic enzyme activities were reduced, while degradation of the remaining recalcitrant soil organic matter by oxidative enzymes was accelerated, demonstrating a severe shift in microbial functioning. This may irreversibly alter the world´s largest alpine pastoral ecosystem by diminishing its C sink function and nutrient cycling dynamics, negatively impacting local food security, regional water quality and climate.

Identification of Feldin, an Antifungal Polyyne from the Beefsteak Fungus Fistulina hepatica.

Fruiting body-forming members of the Basidiomycota maintain their ecological fitness against various antagonists like ascomycetous mycoparasites. To achieve that, they produce myriads of bioactive compounds, some of which are now being used as agrochemicals or pharmaceutical lead structures. Here, we screened ethyl acetate crude extracts from cultures of thirty-five mushroom species for antifungal bioactivity, for their effect on the ascomycete Saccharomyces cerevisiae and the basidiomycete Ustilago maydis. One extract that inhibited the growth of S. cerevisiae much stronger than that of U. maydis was further analyzed. For bioactive compound identification, we performed bioactivity-guided HPLC/MS fractionation. Fractions showing inhibition against S. cerevisiae but reduced activity against U. maydis were further analyzed. NMR-based structure elucidation from one such fraction revealed the polyyne we named feldin, which displays prominent antifungal bioactivity. Future studies with additional mushroom-derived eukaryotic toxic compounds or antifungals will show whether U. maydis could be used as a suitable host to shortcut an otherwise laborious production of such mushroom compounds, as could recently be shown for heterologous sesquiterpene production in U. maydis.

Multiple evolutionary origins of sequestrate species in the agaricoid genus Chlorophyllum.

Chlorophyllum accommodates lamellate agaricoid species as well as sequestrate angiocarpic taxa, which do not form a monophyletic lineage within the genus. To clarify phylogenetic affinities and delimit species boundaries among sequestrate representatives of the genus, we analyzed historical and contemporary material from a broad geographic range, encompassing North America, southern Africa, eastern Asia, the Greek and Iberian peninsulas, and the Mediterranean islands of Cyprus and Lesvos. Six sequestrate lineages of Chlorophyllum were identified, which appear to have evolved in at least three independent gasteromycetation events. Multigene analysis of the nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) and portions of the nuc 28S rDNA (28S), RNA polymerase II second largest subunit (rpb2), and translation elongation factor 1-α (tef1) genes revealed the presence of a previously undescribed species, introduced here as Chlorophyllum levantinum, sp. nov. Its sister species C. lusitanicum, previously known only from Spain, is shown to have a wider distribution throughout the Mediterranean basin. A South African collection of Secotium gueinzii, a poorly known taxon described in 1840 and seldom appearing in literature since, was also shown to nest within Chlorophyllum. An epitype for this rare species is designated, Secotium becomes a priority synonym of Chlorophyllum, and nomenclatural implications are discussed. The phylogenetic placement of C. arizonicum is confirmed after successful sequencing of the century-old holotype and an undescribed sister lineage of this species detected. Emended descriptions of sections Chlorophyllum, Endoptychorum, and Sphaerospororum are provided to reflect current results, along with updated descriptions and extensive imagery for all known sequestrate taxa of Chlorophyllum.

Impact of sea level change on coastal soil organic matter, priming effects and prokaryotic community assembly.

Salt marshes are coastal areas storing high amounts of soil organic matter (SOM) while simultaneously being prone to tidal changes. Here, SOM-decomposition and accompanied priming effects (PE), which describe interactions between labile and old SOM, were studied under controlled flooding conditions. Soil samples from two Wadden Sea salt marsh zones, pioneer (Pio), flooded two times/day, and lower salt marsh (Low), flooded ∼eight times/month, were measured for 56 days concerning CO2-efflux and prokaryotic community shifts during three different inundation-treatments: total-drained (Drained), all-time-flooded (Waterlogged) or temporal-flooding (Tidal). Priming was induced by 14C-glucose addition. CO2-efflux from soil followed Low>Pio and Tidal>Drained>Waterlogged, likely due to O2-depletion and moisture maintenance, two key factors governed by tidal inundation with regard to SOM mineralisation. PEs in both zones were positive (Drained) or absent (Waterlogged, Tidal), presumably as a result of prokaryotes switching from production of extracellular enzymes to direct incorporation of labile C. A doubled amount of prokaryotic biomass in Low compared to Pio probably induced higher chances of cometabolic effects and higher PE. 16S-rRNA-gene-amplicon-based analysis revealed differences in bacterial and archaeal community composition between both zones, revealing temporal niche adaptation with flooding treatment. Strongest alterations were found in Drained, likely due to inundation-mediated changes in C-binding capacities.

