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Agrocybe chaxingu is a widely cultivated edible fungus in China, which is rich in nutrients and medicinal compounds. However, the lack of a high-quality genome hinders further research. In this study, we assembled the telomere-to-telomere genomes of two sexually compatible monokaryons (CchA and CchB) derived from a primarily cultivated strain AS-5. The genomes of CchA and CchB were 50.60 Mb and 51.66 Mb with contig N50 values of 3.95 Mb and 3.97 Mb, respectively. Each contained 13 complete chromosomes with telomeres at both ends. The high mapping rate, uniform genome coverage, high LAI score, all BUSCOs with 98.5%, and all base accuracy exceeding 99.999% indicated the high level of integrity and quality of these two assembled genomes. Comparison of the two genomes revealed that approximately 30% of the nucleotide sequences between homologous chromosomes were non-syntenic, including 19 translocations, 36 inversions, and 15 duplications. An additional gene CchA_000467 was identified at the Mat A locus of CchA, which was observed exclusively in the Cyclocybe cylindracea species complex. A total of 613 (4.26%) and 483 (3.4%) unique genes were identified in CchA and CchB, respectively, with over 80% of these being hypothetical proteins. Transcriptomic analysis revealed that the expression levels of unique genes in CchB were significantly higher than those in CchA, and both CchA and CchB had unique genes specifically expressed at stages of mycelium and fruiting body. It was indicated that the growth and development of the A. chaxingu strain AS-5 required the coordinated action of two different nuclei, with CchB potentially playing a more significant role. These findings contributed to a more profound comprehension of the growth and developmental processes of basidiomycetes. [ABSTRACT FROM AUTHOR]

Mushroom Jin Er has attracted widespread attention in Asia over the past two decades due to its medicinal properties and nutritional values. In the present study, Jin Er basidiocarps were often found to be surrounded by Stereum hirsutum fruiting bodies in their natural habitat and occasionally in artificial cultivation. The observation of two different kinds of mycelia within the hymenium and analyses of ITS sequences confirmed that Jin Er basidiocarps were composed of two fungal species, Naematelia aurantialba and S. hirsutum. This heterogeneity of Jin Er fruiting bodies is indeed distinct from the homogeneous hypha of Tremella fuciformis found in Yin Er mushroom, although its development also requires the presence of another fungus Annulohypoxylon stygium. Basidiospores can germinate on the surface of basidiocarps and produce mycelia. However, basidiospores in PDA medium can only bud into yeast-like conidia. The yeast-like conidia of N. aurantialba can transform into pseudohyphae with a change in temperature from 20 °C to 28 °C or switch into filamentous cells on an induction medium (IDM) at 20 °C, 25 °C and 28 °C. This dimorphic was reported for the first time in N. aurantialba. Haustorium-like structures were abundantly observed both within the hymenium and in the aerial mycelia cultured on the IDM. The developmental process was documented firstly in this study, involving the formation of protuberances with basal clamp connections, elongation at the protuberances, branch production, and eventual maturation. However, further observation is required to determine whether the haustorium-like structures can penetrate S. hirsutum hyphae. These findings are expected to provide valuable insights into the relationship and interaction between these two fungi, thereby advancing the cultivation of fruiting bodies. [ABSTRACT FROM AUTHOR]

Cordyceps militaris is one of the commercially cultivated mushrooms, valued for its medicinal and nutritional benefits. However, the fruiting body development mechanism has remained elusive. Chitin synthases (CHSs) are ubiquitous enzymes involved in the regulation of fungal growth, development and virulence. In this study, a total of eight CmChs genes were identified. Chromosomal localization analysis revealed an uneven distribution of CmCHSs across the C. militaris genome. Based on the phylogenetic analysis, 100 CHSs from Cordyceps sensu lato, encompassing C. militaris, were categorized into three divisions and seven classes, shedding light on their evolutionary relationships. There was no significant difference in the number of CHSs between ascomycetes and basidiomycetes in general (p = 0.067), as well as between pathogenic and saprotrophic fungi in general (p = 0.151 and 0.971 in Ascomycota and Basidiomycota fungi, respectively). This underscored the essential and conserved nature of these CHSs across various fungal lifestyles and ecological niches. The different transcript patterns of the eight CmChss during key life cycle stages, such as conidia germination, infection, and fruiting body development, indicated that each CHS gene may have a distinct role during specific stages of the life cycle. In conclusion, these findings indeed lay the groundwork for a further exploration of the functional roles of CHSs in the regulatory mechanism of fruiting body development in C. militaris. [ABSTRACT FROM AUTHOR]

