Biological characteristics of Cordyceps militaris single mating-type strains.

Cordyceps militaris has been extensively cultivated as a model cordyceps species for commercial purposes. Nevertheless, the problems related to strain degeneration and breeding technologies remain unresolved. This study assessed the physiology and fertility traits of six C. militaris strains with distinct origins and characteristics, focusing on single mating-type strains. The results demonstrated that the three identified strains (CMDB01, CMSY01, and CMJB02) were single mating-type possessing only one mating-type gene (MAT1-1). In contrast, the other three strains (CMXF07, CMXF09, and CMMS05) were the dual mating type. The MAT1-1 strains sourced from CMDB01, CMSY01, and CMJB02 consistently produced sporocarps but failed to generate ascospores. However, when paired with MAT1-2 strains, the MAT1-1 strains with slender fruiting bodies and normal morphology were fertile. The hyphal growth rate of single mating-type strains (CMDB01, CMSY01, and CMJB02) typically surpassed that of dual mating-type strains (CMXF07, CMXF09, and CMMS05). The growth rates of MAT1-2 and MAT1-1 strains were proportional to their ratios, such that a single mating-type strain with a higher ratio exhibited an increased growth rate. As C. militaris matured, the adenosine content decreased. In summary, the C. militaris strains that consistently produce sporocarps and have a single mating type are highly promising for production and breeding.

Selection and validation of reference genes for RT-qPCR in ophiocordyceps sinensis under different experimental conditions.

The Chinese caterpillar mushroom, Ophiocordyceps sinensis (O. sinensis), is a rarely medicinal fungus in traditional chinese herbal medicine due to its unique medicinal values, and the expression stability of reference genes is essential to normalize its gene expression analysis. In this study, BestKeeper, NormFinder and geNorm, three authoritative statistical arithmetics, were applied to evaluate the expression stability of sixteen candidate reference genes (CRGs) in O. sinensis under different stress [low temperature (4°C), light treatment (300 lx), NaCl (3.8%)] and different development stages (mycelia, primordia and fruit bodies) and formation of morphologic mycelium (aeriasubstrate, hyphae knot mycelium). The paired variation values indicated that two genes could be enough to accurate standardization exposed to different conditions of O.sinensis. Among these sixteen CRGs, 18S ribosomal RNA (18S rRNA) and beta-Tubulin (ß-TUB) showed the topmost expression stability in O.sinensis exposed to all conditions, while glutathione hydrolase proenzym (GGT) and Phosphoglucose isomerase (PGI) showed the least expression stability. The optimal reference gene in different conditions was various. ß-TUB and Ubiquitin (UBQ) were identified as the two most stable genes in different primordia developmental stage, while phosphoglucomutase (PGM) with elongation factor 1-alpha (EF1-α) and 18S rRNA with UBQ were the most stably expressed for differentially morphologic mycelium stages and different stresses, respectively. These results will contribute to more accurate evaluation of the gene relative expression levels in O.sinensis under different conditions using the optimal reference gene in real-time quantitative PCR (RT-qPCR) analysis.

First report of emerging fungal pathogens of Cordyceps militaris in Vietnam.

Cultivation of Cordyceps militaris, a valuable medicinal and edible fungus, has dramatically increased in Vietnam since 2010. During industrial production, parasitic white molds were found to infect the mycelia and fruiting bodies of C. militaris causing significant quality and yield losses. Two different fungal strains were obtained from the mycelia and fruiting bodies of C. militaris in Danang mushroom farms and were characterized by morphological and multiple DNA markers analysis. The sequence alignment of ITS, LSU and rpb2 markers revealed that the pathogens are related to the type species Lecanicillium coprophilum and Calcarisporium cordycipiticola with more than 99% sequence identities. The growth characteristics and pathogenic activities of the two isolated species on their host C. militaris were also investigated. The phylogenetic analysis based on the ITS sequences showed that L. coprophilum WF2611 is closer to its host C. militaris than C. cordycipiticola NT1504. To our knowledge, this is the first worldwide report of C. militaris infected by L. coprophilum which would be an useful information on prevention and control of the disease and be helpful for the industrial cultivation of C. militaris.

Safe-Harbor-Targeted CRISPR/Cas9 System and Cmhyd1 Overexpression Enhances Disease Resistance in Cordyceps militaris.