Element distribution in fruiting bodies of Lactarius pubescens with focus on rare earth elements.

During growth and senescence, fungal fruiting bodies accumulate essential and non-essential elements to different extent in their compartments. This study bases on a dataset of 32 basidiocarps of the ectomycorrhizal Lactarius pubescens sampled in a former U mining area. Statistical analyses were combined with rare earth element (REE, La-Lu) patterns to study the element distribution within sporocarp compartments and between three different age classes. For this purpose, fruiting bodies were separated into stipe, pileus trama, pileipelles and lamellae, dried and digested with HNO3. While macronutrient (e.g. K, Mg, P, S) contents resemble those of a non-mining affected site, several elements (e.g. Co, Mn) were site-specifically taken up relative to elevated soil contents. With statistics, two main element distribution groups for L. pubescens were revealed: mainly essential (Cu, Mg, Mn, P, S, Zn, Cd, Co, Ni) and mainly non-essential elements (Al, Ca, Fe, Sr, U, REE). The highest REE contents were found in pileipelles and lamellae, corresponding to relatively small cell sizes. Stipes and pileus trama had low REE contents due to their function as transport systems. During growth, light REE (La-Nd) were strongly enriched in lamellae and pileipelles. Middle REE (Sm-Dy) enrichment was found both in soil and fungal biomass. Contents of nutrients decrease with age, while non-essential elements are enriched especially in pileipelles and lamellae. A weak positive Ce anomaly appeared in the bioavailable soil fraction and in the pileipelles of younger individuals. Substrate dependent uptake thus gets reduced with sporocarp senescence, possibly due to redistribution.

First report of the post-fire morel Morchella exuberans in eastern North America.

Reports of true morels (Morchella) fruiting on conifer burn sites are common in western North America where five different fire-adapted species of black morels (Elata Clade) have been documented based on multilocus phylogenetic analyses. Fruiting of post-fire morels in eastern North America, by comparison, are rare and limited to a report from Minnesota in 1977 and eastern Ontario in 1991. Here, nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) sequences were used to identify the post-fire morel that fruited in great abundance the year following the 2012 Duck Lake Fire in the Upper Peninsula of Michigan and after the 2016 large-scale fire in the Great Smoky Mountains National Park in Tennessee as M. exuberans. A preliminary phylogenetic analysis suggests that the collections from eastern North America may be more closely related to those from Europe than from western North America, Europe, and China.

Hydrophobins in the Life Cycle of the Ectomycorrhizal Basidiomycete Tricholoma vaccinum.

Hydrophobins-secreted small cysteine-rich, amphipathic proteins-foster interactions of fungal hyphae with hydrophobic surfaces, and are involved in the formation of aerial hyphae. Phylogenetic analyses of Tricholoma vaccinum hydrophobins showed a grouping with hydrophobins of other ectomycorrhizal fungi, which might be a result of co-evolution. Further analyses indicate angiosperms as likely host trees for the last common ancestor of the genus Tricholoma. The nine hydrophobin genes in the T. vaccinum genome were investigated to infer their individual roles in different stages of the life cycle, host interaction, asexual and sexual development, and with respect to different stresses. In aerial mycelium, hyd8 was up-regulated. In silico analysis predicted three packing arrangements, i.e., ring-like, plus-like and sheet-like structure for Hyd8; the first two may assemble to rodlets of hydrophobin covering aerial hyphae, whereas the third is expected to be involved in forming a two-dimensional network of hydrophobins. Metal stress induced hydrophobin gene hyd5. In early steps of mycorrhization, induction of hyd4 and hyd5 by plant root exudates and root volatiles could be shown, followed by hyd5 up-regulation during formation of mantle, Hartig' net, and rhizomorphs with concomitant repression of hyd8 and hyd9. During fruiting body formation, mainly hyd3, but also hyd8 were induced. Host preference between the compatible host Picea abies and the low compatibility host Pinus sylvestris could be linked to a stronger induction of hyd4 and hyd5 by the preferred host and a stronger repression of hyd8, whereas the repression of hyd9 was comparable between the two hosts.

Tricholoma vaccinum host communication during ectomycorrhiza formation.