Helvella bachu, a prized edible and medicinal fungus, is primarily found in the forests of Populus euphratica, an ancient and endangered species crucial to desert riparian ecosystems. Despite extensive efforts, the isolation of pure cultures and cultivation of fruiting bodies of H. bachu have remained elusive. While some species within the Helvella genus have been confirmed as ectomycorrhizal fungi, others have been considered either saprotrophic or mycorrhizal. By integrating field observations of H. bachu habitat, macro- and micro-anatomical examination of plant root tips, and molecular data from fruiting bodies, mycorrhizae, and host plants, it has been confirmed that H. bachu forms ectomycorrhizal associations with Populus trees. The mycorrhiza of H. bachu displays a light earth color with a curved smooth cylindrical shape. It features a thick mantle and the presence of a Hartig net, accompanied by a small amount of epitaxy mycelia. Morphological observation of the root tips requires meticulous handling, and the paraffin section technique has yielded noteworthy results. Host plants encompass four Populus species, including P. euphratica, P. pruinosa, P. nigra, and P. alba var. pyramidalis (synonym Populus bolleana). A conservation area was established within the young P. euphratica forest at Tarim University, resulting in a 14.75% increase in the quantity of fruiting bodies during the second year. Establishing a conservation area and in situ propagation of H. bachu holds economic and ecological implications. This study will contribute to the conservation of resources related to H. bachu and P. euphratica. [ABSTRACT FROM AUTHOR]

A deep understanding of the mechanism of fruiting body development is important for mushroom breeding and cultivation. Hydrophobins, small proteins exclusively secreted by fungi, have been proven to regulate the fruiting body development in many macro fungi. In this study, the hydrophobin gene Cmhyd4 was revealed to negatively regulate the fruiting body development in Cordyceps militaris, a famous edible and medicinal mushroom. Neither the overexpression nor the deletion of Cmhyd4 affected the mycelial growth rate, the hydrophobicity of the mycelia and conidia, or the conidial virulence on silkworm pupae. There was also no difference between the micromorphology of the hyphae and conidia in WT and ΔCmhyd4 strains observed by SEM. However, the ΔCmhyd4 strain showed thicker aerial mycelia in darkness and quicker growth rates under abiotic stress than the WT strain. The deletion of Cmhyd4 could promote conidia production and increase the contents of carotenoid and adenosine. The biological efficiency of the fruiting body was remarkably increased in the ΔCmhyd4 strain compared with the WT strain by improving the fruiting body density, not the height. It was indicated that Cmhyd4 played a negative role in fruiting body development. These results revealed that the diverse negative roles and regulatory effects of Cmhyd4 were totally different from those of Cmhyd1 in C. militaris and provided insights into the developmental regulatory mechanism of C. militaris and candidate genes for C. militaris strain breeding. [ABSTRACT FROM AUTHOR]

Light is necessary for primordium differentiation and fruiting body development for most edible fungi; however, light perception and signal transduction have only been well studied in model fungi. In this study, a hierarchical network of transcriptional response to light in Cordyceps militaris, one of the edible fungi, has been described on a genome-wide scale using dynamic transcriptome analysis. It was shown that light regulated the transcript of 1722 genes, making up 18% of the whole genome of C. militaris. Analysis of light-responsive genes in C. militaris identified 4 categories: immediate-early, early, late, and continuous light-responsive genes, and the gene number increased distinctly with prolonged light exposure. Light-responsive genes with distinct functional categories showed specific time-dependent regulation. The target genes of CmWC-1, the most important photoreceptor, were revealed by ChIP-seq. A total of 270 significant peaks corresponding to 427 genes were identified to be directly regulated by CmWC-1, among which 143 genes respond to light. Based on 270 ChIP-seq peaks, the binding site for CmWC-1 was identified as AAATCAGACCAC/GTGGTCTGATTT, differing from the binding site by the homolog in Neurospora crassa. Elucidating the mechanisms of light perception and signal transduction will be helpful for further research on the fruiting body development in edible fungi. [ABSTRACT FROM AUTHOR]

