HyLength: a semi-automated digital image analysis tool for measuring the length of roots and fungal hyphae of dense mycelia.

In plant-fungus phenotyping, determining fungal hyphal and plant root lengths by digital image analysis can reduce labour and increase data reproducibility. However, the degree of software sophistication is often prohibitive and manual measuring is still used, despite being very time-consuming. We developed the HyLength tool for measuring the lengths of hyphae and roots in in vivo and in vitro systems. The HyLength was successfully validated against manual measures of roots and fungal hyphae obtained from all systems. Compared with manual methods, the HyLength underestimated Medicago sativa roots in the in vivo system and Rhizophagus irregularis hyphae in the in vitro system by about 12 cm per m and allowed to save about 1 h for a single experimental unit. As regards hyphae of R. irregularis in the in vivo system, the HyLength overestimated the length by about 21 cm per m compared with manual measures, but time saving was up to 20.5 h per single experimental unit. Finally, with hyphae of Aspergillus oryzae, the underestimation was about 8 cm per m with a time saving of about 10 min for a single germinating spore. By benchmarking the HyLength against the AnaMorf plugin of the ImageJ/Fiji, we found that the HyLength performed better for dense fungal hyphae, also strongly reducing the measuring time. The HyLength can allow measuring the length over a whole experimental unit, eliminating the error due to sub-area selection by the user and allowing processing a high number of samples. Therefore, we propose the HyLength as a useful freeware tool for measuring fungal hyphae of dense mycelia.

Spectral manipulation improves elevation perception with non-individualized head-related transfer functions.

Spatially rendering sounds using head-related transfer functions (HRTFs) is an important part of creating immersive audio experiences for virtual reality applications. However, elevation perception remains challenging when generic, non-personalized HRTFs are used. This study investigated whether digital audio effects applied to a generic set of HRTFs could improve sound localization in the vertical plane. Several of the tested effects significantly improved elevation judgment, and trial-by-trial variability in spectral energy between 2 and 10 kHz correlated strongly with perceived elevation. Digital audio effects may therefore be a promising strategy to improve elevation perception where personalized HRTFs are not available.

Cowpea (Vigna unguiculata L. Walp) hosts several widespread bradyrhizobial root nodule symbionts across contrasting agro-ecological production areas in Kenya.

Cowpea (Vigna unguiculata L. Walp.) is an important African food legume suitable for dry regions. It is the main legume in two contrasting agro-ecological regions of Kenya as an important component of crop rotations because of its relative tolerance to unpredictable drought events. This study was carried out in an effort to establish a collection of bacterial root nodule symbionts and determine their relationship to physicochemical soil parameters as well as any geographical distributional patterns. Bradyrhizobium spp. were found to be widespread in this study and several different types could be identified at each site. Unique but rare symbionts were recovered from the nodules of plants sampled in a drier in-land region, where there were also overall more different bradyrhizobia found. Plants raised in soil from uncultivated sites with a natural vegetation cover tended to also associate with more different bradyrizobia. The occurrence and abundance of different bradyrhizobia correlated with differences in soil texture and pH, but did neither with the agro-ecological origin, nor the origin from cultivated (n = 15) or uncultivated (n = 5) sites. The analytical method, protein profiling of isolated strains by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), provided higher resolution than 16S rRNA gene sequencing and was applied in this study for the first time to isolates recovered directly from field-collected cowpea root nodules. The method thus seems suitable for screening isolate collections on the presence of different groups, which, provided an appropriate reference database, can also be assigned to known species.

Grassland vertical height heterogeneity predicts flower and bee diversity: an UAV photogrammetric approach.