Fungal disease of mushroomCordyceps militaris (CM) caused byCalcarisporium cordycipiticola (CC) is destructive to fruiting body cultivation, resulting in significant economic loss and potential food safety risks. CRISPR/Cas9 genome editing has proven to be a powerful tool for crop improvement but seldom succeeded in mushrooms. Here, the first genomic safe-harbor site, CmSH1 locus, was identified in the CM genome. A safe-harbor-targeted CRISPR/Cas9 system based on an autonomously replicating plasmid was designed to facilitate alien gene integration at the CmSH1 locus. Cmhyd1, one of the hydrophobin genes, was confirmed as a defensive factor against CC infection, and Cmhyd1 overexpression by this system showed enhancement of disease resistance with negligible effect on the agronomic traits of CM. No off-target events and residues of plasmid sequence were tested by PCR and genome resequencing. This study provided the first safe harbor site for genetic manipulations, a safe harbor-targeted CRISPR/Cas9 system, and the first disease-resistant gene-editing breeding system in mushrooms.

Efficient disruption of CmHk1 using CRISPR/Cas9 ribonucleoprotein delivery in Cordyceps militaris.

Cordyceps militaris, an entomopathogenic ascomycete, produces edible medicinal mushrooms known to have medicinal and therapeutic functions. To develop the genetic transformation system in C. militaris, green fluorescent protein (GFP) mutants of C. militaris were generated by PEG-mediated protoplast transformation. The CRISPR/Cas9 ribonucleoprotein (RNP) targeting the class III histidine kinase of C. militaris (CmHk1) was then delivered into protoplasts of C. militaris through the transformation system. Mutations induced by the RNP in selected mutants were detected: 1 nt deletion (6 mutants), 3 nt deletion with substitution of 1 nt (1 mutant), insertion of 85 nts (1 mutant), 41 nts (2 mutants), and 35 nts (5 mutants). An in vitro sensitivity assay of the mutants indicated that knockout of CmHk1 reduced sensitivity to two fungicides, iprodione and fludioxonil, but increased sensitivity to osmotic stresses compared to the wild type. Summing up, the CRISPR/Cas9 RNP delivery system was successfully developed, and our results revealed that CmHk1 was involved in the fungicide resistance and osmotic stress in C. militaris.

Altered GC- and AT-biased genotypes of Ophiocordyceps sinensis in the stromal fertile portions and ascospores of natural Cordyceps sinensis.

OBJECTIVE: To examine multiple genotypes of Ophiocordyceps sinensis in a semi-quantitative manner in the stromal fertile portion (SFP) densely covered with numerous ascocarps and ascospores of natural Cordyceps sinensis and to outline the dynamic alterations of the coexisting O. sinensis genotypes in different developmental phases. METHODS: Mature Cordyceps sinensis specimens were harvested and continuously cultivated in our laboratory (altitude 2,254 m). The SFPs (with ascocarps) and fully and semi-ejected ascospores were collected for histological and molecular examinations. Biochip-based single nucleotide polymorphism (SNP) MALDI-TOF mass spectrometry (MS) was used to genotype multiple O. sinensis mutants in the SFPs and ascospores. RESULTS: Microscopic analysis revealed distinct morphologies of the SFPs (with ascocarps) before and after ascospore ejection and SFP of developmental failure, which, along with the fully and semi-ejected ascospores, were subjected to SNP MS genotyping analysis. Mass spectra showed the coexistence of GC- and AT-biased genotypes of O. sinensis that were genetically and phylogenetically distinct in the SFPs before and after ejection and of developmental failure and in fully and semi-ejected ascospores. The intensity ratios of MS peaks were dynamically altered in the SFPs and the fully and semi-ejected ascospores. Mass spectra also showed transversion mutation alleles of unknown upstream and downstream sequences with altered intensities in the SFPs and ascospores. Genotype #5 of AT-biased Cluster-A maintained a high intensity in all SFPs and ascospores. An MS peak with a high intensity containing AT-biased Genotypes #6 and #15 in pre-ejection SFPs was significantly attenuated after ascospore ejection. The abundance of Genotypes #5‒6 and #16 of AT-biased Cluster-A was differentially altered in the fully and semi-ejected ascospores that were collected from the same Cordyceps sinensis specimens. CONCLUSION: Multiple O. sinensis genotypes coexisted in different combinations with altered abundances in the SFPs prior to and after ejection, the SFP of developmental failure, and the two types of ascospores of Cordyceps sinensis, demonstrating their genomic independence. Metagenomic fungal members present in different combinations and with dynamic alterations play symbiotic roles in different compartments of natural Cordyceps sinensis.

