Tobias Guldberg Frøslev

Improving ITS sequence data for identification of plant pathogenic fungi

SummaryPlant pathogenic fungi are a large and diverse assemblage of eukaryotes with substantial impacts on natural ecosystems and human endeavours. These taxa often have complex and poorly understood life cycles, lack observable, discriminatory morphological characters, and may not be amenable to in vitro culturing. As a result, species identification is frequently difficult. Molecular (DNA sequence) data have emerged as crucial information for the taxonomic identification of plant pathogenic fungi, with the nuclear ribosomal internal transcribed spacer (ITS) region being the most popular marker. However, international nucleotide sequence databases are accumulating numerous sequences of compromised or low-resolution taxonomic annotations and substandard technical quality, making their use in the molecular identification of plant pathogenic fungi problematic. Here we report on a concerted effort to identify high-quality reference sequences for various plant pathogenic fungi and to re-annotate incorrectly or insufficiently annotated public ITS sequences from these fungal lineages. A third objective was to enrich the sequences with geographical and ecological metadata. The results – a total of 31,954 changes – are incorporated in and made available through the UNITE database for molecular identification of fungi (http://unite.ut.ee), including standalone FASTA files of sequence data for local BLAST searches, use in the next-generation sequencing analysis platforms QIIME and mothur, and related applications. The present initiative is just a beginning to cover the wide spectrum of plant pathogenic fungi, and we invite all researchers with pertinent expertise to join the annotation effort.

Phylogenetic relationships of Termitomyces and related taxa

Phylogenetic relationships of termitophilic fungi were estimated with Bayesian as well as other phylogenetic methods from partial sequences of the nuclear encoded large subunit ribosomal DNA (nLSU-rDNA) and the mitochondrial encoded small subunit ribosomal DNA (mtSSU-rDNA). Sequences were obtained from basidiomes covering the morphological, taxonomical, and geographical span of termitophilic mushroom-forming fungi, and analysed together with sequences from termite nests and termite guts from most known genera of fungus growing termites from geographically diverse regions. Topologies of trees resulting from the combined analyses of the two ribosomal genes generally show no positive conflicts with those obtained from separate analyses. We show that termitophilic fungi constitute a strongly supported monophyletic group within lyophylloid species. The genera Sinotermitomyces and Podabrella are derived within Termitomyces, and do not form monophyletic groups. Identical sequences were frequently found among samples of basidiomes from the same continents and among fungi utilized by termites from the same continent. However, only two sequences were identical between basidiome samples and termite nest/gut samples suggesting fruiting species do not form a representative sample of termitophilic fungi. No sequences were identical between samples from Asia and Africa indicating some geographic differentiation between these continents.

Determining threshold values for barcoding fungi: lessons from Cortinarius (Basidiomycota), a highly diverse and widespread ectomycorrhizal genus

Different distance-based threshold selection approaches were used to assess and compare use of the internal transcribed spacer (ITS) region to distinguish among 901 Cortinarius species represented by >3000 collections. Sources of error associated with genetic markers and selection approaches were explored and evaluated using MOTUs from genus and lineage based-alignments. Our study indicates that 1%-2% more species can be distinguished by using the full-length ITS barcode as compared to either the ITS1 or ITS2 regions alone. Optimal threshold values for different picking approaches and genetic marker lengths inferred from a subset of species containing major lineages ranged from 97.0% to 99.5% sequence similarity using clustering optimization and UNITE SH, and from 1% to 2% sequence dissimilarity with CROP. Errors for the optimal cutoff ranged from 0% to 70%, and these can be reduced to a maximum of 22% when excluding species lacking a barcode gap. A threshold value of 99% is suitable for distinguishing species in the majority of lineages in the genus using the entire ITS region but only 90% of the species could be identified using just the ITS1 or ITS2 region. Prior identification of species, lacking barcode gaps and their subsequent separate analyses, maximized the accuracy of threshold approaches.

