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Social Impact Statement Vanilla planifolia is the source of the spice vanilla. This study is part of an international initiative to study the biology, including mycorrhizal fungi and cultivation practices of vanilla to improve its production in Mexico. The study focused on documenting mycorrhizal fungal diversity in vanilla. It also provided preliminary data on differences in mycorrhizal fungal communities associated with cultivation practices. A richer mycorrhizal community was observed in vanilla growing in a wild natural farm compared with those from a highly managed farm. Our research provides insights for sustainable vanilla production that can benefit Mexican farming communities. Summary Relatively little is known regarding differences in root symbionts (i.e., mycorrhizae) between epiphytic and terrestrial orchids. We characterized the mycorrhizal fungal communities of aerial and terrestrial roots of the orchid, Vanilla planifolia, from four Mexican farms representing different management systems. Amplicon sequencing identified 40 putative mycorrhizal fungi based on ITS sequence data, these included traditional orchid mycorrhizal fungi such as Ceratobasidium, and Thanatephorus in the order Cantharellales as well as Serendipitaceae in the order Sebacinales, and species of several genera traditionally considered as ectomycorrhizal fungi. Mycorrhizal fungal communities were similar in aerial and terrestrial roots, but differed in read abundances. Plants growing in wild‐natural conditions hosted a richer, but not statistically different, community of mycorrhizal fungi in comparison with plants in the highly managed farm. Soil characteristics including texture, organic matter, N, P, and K do not explain differences between fungal communities at these farms. This is one of the first reports of a diverse community of fungi traditionally considered to form ectomycorrhizas in association with aerial orchid roots. Further research is needed to understand the functional role of these putative mycorrhizal fungi in the ecology of V. planifolia, and if ectomycorrhizal fungi commonly occur in other hemiepiphytic and epiphytic orchids.

Plant‐associated fungi can ameliorate abiotic stress in their hosts, and changes in these fungal communities can alter plant productivity, species interactions, community structure and ecosystem processes. We investigated the response of root‐associated fungi to experimental drought (66% reduction in growing season precipitation) across six North American grassland ecosystem types to determine how extreme drought alters root‐associated fungi, and understand what abiotic factors influence root fungal community composition across grassland ecosystems. Next generation sequencing of the fungal ITS2 region demonstrated that drought primarily re‐ordered fungal species' relative abundances within host plant species, with different fungal responses depending on host identity. Grass species that declined more under drought trended toward less community re‐ordering of root fungi than species less sensitive to drought. Host identity and grassland ecosystem type defined the magnitude of drought effects on community composition, diversity and root colonization, and the most important factor affecting fungal composition was plant species identity. Fungal diversity was less responsive to drought than fungal composition. Across ecosystems, latitude and soil pH were better predictors of fungal diversity than experimental drought. Drought significantly reduced fungal diversity and evenness only in sideoats grama Bouteloua curtipendula and reduced root colonization only in blue grama B. gracilis. Our results illustrate that predicting the effects of drought on root‐associated fungi will require attention to host plant identity and ecosystem type. By comparing our findings against soil microbe responses in the same experiment, we propose the hypothesis that fungi in plant roots are less sensitive to drought than fungi inhabiting soils. Our study highlights the importance of studying community‐level re‐ordering of fungal composition for understanding plant responses to climate change.

Darksidea is a common genus of dark septate fungi—a group of ascomycetes in semiarid regions. A survey reported D. alpha and a distinct Darksidea lineage as abundant root-associated fungi of foundational grasses in North America. Fungi were isolated, and metabarcode data were obtained from sequencing of fungal communities of grass roots in the United States. During a comprehensive investigation of the Darksidea lineage, we carried out polyphasic taxonomy, genomic characterization, and identification of host associations, geographic distribution, and environmental factors that correlate with its abundance. For molecular phylogenetic studies, seven loci were sequenced. Isolates of the distinct Darksidea had variable colony morphology. No sexual reproductive structures were detected, but chlamydospores were frequently observed. The complete genome of an isolate of the lineage was sequenced with a size of 52.3 Mb including 14 707 gene models. Based on morphology and phylogenetic analysis, we propose the novel species Darksidea phi, sp. nov. Metabarcoding data showed that D. phi distribution and relative abundance were not limited to semiarid regions or a specific grass species, suggesting low host specificity among graminoids. This new species, D. phi, expands the distribution of the genus in the United States beyond prior reports from arid regions.

High temperatures and extended drought in temperate and tropical arid ecosystems promote the colonization of diverse microenvironments by dark septate fungi (DSF). These fungi contribute to soil nutrient cycling, soil stabilization, and plant survival, but the roles of individual DSF species, their distributions, and their community diversity are poorly understood. The objective of this study was to evaluate the distribution, seasonal variation, and potential roles of DSF on plant growth. We collected biocrust (lichen-, moss-, and cyanobacterium-dominated biocrusts) soils at different depths and rhizosphere soils from two grasses, Bromus tectorum and Pleuraphis jamesii, in an arid grassland near Moab, Utah, USA. Seasonal variation of DSF was evaluated using culture-based approaches and compared with fungal community profiles from next-generation sequencing (NGS). Culturing showed that DSF were 30% more abundant in biocrusts compared with the focal rhizospheres. The abundance of DSF varied seasonally in belowground samples (rhizosphere and below-biocrust), with a significant increase during the summer months. Pleosporales was the dominant order (35%) in both biocrust and rhizosphere soils out of 817 isolated fungi. Dominant DSF genera in culture included Alternaria, Preussia, Cladosporium, Phoma, and an unknown Pleosporales. Similar results were observed in biocrust and rhizosphere soils NGS. Further, seed germination experiments using dominant taxa were conducted to determine their potential roles on germination and seedling growth using maize as a model plant. Cladosporium and unknown Pleosporales isolates showed plant growth-promoting ability. The variation in abundance of DSF, their differential occurrence in different microenvironments, and their ability to grow in a xerotolerant medium reflect adaptations to summer environmental conditions and to changes in the abundance of organic matter, as well as a potential increase in plant investment in these fungi when heat and drought stresses are more severe.