The ecosystem services offered by pollinators are vital for supporting agriculture and ecosystem functioning, with bees standing out as especially valuable contributors among these insects. Threats such as habitat fragmentation, intensive agriculture, and climate change are contributing to the decline of natural bee populations. Remote sensing could be a useful tool to identify sites of high diversity before investing into more expensive field survey. In this study, the ability of Unoccupied Aerial Vehicles (UAV) images to estimate biodiversity at a local scale has been assessed while testing the concept of the Height Variation Hypothesis (HVH). This hypothesis states that the higher the vegetation height heterogeneity (HH) measured by remote sensing information, the higher the vegetation vertical complexity and the associated species diversity. In this study, the concept has been further developed to understand if vegetation HH can also be considered a proxy for bee diversity and abundance. We tested this approach in 30 grasslands in the South of the Netherlands, where an intensive field data campaign (collection of flower and bee diversity and abundance) was carried out in 2021, along with a UAV campaign (collection of true color-RGB-images at high spatial resolution). Canopy Height Models (CHM) of the grasslands were derived using the photogrammetry technique "Structure from Motion" (SfM) with horizontal resolution (spatial) of 10 cm, 25 cm, and 50 cm. The accuracy of the CHM derived from UAV photogrammetry was assessed by comparing them through linear regression against local CHM LiDAR (Light Detection and Ranging) data derived from an Airborne Laser Scanner campaign completed in 2020/2021, yielding an [Formula: see text] of 0.71. Subsequently, the HH assessed on the CHMs at the three spatial resolutions, using four different heterogeneity indices (Rao's Q, Coefficient of Variation, Berger-Parker index, and Simpson's D index), was correlated with the ground-based flower and bee diversity and bee abundance data. The Rao's Q index was the most effective heterogeneity index, reaching high correlations with the ground-based data (0.44 for flower diversity, 0.47 for bee diversity, and 0.34 for bee abundance). Interestingly, the correlations were not significantly influenced by the spatial resolution of the CHM derived from UAV photogrammetry. Our results suggest that vegetation height heterogeneity can be used as a proxy for large-scale, standardized, and cost-effective inference of flower diversity and habitat quality for bees.

Shifting carbon flow from roots into associated microbial communities in response to elevated atmospheric CO2.

Rising atmospheric CO(2) levels are predicted to have major consequences on carbon cycling and the functioning of terrestrial ecosystems. Increased photosynthetic activity is expected, especially for C-3 plants, thereby influencing vegetation dynamics; however, little is known about the path of fixed carbon into soil-borne communities and resulting feedbacks on ecosystem function. Here, we examine how arbuscular mycorrhizal fungi (AMF) act as a major conduit in the transfer of carbon between plants and soil and how elevated atmospheric CO(2) modulates the belowground translocation pathway of plant-fixed carbon. Shifts in active AMF species under elevated atmospheric CO(2) conditions are coupled to changes within active rhizosphere bacterial and fungal communities. Thus, as opposed to simply increasing the activity of soil-borne microbes through enhanced rhizodeposition, elevated atmospheric CO(2) clearly evokes the emergence of distinct opportunistic plant-associated microbial communities. Analyses involving RNA-based stable isotope probing, neutral/phosphate lipid fatty acids stable isotope probing, community fingerprinting, and real-time PCR allowed us to trace plant-fixed carbon to the affected soil-borne microorganisms. Based on our data, we present a conceptual model in which plant-assimilated carbon is rapidly transferred to AMF, followed by a slower release from AMF to the bacterial and fungal populations well-adapted to the prevailing (myco-)rhizosphere conditions. This model provides a general framework for reappraising carbon-flow paths in soils, facilitating predictions of future interactions between rising atmospheric CO(2) concentrations and terrestrial ecosystems.

Head-related transfer function interpolation in azimuth, elevation, and distance.

Although distance-dependent head-related transfer function (HRTF) databases provide interesting possibilities, e.g., for rendering virtual sounds in the near-field, there is a lack of algorithms and tools to make use of them. Here, a framework is proposed for interpolating HRTF measurements in 3-D (i.e., azimuth, elevation, and distance) using tetrahedral interpolation with barycentric weights. For interpolation, a tetrahedral mesh is generated via Delaunay triangulation and searched via an adjacency walk, making the framework robust with respect to irregularly positioned HRTF measurements and computationally efficient. An objective evaluation of the proposed framework indicates good accordance between measured and interpolated near-field HRTFs.

Establishment, persistence and effectiveness of arbuscular mycorrhizal fungal inoculants in the field revealed using molecular genetic tracing and measurement of yield components.

• Inoculation of crop plants by non-native strains of arbuscular mycorrhizal (AM) fungi as bio-enhancers is promoted without clear evidence for symbiotic effectiveness and fungal persistence. To address such gaps, the forage legume Medicago sativa was inoculated in an agronomic field trial with two isolates of Funneliformis mosseae differing in their nuclear rDNA sequences from native strains. • The inoculants were traced by PCR with a novel combination of the universal fungal NS31 and Glomeromycota-specific LSUGlom1 primers which target the nuclear rDNA cistron. The amplicons were classified by restriction fragment length polymorphism and sequencing. • The two applied fungal inoculants were successfully traced and discriminated from native strains in roots sampled from the field up to 2 yr post inoculation. Moreover, field inoculation with inocula of non-native isolates of F. mosseae appeared to have stimulated root colonization and yield of M. sativa. • Proof of inoculation success and sustained positive effects on biomass production and quality of M. sativa crop plants hold promise for the role that AM fungal inoculants could play in agriculture.

