Soybean cyst nematode (SCN; Heterodera glycines) is recognized as the most destructive soybean pathogen. Biological control of SCN using fungi associated with cyst nematodes (Heteroderidae) could be a promising strategy. In this study, we screened 75 fungal isolates representing 55 species associated with different cyst nematodes to assess antagonistic effects on SCN. A rapid 24-well plate in vitro assay was used to evaluate fungal cell-free filtrates for their effect on SCN egg viability and hatch. Filtrates of 14 isolates significantly reduced egg viability and hatch by inducing "vacuole-like" structural aberrations in SCN eggs. The 14 fungal isolates were further tested for their effect on SCN second-stage juvenile (J2) viability and direct egg parasitism. Filtrates of Alternaria tenuissima, Fusarium solani, Purpureocillium lilacinum, Debaryomyces hansenii, Plectosphaerella cucumerina, Arthopyrenia salicis, and Cosmospora lavitskiae resulted in the most significant reduction in egg viability. Filtrates of C. lavitskiae, D. hansenii, F. acaciae-mearnsii, and F. solani were the most toxic to SCN J2s in the mortality assay. Trichoderma virens, Alternaria tenuissima, F. acaciae-mearnsii, and Pochonia chlamydosporia had the highest degree of SCN egg parasitism. Based on in vitro assays, 10 promising fungal isolates were selected for evaluation in a soil-based experiment. Trichoderma virens, Purpureocillium lilacinum, and Pochonia chlamydosporia significantly reduced SCN cyst and egg densities, and Talaromyces ramulosus, Plectosphaerella cucumerina, and C. lavitskiae provided moderate SCN suppression. These findings highlight the potential of nematophagous fungi isolated from cyst nematodes for SCN management and offer a screening approach for identifying potent fungal biocontrol agents.

Ustilago esculenta is a smut fungus that parasitizes Zizania latifolia, forming edible fungal galls known as Makomotake. In this study, we quantified indole-3-acetic acid (IAA) produced in liquid-cultured U. esculenta and in fungal galls at different growth stages. IAA was detected in both samples, and its content showed a tendency to increase during the mature growth stage in October. Furthermore, RNA-seq analysis was performed to investigate gene expression changes in U. esculenta during different developmental stages. The gene involved in spore maturation was upregulated in October, consistent with fungal maturation. Moreover, numerous genes with unknown functions were significantly upregulated, highlighting potential targets for further investigation. These findings provide insights into the molecular mechanisms underlying fungal gall development and the symbiotic relationship between U. esculenta and Z. latifolia, providing a foundation for future research on fungal-plant interactions.

Species of Thelephora Ehrh. ex Willd. (Thelephorales, Basidiomycota) are known from all continents where ectomycorrhizal (ECM) host plants occur. Although often poorly represented in sporocarp surveys due to the cryptic basidiomata of the resupinate species, belowground sequencing studies in both temperate and tropical systems have shown that thelephoroid fungi are often the dominant ECM group. In the South American lowland tropics, thelephoroid fungi remain poorly known, and very few species have been described from the region. Long-term surveys in Guyana have revealed a wide diversity of ECM fungal species in association with the ECM trees Dicymbe corymbosa (Fabaceae subfam. Detarioideae) and Aldina insignis (Benth.) Endl. (Fabaceae subfam. Papilionoideae). Thelephoroid species form a prominent component of this ECM fungal assemblage, as evidenced by their dominance in both adult tree and seedling root tip surveys and frequent occurrence as fertile basidiomata on a variety of substrata. Here we describe four new thelephoroid species from Guyana that are among the most frequently collected as basidiomata: Thelephora ascendens, Thelephora compacta, Thelephora singularis, and Thelephora ventricobasidia. Three of these species have resupinate basidiomata and would have formerly been assigned to the genus Tomentella Pers. ex Pat. Morphological descriptions, habit, habitat, and known distribution are provided for each new species. Sequence data for the barcode internal transcribed spacer (ITS) locus is provided for types and most other collections of the new species, and a molecular phylogenetic analysis across the Thelephoraceae corroborates their status as novel taxa.