For decades entomopathogenic fungi have garnered interest as possible alternatives to chemical pesticides. However, their ecology outside of agroecosystems demands further study. We assessed the diversity and abundance of entomopathogenic and insect-associated fungi at a loblolly pine forest in North Carolina, USA using culture-dependent and next-generation sequencing libraries. Fungi were isolated using Galleriamellonella larvae, as well as from soil dilutions plated on a selective medium. Isolates were identified using Sanger sequencing of the ITS and LSU rRNA gene regions, and represented 36 OTUs including Metarhizium, Lecanicillium, and Paecilomyces. Additionally, we assessed the chitinolytic potential of isolates and found widespread, variable ability to degrade chitin within and between genera. Phylogenetic analyses resolved several isolates to genus, with some forming clades with other insect-associated taxa, as well as with fungi associated with plant tissues. Saprophytes were widely distributed in soil, while entomopathogens were less abundant and present primarily in the top two cm of the soil. The similarity between culture-dependent and next-generation sequencing results demonstrates that both methods can be used concurrently in this system to study the ecology of entomopathogenic fungi.

Corn bins in the midwestern United States can reach temperatures up to 52 C. High temperatures combined with sufficient moisture and humidity in bins provide the perfect environment to promote the growth of thermophilic and thermotolerant fungi. In this article, we characterize for the first time thermophilic and thermotolerant fungi in corn grain bins using culture-based methods and pyrosequencing techniques. Corn samples were collected from local farms in western Illinois. Samples were plated and incubated at 50 C using a variety of approaches. Of several hundred kernels examined, more than 90% showed colonization. Species identified using culture methods included Thermomyces lanuginosus, Thermomyces dupontii, Aspergillus fumigatus, Thermoascus crustaceus, and Rhizomucor pusillus. Pyrosequencing was also performed directly on corn grain using fungal-specific primers to determine whether thermophilic fungi could be detected using this technique. Sequences were dominated by pathogenic fungi, and thermophiles were represented by less than 2% of the sequences despite being isolated from 90% of the grain samples using culturing techniques. The high abundance of previously undocumented viable fungi in corn could have negative implications for grain quality and pose a potential risk for workers and consumers of corn-derived products in the food industry. Members of the Sordariales were absent among thermophile isolates and were not represented in nuc rDNA internal transcribed spacer (ITS) sequences. This is in striking contrast with results obtained with other substrates such as litter, dung, and soils, where mesophilic and thermophilic members of the Sordariaceae and Chaetomiaceae are common. This absence appears to reflect an important difference between the ecology of Sordariales and other orders within the Ascomycota in terms of their ability to compete in microhabitats rich in sugars and living tissues.

White-nose syndrome (WNS) is a bat disease caused by the fungal pathogen Pseudogymnoascus destructans, which thrives in cold and very humid environments where bats frequently hibernate. Conidia of Pseudogymnoascus species are often documented on bats prior to the onset of WNS, but characterization of high-risk areas defined by microclimate cave conditions have been lacking. Investigating the occurrence of this fungal genus and appropriate environmental conditions to support P. destructans in southwestern U.S. caves is key to understanding the sites most likely to be impacted by WNS. Microclimate conditions in ten caves at El Malpais (ELMA) National Monument in New Mexico, USA were recorded using i-Button data loggers during the winters of 2011–2014 to assess appropriate environmental conditions (temperature and relative humidity) for P. destructans and other Pseudogymnoascus species. Optimal microclimate conditions for P. destructans and other psychrophilic fungi were found in all the caves with at least 50% of the caves identified as high-risk areas. Pseudogymnoascus species were detected in 70% of the caves using culturing methods and PCR, but no soil samples were positive for P. destructans using real-time PCR in soil and guano samples. Pseudogymnoascus destructans has a recognized range of appropriate temperatures and relative humidity for growth and cave microclimate can help define high-risk areas. This study offers resource managers guidance for establishing priority monitoring areas in their bat caves to determine which bat species are at higher risk.

Four bacterial strains, with the capability of inhibiting Pseudogymnoascus destructans, the causative agent of white-nose syndrome, were isolated from male Townsend’s big-eared bats (Corynorhinus townsendii, Family: Vespertilionidae) in New Mexico. Isolates AC161, AC162, AC208, and AC230T were characterised as a novel clade using morphological, phenotypic and phylogenetic analysis. A draft genome of the type strain was completed to determine its taxonomy and secondary metabolite biosynthetic potential. Multi-locus sequence analysis nests AC230T with neighbours Streptomyces scopuliridis (NRRL B-24574T), Streptomyces lushanensis (NRRL B-24994T), Streptomyces odonnellii (NRRL B-24891T) and Streptomyces niveus (NRRL 2466T). Further phylogenetic analysis showed the MLSA distances between AC230T and its near neighbours are much greater than the generally accepted threshold (> 0.007) for bacterial species delineation. DNA–DNA relatedness between AC230T and its near neighbours ranged between 25.7 ± 2.1 and 29.9 ± 2.4%. The DNA G+C content of the genomic DNA of the type strain is 71.7 mol%. Isolate AC230T presents a white to ivory hue on most ISP media and its micromorphology exhibits ovoid spores with smooth surfaces in flexuous chains. Based on our study of AC230T, the strain warrants the assignment to a novel species, for which the name Streptomyces corynorhini sp. nov. is proposed. The type strain is AC230T (= JCM 33171T, = ATCC TSD155T).

The novel fungal strain, Fusarium sp. strain DS 682, was isolated from the rhizosphere of the perennial grass, Bouteloua gracilis, at the Konza Prairie Biological Station in Kansas. This fungal strain is common across North American grasslands and is resilient to environmental fluctuations. The draft genome is estimated to be 97.2% complete.

