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The increasing multi-drug resistance observed in the turfgrass pathogenClarireediaspp. has emerged as a critical issue. Understanding the mechanisms underlying fungicide resistance is crucial to address this challenge. This study focuses on comparing a highly propiconazole-resistant isolate ofClarireedia jacksonii, HRI11, with a sensitive isolate, HRS10. Genomes were sequenced using the Oxford Nanopore MinION sequencing platform, and hybrid assembly was performed using this data and existing Pacific Biosciences long reads and Illumina short reads. HRI11 genome assembly represents the most contiguous and complete genome assembly reported forClarireediato date, spanning 43.6 MB with 12,831 predicted protein-coding genes across 51 scaffolds. In contrast, the HRS10 had an assembly size of 39.6 MB and encoded 12,161 putative proteins distributed over 100 scaffolds. While the two isolates share substantial sequence similarity and overall protein content, the fungicide resistance observed in HRI11 appears to arise primarily from genetic variants, particularly in genes encoding transcription factors, transporters, and fungicide target genes. These genetic variants establish a foundational resistance level against fungicides. Furthermore, induced resistance in HRI11 involves increased expression of proteins that facilitate fungicide efflux, thereby optimizing energy allocation during fungicide exposures. Together, these mechanisms-inherent genetic variation and adaptive transcriptional responses-contribute to the heightened resilience of HRI11 under fungicide treatment. 

Abstract Traditional fermented foods often contain specialized microorganisms adapted to their unique environments. For example, the filamentous mold Aspergillus oryzae, used in saké fermentation, has evolved to thrive in starch-rich conditions compared to its wild ancestor, Aspergillus flavus. Similarly, Aspergillus sojae, used in soybean-based fermentations like miso and shochu, is hypothesized to have been domesticated from Aspergillus parasiticus. Here, we examined the effects of long-term A. sojae use in soybean fermentation on population structure, genome variation, and phenotypic traits. We analyzed 17 A. sojae and 24 A. parasiticus genomes (23 of which were sequenced for this study), alongside phenotypic traits of 9 isolates. Aspergillus sojae formed a distinct, low-diversity population, suggesting a recent clonal expansion. Interestingly, a population of A. parasiticus was more closely related to A. sojae than other A. parasiticus populations. Genome comparisons revealed loss-of-function mutations in A. sojae, notably in biosynthetic gene clusters encoding secondary metabolites, including the aflatoxin cluster. Interestingly though, A. sojae harbored a partial duplication of a siderophore biosynthetic cluster. Phenotypic assays showed A. sojae lacked aflatoxin production, while it was variable in A. parasiticus isolates. Additionally, certain A. sojae strains exhibited larger colony diameters under miso-like salt conditions. These findings support the hypothesis that A. parasiticus is the progenitor of A. sojae and that domestication significantly reduced genetic diversity. Future research should explore how wild and food-associated strains influence sensory attributes and microbial community dynamics in fermented soy products. 

ABSTRACT Modern taxonomic classification is often based on phylogenetic analyses of a few molecular markers, although single-gene studies are still common. Here, we leverage genome-scale molecular phylogenetics (phylogenomics) of species and populations to reconstruct evolutionary relationships in a dense data set of 710 fungal genomes from the biomedically and technologically important genusAspergillus. To do so, we generated a novel set of 1,362 high-quality molecular markers specific forAspergillusand provided profile Hidden Markov Models for each, facilitating their use by others. Examining the resulting phylogeny helped resolve ongoing taxonomic controversies, identified new ones, and revealed extensive strain misidentification (7.59% of strains were previously misidentified), underscoring the importance of population-level sampling in species classification. These findings were corroborated using the current standard, taxonomically informative loci. These findings suggest that phylogenomics of species and populations can facilitate accurate taxonomic classifications and reconstructions of the Tree of Life.IMPORTANCEIdentification of fungal species relies on the use of molecular markers. Advances in genomic technologies have made it possible to sequence the genome of any fungal strain, making it possible to use genomic data for the accurate assignment of strains to fungal species (and for the discovery of new ones). We examined the usefulness and current limitations of genomic data using a large data set of 710 publicly available genomes from multiple strains and species of the biomedically, agriculturally, and industrially important genusAspergillus. Our evolutionary genomic analyses revealed that nearly 8% of publicly availableAspergillusgenomes are misidentified. Our work highlights the usefulness of genomic data for fungal systematic biology and suggests that systematic genome sequencing of multiple strains, including reference strains (e.g., type strains), of fungal species will be required to reduce misidentification errors in public databases. 