Asian soybean rust (ASR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, was first reported in the continental United States of America (USA) in 2004 and over the years has been of concern to soybean production in the United States. The prevailing hypothesis is that P. pachyrhizi spores were introduced into the United States via hurricanes originating from South America, particularly hurricane Ivan. To investigate the genetic diversity and global population structure of P. pachyrhizi, we employed exome-capture based sequencing on 84 field isolates collected from different geographic regions worldwide. We compared the gene-encoding regions from all these field isolates and found that four major mitochondrial haplotypes are prevalent worldwide. Here, we provide genetic evidence supporting multiple incursions that have led to the currently established P. pachyrhizi population of the United States. Phylogenetic analysis of mitochondrial genes further supports this hypothesis. We observed limited genetic diversity in P. pachyrhizi populations across different geographic regions, suggesting a clonal population structure. Additionally, this study is the first to report the F129L mutation in the Cytb gene outside South America, which is associated with strobilurin tolerance. This study provides the first comprehensive characterisation of P. pachyrhizi population structures defined by genetic evidence from populations across major soybean-growing regions.

Rust fungi (Pucciniales) comprise a large group of ecologically and economically important plant pathogens distributed globally where their hosts grow. The first published study of rusts in Indiana was carried out 131 years ago, and a revised checklist of all Pucciniales for Indiana has not been compiled since the works of Jackson from 1917 to 1920. Efforts to compile a checklist five years ago revealed a dire need for revision due to taxonomic and nomenclatural changes. We examined historical records, including online databases and literature, as well as new and historical collections in the Arthur Fungarium (PUR) at Purdue University. We provide an annotated checklist of all known species of rust fungi occurring in 90/92 Indiana counties. Names in prior records have been updated to reflect the current classifications for this group, with cross-reference to older names. A total of 301 Pucciniales taxa across 32 genera and their host species have been verified. This work serves as a resource for statewide plant disease diagnosticians and records the extant rust fungal biodiversity in Indiana to guide research efforts, resource management, and conservation.

Nucleosomes are the basic repeating unit, each spanning ≈150bp, that structures DNA in the nucleus and their positions have major consequences on gene activity. Here, through analyzing DNA signatures across 1117 microeukaryote genomes, we discovered ≈150bp shifts in A/T content associated with nucleosome organization. Often consecutively arrayed across the genome, A/T peaks were enriched surrounding transcriptional start sites in specific clades. Most nucleosomes (both in vitro and in vivo) across eukaryotes aligned with A/T peaks, even in the presence of DNA modifications. Using artificial intelligence-based approaches, we describe DNA features associated with nucleosomes and construct a deep learning (DL) model for improved nucleosome occupancy prediction. Using this model, we found that ≈70% of "random" transfer DNA inserts from an in vivo fungal RB-TDNAseq library avoided DL predicted nucleosome-bound regions. This study reveals a eukaryote-wide strategy for generating cassettes of nucleosome-favorable DNAs that has a profound impact on nucleosome organization.

Moniliophthora perniciosa C-biotype is the causal agent of witches’ broom disease of cacao (WBD), one of the most important diseases of the crop in Peru. Several questions remain unanswered about this disease in the country, such as the incidence levels, the morphological and genetic diversity, and the dissemination routes of the pathogen. We evaluated fifty isolates from districts located in areas of “Difficult Access” deep in the Amazon, and of “Easy Access” close to urban areas on the Western side of the Amazon. We found higher incidence levels of WBD in districts with historical records of high humidity and precipitation. We also found significant differences in the radial growth of isolates and documented nine mycelial colony growth groups with a varying presence of clamp connections. The microsatellite marker genotyping revealed the highest indexes of diversity in areas of “Difficult Access” and identified these Amazonian areas harbored the ancestral population of M. perniciosa from which the pathogen most likely disseminated towards the “Easy Access” areas. This study lays the foundation for future studies to fully understand the morphological and genetic diversity, and epidemiology of M. perniciosa in the northern Peruvian Amazon.

Trifolium repens, commonly known as white clover, is an herbaceous perennial plant within Fabaceae that is a pasture legume in temperate regions. In September 2023, a rust was observed on T. repens in Iceland. Morphological characteristics and sequencing analysis identified the rust species as Uromyces trifolii-repentis. To our knowledge, this is the first report of U. trifolii-repentis in Iceland.