The genome sequence of Tricholoma vaccinum was obtained to predict its secretome in order to elucidate communication of T. vaccinum with its host tree spruce (Picea abies) in interkingdom signaling. The most prominent protein domains within the 206 predicted secreted proteins belong to energy and nutrition (52%), cell wall degradation (19%) and mycorrhiza establishment (9%). Additionally, we found small secreted proteins that show typical features of effectors potentially involved in host communication. From the secretome, 22 proteins could be identified, two of which showed higher protein abundances after spruce root exudate exposure, while five were downregulated in this treatment. The changes in T. vaccinum protein excretion with first recognition of the partner were used to identify small secreted proteins with the potential to act as effectors in the mutually beneficial symbiosis. Our observations support the hypothesis of a complex communication network including a cocktail of communication molecules induced long before physical contact of the partners.

Dynein Heavy Chain, Encoded by Two Genes in Agaricomycetes, Is Required for Nuclear Migration in Schizophyllum commune.

The white-rot fungus Schizophyllum commune (Agaricomycetes) was used to study the cell biology of microtubular trafficking during mating interactions, when the two partners exchange nuclei, which are transported along microtubule tracks. For this transport activity, the motor protein dynein is required. In S. commune, the dynein heavy chain is encoded in two parts by two separate genes, dhc1 and dhc2. The N-terminal protein Dhc1 supplies the dimerization domain, while Dhc2 encodes the motor machinery and the microtubule binding domain. This split motor protein is unique to Basidiomycota, where three different sequence patterns suggest independent split events during evolution. To investigate the function of the dynein heavy chain, the gene dhc1 and the motor domain in dhc2 were deleted. Both resulting mutants were viable, but revealed phenotypes in hyphal growth morphology and mating behavior as well as in sexual development. Viability of strain Δdhc2 is due to the higher expression of kinesin-2 and kinesin-14, which was proven via RNA sequencing.

Estimating the Phanerozoic history of the Ascomycota lineages: combining fossil and molecular data.

The phylum Ascomycota is by far the largest group in the fungal kingdom. Ecologically important mutualistic associations such as mycorrhizae and lichens have evolved in this group, which are regarded as key innovations that supported the evolution of land plants. Only a few attempts have been made to date the origin of Ascomycota lineages by using molecular clock methods, which is primarily due to the lack of satisfactory fossil calibration data. For this reason we have evaluated all of the oldest available ascomycete fossils from amber (Albian to Miocene) and chert (Devonian and Maastrichtian). The fossils represent five major ascomycete classes (Coniocybomycetes, Dothideomycetes, Eurotiomycetes, Laboulbeniomycetes, and Lecanoromycetes). We have assembled a multi-gene data set (18SrDNA, 28SrDNA, RPB1 and RPB2) from a total of 145 taxa representing most groups of the Ascomycota and utilized fossil calibration points solely from within the ascomycetes to estimate divergence times of Ascomycota lineages with a Bayesian approach. Our results suggest an initial diversification of the Pezizomycotina in the Ordovician, followed by repeated splits of lineages throughout the Phanerozoic, and indicate that this continuous diversification was unaffected by mass extinctions. We suggest that the ecological diversity within each lineage ensured that at least some taxa of each group were able to survive global crises and rapidly recovered.

The anamorphic genus Monotosporella (Ascomycota) from Eocene amber and from modern Agathis resin.

The anamorphic fungal genus Monotosporella (Ascomycota, Sordariomycetes) has been reco-vered from a piece of Early Eocene Indian amber, as well as from the surface of extant resin flows in New Caledonia. The fossil fungus was obtained from the Tarkeshwar Lignite Mine of Gujarat State, western India, and was part of the biota of an early tropical angiosperm rainforest. The amber inclusion represents the second fossil record of Sordariomycetes, as well as the first fossil of its particular order (either Savoryellales or Chaetosphaeriales). The fossil fungus is distinguished from extant representatives by possessing both short conidiophores and small two-septate pyriform conidia, and is described as Monotosporella doerfeltii sp. nov. Inside the amber, the anamorph is attached to its substrate, which is likely the degraded thallus of a cladoniform lichen. The extant New Caledonian species is assigned to Monotosporella setosa. It was found growing on semi-solidified resin flows of Agathis ovata (Araucariaceae), and is the first record of Monotosporella from modern resin substrates.

A consistent phylogenetic backbone for the fungi.