The novel fungal strain, Fusarium sp. strain DS 682, was isolated from the rhizosphere of the perennial grass, Bouteloua gracilis , at the Konza Prairie Biological Station in Kansas. This fungal strain is common across North American grasslands and is resilient to environmental fluctuations. The draft genome is estimated to be 97.2% complete.

Transmission of information has benefitted from a breathtaking level of innovation and change over the past 20 years; however, instructional methods within colleges and universities have been slow to change. In the article, we present a novel framework to structure conversations that encourage innovation, change, and improvement in our system of higher education, in general, and our system of biology education, specifically. In particular, we propose that a conceptual model based on evolutionary landscapes in which fitness is replaced by educational effectiveness would encourage educational improvement by helping to visualize the multidimensional nature of education and learning, acknowledge the complexity and dynamism of the educational landscape, encourage collaboration, and stimulate experimental thinking about how new approaches and methodology could take various fields associated with learning, to more universal fitness optima. The framework also would encourage development and implementation of new techniques and persistence through less efficient or effective valleys of death.

A putative novel clade within the genus Streptomyces was discovered following antifungal screening against Pseudogymnoascus destructans, the causative agent of white-nose syndrome, and described using multi-locus sequencing analysis. Swabs from both the cave myotis bat (Myotis velifer) and the Brazilian free-tailed bat (Tadarida brasiliensis) in southern New Mexico bore isolates AC536, AC541(T) and AC563, which were characterised using phylogenetic, morphological, and phenotypic analyses. Multi-locus sequence analysis positions AC541(T) with neighbors Streptomyces rubidus (NRRL B-24619(T)), Streptomyces guanduensis (NRRL B-24617(T)), and Streptomyces yeochonensis (NRRL B-24245(T)). A complete genome of the type strain was assembled to determine its taxonomy and secondary metabolite potential. ANI comparisons between all closely related types strains are shown to be well below the 95–96% species delineation. DNA-DNA relatedness between AC541(T) and its nearest neighbors ranged between 23.7 and 24.1% confirming novelty. Approximately 1.49 Mb or 17.76% of the whole genome is devoted to natural product biosynthesis. The DNA G + C content of the genomic DNA of the type strain is 73.13 mol %. Micromorphology depicts ovoid spores with smooth surfaces in flexuous chains. Strains presented an ivory to yellow hue on most ISP media except inorganic salts-starch agar (ISP4) and can grow on d-glucose, mannitol, and d-fructose, but exhibited little to no growth on l-arabinose, sucrose, d-xylose, inositol, l-rhamnose, d-raffinose, and cellulose. This clade possesses the capability to grow from 10 to 45 °C and 12.5% (w/v) NaCl. There was strain growth variation in pH, but all isolates thrive at alkaline levels. Based on our polyphasic study of AC541(T), the strain warrants the assignment to a novel species, for which the name Streptomyces buecherae sp. nov. is proposed. The type strain is AC541(T) (= JCM 34263(T), = ATCC TSD201(T)).

Soil fungi in desert ecosystems are adapted to harsh environmental conditions such as high soil surface temperatures and limited organic matter and water. Given limited carbon inputs from plant material, heterotrophic fungi likely use unconventional sources of carbon in these systems. A baiting method was used to culture keratinophilic fungi from biocrust and rhizosphere soils in an arid grassland in Utah, USA. Fungi were baited using llama and sheep wool, horsehair, and snakeskin on two media, and pure cultures were identified using ITS and LSU rRNA sequences. One hundred-eighteen fungal colonies were grown, representing a total of 32 Operational Taxonomic Units (OTUs) at 97 % similarity. Cultures were dominated by the phylum Ascomycota (88 %) followed by Mucoromycota (8.6 %) and Basidiomycota (3.4 %). The orders Pleosporales, Eurotiales, Hypocreales, and Sordariales were commonly isolated, with the dominant taxa Alternaria (27 %), Aspergillus (22 %), Fusarium (11 %), and Chaetomium (8%). Thirty percent of the fungi isolated have the capacity to degrade keratin in vitro using a keratin azure assay, with Penicillium showing the highest degradation followed by Geomyces, Alternaria, and Fusarium. Although keratin degraders can be infectious, dermatophytes associated with skin infections were not isolated in culture or detected in Illumina sequencing. Illumina sequencing was used to determine general patterns in seasonal variation and habitat preference of keratinophiles. Alternaria was the most abundant genus with >70 % of the sequences. The combination of Illumina data with culture-dependent approaches facilitated the characterization of a specialized community and confirmed the low abundance of dermatophytes in this arid site.

Natural soils represent a rich reservoir of novel and diverse microbial communities. Heavy metal tolerant fungi have been described in contaminated soils and water, but it is unknown how abundant and diverse they are in natural systems. We isolated and identified heavy metal tolerant fungi from a temperate pine forest (Duke Forest, NC, USA) and determined their soil abundance and distribution using amplicon sequencing. Soil serial dilutions were inoculated on malt extract agar with metal concentrations between 100 and 1000 ppm of FeSO4, ZnSO4, CuSO4, Al2(SO4)3, Pb(NO3)2, NiCl2, CdCl2, and K2Cr2O7, depending on the metal used, at 25 °C. A total of 425 fungal isolates were obtained, from which the majority were isolated in Pb enriched-medium and the least from Cr and Al enriched media. A total of 62 unique Operational Taxonomic Units (OTUs) were identified at 97% sequence similarity using the ITS nuclear ribosomal RNA. The most common and diverse genera isolated were Penicillium and Trichoderma, with 126 and 92 isolates, representing 10 and 6 unique OTUs, respectively. Umbelopsis, Pochonia, Pseudogymnoascus, Trichocladium, and Ilyonectria are reported for the first time as tolerant taxa to multiple metals. The use of culture-based methods also revealed the presence of taxa that could not be detected using soil amplicon sequencing; these non-detectable OTUs corresponded to cultures with low isolation rates. The most commonly isolated taxa were detected using amplicon sequencing in 90–100% of the soil samples and soil depths using direct sequencing techniques, showing consistent results with the cultured-based methods. Penicillium, Trichoderma, Umbelopsis, and Saitozyma sequences were detected in 100% of the soil samples (156 samples) at all soil depths. Our study shows that this natural environment contained abundant and diverse communities of heavy metal tolerant fungi similar to those reported in metal contaminated sites. These commonly recovered taxa showed wide levels of adaptation to different metals and toxicity levels. The combination of culture-based and direct sequencing approaches were useful to describe the abundance and distribution of heavy metal tolerant fungi in natural soils.

