Pleoardoris graminearum, gen. et sp. nov., a new member of Pleosporales from North American Plains, its biogeography and effects on a foundation grass species.

Diverse fungi colonize plant roots worldwide and include species from many orders of the phylum Ascomycota. These fungi include taxa with dark septate hyphae that colonize grass roots and may modulate plant responses to stress. We describe a novel group of fungal isolates and evaluate their effects on the grass Bouteloua gracilis in vitro. We isolated fungi from roots of six native grasses from 24 sites spanning replicated latitudinal gradients in the south-central US grasslands and characterized isolates phylogenetically using a genome analysis. We analyzed 14 isolates representing a novel clade within the family Montagnulaceae (order Pleosporales), here typified as Pleoardoris graminearum, closely related to the genera Didymocrea and Bimuria. This novel species produces asexual, light brown pycnidium-like conidioma, hyaline hyphae, and chlamydospores when cultured on quinoa and kiwicha agar. To evaluate its effects on B. gracilis, seeds were inoculated with one of three isolates (DS304, DS334, and DS1613) and incubated at 25 C for 20 d. We also tested the effect of volatile organic compounds (VOCs) produced by the same isolates on B. gracilis root and stem lengths. Isolates had variable effects on plant growth. One isolate increased B. gracilis root length up to 34% compared with uninoculated controls. VOCs produced by two isolates increased root and stem lengths (P < 0.05) compared with controls. Internal transcribed spacer ITS2 metabarcode data revealed that P. graminearum is distributed across a wide range of sites in North America (22 of 24 sites sampled), and its relative abundance is influenced by host species identity and latitude. Host species identity and site were the most important factors determining P. graminearum relative abundance in drought experiments at the Extreme Drought in the Grasslands Experiment (EDGE) sites. Variable responses of B. gracilis to inoculation highlight the potential importance of nonmycorrhizal root-associated fungi on plant survival in arid ecosystems.

Genome-scale phylogeny and comparative genomics of the fungal order Sordariales.

The order Sordariales is taxonomically diverse, and harbours many species with different lifestyles and large economic importance. Despite its importance, a robust genome-scale phylogeny, and associated comparative genomic analysis of the order is lacking. In this study, we examined whole-genome data from 99 Sordariales, including 52 newly sequenced genomes, and seven outgroup taxa. We inferred a comprehensive phylogeny that resolved several contentious relationships amongst families in the order, and cleared-up intrafamily relationships within the Podosporaceae. Extensive comparative genomics showed that genomes from the three largest families in the dataset (Chaetomiaceae, Podosporaceae and Sordariaceae) differ greatly in GC content, genome size, gene number, repeat percentage, evolutionary rate, and genome content affected by repeat-induced point mutations (RIP). All genomic traits showed phylogenetic signal, and ancestral state reconstruction revealed that the variation of the properties stems primarily from within-family evolution. Together, the results provide a thorough framework for understanding genome evolution in this important group of fungi.

Darksidea phi, sp. nov., a dark septate root-associated fungus in foundation grasses in North American Great Plains.

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.

Merging Fungal and Bacterial Community Profiles via an Internal Control.

Integrated measurements of fungi and bacteria are critical to understand how interactions between these taxa drive key processes in ecosystems ranging from soils to animal guts. High-throughput amplicon sequencing is commonly used to census microbiomes, but the genetic markers targeted for fungi and bacteria (typically ribosomal regions) are domain-specific so profiling must be performed separately, obscuring relationships between these groups. To solve this problem, we developed a spike-in method with an internal control (IC) construct containing primer sites commonly used for bacterial and fungal taxonomic profiling. The internal control offers several advantages: estimation of absolute abundances, estimation of fungal to bacterial ratios (F:B), integration of bacterial and fungal profiles for holistic community analysis, and lower costs compared to other quantitation methods. To validate the IC as a scaling method, we compared IC-derived measures of F:B to measures from quantitative PCR (qPCR) using a commercial mock community (the ZymoBiomic Microbial Community DNA Standard II, containing two fungi and eight bacteria) and complex environmental samples. For both the mock community and the environmental samples, the IC produced F:B values that were statistically consistent with qPCR. Merging the environmental fungal and bacterial profiles based on the IC-derived F:B values revealed new relationships among samples in terms of community similarity. This IC method is the first spike-in method to employ a single construct for cross-domain amplicon sequencing, offering more reliable measurements.

Diversity of thermophilic and thermotolerant fungi in corn grain.

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.

Genetics of mating in members of the Chaetomiaceae as revealed by experimental and genomic characterization of reproduction in Myceliophthora heterothallica.