Microbes (bacteria, yeasts, molds), in addition to plants and animals, were domesticated for their roles in food preservation, nutrition and flavor. Aspergillus oryzae is a domesticated filamentous fungal species traditionally used during fermentation of Asian foods and beverage, such as sake, soy sauce, and miso. To date, little is known about the extent of genome and phenotypic variation of A. oryzae isolates from different clades. Here, we used long-read Oxford Nanopore and short-read Illumina sequencing to produce a highly accurate and contiguous genome assemble of A. oryzae 14160, an industrial strain from China. To understand the relationship of this isolate, we performed phylogenetic analysis with 90 A. oryzae isolates and 1 isolate of the A. oryzae progenitor, Aspergillus flavus . This analysis showed that A. oryzae 14160 is a member of clade A, in comparison to the RIB 40 type strain, which is a member of clade F. To explore genome variation between isolates from distinct A. oryzae clades, we compared the A. oryzae 14160 genome with the complete RIB 40 genome. Our results provide evidence of independent evolution of the alpha-amylase gene duplication, which is one of the major adaptive mutations resulting from domestication. Synteny analysis revealed that both genomes have three copies of the alpha-amylase gene, but only one copy on chromosome 2 was conserved. While the RIB 40 genome had additional copies of the alpha-amylase gene on chromosomes III, and V, 14160 had a second copy on chromosome II and an third copy on chromosome VI. Additionally, we identified hundreds of lineage specific genes, and putative high impact mutations in genes involved in secondary metabolism, including several of the core biosynthetic genes. Finally, to examine the functional effects of genome variation between strains, we measured amylase activity, proteolytic activity, and growth rate on several different substrates. RIB 40 produced significantly higher levels of amylase compared to 14160 when grown on rice and starch. Accordingly, RIB 40 grew faster on rice, while 14160 grew faster on soy. Taken together, our analyses reveal substantial genome and phenotypic variation within A. oryzae . 

Lentinulais a broadly distributed group of fungi that contains the cultivated shiitake mushroom,L. edodes. We sequenced 24 genomes representing eight described species and several unnamed lineages ofLentinulafrom 15 countries on four continents.Lentinulacomprises four major clades that arose in the Oligocene, three in the Americas and one in Asia–Australasia. To expand sampling of shiitake mushrooms, we assembled 60 genomes ofL. edodesfrom China that were previously published as raw Illumina reads and added them to our dataset.Lentinula edodessensu lato (s. lat.) contains three lineages that may warrant recognition as species, one including a single isolate from Nepal that is the sister group to the rest ofL. edodess. lat., a second with 20 cultivars and 12 wild isolates from China, Japan, Korea, and the Russian Far East, and a third with 28 wild isolates from China, Thailand, and Vietnam. Two additional lineages in China have arisen by hybridization among the second and third groups. Genes encoding cysteine sulfoxide lyase (lecsl) and γ-glutamyl transpeptidase (leggt), which are implicated in biosynthesis of the organosulfur flavor compound lenthionine, have diversified inLentinula. Paralogs of both genes that are unique toLentinula(lecsl3 andleggt5b) are coordinately up-regulated in fruiting bodies ofL. edodes. The pangenome ofL. edodess. lat. contains 20,308 groups of orthologous genes, but only 6,438 orthogroups (32%) are shared among all strains, whereas 3,444 orthogroups (17%) are found only in wild populations, which should be targeted for conservation.