Both Glycine max (soybean) and Thlaspi arvense (pennycress) are valuable resources for renewable biofuels and industrial products. Pennycress, a member of the Brassicaceae, can be grown over the winter months providing soil cover and thus reducing erosion. In addition, pennycress does not compete with food production making it an appealing option for biofuels. The objective of this study was to determine the effect of different agricultural practices on fungal communities associated with soybean roots and their interactions with an emerging crop/cover crop, pennycress. We examined roots collected in the summer of 2013 and 2014 from three treatment plots (organic, conventional soybeans, and conventional soybean with pennycress as a cover crop). Root DNA was amplified using fungal primers followed by 454 pyrosequencing. A total of 560,948 sequences were obtained with Ascomycota as the dominant fungal phylum in all plot treatments. Soybean roots in all treatments were dominated by the order Hypocreales with Fusarium as the most abundant genus. Fungal community structure was affected by host and farming practice. The magnitude of the effect was determined by sampling year. The cover crop, pennycress, had a distinct fungal assembly from soybean and did not affect the structure of fungal communities in soybean when used as a cover crop. The organic soybean fungal community was significantly different and contained lower diversity than the conventional soybean, with the greatest differences observed in 2014. This study shows that the structure of fungal communities associated with plants in agroecosystems are regulated by a complex number of abiotic and biotic factors including weather conditions, farming practice, and host.

BackgroundThe dominant fungi in arid grasslands and shrublands are members of the Ascomycota phylum. Ascomycota fungi are important drivers in carbon and nitrogen cycling in arid ecosystems. These fungi play roles in soil stability, plant biomass decomposition, and endophytic interactions with plants. They may also form symbiotic associations with biocrust components or be latent saprotrophs or pathogens that live on plant tissues. However, their functional potential in arid soils, where organic matter, nutrients and water are very low or only periodically available, is poorly characterized.ResultsFive Ascomycota fungi were isolated from different soil crust microhabitats and rhizosphere soils around the native bunchgrassPleuraphis jamesiiin an arid grassland near Moab, UT, USA. Putative genera wereConiochaeta, isolated from lichen biocrust,Embellisiafrom cyanobacteria biocrust, Chaetomiumfrom below lichen biocrust,Phomafrom a moss microhabitat, andAspergillusfrom the soil. The fungi were grown in replicate cultures on different carbon sources (chitin, native bunchgrass or pine wood) relevant to plant biomass and soil carbon sources. Secretomes produced by the fungi on each substrate were characterized. Results demonstrate that these fungi likely interact with primary producers (biocrust or plants) by secreting a wide range of proteins that facilitate symbiotic associations. Each of the fungal isolates secreted enzymes that degrade plant biomass, small secreted effector proteins, and proteins involved in either beneficial plant interactions or virulence.AspergillusandPhomaexpressed more plant biomass degrading enzymes when grown in grass- and pine-containing cultures than in chitin.ConiochaetaandEmbellisiaexpressed similar numbers of these enzymes under all conditions, whileChaetomiumsecreted more of these enzymes in grass-containing cultures.ConclusionsThis study of Ascomycota genomes and secretomes provides important insights about the lifestyles and the roles that Ascomycota fungi likely play in arid grassland, ecosystems. However, the exact nature of those interactions, whether any or all of the isolates are true endophytes, latent saprotrophs or opportunistic phytopathogens, will be the topic of future studies.

Fungi are key organisms in many ecological processes and communities. Rapid and low cost surveys of the fungal members of a community can be undertaken by isolating and sequencing a taxonomically informative genomic region, such as the ITS (internal transcribed spacer), from DNA extracted from a metagenomic sample, and then classifying these sequences to determine which organisms are present. This paper announces the availability of the Warcup ITS training set and shows how it can be used with the Ribosomal Database Project (RDP) Bayesian Classifier to rapidly and accurately identify fungi using ITS sequences. The classifications can be down to species level and use conventional literature-based mycological nomenclature and taxonomic assignments.

Illumina amplicon sequencing of soil in a temperate pine forest in the southeastern United States detected an abundant, nitrogen (N)-responsive fungal genotype of unknown phylogenetic affiliation. Two isolates with ribosomal sequences consistent with that genotype were subsequently obtained. Examination of records in GenBank revealed that a genetically similar fungus had been isolated previously as an endophyte of moss in a pine forest in the southwestern United States. The three isolates were characterized using morphological, genomic, and multilocus molecular data (18S, internal transcribed spacer [ITS], and 28S rRNA sequences). Phylogenetic and maximum likelihood phylogenomic reconstructions revealed that the taxon represents a novel lineage in Mucoromycotina, only preceded by Calcarisporiella, the earliest diverging lineage in the subphylum. Sequences for the novel taxon are frequently detected in environmental sequencing studies, and it is currently part of UNITE's dynamic list of most wanted fungi. The fungus is dimorphic, grows best at room temperature, and is associated with a wide variety of bacteria. Here, a new monotypic genus, Bifiguratus, is proposed, typified by Bifiguratus adelaidae.