AbstractThe fungusFusarium xyrophilumproduces flower-like structures (i.e., pseudoflowers) that were recently discovered on yellow-eyed grasses (Xyrisspp.) in Guyana. It is unknown whether these pseudoflowers, which are composed entirely of fungal tissue, are true mimics that attract insects as a means of fungal dispersal. We evaluated the potential ofF. xyrophilumto affect insect visitation patterns to flowers and pseudoflowers by 1) documenting insect visitation toX. surinamensisin Guyana, 2) measuring the presence ofF. xyrophilumDNA on insects, and 3) evaluating fluorescence and volatile production on flowers and pseudoflowers. We report for the first time Vespidae, Formicidae, Salticidae, Acrididae, and Tetrigidae visitingXyris. Diverse insects, including Conocephalini spp. (meadow katydids; Tettigoniidae),Camponotusspp. (carpenter ants; Formicidae), and a Geometridae sp. (geometer moths) were found to visit flowers and pseudoflowers.Fusarium xyrophilumDNA was detected on 3/12 (25%) of captured insect bodies using conventional and quantitative PCR. Volatiles produced in the field by pseudoflowers and flowers were similar, except for the presence of a sesquiterpene, putatively identified here as α-gurjunene, which was detected both inF. xyrophilumpure cultures and field-collected pseudoflower samples, but not from flowers. The production of this sesquiterpene byF. xyrophilumand the fluorescence ofX. surinamensispeduncles represent potential signals involved in insect attraction for this system. These observations, along with the overlap in insect visitors of flowers and pseudoflowers and the detection ofF. xyrophilumDNA on insect bodies, are consistent with insect visitors being vectors ofXyrispollen andF. xyrophilumpropagules between host plants.

Species of the ectomycorrhizal (ECM) family Cortinariaceae (Agaricales, Agaricomycetes, Basidiomycota) have long been considered impoverished or absent from lowland tropical rainforests. Several decades of collecting in forests dominated by ECM trees in South America's Guiana Shield is countering this view, with discovery of numerous Cortinariaceae species. To date, ~12 morphospecies of this family have been found in the central Pakaraima Mountains of Guyana. Here, we describe three of these as new species of Cortinarius and two as new species of Phlegmacium from forests dominated by the ECM tree genera Dicymbe (Fabaceae subfam. Detarioideae), Aldina (Fabaceae subfam. Papilionoideae), and Pakaraimaea (Cistaceae). Macromorphological, micromorphological, habitat, and DNA sequence data are provided for each new species.

Chlorosplenium is a small genus comprising five species of inoperculate discomycetes in the order Helotiales (Leotiomycetes) often recognizable by their bright yellowish-green colors and gregarious growth on wood. In this study, we describe five new species-C. aotearoa, C. australiense, C. cusucoense, C. epimorsicum, and C. hawaiiense-based on a combination of recent fieldwork and examination of previously collected fungarium specimens. We use an integrative taxonomic approach to support the distinction of new species, incorporating morphology and DNA sequence data with biogeography. Macro- and micromorphological features of apothecia for all species and culture characteristics for four of the five new species are documented. A multilocus phylogeny based on nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2, partial large subunit nuc ribosomal DNA (28S nuc rDNA), and A-B regions of the largest subunit of RNA polymerase II (RPB1) gene is presented. Additionally, we report Chlorosplenium chlora from Europe for the first time and expand our knowledge of the diversity and distributions of species in this genus in America, Australia, and New Zealand.

Fungi are arguably the most diverse eukaryotic kingdom of organisms in terms of number of estimated species, trophic and life history strategies, and their functions in ecosystems. However, our knowledge of fungi is limited due to a distributional bias; the vast majority of available data on fungi have been compiled from non-tropical regions. Far less is known about fungi from tropical regions, with the bulk of these data being temporally limited surveys for fungal species diversity. Long-term studies (LTS), or repeated sampling from the same region over extended periods, are necessary to fully capture the extent of species diversity in a region, but LTS of fungi from tropical regions are almost non-existent. In this paper, we discuss the contributions of LTS of fungi in tropical regions to alpha diversity, ecological and functional diversity, biogeography, hypothesis testing, and conservation—with an emphasis on an ongoing tropical LTS in the Pakaraima Mountains of Guyana. We show how these contributions refine our understanding of Fungi. We also show that public data repositories such as NCBI, IUCN, and iNaturalist contain less information on tropical fungi compared to non-tropical fungi, and that these discrepancies are more pronounced in fungi than in plants and animals.