[Differential transcription of mating-type genes during sexual reproduction of natural Cordyceps sinensis].

Natural Cordyceps sinensis as an insect-fungal complex, which is developed after Ophiocordyceps sinensis infects a larva of Hepialidae family. Seventeen genotypes of O. sinensis have been identified in natural C. sinensis. This paper summarized the literature reports and GenBank database regarding occurrence and transcription of the mating-type genes of MAT1-1 and MAT1-2 idiomorphs in natural C. sinensis, in Hirsutella sinensis(GC-biased Genotype #1 of O. sinensis), to infer the mating pattern of O. sinensis in the lifecycle of natural C. sinensis. The mating-type genes and transcripts of MAT1-1 and MAT1-2 idiomorphs were identified in the metagenomes and metatranscriptomes of natural C. sinensis. However, their fungal sources are unclear because of co-colonization of several genotypes of O. sinensis and multiple fungal species in natural C. sinensis. The mating-type genes of MAT1-1 and MAT1-2 idiomorphs were differentially present in 237 H. sinensis strains, constituting the genetic control of the O. sinensis reproduction. Transcriptional control of the O. sinensis reproduction includes: differential transcription or silencing of the mating-type genes of MAT1-1 and MAT1-2 idiomorphs, and the MAT1-2-1 transcript with unspliced intron I that contains 3 stop codons. Research on the H. sinensis transcriptome demonstrated differential and complementary transcriptions of the mating-type genes of MAT1-1 and MAT1-2 idiomorphs in Strains L0106 and 1229, which may become mating partners to accomplish physiological heterothallism. The differential occurrence and transcription of the mating-type genes in H. sinensis are inconsistent with the self-fertilization hypothesis under homothallism or pseudohomothallism, but instead indicate the need of mating partners of the same H. sinensis species, either monoecious or dioecious, for physiological heterothallism, or heterospecific species for hybridization. Multiple GC-and AT-biased genotypes of O. sinensis were identified in the stroma, stromal fertile portion(densely covered with numerous ascocarps) and ascospores of natural C. sinensis. It needs to be further explored if the genome-independent O. sinensis genotypes could become mating partners to accomplish sexual reproduction. S. hepiali Strain FENG experienced differential transcription of the mating-type genes with a pattern complementary to that of H. sinensis Strain L0106. Additional evidence is needed to explore a hybridization possibility between S. hepiali and H. sinensis, whether they are able to break the interspecific reproductive isolation. Genotypes #13～14 of O. sinensis feature large DNA segment reciprocal substitutions and genetic material recombination between 2 heterospecific parental fungi, H. sinensis and an AB067719-type fungus, indicating a possibility of hybridization or parasexuality. Our analysis provides important information at the genetic and transcriptional levels regarding the mating-type gene expression and reproduction physiology of O. sinensis in the sexual life of natural C. sinensis and offers crucial reproductive physiology evidence, to assist in the design of the artificial cultivation of C. sinensis to supplement the increasing scarcity of natural resource.

Diversity of anamorphic Cordyceps (formerly Isaria) isolated from Brazilian agricultural sites.

A total of 53 anamorphic strains of Brazilian Cordyceps species currently maintained in a government-owned culture collection, were reassessed for diversity and species identity using multi-loci-based phylogenetic methods. The strains used in this study were originally obtained from soil samples or were isolated from insects of the orders Hemiptera, Lepidoptera, Coleoptera and Diptera, mostly from agricultural sites. A Bayesian phylogenetic tree was constructed based on a concatenation of five loci (ITS, LSU, RPB1, RPB2 and TEF). In a few cases of ambiguity, morphological traits were also considered for species delimitations. Considerable variability within the set of strains was detected and six Cordyceps species were identified: C. amoenerosea, C. fumosorosea, C. javanica, C. tenuipes and, for the first time, C. brevistroma and C. spegazzinii are reported in Brazil. Four other taxonomically equivocal groups, closely related to other known taxa (C. amoenerosea, C. cateniannulata, C. polyarthra and C. spegazzinii), were also recognized, although further studies will be required to confirm their identifications or their descriptions as new species. Cordyceps javanica was the most common species in our dataset, originally isolated from soil and several different insect orders, and includes 17 strains from the whitefly, Bemisia tabaci. Interestingly, strains previously identified as C. fumosorosea based on morphology and growth characteristics, were shown to be C. javanica, including the active ingredients of some commercial mycoinsecticides. Cordyceps farinosa, usually mentioned in the literature as occurring in Brazil, was not found in our study. Since most strains were from insect crop pests, further studies with hosts from non-agricultural settings or from environmental samples would be advisable for a deeper understanding of the occurrence of anamorphic Cordyceps in Brazil.