Vanilla is an orchid of economic importance widely cultivated in tropical regions and native to Mexico. We sampled three species of Vanilla (V. planifolia, V. pompona, and V. insignis) in different crop systems. We studied the effect of crop system on the abundance, type of fungi, and quality of pelotons found in the roots using light and electron microscopy and direct sequencing of mycorrhizal structures. Fungi were identified directly from pelotons obtained from terrestrial roots of vanilla plants in the flowering stage. Root samples were collected from plants in crop systems located in the Totonacapan area in Mexico (states of Puebla and Veracruz). DNA was extracted directly from 40 pelotons and amplified using ITS rRNA sequencing. Peloton-like structures were observed, presenting a combination of active pelotons characterized by abundant hyphal coils and pelotons in various stages of degradation. The most active pelotons were observed in crop systems throughout living tutors (host tree) in comparison with roots collected from dead or artificial tutors. Fungi identified directly from pelotons included Scleroderma areolatum, a common ectomycorrhizal fungus that has not been reported as a mycorrhizal symbiont in orchids. Direct amplification of pelotons also yielded common plant pathogens, including Fusarium and Pyrenophora seminiperda, especially in those sites with low colonization rates, and where large numbers of degraded pelotons were observed. This research reports for the first time the potential colonization of Vanilla by Scleroderma, as a putative orchid mycorrhizal symbiont in four sites in Mexico and the influence of crop system on mycorrhizal colonization on this orchid.

At least two-thirds of commercial antibiotics today are derived from Actinobacteria , more specifically from the genus Streptomyces . Antibiotic resistance and new emerging diseases pose great challenges in the field of microbiology. Cave systems, in which actinobacteria are ubiquitous and abundant, represent new opportunities for the discovery of novel bacterial species and the study of their interactions with emergent pathogens. White-nose syndrome is an invasive bat disease caused by the fungus Pseudogymnoascus destructans , which has killed more than six million bats in the last 7 years. In this study, we isolated naturally occurring actinobacteria from white-nose syndrome (WNS)-free bats from five cave systems and surface locations in the vicinity in New Mexico and Arizona, USA. We sequenced the 16S rRNA region and tested 632 isolates from 12 different bat species using a bilayer plate method to evaluate antifungal activity. Thirty-six actinobacteria inhibited or stopped the growth of P. destructans , with 32 (88.9%) actinobacteria belonging to the genus Streptomyces . Isolates in the genera Rhodococcus , Streptosporangium , Luteipulveratus , and Nocardiopsis also showed inhibition. Twenty-five of the isolates with antifungal activity against P. destructans represent 15 novel Streptomyces spp. based on multilocus sequence analysis. Our results suggest that bats in western North America caves possess novel bacterial microbiota with the potential to inhibit P. destructans . IMPORTANCE This study reports the largest collection of actinobacteria from bats with activity against Pseudogymnoascus destructans , the fungal causative agent of white-nose syndrome. Using multigene analysis, we discovered 15 potential novel species. This research demonstrates that bats and caves may serve as a rich reservoir for novel Streptomyces species with antimicrobial bioactive compounds.

Healthy plant roots usually host heterogeneous communities of root endophytes that are abundant in all plants and ecosystems. Despite their abundance, current understanding of the distribution of these endophytes is superficial at the best. A comprehensive treatise of the distribution of variety of fungi described as root endophytes would most likely prove an exhausting exercise in futility, given the large gaps in current data. As a result, we broadly target the helotialean endophytes that are commonly observed in temperate and boreal, forested ecosystems and the pleosporalean endophytes commonly observed in grassland ecosystems. To explore the sparse data available for the distribution of these root-associated fungal endophytes, we combine references from current literature and a case study to evaluate whether or not the endophyte communities (1) shift over environmental gradients, (2) respond to some specific drivers, or (3) seem limited by dispersal by showing distance-decay responses. The data indicate that communities of endophytic fungi indeed change over large geographical scales. The available data suggest a Helotiales-dominated endophyte guild in forested ecosystems and a Pleosporales-dominated assemblage in grassland ecosystems. Further, large-scale studies and our preliminary analyses of communities across North American grassland ecosystems suggest some shifts corresponding to geographical/environmental gradients. However, specific drivers are difficult to distinguish because abiotic gradients are often correlated, resulting in complex model selection problems. Finally, we observed that our own and published data reach no agreement on distance decay, even though both agree that the distance decay may be of rather minor importance.

Nitrogen (N) deposition in many areas of the world is over an order of magnitude greater than it would be in absence of human activity. We ask how abiotic (N)and biotic (plant host and neighborhood) effects interact to influence root-associated bacterial (RAB)community assembly. Using 454 pyrosequencing, we examined RAB communities from two dominantal pine tundra plants, Geum rossii and Deschampsia cespitosa, under control, N addition and D. cespitosa removal treatments, implemented in a factorial design. We hypothesized that host would have the strongest effect on RAB assembly, followed by N,then neighbor effects.The most dominant phyla were Proteobacteria (mostly Gammaproteobacteria), Actinobacteria,Bacteroidetes and Acidobacteria. We found RAB communities were host specific, with only 17% overlap in operational taxonomic units. Host effects on composition were over twice as strong as Neffects. D. cespitosa RAB diversity declined with N, while G. rossii RAB did not. D. cespitosa removal did not influence G. rossii RAB community composition, but G. rossii RAB diversity declined with N only when D. cespitosa was absent. We conclude that RAB of both hosts are sensitive to N enrichment, and RAB response to N is influenced by host identity and plant neighborhood.

