Abstract

Availability of DNA sequencing approaches to identify fungi in samples from nature revolutionized the research on fungi and are now standard. DNA sequencing of fungal specimens and cultures, and natural soil and root samples has considerably changed the understanding of the diversity of Glomeromycotina, fungi forming arbuscular mycorrhiza (AM). It is now possible to identify AM fungal taxa by DNA sequencing irrespective of the presence of microscopically identifiable structures. Further developments of species proxies (Virtual Taxa, VT; Species Hypotheses, SH) and concurrent systematic organization of information about their occurrences in databases provides tools and data for broad research community to target these fungi in studies from taxonomy to physiology and genomics or to ecosystem sciences and beyond. However, several questions remain unsolved. How to handle “unnamed” diversity? How to appropriately delimit DNA sequence based species proxies? How to maintain databases? Do the unnamed AM fungi carry biological properties that differ from those of cultured “lab rats”? To illustrate some of these issues, I will tackle Rhizophagus intraradices-irregularis species group. I’ll present the current knowledge of the phylogenetic diversity within the group in the context of recent genotyping data, the global geographical and habitat-wise distribution in the group, and the share of data in culture collections vs DNA sequences from various samples. I will conclude with future prospects in the direction of unravelling the AM fungal diversity and its patterns in the nature.

Abstract

Mycorrhizal and pathogenic fungi dominate curated databases of root-associated fungi limiting our capacity to identify and define specific functions of other fungal symbionts found in plant roots. Next generation sequencing and the use of fungal-plant bioassays can guide studies for the discovery and characterization of novel and specialized fungal species across multiple environments. This talk will discuss the importance of technology integration and collaborations to advance our understanding of plant-fungal interactions, the discovery of dark taxa, and the emergent properties that result from complex microbial interactions. I will focus on the use of targeted culturing efforts based on Illumina sequencing data to discover and characterize abundant but unknown fungal species.

Abstract

The increasing numbers of studies using environmental nucleic acid sequences (ENAS) to assess fungal diversity have revealed the existence of thousands of previously unknown, and in many cases unculturable, species. It is estimated that more than a third of all fungal DNA sequences in GenBank are of environmental origin. But inconsistent annotation of these undescribed, sequence-based taxa limits functional access to the data. Consequently, these ENAS are rarely considered in other studies, especially not in taxonomic treatments. This problem is confound by the fact that the International Code of Nomenclature for Algae, Fungi, and Plants at present prohibits the description of novel taxa known only from ENAS, which discourages taxonomists to include these sequences in their studies. Various options have been suggested by members of the mycological community to amend the Code to allow the systematic nomenclatural treatment of these ‘orphan’ taxa. One possibility would be to allow DNA sequences as types instead of the typical herbarium specimens, graphic representations or living cultures. As an example, a new species with an ITS sequence as type was recently described in a study based on two matching ITS sequences of fungi inhabiting conifer wood, but that was generated in two earlier, independent studies. One came from an uncultured fungus clone from spruce in Sweden, and the other from a culture from cedar wood in Canada, that later died. The lineage containing these two sequences was phylogenetically different from related species in the Ophiostomatales and was described as “Hawksworthiomyces sequentia sp. nov. ENAS”. It was suggested that in cases like these the ENAS acronym should be used with the species name until a specimen is found and designated as type, after which it can be omitted. Of importance is that this novel ENAS species was described adhering to currently accepted phylogenetic standards in the Ophiostomatales. The inclusion of environmental LSU sequences in the same study, furthermore confirmed the presence of a novel genus in the order that was represented previously only by a single taxon. This genus seems to be biologically and ecologically different from other genera in the order, and thus enhances our understanding of evolutionary processes in this group of fungi. In this rather modest case study we showed that much value can be added by including environmental sequences in taxonomic studies. What is clear is that a decision and guidance is needed from the mycological community that will allow for and enable the systematic naming of sequence-based taxa.