Stable reference gene selection for Ophiocordyceps sinensis gene expression studies under different developmental stages and light-induced conditions.

The molecular mechanism of Chinese cordyceps formation has received a substantial amount of attention because of its usage as traditional Chinese medicine. The formation process of Chinese cordyceps includes two parts: asexual proliferation (Ophiocordyceps sinensis proliferates in the hemolymph of Thitarodes armoricanus larvae) and sexual development (formation and development of fruiting bodies). Therefore, validation of reference genes under different development stages and experimental conditions is crucial for RT-qPCR analysis. However, there is no report on stable reference genes at the development stage of O. sinensis fruiting body. In this study, 10 candidate reference genes, Actin, Cox5, Tef1, Ubi, 18s, Gpd, Rpb1, Try, Tub1 and Tub2, were selected and calculated their expression stability using four methods: geNorm, NormFinder, BestKeeper, and Comparative △Ct. After comprehensive analysis of the results of these four methods with RefFinder, we determined that the most stable reference genes during asexual reproduction of O. sinensis were Tef1 and Tub1, while the most stable reference genes during fruiting body development were Tyr and Cox5, and the most stable reference genes under light-induced conditions were Tyr and Tef1. Our study provides a guidance for reference genes selections at different proliferation processes with light stress of O. sinensis, and represents a foundation for studying the molecular mechanism of Chinese cordyceps formation.

Functional genomics and systems biology of Cordyceps species for biotechnological applications.

The use of Cordyceps species for the manufacture of natural products has been established; however, the tremendous advances observed in recent years in genetic engineering and molecular biology have revolutionized the optimization of Cordyceps as cell factories and drastically expanded the biotechnological potential of these fungi. Here, we present a review of systems and synthetic biology studies of Cordyceps and their implications for fungal biology and industrial applications. We summarize the current status of synthetic biology for enhancing targeted metabolites in Cordyceps species, such as cordycepin, adenosine, polysaccharide, and pentostatin. Progress in the systems and synthetic biology of Cordyceps provides a strategy for comprehensively comprehensive controlling efficient cell factories of natural bioproducts and novel synthetic biology toolbox for targeted engineering.

Heterologous expression of the lectin CmRlec from Cordyceps militaris (Cordycipitaceae, Ascomycota) in Escherichia coli.

Ascomycete lectins may play an important role in their life cycle. In this report, we mined a ricin B-type lectin, named CmRlec, from the Cordyceps militaris genome by homology search. Furthermore, we succeeded in the soluble expression of CmRlec using ß-glucuronidase as a solubilization tag and demonstrated that this lectin is a novel chitin-recognizing lectin.

Uncovering Gene Expression Profiles of Three Morphologic Mycelium Forms of the Chinese Caterpillar Mushroom Ophiocordyceps sinensis (Ascomycota) Using High-Throughput Sequencing.

The asexual form of Ophiocordyceps sinensis has been controversial, but various morphologic mycelium appeared when O. sinensis was cultured under experimental conditions. To explore the generation mechanism of morphologic mycelium, developmental transcriptomes were analyzed from three kinds of mycelium (aerial mycelium, hyphae knot, and substrate mycelium). The results showed that diameter and morphology of these three kinds of mycelium were obviously different. KEGG functional enrichment analysis showed that the differential expressed genes (DEGs) of substrate mycelium were enriched in ribosomes and peroxisomes, indicating that prophase culture was rich in nutrients and the metabolism of substrate mycelium cells was vigorous in the stage of nutrient absorption. The up-DEGs of hyphae knot were mainly enriched in the oxidative phosphorylation pathway, indicating that oxidative phosphorylation was the main energy source for mycelium formation in the stage of nutrient accumulation and reproductive transformation. The up-DEGs of aerial mycelium were mainly enriched in the synthesis and degradation pathways of valine, leucine, and isoleucine, suggesting that the occurrence of aerial mycelium was related to amino acid metabolism at the later stage of culture, and nutritional stress accelerated the reproduction of asexual spores. In addition, the important roles of mycelium formation related genes were verified by combined analysis of qRT-PCR and transcriptome sequencing. Collectively, this study will provide theoretical guidance for inhibiting the occurrence of aerogenous mycelium and promoting the development of mycelium into pinhead primordia in the culture of O. sinensis in the future.