Fungal taxonomy and ecology have been revolutionized by the application of molecular methods and both have increasing connections to genomics and functional biology. However, data streams from traditional specimen- and culture-based systematics are not yet fully integrated with those from metagenomic and metatranscriptomic studies, which limits understanding of the taxonomic diversity and metabolic properties of fungal communities. This article reviews current resources, needs, and opportunities for sequence-based classification and identification (SBCI) in fungi as well as related efforts in prokaryotes. To realize the full potential of fungal SBCI it will be necessary to make advances in multiple areas. Improvements in sequencing methods, including long-read and single-cell technologies, will empower fungal molecular ecologists to look beyond ITS and current shotgun metagenomics approaches. Data quality and accessibility will be enhanced by attention to data and metadata standards and rigorous enforcement of policies for deposition of data and workflows. Taxonomic communities will need to develop best practices for molecular characterization in their focal clades, while also contributing to globally useful datasets including ITS. Changes to nomenclatural rules are needed to enable validPUBLICation of sequence-based taxon descriptions. Finally, cultural shifts are necessary to promote adoption of SBCI and to accord professional credit to individuals who contribute to community resources.

Soil fungal communities are responsible for carbon and nitrogen (N) cycling. The high complexity of the soil fungal community and the high proportion of taxonomically unidentifiable sequences confound ecological interpretations in field studies because physiological information is lacking for many organisms known only by their rRNA sequences. This situation forces experimental comparisons to be made at broader taxonomic racks where functions become difficult to infer. The objective of this study was to determine OTU (operational taxonomic units) level responses of the soil fungal community to N enrichment in a temperate pine forest experiment and to use the sequencing data to guide culture efforts of novel N-responsive fungal taxa. Replicate samples from four soil horizons (up to 10 cm depth) were obtained from ambient, enriched CO

Orchid mycorrhizal fungi are characterized by the presence of complex, globose masses of hyphe, called pelotons, in the cortex of the root (Hadley et al. 1989, Esnault et al. 1994). Extent and patterns of infections are very variable, and this variation is observed in many epiphytic orchids and some terrestrial tropical orchids (Hadley et al. 1989, Allen 1991). Orchid mycorrhizal fungi are characterized by the presence of complex, globose masses of hyphe, called pelotons, in the cortex of the root (Hadley et al. 1989, Esnault et al. 1994). Extent and patterns of infections are very variable, and this variation is observed in many epiphytic orchids and some terrestrial tropical orchids (Hadley et al. 1989, Allen 1991).

Endophytes are microorganisms that live within plant tissues without causing symptoms of disease. They are important components of plant microbiomes. Endophytes interact with, and overlap in function with, other core microbial groups that colonize plant tissues, e.g., mycorrhizal fungi, pathogens, epiphytes, and saprotrophs. Some fungal endophytes affect plant growth and plant responses to pathogens, herbivores, and environmental change; others produce useful or interesting secondary metabolites. Here, we focus on new techniques and approaches that can provide an integrative understanding of the role of fungal endophytes in the plant microbiome. Clavicipitaceous endophytes of grasses are not considered because they have unique properties distinct from other endophytes. Hidden from view and often overlooked, endophytes are emerging as their diversity, importance for plant growth and survival, and interactions with other organisms are revealed.

The fungal loop model of semiarid ecosystems integrates microtopographic structures and pulse dynamics with key microbial processes. However limited data exist about the composition and structure of fungal communities in these ecosystems. The goal of this study was to characterize diversity and structure of soil fungal communities in a semiarid grassland. The effect of long-term nitrogen fertilization on fungi also was evaluated. Samples of rhizosphere (soil surrounding plant roots) and biological soil crust (BSC) were collected in central New Mexico, USA. DNA was amplified from the samples with fungal specific primers. Twelve clone libraries were generated with a total of 307 (78 operational taxonomic units, OTUs) and 324 sequences (67 OTUs) for BSC and rhizosphere respectively. Approximately 40% of soil OTUs were considered novel (less than 97% identity when compared to other sequences in NCBI using BLAST). The dominant organisms were dark-septate (melanized fungi) ascomycetes belonging to Pleosporales. Effects of N enrichment on fungi were not evident at the community level; however the abundance of unique sequences, sampling intensity and temporal variations may be uncovering the effect of N in composition and diversity of fungal communities. The fungal communities of rhizosphere soil and BSC overlapped substantially in composition, with a Jaccard abundance similarity index of 0.75. Further analyses are required to explore possible functions of the dominant species colonizing zones of semiarid grassland soils.

Although research has shown that root associated fungi (RAF) are necessary for plant success in harsh environments, few studies have examined RAF community variability between different plant species coexisting in arid habitats. We compared the diversity and composition of the fungal communities colonizing dominant and important forage grasses, Bouteloua gracilis and Sporobolus cryptandrus, inhabiting the Sevilleta National Wildlife Refuge, a semiarid grassland in New Mexico. A third sympatric plant, Yucca glauca (Agavaceae), also was analyzed. ITS rDNA from roots, collected and amplified in 2007, yielded 447 fungal sequences. Sequences obtained from all three species suggest that grasses share a core group of RAF (90% of sequences representing the orders Pleosporales, Agaricales, and Sordariales). Conversely, 57% of the fungal community within Y. glauca was dominated by a different RAF cohort. The two most common OTUs within the grasses are related to Paraphaeospheria sp. and Moniliophthora sp. (91 and 85 of 331 sequences, respectively). These same two species accounted for 58% of isolates in culture-based analysis of the same grass root tissue. The presence of these RAF in several grass species, over several years, and at several sites suggests that grasses in semiarid landscapes share a similar cohort of fungal dominants.