Automated Extraction of DNA from Blood and PCR Setup using a Tecan Freedom EVO Liquid Handler for Forensic Genetic STR Typing of Reference Samples

We have implemented and validated automated protocols for DNA extraction and PCR setup using a Tecan Freedom EVO liquid handler mounted with the Te-MagS magnetic separation device (Tecan, Mannedorf, Switzerland). The protocols were validated for accredited forensic genetic work according to ISO 17025 using the Qiagen MagAttract DNA Mini M48 kit (Qiagen GmbH, Hilden, Germany) from fresh whole blood and blood from deceased individuals. The workflow was simplified by returning the DNA extracts to the original tubes minimizing the risk of misplacing samples. The tubes that originally contained the samples were washed with MilliQ water before the return of the DNA extracts. The PCR was setup in 96-well microtiter plates. The methods were validated for the kits: AmpFSTR Identifier, SGM Plus and Yfiler (Applied Biosystems, Foster City, CA), GenePrint FFFL and PowerPlex Y (Promega, Madison, Wl). The automated protocols allowed for extraction and addition of PCR master mix of 96 samples within 3.5 h. In conclusion, we demonstrated that (1) DNA extraction with magnetic beads and (2) PCR setup for accredited, forensic genetic short tandem repeat typing can be implemented on a simple automated liquid handler leading to the reduction of manual work, and increased quality and throughput.

A simple method for validation and verification of pipettes mounted on automated liquid handlers

We have implemented a simple, inexpensive, and fast procedure for validation and verification of the performance of pipettes mounted on automated liquid handlers (ALHs) as necessary for laboratories accredited under ISO 17025. A six- or seven-step serial dilution of OrangeG was prepared in quadruplicates in a flat-bottom 96-well microtiter plate, manually using calibrated pipettes. Each pipette of the liquid handler (1–8) dispensed a selected volume (1–200μL) of OrangeG eight times into the wells of the microtiter plate. All wells contained a total of 200μL liquid. The absorbance was read, and the dispensed volume of each pipette was calculated based on a plot of volume and absorbance of a known set of OrangeG dilutions. Finally, the percent inaccuracy (%d) and the imprecision (%CV) of each pipette were calculated. Using predefined acceptance criteria, each pipette was then either approved or failed. Failed pipettes were either repaired or the volume deviation was compensated for by applying a calibration curve in the liquid-handler software. We have implemented the procedure on a Sias Xantus, an MWGt The ONYX, four Tecan Freedom EVO, a Biomek NX Span-8, and four Biomek 3000 robots, and the methods are freely available. In conclusion, we have set up a simple, inexpensive, and fast solution for the continuous validation of ALHs used for accredited work according to the ISO 17025 standard. The method is easy to use for aqueous solutions but requires a spectrophotometer that can read microtiter plates.

Algorithm for post-clustering curation of DNA amplicon data yields reliable biodiversity estimates

DNA metabarcoding is promising for cost-effective biodiversity monitoring, but reliable diversity estimates are difficult to achieve and validate. Here we present and validate a method, called LULU, for removing erroneous molecular operational taxonomic units (OTUs) from community data derived by high-throughput sequencing of amplified marker genes. LULU identifies errors by combining sequence similarity and co-occurrence patterns. To validate the LULU method, we use a unique data set of high quality survey data of vascular plants paired with plant ITS2 metabarcoding data of DNA extracted from soil from 130 sites in Denmark spanning major environmental gradients. OTU tables are produced with several different OTU definition algorithms and subsequently curated with LULU, and validated against field survey data. LULU curation consistently improves α-diversity estimates and other biodiversity metrics, and does not require a sequence reference database; thus, it represents a promising method for reliable biodiversity estimation.A central problem in biodiversity estimation from genetic markers is the ability of algorithms to retain ‘true’ species while discarding artefacts. Here, the authors present a new post-clusturing curation algorithm using OTU co-occurrences to estimate plant biodiversity from soil samples.

A comparison between ITS phylogenetic relationships and morphological species recognition within Mycena sect. Calodontes in Northern Europe

The members of Mycena sect. Calodontes (Tricholomataceae s.l., Basidiomycota) are characterised by a collybioid aspect and more or less purplish to reddish colours and a distinct raphanoid odour. In Europe, nine species have been recognised though some of these based on somewhat dubious morphological differences. Historically, most were assigned to Mycena pura. However, since Mycena pura displays one of the most striking colour variabilities within European agarics, many attempts have been made to subdivide it into independent entities, and several forms, varieties and species have been split from Mycena pura s.l. based largely on differences in colouration, gross macromorphology or other phenetic traits. We developed a large sample of ITS sequences of all species of sect. Calodontes known from Europe for which vouchers exist. Furthermore, partial LSU data were developed and additional sequences downloaded from GENBANK to assess the relationship of Calodontes with other Mycena spp. We show that most Calodontes form a monophyletic group including a few North and South American collections, but that this cannot be conclusively shown when an additional North American sequence is added. For all other species than M. pura and M. diosma, we found morphological species recognition to be in agreement with the ITS data. Several significantly different clades can be recognised within the M. pura morphospecies, none of which can be linked to the observed (and described by proxy) colour varieties/forms. Indications of a possible environmental basis of the colour differentiation in the M. pura morphospecies are discussed.