The kingdom of fungi provides model organisms for biotechnology, cell biology, genetics, and life sciences in general. Only when their phylogenetic relationships are stably resolved, can individual results from fungal research be integrated into a holistic picture of biology. However, and despite recent progress, many deep relationships within the fungi remain unclear. Here, we present the first phylogenomic study of an entire eukaryotic kingdom that uses a consistency criterion to strengthen phylogenetic conclusions. We reason that branches (splits) recovered with independent data and different tree reconstruction methods are likely to reflect true evolutionary relationships. Two complementary phylogenomic data sets based on 99 fungal genomes and 109 fungal expressed sequence tag (EST) sets analyzed with four different tree reconstruction methods shed light from different angles on the fungal tree of life. Eleven additional data sets address specifically the phylogenetic position of Blastocladiomycota, Ustilaginomycotina, and Dothideomycetes, respectively. The combined evidence from the resulting trees supports the deep-level stability of the fungal groups toward a comprehensive natural system of the fungi. In addition, our analysis reveals methodologically interesting aspects. Enrichment for EST encoded data-a common practice in phylogenomic analyses-introduces a strong bias toward slowly evolving and functionally correlated genes. Consequently, the generalization of phylogenomic data sets as collections of randomly selected genes cannot be taken for granted. A thorough characterization of the data to assess possible influences on the tree reconstruction should therefore become a standard in phylogenomic analyses.

Polyphasic study of plant- and clinic-associated Pantoea agglomerans strains reveals indistinguishable virulence potential.

Pantoea species are ubiquitous in nature and occasionally associated with infections caused by contaminated clinical material. Hence, Pantoea agglomerans is considered as an opportunistic pathogen of humans. Since species of the genus Pantoea and closely related species of other Enterobacteriaceae genera are phenotypically very similar, many clinical isolates are misassigned into P. agglomerans based on the use of quick commercial-offered biochemical tests. Our objective was to find markers enabling discrimination between clinical and plant isolates and to assess their virulence potential. We characterized 27 Pantoea strains, including 8 P. agglomerans isolates of clinical, and 11 of plant origin by biochemical tests and genotyping, including analysis of 16S rDNA and gapA gene sequences, and pattern polymorphisms of ITS- and ERIC/REP-DNA. All data showed that no discrete evolution occurred between plant-associated and clinical P. agglomerans isolates. Based on the typing results, five clinical- and five plant-associated P. agglomerans strains representing the majority of clades were tested on a model plant and in embryonated eggs. On soybean plants P. agglomerans strains independent of their origin could develop stable epiphytic populations. Surprisingly, in the embryonated egg model there was no difference of virulence between clinical and vegetable P. agglomerans isolates. However, these strains were significantly less virulent than a phytopathogenic P. ananatis isolate. We suggest that, independent of their origin, all P. agglomerans strains might possess indistinguishable virulence potential.

In silico analysis of nickel containing superoxide dismutase evolution and regulation.

Superoxide dismutases are essential enzymes involved in detoxification of reactive oxygen by dismutation of the superoxide radical anion. A class of nickel containing superoxide dismutases has been described for streptomycetes and cyanobacteria. In silico analysis was used to study the distribution of genes coding for NiSOD in other taxa and to elucidate signals linked to nickel incorporation and maturation of NiSOD. Data mining revealed homologous proteins from actinobacteria, proteobacteria, chlamydiae, and eukarya (green algae) thus allowing a comparison of protein structural elements. Nickel ligands and maturation signals for N-terminal proteolysis were highly conserved. Genomic sequences surrounding genes encoding NiSOD homologs were compared in order to detect putative accessory enzymes involved in maturation. An endopeptidase gene linked to sodN coding for NiSOD was found in actinobacteria and cyanobacteria, but not in other taxa. The distribution of NiSOD encoding sequences showed four clusters which are not consistent with the phylogeny of the species. In addition, the different genomic context argues for heterologous gene transfer, most likely from actinobacteria to other taxa. In order to address regulation by nickel availability and incorporation into the mature protein, we present first evidence for putative regulatory nucleotide sequences which will be useful in future studies on nickel uptake and incorporation.

The gleba development of Langermannia gigantea (Batsch: Pers.) Rostk. (Basidiomycetes) compared to other Lycoperdaceae, and some systematic implications.

The fruit body and in particular the gleba development of several species of Lycoperdaceae has been examined by light microscopic analysis of microtome sections of fruit body primordia in different ontogenetic stages. The gleba of Langermannia gigantea develops in a unique and previously unknown manner: the hymenium-forming palisade structures are borne on fan-like branches of hyphae. Hence the term of flabelloid glebal development is introduced. The other genera of Lycoperdaceae, including the genus Calvatia, are characterized by both lacunar and coralloid development of the gleba. Because these features cannot be distinguished clearly, this type of glebal development is referred to as coralloid-lacunar. Due to the peculiar ontogeny I suggest keeping the genus Langermannia separate from Calvatia.