Multicopy nuclear ribosomal (rDNA) genes have been used as markers for fungal identification for three decades. The rDNA sequences in a genome are thought to be homogeneous due to concerted evolution. However, intragenomic variation of rDNA sequences has recently been observed in many fungi, which cause problems in fungal identification and species abundance estimation. Various sequence-based methods have been used to demonstrate rDNA sequence heterogeneity, but there is no technical assessment of the comparability of results from these methods. In this article, we sampled smut fungi representing all major lineages of subphylum Ustilaginomycotina as a system to examine sequence heterogeneity in the rDNA repeats. Three methods were used: PCR-cloning-Sanger sequencing, targeted amplicon high-throughput sequencing, and WGS high-throughput sequencing. Based on our analyses, Ceraceosorus is the only sampled fungal genus in Ustilaginomycotina showing intragenomic variation, with up to 27 nucleotide variant sites in the ITS1–5.8S–ITS2 region and 2.6% divergence among analyzed ITS haplotypes. We found many conflicting patterns across the three detection methods, with up to 28 conflicting variant sites in one sample. Surprisingly, at least 40% of these conflicts are due to PCR-cloning-sequencing errors, as the corresponding variant sites were not observed in the other methods. Based on our data and the literature, we evaluated the characteristics and advantages/disadvantages of each detection method. A model for how intragenomic variation may arise in the rDNA region is presented. Finally, we describe the fourth known species of Ceraceosorus , C. americanus , isolated from an asymptomatic rosemary leaf collected in Louisiana, USA. We anticipate that our study will provide a framework for future research in rDNA regions as well as other similar multicopy genes.Author Summary Ribosomal DNA (rDNA) genes are one of the most ancient multicopy genes in cellular organisms. They function as a part of the protein synthesis machinery in a cell. The rDNA sequences have also been used in species identification and microbial community profiling. Despite these utilities, little is known how the rDNA genes have evolved. Biologists initially thought the sequences among rDNA copies are homogeneous, but many recent cases illustrated rDNA sequence heterogeneity. In this article, we utilized the fungal genus Ceraceosorus together with allied smut fungi as a system to study sequence heterogeneity in the rDNA genes using various detection methods. Our system found rDNA sequence homogeneity as a common form, while sequence heterogeneity is taxon-specific. Based on our data and literature review, we explained possible sources for sequence heterogeneity in the rDNA genes. Our study also noticed result discrepancies across variant detection methods. These include artefactual variants from the PCR-cloning-sequencing method, inconsistent detected variants from the independent runs of high-throughput sequencing, and technical errors in bioinformatic analyses. We therefore emphasize the importance of methodological choices which have different pros and cons for studying intragenomic variation of rDNA genes, as well as other multicopy gene families.### Competing Interest StatementThe authors have declared no competing interest.

The porcini mushroom family Boletaceae is a diverse, widespread group of ectomycorrhizal (ECM) mushroom-forming fungi that so far has eluded intrafamilial phylogenetic resolution based on morphology and multilocus data sets. In this study, we present a genome-wide molecular data set of 1764 single-copy gene families from a global sampling of 418 Boletaceae specimens. The resulting phylogenetic analysis has strong statistical support for most branches of the tree, including the first statistically robust backbone. The enigmatic Phylloboletellus chloephorus from non-ECM Argentinian subtropical forests was recovered as a new subfamily sister to the core Boletaceae. Time-calibrated branch lengths estimate that the family first arose in the early to mid-Cretaceous and underwent a rapid radiation in the Eocene, possibly when the ECM nutritional mode arose with the emergence and diversification of ECM angiosperms. Biogeographic reconstructions reveal a complex history of vicariance and episodic long-distance dispersal correlated with historical geologic events, including Gondwanan origins and inferred vicariance associated with its disarticulation. Together, this study represents the most comprehensively sampled, data-rich molecular phylogeny of the Boletaceae to date, establishing a foundation for future robust inferences of biogeography in the group.