Large-scale simulation of bird localization systems in forests with distributed microphone arrays.

Acoustic wildlife monitoring systems are important tools for capturing information about animal habitation in ecosystems. Previous work has demonstrated the effectiveness of audio-based bird localization techniques. However, few studies have investigated the performance and robustness of distributed systems in large forests. Here, the performance of distributed microphone arrays for localizing birds is examined by simulating forest scenes with added reverberation, ambient noise, and measurement errors. The simulation revealed the importance of the signal-to-noise ratio and the spectral weighting in the localization algorithm. These results may guide the design of large-scale wildlife monitoring systems and suggest promising directions for further improvements.

New pedotransfer approaches to predict soil bulk density using WoSIS soil data and environmental covariates in Mediterranean agro-ecosystems.

For the estimation of the soil organic carbon stocks, bulk density (BD) is a fundamental parameter but measured data are usually not available especially when dealing with legacy soil data. It is possible to estimate BD by applying pedotransfer function (PTF). We applied different estimation methods with the aim to define a suitable PTF for BD of arable land for the Mediterranean Basin, which has peculiar climate features that may influence the soil carbon sequestration. To improve the existing BD estimation methods, we used a set of public climatic and topographic data along with the soil texture and organic carbon data. The present work consisted of the following steps: i) development of three PTFs models separately for top (0-0.4 m) and subsoil (0.4-1.2 m), ii) a 10-fold cross-validation, iii) model transferability using an external dataset derived from published data. The development of the new PTFs was based on the training dataset consisting of World Soil Information Service (WoSIS) soil profile data, climatic data from WorldClim at 1 km spatial resolution and Shuttle Radar Topography Mission (SRTM) digital elevation model at 30 m spatial resolution. The three PTFs models were developed using: Multiple Linear Regression stepwise (MLR-S), Multiple Linear Regression backward stepwise (MLR-BS), and Artificial Neural Network (ANN). The predictions of the newly developed PTFs were compared with the BD calculated using the PTF proposed by Manrique and Jones (MJ) and the modelled BD derived from the global SoilGrids dataset. For the topsoil training dataset (N = 129), MLR-S, MLR-BS and ANN had a R2 0.35, 0.58 and 0.86, respectively. For the model transferability, the three PTFs applied to the external topsoil dataset (N = 59), achieved R2 values of 0.06, 0.03 and 0.41. For the subsoil training dataset (N = 180), MLR-S, MLR-BS and ANN the R2 values were 0.36, 0.46 and 0.83, respectively. When applied to the external subsoil dataset (N = 29), the R2 values were 0.05, 0.06 and 0.41. The cross-validation for both top and subsoil dataset, resulted in an intermediate performance compared to calibration and validation with the external dataset. The new ANN PTF outperformed MLR-S, MLR-BS, MJ and SoilGrids approaches for estimating BD. Further improvements may be achieved by additionally considering the time of sampling, agricultural soil management and cultivation practices in predictive models.

Molecular trait indicators: moving beyond phylogeny in arbuscular mycorrhizal ecology.

Arbuscular mycorrhizal (AM) fungi form symbiotic associations with the roots of most plants, thereby mediating nutrient and carbon fluxes, plant performance, and ecosystem dynamics. Although considerable effort has been expended to understand the keystone ecological position of AM symbioses, most studies have been limited in scope to recording organism occurrences and identities, as determined from morphological characters and (mainly) ribosomal sequence markers. In order to overcome these restrictions and circumvent the shortcomings of culture- and phylogeny-based approaches, we propose a shift toward plant and fungal protein-encoding genes as more immediate indicators of mycorrhizal contributions to ecological processes. A number of candidate target genes, involved in the uptake of phosphorus and nitrogen, carbon cycling, and overall metabolic activity, are proposed. We discuss the advantages and disadvantages of future protein-encoding gene marker and current (phylo-) taxonomic approaches for studying the impact of AM fungi on plant growth and ecosystem functioning. Approaches based on protein-encoding genes are expected to open opportunities to advance the mechanistic understanding of ecological roles of mycorrhizas in natural and managed ecosystems.