Unexpectedly High Beta-Diversity of Root-Associated Fungal Communities in the Bolivian Andes

Bolivia is one of the most biologically diverse countries on the planet. Between the Andes and the Amazon drainage basin spans the Yungas, a vast forested region shown to be extremely species rich in macro-organisms. However, it remains unclear whether this high diversity is also reflected in microbial diversity. Here we assess the genetic, taxonomic and functional diversity of root-associated fungi surrounding Cinchona calisaya trees, a typical element of the intermediate altitudes of the Bolivian Yungas. We determine the relative effects of edaphic properties, climate, and geography in regulating fungal community assembly. We show that α-diversity for these fungal communities was similar to temperate and arid ecosystems, averaging 90.1 operational taxonomic units (OTUs) per sample, with reads predominantly assigned to the Ascomycota phylum and with a saprotrophic lifestyle. ß-diversity was calculated as the distance-decay rate, and in contrast to α-diversity, was exceptionally high with a rate of −0.407. Soil properties (pH and P) principally regulated fungal community assembly in an analogous manner to temperate environments, with pH and phosphorus explaining 7.8 and 7.2% of community variation respectively. Surprisingly, altitude does not influence community formation, and there is limited evidence that climate (precipitation and temperature) play a role. Our results suggest that sampling should be performed over a wide geographical and environmental range in order to capture the full root-associated fungal diversity in subtropical regions. This study sheds further light on the diversity and distribution of the worlds “hidden biodiversity.”

New Type of Papillomavirus and Novel Circular Single Stranded DNA Virus Discovered in Urban Rattus norvegicus Using Circular DNA Enrichment and Metagenomics

Rattus norvegicus (R. norvegicus) are ubiquitous and their presence has several effects on the human populations in our urban areas on a global scale. Both historically and presently, this close interaction has facilitated the dissemination of many pathogens to humans, making screening for potentially zoonotic and emerging viruses in rats highly relevant. We have investigated faecal samples from R. norvegicus collected from urban areas using a protocol based on metagenomic enrichment of circular DNA genomes and subsequent sequencing. We found a new type of papillomavirus, with a L1 region 82% identical to that of the known R. norvegicus Papillomavirus 2. Additionally, we found 20 different circular replication associated protein (Rep)-encoding single stranded DNA (CRESS-DNA) virus-like genomes, one of which has homology to the replication-associated gene of Beak and feather disease virus. Papillomaviruses are a group of viruses known for their carcinogenic potential, and although they are known to infect several different vertebrates, they are mainly studied and characterised in humans. CRESS-DNA viruses are found in many different environments and tissue types. Both papillomaviruses and CRESS-DNA viruses are known to have pathogenic potential and screening for novel and known viruses in R. norvegicus could help identify viruses with pathogenic potential.

Amyloidity is not diagnostic for species in the Mycena pearsoniana complex (Mycena sectio Calodontes)

In Mycena sectio Calodontes with otherwise amyloid spores, the inamyloid spores of Mycena pearsoniana Dennis ex Singer were a distinguishing feature for this species and its subsection Violacella. Although the original concept of this species was European, Singer chose to typify it with material collected in Mexico. The name has since been applied to all European collections with inamyloid spores and decurrent lamellae. Our phylogenetic analysis of 91 ITS sequences from European, North and South American Calodontes collections shows that European collections identified as M. pearsoniana fall into two well-supported sibling clades together with both inamyloid and weakly amyloid North American collections. Since the holotype of M. pearsoniana is in an advanced state of decay, we have selected an epitype from a North American locality with a climate comparable to the Mexican type locality. Our results show weakly and inamyloid spore reactions to be homoplastic in Calodontes, and furthermore that spores of M. pearsoniana can show either amyloid or inamyloid reactions interchangeably. This raises doubt about the taxonomic value of this trait in Mycena systematics.