Abstract

The number of sequences in public sequence databases without specific taxon name are steadily increasing. It is clear that many of these sequences represent taxa that have previously not been recognized by the scientific community, but that are important for our understanding of fungal diversity, ecology, and evolution. For example, Archaeorhizomyces is a group with world wide distribution and many species, but searching biodiversity databases such as GBIF and Species Fungorum there are only two species listed, since they are the only that have been possible to typify and name according to the rules of the International Code of Nomenclature of algae, fungi, and plants (ICN). Even if sequences without specific taxon names are now being included into some taxonomic studies, and species are being described to accommodate them, they are usually ignored due to the lack of valid name under the ICN. What is included in biodiversity datasets does therefore often depend on nomenclature issues rather than taxonomic issues, even if taxa is what are of interest. There are many possible ways to amend this problem, for example: 1) We can separate our biodiversity data from the dependence of names. We then need some other unique identifier, preferably global identifiers that can be used to link different datasets. UNITE provides identifiers in the form of DOIs for species hypotheses, but not species. The identifiers consequently change with the dataset on which the hypotheses are based, and they are not stable taxon identifiers. We could construct another system based for example on the identifiers provided by taxonomic databases. However, DOIs and accession numbers alike may be suitable for database handling, but are less suitable for human communication. 2) We can use names not valid under the code of nomenclature, which are now being provided for some taxa, and include these in biodiversity datasets. The downside with this is that there is no available, agreed upon, rules to govern these names and resolve conflicts between them, i.e. nothing to fill the very function ICN was designed for. 3) We can amend the ICN to include names for taxa without any physical voucher. One possible downside with this is that the number of descriptions of taxa that are in fact not new to sciences may increase significantly, and thereby the number of synonyms, making nomenclature and consequently taxonomy more cumbersome. The extent of the problems with names outside the code and names governed by the code will depends on the actions of the community in promoting good taxonomy and nomenclature through discussions, training and peer-review, but also on the actions of individual researchers. There is likely no system without any risks or drawbacks to deal with the issues of species known only from sequences, but we urgently need a solution to be able to present an as true picture of the fungal diversity as possible.

Abstract

Today, species are increasingly identified by their ITS sequence. Accordingly, any taxon that can be recognized by its ITS alone can hardly be viewed a “dark taxon” and such taxa are likely to be discovered by concurrently widely applied methods such as barcoding or metagenomics. A different situation applies to taxa that do not have a distinct ITS sequence; these are unlikely to be discovered by these methods. Further, taxa that are represented by types that are not readily accessible to molecular methods often remain neglected. Here, we present an approach that links taxonomy, morphology and molecular data through the use of modern database functions, type studies and multi-locus-sequencing. The use of a digital database allows collection data and species morphology to be recorded as a set of parameters. This facilitates easy comparison of collections. Database queries may be built representing putative species profiles which may then be compared with the results of molecular analyses. Conversely, sets of collections forming clades in molecular analyses may be compared and analysed to determine which characters they have in common and the variation within those characters. After several rounds of comparing results from molecular analyses with sets of collections exhibiting common characters and refining the characters and character value ranges to a query, species descriptions may be assembled automatically from the database, corresponding to clades generated within phylogenetic trees. These descriptions have two major advantages over traditional methods: (i) they are based on easily traceable data, and (ii) they can be re-adjusted if more material becomes available. Keys may also be built, tested and refined through the same process. By entering into the database character values for types, existing names may be linked to sets of collections representing putative taxa. If molecular data is available for a type, this provides another line of evidence. More often than not, as the data sets are enriched, ecological and biogeographical patterns emerge to support the validity of the delimited taxa. Furthermore, multi-locus sequencing can also provide additional evidence on the species delimitation. This methodology allows the identification of taxa that could not be recognized by ITS alone. Our subject is Hebeloma, a genus of ectomycorrhizal fungi, which are often similar in appearance and for which molecular species divergence is low. In Europe, we managed to link 55 species to existing names and discover 29 new species through this approach. In North America, based on 944 collections contributed through the mycologist community and type collections loaned from herbaria, we have discovered 38 species not known from Europe. For 18 of these, we have been able to establish that they have been described before under at least one legitimate name.