Positive effects of organic farming on below-ground mutualists: large-scale comparison of mycorrhizal fungal communities in agricultural soils.

*The impact of various agricultural practices on soil biodiversity and, in particular, on arbuscular mycorrhizal fungi (AMF), is still poorly understood, although AMF can provide benefit to plants and ecosystems. Here, we tested whether organic farming enhances AMF diversity and whether AMF communities from organically managed fields are more similar to those of species-rich grasslands or conventionally managed fields. *To address this issue, the AMF community composition was assessed in 26 arable fields (13 pairs of organically and conventionally managed fields) and five semi-natural grasslands, all on sandy soil. Terminal restriction fragment length polymorphism community fingerprinting was used to characterize AMF community composition. *The average number of AMF taxa was highest in grasslands (8.8), intermediate in organically managed fields (6.4) and significantly lower in conventionally managed fields (3.9). Moreover, AMF richness increased significantly with the time since conversion to organic agriculture. AMF communities of organically managed fields were also more similar to those of natural grasslands when compared with those under conventional management, and were less uniform than their conventional counterparts, as expressed by higher beta-diversity (between-site diversity). *We suggest that organic management in agro-ecosystems contributes to the restoration and maintenance of these important below-ground mutualists.

Comparing arbuscular mycorrhizal communities of individual plants in a grassland biodiversity experiment.

Plants differ greatly in the soil organisms colonizing their roots. However, how soil organism assemblages of individual plant roots can be influenced by plant community properties remains poorly understood. We determined the composition of arbuscular mycorrhizal fungi (AMF) in Jacobaea vulgaris plants, using terminal restriction fragment length polymorphism (T-RFLP). The plants were collected from an experimental field site with sown and unsown plant communities. Natural colonization was allowed for 10 yr in sown and unsown plots. Unsown plant communities were more diverse and spatially heterogeneous than sown ones. Arbuscular mycorrhizal fungi diversity did not differ between sown and unsown plant communities, but there was higher AMF assemblage dissimilarity between individual plants in the unsown plant communities. When we grew J. vulgaris in field soil that was homogenized after collection in order to rule out spatial variation, no differences in AMF dissimilarity between sown and unsown plots were found. Our study shows that experimental manipulation of plant communities in the field, and hence plant community assembly history, can influence the AMF communities of individual plants growing in those plant communities. This awareness is important when interpreting results from field surveys and experimental ecological studies in relation to plant-symbiont interactions.

Geographical patterns of root nodule bacterial diversity in cultivated and wild populations of a woody legume crop.

There is interest in understanding how cultivation, plant genotype, climate and soil conditions influence the biogeography of root nodule bacterial communities of legumes. For crops from regions with relict wild populations, this is of even greater interest because the effects of cultivation on symbiont communities can be revealed, which is of particular interest for bacteria such as rhizobia. Here, we determined the structure of root nodule bacterial communities of rooibos (Aspalathus linearis), a leguminous shrub endemic to South Africa. We related the community dissimilarities of the root nodule bacteria of 18 paired cultivated and wild rooibos populations to pairwise geographical distances, plant ecophysiological characteristics and soil physicochemical parameters. Using next-generation sequencing data, we identified region-, cultivation- and farm-specific operational taxonomic units for four distinct classes of root nodule bacterial communities, dominated by members of the genus Mesorhizobium. We found that while bacterial richness was locally increased by organic cultivation, strong biogeographical differentiation in the bacterial communities of wild rooibos disappeared with cultivation of one single cultivar across its entire cultivation range. This implies that expanding rooibos farming has the potential to endanger wild rooibos populations through the homogenisation of root nodule bacterial diversity.

Experimental assembly reveals ecological drift as a major driver of root nodule bacterial diversity in a woody legume crop.

Understanding how plant-associated microbial communities assemble and the role they play in plant performance are major goals in microbial ecology. For nitrogen-fixing rhizobia, community assembly is generally driven by host plant selection and soil conditions. Here, we aimed to determine the relative importance of neutral and deterministic processes in the assembly of bacterial communities of root nodules of a legume shrub adapted to extreme nutrient limitation, rooibos (Aspalathus linearis Burm. Dahlgren). We grew rooibos seedlings in soil from cultivated land and wild habitats, and mixtures of these soils, sampled from a wide geographic area, and with a fertilization treatment. Bacterial communities were characterized using next generation sequencing of part of the nodA gene (i.e. common to the core rhizobial symbionts of rooibos), and part of the gyrB gene (i.e. common to all bacterial taxa). Ecological drift alone was a major driver of taxonomic turnover in the bacterial communities of root nodules (62.6% of gyrB communities). In contrast, the assembly of core rhizobial communities (genus Mesorhizobium) was driven by dispersal limitation in concert with drift (81.1% of nodA communities). This agrees with a scenario of rooibos-Mesorhizobium specificity in spatially separated subpopulations, and low host filtering of other bacteria colonizing root nodules in a stochastic manner.

