Experimental evolution of extremotolerant and extremophilic fungi under osmotic stress.

Experimental evolution was carried out to investigate the adaptive responses of extremotolerant fungi to a stressful environment. For 12 cultivation cycles, the halotolerant black yeasts Aureobasidium pullulans and Aureobasidium subglaciale were grown at high NaCl or glycerol concentrations, and the halophilic basidiomycete Wallemia ichthyophaga was grown close to its lower NaCl growth limit. All evolved Aureobasidium spp. accelerated their growth at low water activity. Whole genomes of the evolved strains were sequenced. No aneuploidies were detected in any of the genomes, contrary to previous studies on experimental evolution at high salinity with other species. However, several hundred single-nucleotide polymorphisms were identified compared with the genomes of the progenitor strains. Two functional groups of genes were overrepresented among the genes presumably affected by single-nucleotide polymorphisms: voltage-gated potassium channels in A. pullulans at high NaCl concentration, and hydrophobins in W. ichthyophaga at low NaCl concentration. Both groups of genes were previously associated with adaptation to high salinity. Finally, most evolved Aureobasidium spp. strains were found to have increased intracellular and decreased extracellular glycerol concentrations at high salinity, suggesting that the strains have optimised their management of glycerol, their most important compatible solute. Experimental evolution therefore not only confirmed the role of potassium transport, glycerol management, and cell wall in survival at low water activity, but also demonstrated that fungi from extreme environments can further improve their growth rates under constant extreme conditions in a relatively short time and without large scale genomic rearrangements.

High-resolution single-molecule long-fragment rRNA gene amplicon sequencing of bacterial and eukaryotic microbial communities.

Sequencing of hypervariable regions as well as internal transcribed spacer regions of ribosomal RNA genes (rDNA) is broadly used to identify bacteria and fungi, but taxonomic and phylogenetic resolution is hampered by insufficient sequencing length using high throughput, cost-efficient second-generation sequencing. We developed a method to obtain nearly full-length rDNA by assembling single DNA molecules combining DNA co-barcoding with single-tube long fragment read technology and second-generation sequencing. Benchmarking was performed using mock bacterial and fungal communities as well as two forest soil samples. All mock species rDNA were successfully recovered with identities above 99.5% compared to the reference sequences. From the soil samples we obtained good coverage with identification of more than 20,000 unknown species, as well as high abundance correlation between replicates. This approach provides a cost-effective method for obtaining extensive and accurate information on complex environmental microbial communities.

Clonality, inbreeding, and hybridization in two extremotolerant black yeasts.

BACKGROUND: The great diversity of lifestyles and survival strategies observed in fungi is reflected in the many ways in which they reproduce and recombine. Although a complete absence of recombination is rare, it has been reported for some species, among them 2 extremotolerant black yeasts from Dothideomycetes: Hortaea werneckii and Aureobasidium melanogenum. Therefore, the presence of diploid strains in these species cannot be explained as the product of conventional sexual reproduction. RESULTS: Genome sequencing revealed that the ratio of diploid to haploid strains in both H. werneckii and A. melanogenum is about 2:1. Linkage disequilibrium between pairs of polymorphic loci and a high degree of concordance between the phylogenies of different genomic regions confirmed that both species are clonal. Heterozygosity of diploid strains is high, with several hybridizing genome pairs reaching the intergenomic distances typically seen between different fungal species. The origin of diploid strains collected worldwide can be traced to a handful of hybridization events that produced diploids, which were stable over long periods of time and distributed over large geographic areas. CONCLUSIONS: Our results, based on the genomes of over 100 strains of 2 black yeasts, show that although they are clonal, they occasionally form stable and highly heterozygous diploid intraspecific hybrids. The mechanism of these apparently rare hybridization events, which are not followed by meiosis or haploidization, remains unknown. Both extremotolerant yeasts, H. werneckii and even more so A. melanogenum, a close relative of the intensely recombining and biotechnologically relevant Aureobasidium pullulans, provide an attractive model for studying the role of clonality and ploidy in extremotolerant fungi.

Leguminous cover crops and soya increased soil fungal diversity and suppressed pathotrophs caused by continuous cereal cropping.