In arid ecosystems, abiotic processes facilitate the physical and chemical degradation of plant litter to the extent that decomposition models that use climatic and litter composition variables as surrogates for microbial activity are not predictive. The purpose of this study was to estimate the potential contribution of photodegradation to the decomposition of plant litters that varies in architecture and chemical composition. Litter of Pinus edulis, Juniperus monosperma and Populus deltoides were exposed to ambient and attenuated sunlight, with and without supplemental water additions, at a riparian forest site along the Middle Rio Grande (New Mexico, USA). Mass loss, elemental composition, and microbial extracellular enzyme activities (EEA) were measured over 639 days. The composition of the fungal communities associated with the decomposing litters was compared by analyses of fungal ITS nrDNA sequences. Litter exposed to ambient sunlight had greater mass loss rates than shaded litter, independent of the water treatment: Populus increased by 100%, Pinus by 86% and Juniperus by 46%. The increases were proportional to exposed litter surface area per g dry mass. EEA potentials, particularly oxidative activities, were low in comparison to those measured in mesic ecosystems. For Populus litter, the principal driver of photoacceleration appeared to be photodegradation of cellulose; for Pinus, it was photodegradation of polyphenols; for Juniperus accelerated mass loss was associated with photodegradation of both polysaccharides and polyphenols. Fungal community composition varied by litter type, but the dominant colonizers were yeasts and dark-septate hyphal taxa; a finding consistent with the low enzymatic oxidation potential. This study shows that photochemical oxidation can supplement enzymatic oxidation and increase decomposition rates. As a result, organic matter decomposition in arid ecosystems is not restricted to periods of high moisture availability as is plant production. This decoupling may partly account for the low soil organic matter content of these ecosystems.

1. Arid ecosystems are a patch mosaic of plants and biological soil crusts that have been described as islands and mantles of fertility, respectively. To determine whether these patches are metabolically linked by a fungal network of dark septate and arbuscular mycorrhizal fungi (AMF), we measured translocation of nitrogen (N) and carbon (C) in a desert grassland using 15 N-NO 3 and 13 C 5 , 15 N-glutamic acid as substrates. 2. Substrates were applied as point sources to either small patches of biotic crust or to a subset of leaves within a bunch grass tussock. 3. Both substrates were translocated over approximately 1 m 2 areas (approximately 20 patches) at rates up to 100 cm day -1 during a 4-day period following a natural rainfall event. Foliar uptake of 15 N from glutamate was initially more rapid than 15 N uptake from nitrate, and translocation from foliage was initially more rapid than translocation from soil crust. Rates of 15 N translocation between patches were similar to rates of atmospheric N deposition and denitrification. 4. 13 C from 13 C 5 , 15 N-glutamic acid applied to leaves was translocated to crust suggesting that plant C may provide metabolic support to biological soil crusts during periods of active growth. 13 C from 13 C 5 , 15 N-glutamic acid applied to soil crusts did not enter plants, in contrast to the 15 N from this substrate, indicating that glutamate was not translocated intact. 5. The dominant fungi of roots, rhizosphere soil and biological soil crusts are dark septate ascomycetes, most classified as Pleosporales, and AMF are rare. Phylogenetic analyses indicate substantial overlap in fungal community composition between roots and crusts, which may facilitate nutrient transfers. 6. Synthesis. This study supports the hypothesis that the spatial structure of semi-arid ecosystems, a patch mosaic of grasses and biological soil crust described, respectively, by the island of fertility and mantle of fertility paradigms, is functionally integrated by exchanges of C and N through a symbiotic fungal network dominated by dark-septate fungi.

Summary 1 Aridland ecosystems cover about one-third of terrestrial environments globally, yet the extent to which models of carbon (C) and nitrogen (N) cycling, developed largely from studies of mesic ecosystems, apply to aridland systems remains unclear. 2 Within aridland ecosystems, C and N dynamics are often described by a pulse-reserve model in which episodic precipitation events stimulate biological activity that generate reserves of biomass, propagules and organic matter that prime the ecosystem to respond rapidly to subsequent precipitation events. 3 The role of microbial C and N processing within the pulse-reserve paradigm has not received much study. We present evidence suggesting that fungi play a critical and underappreciated role in aridland soils, including efficient decomposition of recalcitrant C compounds, N-transformations such as nitrification, and nutrient storage and translocation of C and N between plants and biotic soil crusts. While fungi may perform some of these functions in other ecosystems, this ‘fungal loop’ assumes particular importance in the N cycle in aridlands because water availability imposes even greater restrictions on bacterial activity and physicochemical processes limit accumulation of soil organic matter (SOM). 4 We incorporate these findings into a Threshold-Delay Nutrient Dynamics (TDND) model for aridland ecosystems in which plant responses to pulsed precipitation events are mediated by a fungal loop that links C and N cycling, net primary production (NPP) and decomposition in aridland soils. 5 Synthesis. Arid ecosystems are highly sensitive to global environmental change including N deposition and altered precipitation patterns; yet, models from mesic ecosystems do not adequately apply to aridland environments. Our ‘fungal loop’ N cycle model integrates spatial structure with pulse dynamics and extends the pulse-reserve paradigm to include the key role of microbial processes in aridland ecosystem dynamics.

Mycorrhizal fungi are essential for the germination of orchid seeds. However, the specificity of orchids for their mycorrhizal fungi and the effects of the fungi on orchid growth are controversial. Mycorrhizal fungi have been studied in some temperate and tropical, epiphytic orchids, but the symbionts of tropical, terrestrial orchids are still unknown. Here we study diversity, specificity and function of mycorrhizal fungi in Vanilla, a pantropical genus that is both terrestrial and epiphytic. Mycorrhizal roots were collected from four Vanilla species in Puerto Rico, Costa Rica and Cuba. Cultured and uncultured mycorrhizal fungi were identified by sequencing the internal transcribed spacer region of nuclear rDNA (nrITS) and part of the mitochondrial ribosomal large subunit (mtLSU), and by counting number of nuclei in hyphae. Vanilla spp. were associated with a wide range of mycorrhizal fungi: Ceratobasidium, Thanatephorus and Tulasnella. Related fungi were found in different species of Vanilla, although at different relative frequencies. Ceratobasidium was more common in roots in soil and Tulasnella was more common in roots on tree bark, but several clades of fungi included strains from both substrates. Relative frequencies of genera of mycorrhizal fungi differed significantly between cultured fungi and those detected by direct amplification. Ceratobasidium and Tulasnella were tested for effects on seed germination of Vanilla and effects on growth of Vanilla and Dendrobium plants. We found significant differences among fungi in effects on seed germination and plant growth. Effects of mycorrhizal fungi on Vanilla and Dendrobium were similar: a clade of Ceratobasidium had a consistently positive effect on plant growth and seed germination. This clade has potential use in germination and propagation of orchids. Results confirmed that a single orchid species can be associated with several mycorrhizal fungi with different functional consequences for the plant.