Differential coexistence of multiple genotypes of Ophiocordyceps sinensis in the stromata, ascocarps and ascospores of natural Cordyceps sinensis.

OBJECTIVE: To examine the differential occurrence of Ophiocordyceps sinensis genotypes in the stroma, stromal fertile portion (SFP) densely covered with numerous ascocarps, and ascospores of natural Cordyceps sinensis. METHODS: Immature and mature C. sinensis specimens were harvested. Mature C. sinensis specimens were continuously cultivated in our laboratory (altitude 2,200 m). The SFPs (with ascocarps) and ascospores of C. sinensis were collected for microscopic and molecular analyses using species-/genotype-specific primers. Sequences of mutant genotypes of O. sinensis were aligned with that of Genotype #1 Hirsutella sinensis and compared phylogenetically using a Bayesian majority-rule method. RESULTS: Fully and semiejected ascospores were collected from the same specimens. The semiejected ascospores tightly adhered to the surface of the asci as observed by the naked eye and under optical and confocal microscopies. The multicellular heterokaryotic ascospores showed uneven staining of nuclei. The immature and mature stromata, SFPs (with ascocarps) and ascospores were found to differentially contain several GC- and AT-biased genotypes of O. sinensis, Samsoniella hepiali, and an AB067719-type fungus. The genotypes within AT-biased Cluster-A in the Bayesian tree occurred in all compartments of C. sinensis, but those within AT-biased Cluster-B were present in immature and mature stromata and SPFs but absent in the ascospores. Genotype #13 of O. sinensis was present in semi-ejected ascospores and Genotype #14 in fully ejected ascospores. GC-biased Genotypes #13-14 featured large DNA segment substitutions and genetic material recombination between the genomes of the parental fungi (H. sinensis and the AB067719-type fungus). These ascosporic offspring genotypes combined with varying abundances of S. hepiali in the 2 types of ascospores participated in the control of the development, maturation and ejection of the ascospores. CONCLUSION: Multiple genotypes of O. sinensis coexist differentially in the stromata, SFPs and 2 types of C. sinensis ascospores, along with S. hepiali and the AB067719-type fungus. The fungal components in different combinations and their dynamic alterations in the compartments of C. sinensis during maturation play symbiotic roles in the lifecycle of natural C. sinensis.

The phylogeny and divergence time of Ophiocordyceps sinensis and its host insects based on elongation factor 1 alpha.

Ophiocordyceps sinensis Berk. is a fungal parasite that parasitizes the larvae of Hepialidae and is endemic to the Qinghai-Tibet Plateau (QTP). The phylogeny and divergence time of O. sinensis and its host insects were analyzed for 137 individuals from 48 O. sinensis populations based on the elongation factor 1 alpha (EF-1α) gene. Lower nucleotide variation, with only 7 and 16 EF-1α haplotypes, was detected in O. sinensis and its host insects, respectively. The isolated and broad distribution patterns coexisted in both O. sinensis and its host insects on the QTP. The divergence time estimates show that O. sinensis and its host insects originated later than 14.33 million years (Myr) and earlier than 23.60 Myr in the Miocene period, and the major differentiation occurred later than 4 Myr. Their origin and differentiation match well with the second and third uplifts of the QTP, respectively. The host insects from the O. sinensis populations distributed around Qinghai Lake are inferred as an ancient and relict species that has survived various geological events of the QTP. It is suitable to estimate the divergence times of both O. sinensis and its host insects from the same individuals using one gene: EF-1α. Our findings of the origin, phylogeny, and evolution of the endemic species also support the epoch of geological events on the QTP.

Detection of the pathogenic fungus Cordyceps farinosa in the Thitarodes armoricanus soil-rearing environment based on nucleic acid targets.

Cordyceps farinosa, an entomopathogenic fungus, infects and leads to high mortality of Thitarodes armoricanus larvae, which die soon after the infection of C. farinose, usually before the colonization of Ophiocordyceps sinensis owing to competitive inhibition and fruiting body formation. Therefore, monitoring C. farinosa in the O. sinensis cultivation environment is critical for minimizing the C. farinosa infection-induced losses. In this study, we initially designed a PCR primer pair (Tar-1F/Tar-1R) through open reading frame prediction and homology comparison of the C. farinosa genome sequence. This primer pair can detect both C. farinosa and Samsoniella hepiali. To further distinguish, primers (ITS5-172/ITS4-95) were then designed to selectively amplify the large ribosomal subunit sequences in the C. farinosa genome. All these primers were applied in combination for detection of C. farinosa in soil samples. The sensitivity reached a detection limit of 1 × 106 spores/g soil. In addition, these primers can detect the presence of C. farinosa in dead T. armoricanus larval samples. This newly established rapid detection method provides important information for C. farinosa control during O. sinensis cultivation.