Diversispora celata sp. nov: molecular ecology and phylotaxonomy of an inconspicuous arbuscular mycorrhizal fungus.

The increasing numbers of taxonomically unassigned phylotypes reported in molecular ecological studies contrast with the few formally described arbuscular mycorrhizal fungi (AMF; Glomeromycota). Here, a species new to science with Glomus-like spores is phylogenetically, morphologically and ecologically characterized. From single spore isolates of a previously recognized member of the Diversisporaceae from Swiss agricultural grassland, 17 new nuclear internal transcribed spacer (ITS), large subunit (LSU) and small subunit (SSU) ribosomal RNA (rRNA) gene sequences were determined and compared with 14 newly generated sequences of two close relatives and public database sequences, including environmental sequences, of known geographic origin. SSU ribosomal DNA (rDNA) sequence signatures and phylogenies based on ITS, LSU and SSU rDNA sequences show that the fungus belongs to the genus Diversispora. It is described as Diversispora celata sp. nov. Comparison with environmental sequences in the public domain confirmed its molecular genetic distinctiveness and revealed a cross-continental distribution of close relatives. The value of combining morphology and phylogeny to characterize AMF was reinforced by the morphological similarity to other species and the inconspicuous nature of D. celata spores and mycorrhizas. Inclusion of all three nuclear rDNA regions in species descriptions will facilitate species determination from environmental phylotypes.

Forage Rotations Conserve Diversity of Arbuscular Mycorrhizal Fungi and Soil Fertility.

In the Mediterranean, long-term impact of typical land uses on soil fertility have not been quantified yet on replicated mixed crop-livestock farms and considering the variability of soil texture. Here, we report the effects, after 15 years of practice, of two legume-winter cereal rotations, olive orchards and vineyards on microbiological and chemical indicators of soil fertility and the communities of arbuscular mycorrhizal fungi (AMF). We compare the changes among these four agricultural land-use types to woodland reference sites. Root colonization by AMF of English ryegrass (Lolium perenne L.), a grass that occurred under all land use types, was only half as heavy in biannual berseem clover (Trifolium alexandrinum L.)-winter cereal rotations than in 4-year alfalfa (Medicago sativa L.)-winter cereal rotations. In olive (Olea europaea L.) orchards and vineyards (Vitis vinifera L.), where weeds are controlled by frequent surface tillage, the AMF root colonization of ryegrass was again much lower than in the legume-cereal rotations and at the woodland reference sites. All the microbial parameters and soil organic carbon correlated most strongly with differences in occurrence and relative abundance (ß-diversity) of AMF genera in soil. The soil pH and mineral nutrients in soil strongly correlated with differences in AMF root colonization and AMF genus richness (α-diversity) in soil. Diversity of AMF was much less affected by soil texture than land use, while the opposite was true for microbial and chemical soil fertility indicators. Land uses that guaranteed a continuous ground cover of herbaceous plants and that involved only infrequent tillage, such as multiyear alfalfa-winter cereal rotation, allowed members of the AMF genus Scutellospora to persist and remain abundant. On the contrary, under land uses accompanied by frequent tillage and hence discontinuous presence of herbaceous plants, such as tilled olive orchard and vineyard, members of the genus Funneliformis dominated. These results suggest that multiyear alfalfa-winter cereal rotation with active plant growth throughout the year is the least detrimental agricultural land use in soil carbon and AMF abundance and diversity, relative to the woodland reference.

Insights from invasion ecology: Can consideration of eco-evolutionary experience promote benefits from root mutualisms in plant production?