As an agroforestry resource willow species (Salix spp.) becoming increasingly important in the protection of riverbanks, recovery of floodable lands, soil bioremediation, biomass production, and wicker furniture manufacture, Babylon willow (Salix babylonica) is widely planted all over the world and is found throughout Iran as part of the rural landscape and is mostly planted for ornamental or bioremediation purposes. Rust fungi in the genus Melampsora usually cause disease on Salix species. Identification of Melampsora species is often complicated because there are few differences in spore morphology and little is publicly available in comparative sequence data. Willow trees in Iran have been reported to be infected by 15 Melampsora species; however, most of these records are based on morphological characterization. During surveillance activities carried out in June 2007, a rust species was detected on isolated Babylon willow trees in an area in Northern Iran. Both the rust fungus and the host plant were identified on the basis of morphological features and DNA sequencing. Based on a combination of morphology, large subunit (LSU) rDNA sequencing and phylogenetic analyses, our rust specimen was identified as Melampsora ferrinii, which is newly reported from Iran on Salix babylonica. Melampsora ferrinii was originally described from the Americas and this is the first time that this rust has been observed on its telial host, Babylon willow, in Asia. The documentation of the rust fungus was performed using both scanning electron microscopy (SEM) and light microscopy. Additionally, an identification key for Melampsora species reported on Salix spp. in Iran is included.

Multicopy nuclear ribosomal DNA (rDNA) genes have been used as markers for fungal identification for three decades. The rDNA sequences in a genome are thought to be homogeneous due to concerted evolution. However, intragenomic variation of rDNA sequences has recently been observed in many fungi, which may make fungal identification and species abundance estimation based on these loci problematic. Ceraceosorus is an enigmatic genus in the smut lineage Ustilaginomycotina for which very limited distribution data exist. Our previous research demonstrated intragenomic variation in the internal transcribed spacer (ITS1-5.8S-ITS2) region of two Ceraceosorus species. In this study, we described the fourth known species of Ceraceosorus, C. americanus, isolated from an asymptomatic rosemary leaf collected in Louisiana, USA. This is the first report of this genus in the Americas. We then selected all four known Ceraceosorus species, plus exemplar smut fungi representing all major lineages of subphylum Ustilaginomycotina, to examine sequence heterogeneity in three regions of the rDNA repeat (partial 18S, ITS, and partial 28S regions). Three methods were used: PCR-cloning-Sanger sequencing, targeted amplicon high-throughput sequencing, and whole-genome shotgun high-throughput sequencing. Our results show that Ceraceosorus is the only sampled fungal genus in Ustilaginomycotina with significant intragenomic variation at the ITS, with up to 25 nucleotide variant sites in the ITS1-5.8S-ITS2 region and 2.6% divergence among analyzed ITS haplotypes. We found many conflicting patterns across the three detection methods, with up to 27 conflicting variant sites recorded from a single individual. At least 40% of the conflicting patterns are possibly due to PCR-cloning-sequencing errors, as the corresponding variant sites were not observed in the other detection methods. Based on our data and the literature, we evaluated the characteristics and advantages/disadvantages of each detection method. Finally, a model for how intragenomic variation in the rDNA copies within a genome may arise is presented.

Plant-parasitic nematodes are important pathogens of agricultural crops. Of particular importance are cyst nematode species of Globodera and Heterodera, which remain a major constraint to global production of crops such as potato, soybean, and wheat. Recent restrictions on the use of some synthetic nematicides has created an urgent need for alternative methods of controlling cyst nematodes. This project aimed to (i) document fungi found associated with cyst nematodes, (ii) determine if the fungi associated with cyst nematodes are dependent on growth conditions or nematode species, (iii) identify core mycobiomes for each of the eight cyst nematode species, (iv) identify the core mycobiome across all cyst nematode species studied, and (v) determine if the fungi associated with Globodera species are structured geographically. Sixty cyst nematode populations from eight species-Globodera sp., G. ellingtonae, G. pallida, G. rostochiensis, H. avenae, H. carotae, H. glycines, and H. schachtii-were collected from several geographic locations globally and screened for fungi using both culture-dependent and -independent methods. The core mycobiome was determined for each species. In total, 744 species of fungi were found to be associated with cyst nematodes. Fungal species shared across all cyst nematode species included Pochonia sp., Sporothrix sp., Neocosmospora solani, Exophiala equina, Mortierella sp., and Sarocladium kiliense. The core mycobiomes were conserved between cyst nematode species irrespective of their geographic origin. Cyst nematodes collected from the field or from greenhouse cultures also recruited similar core mycobiomes. These findings could be relevant when choosing fungi to evaluate as biocontrol agents against cyst nematodes.