Abstract

High-throughput sequencing (HTS) has ushered in a new age of mycological exploration, revealing uncharacterized fungal diversity across disparate habitats. However, efforts to infer the true phylogenetic affinities of many novel fungal phylotypes are hampered by the maximum read lengths of most sequencing platforms (< 500 bp), which often provide little phylogenetic signal. Moreover, the increasing adoption of next-generation sequencing methods to characterize fungal communities supplants traditional culturing surveys, decreasing the generation and availability of high-quality reference sequences. Taxonomic identification of OTUs observed in environmental surveys hinges on well-curated databases of complete or nearly-complete reference sequences. Consequently, despite the detection of wholly unknown groups among the fungi, we are unable to put these taxa into a robust phylogenetic framework, nor can we add them to existing databases to inform future HTS studies. For this study, I explored using a third-generation sequencing platform (PacBio) to generate long reads suitable for phylogenetic analysis and improved taxonomic identification of uncultured fungi from poorly sampled habitats. I targeted a nearly 2,000-bp region of fungal rDNA spanning three loci commonly employed in environmental surveys of fungi: ITS1, ITS2, and LSU. Using a mock community approach, I calculated the error profile for PacBio’s default parameters, from which I developed an analysis pipeline for environmental samples. Amplicon libraries from water and sediment samples originating from diverse marine habitats were then sequenced on the PacBio platform. I observed over 200 OTUs, with Ascomycota and Chytridiomycota exhibiting the highest diversity. Phylogenetic analyses placed most OTUs in the Dikarya to known marine genera, but marine OTUs allied to the zoosporic lineages represented novel clades. Locus-specific databases were shown to vary widely in their ability to assign the correct phylum-level taxonomy to marine OTUs outside of the Dikarya, demonstrating that biases in the taxonomic composition of extant reference databases can result in the failure to recognize OTUs from the early-diverging phyla in environmental sequence datasets.

Abstract

Microorganisms evolve via sexual/parasexual reproduction, mutators, aneuploidy, Hsp90, or prions. Mechanisms that are detrimental can be repurposed to generate diversity. Microbes are known to evolve resistance to antimicrobial agents via pathways involving both stable and unstable genetic mechanisms, such as aneuploidy underlying azole resistance in Candida albicans and Cryptococcus neoformans. We discovered a new mechanism conferring antifungal drug resistance in the human fungal pathogen Mucor circinelloides. Spontaneous resistance to the antifungal drug FK506 was found to evolve via two distinct mechanisms. One involves Mendelian mutations conferring stable irreversible drug resistance; the other occurs via an epigenetic RNA interference (RNAi)-mediated pathway resulting in unstable, transient drug resistance. The peptidyl-prolyl isomerase FKBP12 interacts with FK506 forming a complex that inhibits the protein phosphatase calcineurin. Calcineurin inhibition by FK506 blocks M. circinelloides dimorphic transition to hyphae and enforces growth as yeasts. In some FK506 resistant isolates, mutations in the fkbA gene encoding FKBP12 or the calcineurin cnbR or cnaA genes confer FK506 resistance and restore hyphal growth. In other resistant isolates, no mutations are found in the known drug targets. Instead, RNAi has been triggered to silence the fkbA gene, yielding drug-resistant epimutants. FK506-resistant epimutants readily reverted to drug-sensitivity in the absence of FK506. The establishment of epimutants is accompanied by generation of abundant fkbA small RNAs and requires some known RNAi pathway components whereas others are dispensable. Surprisingly, epimutants occur at a higher frequency and are more stable in mutants lacking RNA-dependent RNA polymerase 1 (Rdrp1), revealing some RNAi components constrain or reverse epimutation. Silencing of the drug target FKBP12 appears to involve generation of a double-stranded RNA trigger intermediate using the fkbA mature mRNA as template to produce antisense fkbA RNA. Epimutational silencing may be stochastic, similar to Mendelian mutations, but differs as the altered phenotype is reversible in response to fluctuating environmental conditions. Our recent studies reveal novel components required for epimutation, including orthologs of the Neurospora crassa quelling inducing protein (QIP) and Sad-3 helicase (RnhA); interestingly, the rnhA gene is linked to the Mucor sex locus, suggesting sexual reproduction may activate epimutation similar to sex-induced-silencing in Cryptococcus. We found epimutants occur at a higher frequency in mutants lacking RNA-dependent RNA polymerase 3 (Rdrp3) or the RNaseIII-like protein R3B2. Rdrp1, Rdrp3, and R3B2 operate a non-canonical RNA degradation pathway suppressing RNAi-dependent epimutation by competition for targets. We generalized these findings by showing epimutations occur in a second species of Mucor, and identifying epimutations in the pyrF or pyrG genes conferring 5-fluoroorotic acid (5-FOA) resistance. These studies uncover a novel, reversible, transient epigenetic RNAi-based epimutation mechanism controlling phenotypic plasticity, with implications for antimicrobial drug resistance and RNAi-regulatory mechanisms in fungi and other eukaryotes. The full impact of epimutations in this and other genetic systems may have eluded discovery previously given their inherently unstable nature.