Mutualistic plant-microbial functioning relies on co-adapted symbiotic partners as well as conducive environmental conditions. Choosing particular plant genotypes for domestication and subsequent cultivar selection can narrow the gene pools of crop plants to a degree that they are no longer able to benefit from microbial mutualists. Elevated mineral nutrient levels in cultivated soils also reduce the dependence of crops on nutritional support by mutualists such as mycorrhizal fungi and rhizobia. Thus, current ways of crop production are predestined to compromise the propagation and function of microbial symbionts, limiting their long-term benefits for plant yield stability. The influence of mutualists on non-native plant establishment and spread, i.e. biological invasions, provides an unexplored analogue to contemporary crop production that accounts for mutualistic services from symbionts like rhizobia and mycorrhizae. The historical exposure of organisms to biotic interactions over evolutionary timescales, or so-called eco-evolutionary experience (EEE), has been used to explain the success of such invasions. In this paper, we stress that consideration of the EEE concept can shed light on how to overcome the loss of microbial mutualist functions following crop domestication and breeding. We propose specific experimental approaches to utilize the wild ancestors of crops to determine whether crop domestication compromised the benefits derived from root microbial symbioses or not. This can predict the potential for success of mutualistic symbiosis manipulation in modern crops and the maintenance of effective microbial mutualisms over the long term.

Real-time PCR and microscopy: are the two methods measuring the same unit of arbuscular mycorrhizal fungal abundance?

To enable quantification of mycelial abundance in mixed-species environments, eight new TaqMan((R)) real-time PCR assays were developed for five arbuscular mycorrhizal fungal (AMF, Glomeromycota) taxa. The assays targeted genes encoding 18S rRNA or actin, and were tested on DNA from cloned gene fragments, from spores, mycelia, and from root-free soil, and on reverse-transcribed rRNA templates from entire mycelia and from colonized roots. The assays showed high specificity, sensitivity, and reproducibility, enabling reliable quantitation over broad ranges of template molecules. From cultured mycelia, DNA and RNA measures both correlated with spore number rather than extraradical hyphal length, and epifluorescence microscopy identified pronounced heterogeneity in vitality and nuclear distribution in hyphae. Root colonization was also spatially heterogeneous, as shown by a mixing experiment with root fragments of different length. Therefore, although real-time PCR can reproducibly and accurately quantify AMF nucleic acids, these are poorly correlated with visual measures because of spatial heterogeneity.

Genetic diversity and host plant preferences revealed by simple sequence repeat and mitochondrial markers in a population of the arbuscular mycorrhizal fungus Glomus intraradices.

Arbuscular mycorrhizal fungi (AMF) are important symbionts of plants that improve plant nutrient acquisition and promote plant diversity. Although within-species genetic differences among AMF have been shown to differentially affect plant growth, very little is actually known about the degree of genetic diversity in AMF populations. This is largely because of difficulties in isolation and cultivation of the fungi in a clean system allowing reliable genotyping to be performed. A population of the arbuscular mycorrhizal fungus Glomus intraradices growing in an in vitro cultivation system was studied using newly developed simple sequence repeat (SSR), nuclear gene intron and mitochondrial ribosomal gene intron markers. The markers revealed a strong differentiation at the nuclear and mitochondrial level among isolates. Genotypes were nonrandomly distributed among four plots showing genetic subdivisions in the field. Meanwhile, identical genotypes were found in geographically distant locations. AMF genotypes showed significant preferences to different host plant species (Glycine max, Helianthus annuus and Allium porrum) used before the fungal in vitro culture establishment. Host plants in a field could provide a heterogeneous environment favouring certain genotypes. Such preferences may partly explain within-population patterns of genetic diversity.

A Comprehensive, Automatically Updated Fungal ITS Sequence Dataset for Reference-Based Chimera Control in Environmental Sequencing Efforts.

The nuclear ribosomal internal transcribed spacer (ITS) region is the most commonly chosen genetic marker for the molecular identification of fungi in environmental sequencing and molecular ecology studies. Several analytical issues complicate such efforts, one of which is the formation of chimeric-artificially joined-DNA sequences during PCR amplification or sequence assembly. Several software tools are currently available for chimera detection, but rely to various degrees on the presence of a chimera-free reference dataset for optimal performance. However, no such dataset is available for use with the fungal ITS region. This study introduces a comprehensive, automatically updated reference dataset for fungal ITS sequences based on the UNITE database for the molecular identification of fungi. This dataset supports chimera detection throughout the fungal kingdom and for full-length ITS sequences as well as partial (ITS1 or ITS2 only) datasets. The performance of the dataset on a large set of artificial chimeras was above 99.5%, and we subsequently used the dataset to remove nearly 1,000 compromised fungal ITS sequences from public circulation. The dataset is available at http://unite.ut.ee/repository.php and is subject to web-based third-party curation.