Researchers employ high-throughput sequencing technologies to characterize mycobiome community composition and structure. However, the short sequences generated by these technologies can be limiting and may result in an inability to identify species at lower ranks, and to exclude taxa due to primer bias. Consequently, we introduce a novel protocol that (1) greatly decreases the number of unclassified sequences by providing three methods for determining the identity of each ASV or OTU and (2) sequences both dikaryon and early-diverging fungal lineages. This new protocol was tested specifically on the mycobiome of rock face colonizers. Samples were collected from two adjacent rock faces of varying colonization ages and subjected to DNA extraction, PCR, and library preparation, followed by Illumina MiSeq sequencing. Results consisted of 4885 ASVs of which were further reduced to 2987 operational taxonomic units (97

Rust fungi (Pucciniales) are plant pathogens that can cause devastating yield losses to economically important crops and threaten native plants with extinction. Rusts are usually controlled with fungicides when rust-resistant plant varieties are unavailable. However, natural enemies may offer an alternative to chemicals by acting as biological controls. The larvae of Mycodiplosis Rübsaamen (49 spp.) feed on the spores of rusts and powdery mildew fungi and have been suggested as a potential biocontrol candidate for disease-causing rusts. However, little is known about the phylogenetic relationships, biogeography, and host range of this genus. We screened 5,665 rust specimens from fungarium specimens and field collections and recovered a total of 363 larvae on 315 rust specimens from 17 countries. Three mitochondrial and 2 nuclear loci were amplified and sequenced for the phylogenetic reconstruction of 129 individuals. We recovered 12 clades, of which 12 and 10 were supported with maximum likelihood and Bayesian inference, respectively. Of the 12 clades, 7 comprised species from multiple continents and climatic regions, and 5 comprised species from a single region. Individuals forming clades were collected from 2 to 18 rust species, suggesting that Mycodiplosis species have a broad host range. In total, Mycodiplosis larvae were identified on 44 different rust species collected from 18 plant families. Future studies should focus on expanding field sampling efforts, including data from additional gene regions, and incorporating morphological data to further elucidate species diversity and distribution patterns.

South Africa has an indigenous rust (Pucciniales) funga of approximately 460 species. This funga was sampled with species from as many genera as possible. The nuclear ribosomal large subunit (28S) region was amplified from samples representing 110 indigenous species, as well as the small subunit (18S) region and the cytochrome c oxidase subunit 3 (CO3) in some cases, and these were used in phylogenetic analyses. One new species is described, 12 new combinations made, six names reinstated, and two life history connections made. The life histories of this funga were summarized; it is dominated by species with contracted life histories. The majority of species are autoecious, with a small proportion being heteroecious. Of the autoecious species, many will likely be homothallic with no spermagonia. A shortened life history with homothallism allows for a single basidiospore infection to initiate a local population buildup under the prevailing unpredictable climatic conditions. Suggestions are made as to the possible origin of this funga based on the development of the modern South African flora. It is postulated that the rusts of South Africa are of relatively recent origin, consisting of three groups. Firstly, there is an African tropical element with members of the Mikronegerineae (Hemileia), the Sphaerophragmiaceae (Puccorchidium, Sphaerophragmium), and certain Uredinineae (Stomatisora). Their immediate ancestors likely occurred in the tropical forests of Africa during the Paleogene. Secondly, there is a pantropical element including the Raveneliaceae (e.g., Diorchidium, Maravalia, Ravenelia sensu lato, Uropyxis). This likely diversified during the Neogene, when the mimosoids became the dominant trees of the developing savannas. Thirdly, the Pucciniaceae invaded Africa as this continent pushed northward closing the Tethys Sea. They diversified with the development of the savannas as these become the dominant habitat in most of Africa, and are by far the largest component of the South African rust funga.