Abstract

Fusarium head blight, Fusarium ear rot, and vascular wilting are –despite their names- typical diseases caused by Fusarium species in different plant hosts, including many economically important plant species. Fusaria have often specialised on a certain host and in some cases this virulence seems to be organised on supernumerary chromosomes containing the essential information for pathogenicity on one or a limited number of host species. As added bonus, Fusarium is capable of the production of a large variety of mycotoxins, often acting as virulence factors, contaminating harvested products.

However, over the past years we also see Fusarium species as emerging pathogens both in human and animal. There they cause from relatively innocent, but actual quite frequent, nail and skin infections to more rare local, deep and in the growing group of immuno-compromised hosts even disseminated infections. These latter infections are connected with high mortality rates. In Fusarium, it has become customary to cluster closely related sibling species or lineages with little to no morphological differences in so-called species complexes: The opportunists on human and animal group into seven main species complexes: the Fusarium solani, F. oxysporum, F. incarnatum-equiseti, F. fujikuroi, F. clamydosporum, F. dimerum and F. sporotrichioides species complexes. In some cases the human infections can directly be linked to trauma with infected plant materials, making Fusarium a true trans-kingdom pathogen.

With the control of both plant and human pathogenic Fusarium, we are faced with several major challenges: One foremost being the limited availability of effective treatments as the species are generally very resistant to the available antifungals. Also some species prove to vary in their susceptibility to those few effective drugs available, emphasizing the need for fast identification and suitable diagnostic tools which are at the moment limited available. Additional confusing factors are that especially for some of the clinically important species there has been limited nomenclatural stability over the years, obscuring at times available data, and the recent discovery of many new species.

Abstract

Species in the genus Coccidioides, C. posadasii and C. immitis, are the causative agents for coccidioidomycosis, the disease commonly known as Valley Fever. Coccidioidomycosis is estimated to affect more than 150,000 humans each year and is one of the few fungal diseases to affect otherwise healthy individuals. While progress has been made in the clinical understanding of the disease, little is known of the natural biology. Eighteen clinical isolates derived from 17 individuals diagnosed with coccidioidomycosis collected from New Mexico were used in a multi-locus sequencing analysis to explore genetic variation within the state and between neighboring states. While New Mexico is predicted to have C. posadasii, results of our analysis indicate that both C. immitis and C. posadasii are present among clinical isolates in New Mexico. Five of eight infections for which patient ethnicity was known occurred in Native Americans, suggesting that further studies should be conducted to determine if American Indians represent a risk group for coccidioidomycosis. We are also taking a novel approach to screen small rodents for exposure to Coccidioides by two means: 1) a survey of frozen mammal lung tissue for fungal infections, and 2) an enzyme immunoassay that detects IgG antibodies against Coccidioides in a variety of mammalian species. This will produce critical information regarding animal infection rates, the geographical distribution of infected animals, and relative spore loads in soils. Characterization of clinical and environmental isolates will allow us to understand the genetic variation that affects the virulence of these pathogens.