The enrichment of soil-borne fungal pathogens and a high input of mineral fertilizer in the continuous cropping of cereal crops have raised a concern about soil health deterioration. Conversion of continuous cereal cropping to a legume-involved system alters the soil fungal community. However, when a leguminous cover crop is grown with a succeeding legume grain crop such as soya (Glycine max L. Merril), the effects on the soil fungal community when two legumes are involved in the crop system remain unclear. Thus, the effects of the cover crop on the soil fungal community under a succession of soya and a succession of maize (Zea mays L.) were clarified: a continuous wheat (Triticum aestivum L.)-maize cropping system was converted to new rotation systems with three cover crop treatments: leguminous vetch (Vicia sativa L.), a mixture of vetch and rye (Secale cereale L.), and fallow, succeeded by soya or maize in this study. The soil fungal community at the harvest of soya and maize were determined using high-throughput sequencing of ITS2 amplicons. Compared to a wheat-maize rotation system, all of the new rotation systems that involved leguminous crops or fallow increased the soil fungal diversity and suppressed pathotrophs by reducing the soil NH4 +, NO3 -, available K, and available P concentrations. Different cover crops changed the fungal community composition, but their effect was overwhelmed by the strong effect of succeeding soya, which induced minor shifts among the cover crop treatments under soya than maize. The Vetch-Soya system exhibited the highest fungal diversity, which have been due to an increase of symbiotrophs. Replacing wheat with mixed vetch and rye most greatly suppressed the pathotrophs, and this suppression effect was stronger when succeeded by maize than by soya. These results showed the short-term benefits of legume-legume succession and legume-cereal mixed cover crops for increasing fungal diversity and suppressing pathotrophs. Further study is needed to examine the long-term effects of Vetch-Soya on the accumulation of legume-associated pathogens.

From Glaciers to Refrigerators: the Population Genomics and Biocontrol Potential of the Black Yeast Aureobasidium subglaciale.

Apples are affected by numerous fungi known as storage rots, which cause significant losses before and after harvest. Concerns about increasing antimicrobial resistance, bans on various fungicides, and changing consumer preferences are motivating the search for safer means to prevent fruit rot. The use of antagonistic microbes has been shown to be an efficient and environmentally friendly alternative to conventional phytopharmaceuticals. Here, we investigate the potential of Aureobasidium subglaciale for postharvest rot control. We tested the antagonistic activity of 9 strains of A. subglaciale and 7 closely related strains against relevant phytopathogenic fungi under conditions simulating low-temperature storage: Botrytis cinerea, Penicillium expansum, and Colletotrichum acutatum. We also investigated a selection of phenotypic traits of all strains and sequenced their whole genomes. The tested strains significantly reduced postharvest rot of apples at low temperatures caused by B. cinerea, C. acutatum (over 60%), and P. expansum (about 40%). Several phenotypic traits were observed that may contribute to this biocontrol capacity: growth at low temperatures, tolerance to high temperatures and elevated solute concentrations, and strong production of several extracellular enzymes and siderophores. Population genomics revealed that 7 of the 15 strains originally identified as A. subglaciale most likely belong to other, possibly undescribed species of the same genus. In addition, the population structure and linkage disequilibrium of the species suggest that A. subglaciale is strictly clonal and therefore particularly well suited for use in biocontrol. Overall, these data suggest substantial biological control potential for A. subglaciale, which represents another promising biological agent for disease control in fresh fruit. IMPORTANCE After harvest, fruits are often stored at low temperatures to prolong their life. However, despite the low temperatures, much of the fruit is lost to rot caused by a variety of fungi, resulting in major economic losses and food safety risks. An increasingly important environmentally friendly alternative to conventional methods of mitigating the effects of plant diseases is the use of microorganisms that act similarly to probiotics-occupying the available space, producing antimicrobial compounds, and consuming the nutrients needed by the rot-causing species. To find a new microorganism for biological control that is particularly suitable for cold storage of fruit, we tested different isolates of the cold-loving yeast Aureobasidium subglaciale and studied their phenotypic characteristics and genomes. We demonstrated that A. subglaciale can significantly reduce rotting of apples caused by three rot-causing molds at low temperatures and thus has great potential for preventing fruit rot during cold storage.