True morels (Morchella spp.) are edible mushrooms that are commercially important worldwide due to their rich nutrition and unique appearance. In recent years, outdoor cultivation has been achieved and expanded on a large scale in China. However, the mechanisms of fruiting body development in morels are poorly understood. In this study, the role of mating-type genes in fruiting body development was researched. Fruiting bodies cultivated with different mating-type strains showed no difference in appearance, but the ascus and ascospores were slightly malformed in fruiting bodies obtained from the MAT1-1 strains. The transcript levels of mating-type genes and their target genes revealed that the regulatory mechanisms were conserved in ascomycetes fungi. The silencing of mat1-2-1 by RNA interference verified the direct regulatory effect of mat1-2-1 on its target genes at the asexual stage. When cultivated with the spawn of single mating-type strains of MAT1-1 or MAT1-2, only one corresponding mating-type gene was detected in the mycelial and conidial samples, but both mat1-1-1 and mat1-2-1 were detected in the samples of primordium, pileus, and stipe. An understanding of the mating-type genes' role in fruiting body development in M. sextelata may help to understand the life cycle and facilitate artificial cultivation. [ABSTRACT FROM AUTHOR]

Chinese cordyceps, an entity of the Chinese caterpillar fungus (Ophiocordyceps sinensis, syn. Cordyceps sinensis) that parasitizes ghost moth larvae, is one of the best known traditional Chinese medicines and is found exclusively on the Tibetan Plateau with limited natural resources. Although the fungus O. sinensis can grow on artificial substrates and the ghost moth has been successfully reared, the large-scale artificial cultivation of Chinese cordyceps has only recently been accomplished after several decades of efforts and attempts. In this article, research progress related to this breakthrough from living habitats, the life history of the fungus, its host insect, fungal isolation and culture, host larvae rearing, infection cycle of the fungus to the host, primordium induction, and fruiting body development have been reviewed. An understanding of the basic biology of O. sinensis, its host insect and the simulation of the Tibetan alpine environment resulted in the success of artificial cultivation on a large scale. Practical workshop production has reached annual yields of 2.5, 5, and 10 tons in 2014, 2015, and 2016, respectively. There was no difference in the chemical components detected between the cultivated and natural Chinese cordyceps. However, the artificial cultivation system can be controlled to avoid heavy metal contamination and results in high-quality products. Although omics studies, including genomic, transcriptomic, proteomic, and metabolomic studies, have helped to understand the biology of the fungus, the success of the artificial cultivation of the Chinese cordyceps is clearly a milestone and provides the possibility for research on the in-depth mechanisms of the interaction between the fungus and host insects and their adaptation to the harsh habitats. This cultivation will not only result in a large industry to alleviate the pressure of human demand but also protect the limited natural resources for sustainable utilization. [ABSTRACT FROM AUTHOR]

Light is an essential factor for pigment formation and fruit body development in Cordyceps militaris, a well-known edible and medicinal fungus. Cmwc-1, a homolog of the blue-light receptor gene white collar-1 ( wc-1) in Neurospora crassa, was cloned from the C. militaris genome in our previous study. Here, Cmwc-1 gene inactivation results in thicker aerial hyphae, disordered fruit body development, a significant reduction in conidial formation, and carotenoid and cordycepin production. These characteristics were restored when the Δ Cmwc- 1 strains were hybridized with wild-type strains of the opposite mating type. A genome-wide expression analysis revealed that there were 1042 light-responsive genes in the wild-type strain and only 458 in the Δ Cmwc- 1 strain. Among five putative photoreceptors identified, Vivid, cryptochrome-1, and cyclobutane pyrimidine dimer photolyase are strongly induced by light in a Cmwc-1-dependent manner, while phytochrome and cryptochrome-2 were not induced. The transcription factors involved in the fungal light reaction were mainly of the ZnCys type. CmWC-1 regulates adenylosuccinate synthase, an important enzyme for adenosine de novo synthesis, which could explain the reduction in cordycepin production. Some G protein-coupled receptors that control fungal fruit body formation and the sexual cycle were regulated by CmWC-1, and the cAMP pathway involved in light signal transduction in N. crassa was not critical for the photoreaction in the fungus here. A transcriptional analysis indicated that steroid biosynthesis was more active in the Δ Cmwc-1 strain, suggesting that CmWC-1 might switch the vegetative growth state to primordia differentiation by suppressing the expression of related genes. [ABSTRACT FROM AUTHOR]