Abstract

Introduction. Candida auris, an emerging fungal pathogen, causes hospital-associated outbreaks of invasive candidiasis with mortality near 60%. Little is known about the pathogenesis of this species that has newly arisen in the last 10 years, and it is unclear why this species is rapidly spreading worldwide. Neutrophils are critical for control of numerous invasive fungal infections, including candidiasis. These leukocytes kill fungi through phagocytosis or the release of neutrophil extracellular traps (NETs), which are structures of DNA, histones, and proteins with antimicrobial activity. Objective. The objective of this study was to delineate the neutrophil response to C. auris. We hypothesized that an ineffective neutrophil response may account for the poor outcomes observed in patients. Methods. We examined interactions of human neutrophils with C. auris and included C. albicans for comparison. Neutrophil-Candida interactions were visualized by time-lapse fluorescent microscopy and scanning electron microscopy (SEM). We utilized oxidative stress indicator CM-H2DCFDA to measure the generation of reactive oxygen species (ROS) in neutrophils. NET formation was quantified by Sytox Green staining and assessed by SEM and immunofluorescent labeling of NET-associated proteins. Fungal viability was evaluated using microbiological counts and viability stains. We utilized a zebrafish larval infection model to evaluate neutrophil-Candida interactions in vivo. Results. Imaging revealed the phagocytosis of C. albicans by human neutrophils at 1 h, followed by the formation of NETs by 4 h. In contrast, neutrophils appeared rounded upon encountering C. auris and rarely engaged in phagocytosis or produced NETs. As shown by Sytox Green staining, C. auris triggered negligible NET release by human neutrophils, with levels 7-fold lower when compared to C. albicans. C. auris did not induce neutrophils to generate ROS, a key signaling mechanism for NET formation. The ineffective neutrophil response to C. auris correlated with diminished fungal killing. Imaging of neutrophils in a zebrafish model of invasive candidiasis revealed the recruitment of approximately 50% fewer neutrophils in response to C. auris as compared to C. albicans. Conclusion. C. auris evades neutrophils by altering multiple aspects of their usual anti-candidal responses. This is linked to improved fungal survival. We propose that this diminished innate immune response may contribute to the unexpected virulence of C. auris.

Abstract

Abstract

Mucoralean fungi can cause mucormycosis, a life-threatening disease in immunocompromised patients. In our study, we analysed the influence of different enzymatic treatments of the spore surface alterations on the phagocytosis by murine alveolar macrophages. Two strains which were shown to be virulent and attenuated in avian, invertebrate and murine infection models were used in this study. The spore surface was treated with different cell wall-degrading cell wall enzymes targeting carbohydrate and protein cell wall components. The highest phagocytosis index was achieved with the proteolytic treatments which encouraged us to do focus our research on the protein surface of spores. Proteomic analysis of the spore surface was conducted for both strains. About four-teen candidate proteins were found which were differentially abundant in either the virulent strain or in the attenuated strain leading to the hypothesis that these proteins may play a role in virulence. One of these candidate proteins is the hydrophobic surface binding protein A (HsbA) which was first found in higher abundance in the virulent strain of L. corymbifera. HsbA was first described as an adhesin in Aspergillus oryzae. Additionally, HsbA was purified from the insect-killing ascomycete Beauveria bassiana and identified to play a role in immunogenicity. The HsbA protein from the virulent strain of L. corymbifera was heterologously overexpressed in Pichia pastoris. After pretreatment of murine alveolar macrophages and spores with purified fractions of the HsbA protein, the phagocytic index was found to be enhanced in comparison with unstimulated host cells. The findings presented in this study will open the door for the role of surface protein in the recognition of L. corymbifera by phagocytes of the innate immune system which raise important measures to mammalian infection models. Our prospect for the future research will focus on the identification of potential stimulatory effects of L. corymbifera surface proteins and their putative receptors on the surface of macrophages which possibly contribute to virulence.