Network structure of resource use and niche overlap within the endophytic microbiome.

Endophytes often have dramatic effects on their host plants. Characterizing the relationships among members of these communities has focused on identifying the effects of single microbes on their host, but has generally overlooked interactions among the myriad microbes in natural communities as well as potential higher-order interactions. Network analyses offer a powerful means for characterizing patterns of interaction among microbial members of the phytobiome that may be crucial to mediating its assembly and function. We sampled twelve endophytic communities, comparing patterns of niche overlap between coexisting bacteria and fungi to evaluate the effect of nutrient supplementation on local and global competitive network structure. We found that, despite differences in the degree distribution, there were few significant differences in the global network structure of niche-overlap networks following persistent nutrient amendment. Likewise, we found idiosyncratic and weak evidence for higher-order interactions regardless of nutrient treatment. This work provides a first-time characterization of niche-overlap network structure in endophytic communities and serves as a framework for higher-resolution analyses of microbial interaction networks as a consequence and a cause of ecological variation in microbiome function.

Virulence Traits and Population Genomics of the Black Yeast Aureobasidium melanogenum.

The black yeast-like fungus Aureobasidium melanogenum is an opportunistic human pathogen frequently found indoors. Its traits, potentially linked to pathogenesis, have never been systematically studied. Here, we examine 49 A. melanogenum strains for growth at 37 °C, siderophore production, hemolytic activity, and assimilation of hydrocarbons and human neurotransmitters and report within-species variability. All but one strain grew at 37 °C. All strains produced siderophores and showed some hemolytic activity. The largest differences between strains were observed in the assimilation of hydrocarbons and human neurotransmitters. We show for the first time that fungi from the order Dothideales can assimilate aromatic hydrocarbons. To explain the background, we sequenced the genomes of all 49 strains and identified genes putatively involved in siderophore production and hemolysis. Genomic analysis revealed a fairly structured population of A.melanogenum, raising the possibility that some phylogenetic lineages have higher virulence potential than others. Population genomics indicated that the species is strictly clonal, although more than half of the genomes were diploid. The existence of relatively heterozygous diploids in an otherwise clonal species is described for only the second time in fungi. The genomic and phenotypic data from this study should help to resolve the non-trivial taxonomy of the genus Aureobasidium and reduce the medical hazards of exploiting the biotechnological potential of other, non-pathogenic species of this genus.

Efficient and stable metabarcoding sequencing data using a DNBSEQ-G400 sequencer validated by comprehensive community analyses.

Metabarcoding is a widely used method for fast characterization of microbial communities in complex environmental samples. However, the selction of sequencing platform can have a noticeable effect on the estimated community composition. Here, we evaluated the metabarcoding performance of a DNBSEQ-G400 sequencer developed by MGI Tech using 16S and internal transcribed spacer (ITS) markers to investigate bacterial and fungal mock communities, as well as the ITS2 marker to investigate the fungal community of 1144 soil samples, with additional technical replicates. We show that highly accurate sequencing of bacterial and fungal communities is achievable using DNBSEQ-G400. Measures of diversity and correlation from soil metabarcoding showed that the results correlated highly with those of different machines of the same model, as well as between different sequencing modes (single-end 400 bp and paired-end 200 bp). Moderate, but significant differences were observed between results produced with different sequencing platforms (DNBSEQ-G400 and MiSeq); however, the highest differences can be caused by selecting different primer pairs for PCR amplification of taxonomic markers. These differences suggested that care is needed while jointly analyzing metabarcoding data from differenet experiments. This study demonstrated the high performance and accuracy of DNBSEQ-G400 for short-read metabarcoding of microbial communities. Our study also produced datasets to allow further investigation of microbial diversity.

Inhibitory interaction networks among coevolved Streptomyces populations from prairie soils.