We studied the diversity of arbuscular mycorrhizal fungi (AMF) in semiarid grassland and the effect of long-term nitrogen (N) fertilization on this fungal community. Root samples of Bouteloua gracilis were collected at the Sevilleta National Wildlife Refuge (New Mexico, USA) from control and N-amended plots that have been fertilized since 1995. Small subunit rDNA was amplified using AMF specific primers NS31 and AM1. The diversity of AMF was low in comparison with other ecosystems, only seven operational taxonomic units (OTU) were found in B. gracilis and all belong to the genus Glomus. The dominant OTU was closely related to the ubiquitous G. intraradices/G. fasciculatum group. N-amended plots showed a reduction in the abundance of the dominant OTU and an increase in AMF diversity. The greater AMF diversity in roots from N-amended plots may have been the result of displacement of the dominant OTU, which facilitated detection of uncommon AMF. The long-term implications of AMF responses to N enrichment for plant carbon allocation and plant community structure remain unclear.

Nitrogen (N) deposition in many areas of the world is over an order of magnitude greater than it would be in absence of human activity. We ask how abiotic (N)and biotic (plant host and neighborhood) effects interact to influence root-associated bacterial (RAB)community assembly. Using 454 pyrosequencing, we examined RAB communities from two dominantal pine tundra plants, Geum rossii and Deschampsia cespitosa, under control, N addition and D. cespitosa removal treatments, implemented in a factorial design. We hypothesized that host would have the strongest effect on RAB assembly, followed by N,then neighbor effects.The most dominant phyla were Proteobacteria (mostly Gammaproteobacteria), Actinobacteria,Bacteroidetes and Acidobacteria. We found RAB communities were host specific, with only 17% overlap in operational taxonomic units. Host effects on composition were over twice as strong as Neffects. D. cespitosa RAB diversity declined with N, while G. rossii RAB did not. D. cespitosa removal did not influence G. rossii RAB community composition, but G. rossii RAB diversity declined with N only when D. cespitosa was absent. We conclude that RAB of both hosts are sensitive to N enrichment, and RAB response to N is influenced by host identity and plant neighborhood.

White-nose syndrome (WNS), a disease caused by the fungus Pseudogymnoascus destructans1, has spread west from New York to Missouri and has killed more than six million bats2. In bat hibernacula where WNS is present, mass mortality has been observed and there is a high potential for population collapse or extinction of some species at a regional level. Although WNS is not yet present in the western U.S. , the high diversity of bat species3 and appropriate conditions for P. destructans in area caves may put these populations at risk. The absence of WNS in western caves provides a unique opportunity to ask questions about how bat species, geographic location, and habitat shape pre-WNS microbiota. The importance of microbiota is shown in many organisms, including amphibians, where individuals that survive a chytrid infection carry a higher prevalence of Janthinobacterium lividum4. The establishment of a pre-WNS baseline microbiota of western bats is critical to understanding how P. destructans may impact the native microbiota of the bats. Previous studies5,6 that identified the microbiota of bats have focused on gut and fecal microbiota, with little attention given to the external microbiota. Here we show for the first time that there are biogeographic differences in the abundance and diversity of external bat microbiota. From our 202 (62 cave-netted, 140 surface-netted) bat samples belonging to 13 species of western bats uninfected with WNS, we identified differences in microbiota diversity among sites, and between cave bats versus surface-netted bats, regardless of sex and species. These results present novel information about the factors that shape external microbiota of bats providing new insights into potential vulnerability of different bat species to WNS.

Recent droughts in southwestern USA have led to large-scale mortality of pinon (Pinus edulis) in pinon-juniper woodlands. Pinon mortality alters soil moisture, nutrient and carbon availability, which could affect the root-associated fungal (RAF) communities and therefore the fitness of the remaining plants. We collected fine root samples at a pinon-juniper woodland and a juniper savannah site in central New Mexico. Roots were collected from pinon and juniper (Juniperus monosperma) trees whose nearest neighbors were live pinon, live juniper or dead pinon. RAF communities were analyzed by 454 pyrosequencing of the universal fungal ITS region. The most common taxa were Hypocreales and Chaetothyriales. More than 10% of ITS sequences could not be assigned taxonomy at the phylum level. Two of the unclassified OTUs significantly differed between savanna and woodland, had few like sequences in GenBank and formed new fungal clades with other unclassified RAF from arid plants, highlighting how little study has been done on the RAF of arid ecosystems. Plant host or neighbor did not affect RAF community composition. However, there was a significant difference between RAF communities from woodland vs. savanna, indicating that abiotic factors such as temperature and aridity might be more important in structuring these RAF communities than biotic factors such as plant host or neighbor identity. Ectomycorrhizal fungi (EM) were present in juniper as well as pinon in the woodland site, in contrast with previous research, but did not occur in juniper savanna, suggesting a potential shared EM network with juniper. RAF richness was lower in hosts that were neighbors of the opposite host. This may indicate competitive exclusion between fungi from different hosts. Characterizing these communities and their responses to environment and plant neighborhood is a step toward understanding the effects of drought on a biome that spans 19,000,000 ha of southwestern USA.