Members of the Chaetomiaceae are among the most studied fungi in industry and among the most reported in investigations of biomass degradation in both natural and laboratory settings. The family is recognized for production of carbohydrate-active enzymes and antibiotics. Thermophilic species are of special interest for their abilities to produce thermally stable enzymes and to be grown under conditions that are unsuitable for potential contaminant microorganisms. Such interests led to the recent acquisition of genome sequences from several members of the family, including thermophilic species, several of which are reported here for the first time. To date, however, thermophilic fungi in industry have served primarily as parts reservoirs and there has been no good genetic model for species in the family Chaetomiaceae or for thermophiles in general. We report here on the reproductive biology of the thermophile Myceliophthora heterothallica, which is heterothallic, unlike most described species in the family. We confirmed heterothallism genetically by following the segregation of mating type idiomorphs and other markers. We have expanded the number of known sexually-compatible individuals from the original isolates from Indiana and Germany to include several isolates from New Mexico. An interesting aspect of development in M. heterothallica is that ascocarp formation is optimal at approximately 30 °C, whereas vegetative growth is optimal at 45 °C. Genome sequences obtained from several strains, including isolates of each mating type, revealed mating-type regions whose genes are organized similarly to those of other members of the Sordariales, except for the presence of a truncated version of the mat A-1 (MAT1-1-1) gene in mating-type a (MAT1-2) strains. In M. heterothallica and other Chaetomiaceae, mating-type A (MAT1-1) strains have the full-length version of mat A-1 that is typical of mating-type A strains of diverse Ascomycota, whereas a strains have only the truncated version. This truncated mat A-1 has an intact open reading frame and a derived start codon that is not present in mat A-1 from A strains. The predicted protein contains a region that is conserved across diverse mat A-1 genes, but it lacks the major alpha1 domain, which characterizes proteins in this family and is known to be required for fertility in A strains from other Ascomycota. Finally, we have used genes from M. heterothallica to probe for mating genes in other homothallic and heterothallic members of the Chaetomiaceae. The majority of homothallic species examined have a typical mat A-1,2,3 (MAT1-1-1,2,3) region in addition to an unlinked mat a-1 (MAT1-2-1) gene, reflecting one type of homothallism commonly observed in diverse Ascomycota.

Lignin-modifying processes in the rhizosphere of arid land grasses.

Genes associated with elevated oxidative enzyme activities in arid systems have not been well characterized. To link measured oxidative activities with specific enzymes, we assembled protein-coding reads from the rhizospheres (RHZ) of two arid land grasses. Targeted gene scans for open reading frames, encoding genes potentially involved in lignin modification, resulted in 127 distinct assembly products. The putative genes included those significantly similar to Class II secretory fungal peroxidases. These genes are expressed at sufficiently high levels for assembly, annotation and differentiation across experimental conditions, and they demonstrate the interplay of root systems, environment and plant microbiomes. The genes assembled also included copper-dependent lytic polysaccharide monooxygenases. We detail the enzymes in the host grass RHZs and present a preliminary taxonomic microhabitat characterization. Our findings provide support for biologically mediated Fenton chemistry in the root zones of desert grasses, and provide insight into arid land carbon flow. These results also demonstrate a hyperdiverse microbial community. Both ribosomal RNA and messenger RNA sequences were dominated by bacteria, followed by fungal sequence abundance. Among the notable fungal sequences were those from the members of the arbuscular mycorrhizal fungi (Glomeromycota), which though abundant in this study, we rarely observed in previous PCR-based surveys.

Mycothermus thermophilus gen. et comb. nov., a new home for the itinerant thermophile Scytalidium thermophilum (Torula thermophila).

Thermophilic fungi have received substantial attention in industry for their potential to produce thermostable enzymes and as production platforms tolerant of high temperature. Studies exploring the ecology and biosystematics of thermophilic fungi have lagged behind studies in applied biology. The species commonly known as Scytalidium thermophilum (Chaetomiaceae) is one of the most frequently encountered organisms in surveys of thermophilic fungi. There is evidence that it is ecologically and economically important, for example in the context of commercial mushroom growing. As described here, this species should not be placed in the genus Scytalidium or any other existing genus. We propose a new genus and combination, Mycothermus thermophilus.

Thermophilic fungi in an aridland ecosystem.

We report a comprehensive multi-year study of thermophilic fungi at the Sevilleta National Wildlife Refuge in central New Mexico. Recovery of thermophilic fungi from soils showed seasonal fluctuations, with greater abundance correlating with spring and summer precipitation peaks. In addition to grassland soils, we obtained and characterized isolates from grassland and riparian litter, herbivore dung and biological soil crusts. All strains belonged to either the Eurotiales or Sordariales (Chaetomiaceae). No particular substrate or microhabitat associations were detected. Molecular typing of strains revealed substantial phylogenetic diversity, eight ad hoc phylogroups across the two orders were identified and genetic diversity was present within each phylogroup. Growth tests over a range of temperatures showed substantial variation in maximum growth rates among strains and across phylogroups but consistency within phylogroups. Results demonstrated that 45-50 C represents the optimal temperature for growth of most isolates, with a dramatic decline at 60 C. Most strains grew at 60 C, albeit slowly, whereas none grew at 65 C, providing empirical confirmation that 60 C presents an evolutionary threshold for fungal growth. Our results support the hypothesis that fungal thermophily is an adaptation to transient seasonal and diurnal high temperatures, rather than simply an adaptation to specialized high-temperature environments. We note that the diversity observed among strains and the frequently confused taxonomy within these groups highlight the need for comprehensive biosystematic revision of thermophilic taxa in both orders.