Soil microbes live within highly complex communities, where community composition, function, and evolution are the product of diverse interactions among community members. Analysis of the complex networks of interactions within communities has the potential to shed light on community stability, functioning, and evolution. However, we have little understanding of the variation in interaction networks among coevolved soil populations. We evaluated networks of antibiotic inhibitory interactions among sympatric Streptomyces communities from prairie soil. Inhibition networks differed significantly in key network characteristics from expectations under null models, largely reflecting variation among Streptomyces in the number of sympatric populations that they inhibited. Moreover, networks of inhibitory interactions within Streptomyces communities differed significantly from each other, suggesting unique network structures among soil communities from different locations. Analyses of tri-partite interactions (triads) showed that some triads were significantly over- or under- represented, and that communities differed in 'preferred' triads. These results suggest that local processes generate distinct structures among sympatric Streptomyces inhibition networks in soil. Understanding the properties of microbial interaction networks that generate competitive and functional capacities of soil communities will shed light on the ecological and coevolutionary history of sympatric populations, and provide a foundation for more effective management of inhibitory capacities of soil microbial communities.

Population Genomics of an Obligately Halophilic Basidiomycete Wallemia ichthyophaga.

BACKGROUND: Wallemia ichthyophaga is a highly specialized basidiomycetous fungus. It is one of the most halophilic fungi ever described, only able to grow at low water activity. This specialization is thought to explain why it is only rarely isolated from nature. RESULTS: Genomes of 21 W. ichthyophaga strains were sequenced with PE150 reads on BGISEQ500 platform. The genomes shared high similarity with the reference genome of the species, they were all smaller than 10 Mbp and had a low number of predicted genes. Groups of strains isolated in the same location encompassed clones as well as very divergent strains. There was little concordance between phylogenies of predicted genes. Linkage disequilibrium of pairs of polymorphic loci decayed relatively quickly as a function of distance between the loci (LD decay distance 1270 bp). For the first time a putative mating-type locus was identified in the genomes of W. ichthyophaga. CONCLUSION: Based on the comparison of W. ichthyophaga genomes it appears that some phylogenetic lineages of the species can persist in the same location over at least several years. Apart from this, the differences between the strains do not reflect the isolation habitat or geographic location. Together with results supporting the existence of (sexual) recombination in W. ichthyophaga, the presented results indicate that strains of W. ichthyophaga can form a single recombining population even between different habitats and over large geographical distances.

Genomic Evidence of Recombination in the Basidiomycete Wallemia mellicola.

One of the most commonly encountered species in the small basidiomycetous sub-phylum Wallemiomycotina is Wallemiamellicola, a xerotolerant fungus with a widespread distribution. To investigate the population characteristics of the species, whole genomes of twenty-five strains were sequenced. Apart from identification of four strains of clonal origin, the distances between the genomes failed to reflect either the isolation habitat of the strains or their geographical origin. Strains from different parts of the world appeared to represent a relatively homogenous and widespread population. The lack of concordance between individual gene phylogenies and the decay of linkage disequilibrium indicated that W. mellicola is at least occasionally recombining. Two versions of a putative mating-type locus have been found in all sequenced genomes, each present in approximately half of the strains. W. mellicola thus appears to be capable of (sexual) recombination and shows no signs of allopatric speciation or specialization to specific habitats.

Effort versus Reward: Preparing Samples for Fungal Community Characterization in High-Throughput Sequencing Surveys of Soils.

Next generation fungal amplicon sequencing is being used with increasing frequency to study fungal diversity in various ecosystems; however, the influence of sample preparation on the characterization of fungal community is poorly understood. We investigated the effects of four procedural modifications to library preparation for high-throughput sequencing (HTS). The following treatments were considered: 1) the amount of soil used in DNA extraction, 2) the inclusion of additional steps (freeze/thaw cycles, sonication, or hot water bath incubation) in the extraction procedure, 3) the amount of DNA template used in PCR, and 4) the effect of sample pooling, either physically or computationally. Soils from two different ecosystems in Minnesota, USA, one prairie and one forest site, were used to assess the generality of our results. The first three treatments did not significantly influence observed fungal OTU richness or community structure at either site. Physical pooling captured more OTU richness compared to individual samples, but total OTU richness at each site was highest when individual samples were computationally combined. We conclude that standard extraction kit protocols are well optimized for fungal HTS surveys, but because sample pooling can significantly influence OTU richness estimates, it is important to carefully consider the study aims when planning sampling procedures.

Signature wood modifications reveal decomposer community history.