Cordyceps militaris (L.) Fr. Has long been recognized as a valuable functional food consumed in numerous countries. However, biosynthetic gene clusters of this species and safety regarding mycotoxin production remain largely unexplored. In this study, a ribosomally synthesized and post-translationally modified peptide (RiPP) cluster responsible for the production of cyclopeptide mycotoxins in Cordyceps was unveiled via genome mining. Ustiloxin B and a novel, predominant and Cordyceps specific ustiloxin I were confirmed by extraction and structural analysis. The difference between Ustiloxins I and B lied in the side chain at C19, where an additional methyl substituent in Ustiloxin I resulted in an alanine moiety substitution for glycine of Ustiloxin B. The simultaneous deletion of the two adjacent core genes, CmustYb and CmustYa , using a single guide RNA designed in the intergenic region, and subsequent in-situ complementation via AMA-mediated CRISPR/Cas9 system confirmed the RiPP cluster's responsibility for ustiloxin production. The cultivation of the edited strain yielded ustiloxin-free fruiting bodies without affecting agronomic characters. PCR and genome resequencing confirmed the absence of any off-target events or foreign sequence remnants. This study marks a significant advancement in utilizing CRISPR technology to control ustiloxins in food, underscoring its broader implications for food safety and quality improvement. [Display omitted] • Cordyceps militaris produce cyclopeptide mycotoxin, ustiloxins B and I. • Ustiloxin I was a new kind of ustiloxin, predominant and Cordyceps specific. • A RiPP was responsible for ustiloxin production confirmed by CRISPR/Cas9 editing. • Cultivation of the edited strain yielded ustiloxin-free fruiting bodies. • CRISPR-based technology is useful in improvement of food quality and safety. [ABSTRACT FROM AUTHOR]

Fungal sexual lives are considerably diversified in terms of the types of mating systems and mating-control gene functions. Sexual fruiting bodies of the ascomycete fungus Cordyceps militaris have been widely consumed as edible and medicinal mushrooms, whereas the regulation of fruiting-body development and sex in this fungus remain elusive. Herein, we performed the comprehensive functional analyses of mating-type (MAT) genes in C. militaris . Interspecies functional convergence was evident that MAT1-1 and MAT1-2-1 null mutants were sterile and lost the ability to produce stromata in outcrosses with the opposite mating-type partner. In contrast to other fungal species, functional divergence of MAT1-1-1 and MAT1-1-2 was also observed that Δ MAT1-1-1 produced barren stromata in outcrosses, whereas Δ MAT1-1-2 generated fruiting bodies morphologically similar to that of the parental strain but with sterile perithecia. The homothallic-like transformants MAT1-2:: MAT1-1-1 (haploidic MAT1-2 isolate transformed with the MAT1-1-1 gene) produced sterile stromata, whereas the MAT1-1:: MAT1-2-1 (haploidic MAT1-1 isolate transformed with the MAT1-2-1 gene) mutant was determined to be completely fruitless. The findings relating to the fully fertile gene-complementation mutants suggest that the genomic location is not essential for the MAT genes to fulfill their functions in C. militaris . Comparison of the production of bioactive constituents cordycepin and adenosine provides experimental support that the fungal sexual cycle is an energy consuming process. The results of the present study enrich our knowledge of both convergent and divergent controls of fungal sex. [ABSTRACT FROM AUTHOR]