Correlating plant litter decay rates with initial tissue traits (e.g. C, N contents) is common practice, but in woody litter, predictive relationships are often weak. Variability in predicting wood decomposition is partially due to territorial competition among fungal decomposers that, in turn, have a range of nutritional strategies (rot types) and consequences on residues. Given this biotic influence, researchers are increasingly using culture-independent tools in an attempt to link variability more directly to decomposer groups. Our goal was to complement these tools by using certain wood modifications as 'signatures' that provide more functional information about decomposer dominance than density loss. Specifically, we used dilute alkali solubility (DAS; higher for brown rot) and lignin:density loss (L:D; higher for white rot) to infer rot type (binary) and fungal nutritional mode (gradient), respectively. We first determined strength of pattern among 29 fungi of known rot type by correlating DAS and L:D with mass loss in birch and pine. Having shown robust relationships for both techniques above a density loss threshold, we then demonstrated and resolved two issues relevant to species consortia and field trials, 1) spatial patchiness creating gravimetric bias (density bias), and 2) brown rot imprints prior or subsequent to white rot replacement (legacy effects). Finally, we field-tested our methods in a New Zealand Pinus radiata plantation in a paired-plot comparison. Overall, results validate these low-cost techniques that measure the collective histories of decomposer dominance in wood. The L:D measure also showed clear potential in classifying 'rot type' along a spectrum rather than as a traditional binary type (brown versus white rot), as it places the nutritional strategies of wood-degrading fungi on a scale (L:D=0-5, in this case). These information-rich measures of consequence can provide insight into their biological causes, strengthening the links between traits, structure, and function during wood decomposition.

Influence of Hyphal Inoculum potential on the Competitive Success of Fungi Colonizing Wood.

The relative amounts of hyphal inoculum in forest soils may determine the capacity for fungi to compete with and replace early colonizers of wood in ground contact. Our aim in this study was to test the flexibility of priority effects (colonization timing) by varying the timing of inoculum introduction (i.e., precolonization) and amount of inoculum (i.e., inoculum potential). We controlled these variables in soil-block microcosms using fungi with known competitive outcomes in similar conditions, tracking isolate-specific fungal biomass, and residue physiochemistry over time. In the precolonization trial (experiment I), a brown rot fungus Gloeophyllum trabeum was given 1, 3, or 5 weeks to precolonize wood blocks (oak, birch, pine, and spruce) prior the introduction of a white rot fungus, Irpex lacteus, a more aggressive colonizer in this set-up. In the inoculum potential trial (experiment II), the fungi were inoculated simultaneously, but with eightfold higher brown rot inoculum than that of experiment I. As expected, longer precolonization duration increased the chance for the less-competitive brown rot fungus to outcompete its white rot opponent. Higher brown rot fungal inoculum outside of the wood matrix also resulted in competitive success for the brown rot isolate in most cases. These temporal shifts in fungal dominance were detectable in a 'community snapshot' as isolate-specific quantitative PCR, but also as functionally-relevant consequences of wood rot type, including carbohydrate depolymerization and pH. These results from a controlled system reinforce fungal-fungal interaction and suggest that relative inoculum availability beyond the wood matrix (i.e., soils) might regulate the duration of priority effects and shift the functional trajectory of wood decomposition.

Competition between two wood-degrading fungi with distinct influences on residues.

Many wood-degrading fungi colonize specific types of forest trees, but often lack wood specificity in pure culture. This suggests that wood type affects competition among fungi and indirectly influences the soil residues generated. While assessing wood residues is an established science, linking this information to dominant fungal colonizers has proven to be difficult. In the studies presented here, we used isolate-specific quantitative PCR to quantify competitive success between two distinct fungi, Gloeophyllum trabeum and Irpex lacteus, brown and white rot fungi, respectively, colonizing three wood types (birch, pine, oak). Ergosterol (fungal biomass), fungal species-specific DNA copy numbers, mass loss, pH, carbon fractions, and alkali solubility were determined 3 and 8 weeks postinoculation from replicate wood sections. Quantitative PCR analyses indicated that I. lacteus consistently outcompeted G. trabeum, by several orders of magnitude, on all wood types. Consequently, wood residues exhibited distinct characteristics of white rot. Our results show that competitive interactions between fungal species can influence colonization success, and that this can have significant consequences on the outcomes of wood decomposition.