Integrated metabolomics and transcriptomics reveal metabolites difference between wild and cultivated Ophiocordyceps sinensis.

Ophiocordyceps sinensis is a traditional medicinal fungus endemic to the alpine and high-altitude areas of the Qinghai-Tibet plateau. The scarcity of the wild resource has led to increased attention to artificially cultivated O. sinensis. However, little is known about the metabolic differences and the regulatory mechanisms between cultivated and wild O. sinensis. This study exploited untargeted metabolomics and transcriptomics to uncover the differences in accumulated metabolites and expressed genes between wild and cultivated O. sinensis. Metabolomics results revealed that 368 differentially accumulated metabolites were mainly enriched in biosynthesis of amino acids, biosynthesis of plant secondary metabolites and purine nucleotide metabolism. Cultivated O. sinensis contained more amino acids and derivatives, carbohydrates and derivatives, and phenolic acids than wild O. sinensis, whereas the contents of most nucleosides and nucleotides in wild O. sinensis were significantly higher than in cultivated O. sinensis. Transcriptome analysis indicated that 4430 annotated differentially expressed genes were identified between two types. Integrated metabolomics and transcriptomics analyses suggested that IMPDH, AK, ADSS, guaA and GUK genes might be related to the synthesis of purine nucleotides and nucleosides. Our findings will provide a new insight into the molecular basis of metabolic variations of this medicinal fungus.

Differential transcription of mating-type genes during sexual reproduction of natural Cordyceps sinensis / 中国中药杂志

Natural Cordyceps sinensis as an insect-fungal complex, which is developed after Ophiocordyceps sinensis infects a larva of Hepialidae family. Seventeen genotypes of O. sinensis have been identified in natural C. sinensis. This paper summarized the literature reports and GenBank database regarding occurrence and transcription of the mating-type genes of MAT1-1 and MAT1-2 idiomorphs in natural C. sinensis, in Hirsutella sinensis(GC-biased Genotype #1 of O. sinensis), to infer the mating pattern of O. sinensis in the lifecycle of natural C. sinensis. The mating-type genes and transcripts of MAT1-1 and MAT1-2 idiomorphs were identified in the metagenomes and metatranscriptomes of natural C. sinensis. However, their fungal sources are unclear because of co-colonization of several genotypes of O. sinensis and multiple fungal species in natural C. sinensis. The mating-type genes of MAT1-1 and MAT1-2 idiomorphs were differentially present in 237 H. sinensis strains, constituting the genetic control of the O. sinensis reproduction. Transcriptional control of the O. sinensis reproduction includes: differential transcription or silencing of the mating-type genes of MAT1-1 and MAT1-2 idiomorphs, and the MAT1-2-1 transcript with unspliced intron I that contains 3 stop codons. Research on the H. sinensis transcriptome demonstrated differential and complementary transcriptions of the mating-type genes of MAT1-1 and MAT1-2 idiomorphs in Strains L0106 and 1229, which may become mating partners to accomplish physiological heterothallism. The differential occurrence and transcription of the mating-type genes in H. sinensis are inconsistent with the self-fertilization hypothesis under homothallism or pseudohomothallism, but instead indicate the need of mating partners of the same H. sinensis species, either monoecious or dioecious, for physiological heterothallism, or heterospecific species for hybridization. Multiple GC-and AT-biased genotypes of O. sinensis were identified in the stroma, stromal fertile portion(densely covered with numerous ascocarps) and ascospores of natural C. sinensis. It needs to be further explored if the genome-independent O. sinensis genotypes could become mating partners to accomplish sexual reproduction. S. hepiali Strain FENG experienced differential transcription of the mating-type genes with a pattern complementary to that of H. sinensis Strain L0106. Additional evidence is needed to explore a hybridization possibility between S. hepiali and H. sinensis, whether they are able to break the interspecific reproductive isolation. Genotypes #13～14 of O. sinensis feature large DNA segment reciprocal substitutions and genetic material recombination between 2 heterospecific parental fungi, H. sinensis and an AB067719-type fungus, indicating a possibility of hybridization or parasexuality. Our analysis provides important information at the genetic and transcriptional levels regarding the mating-type gene expression and reproduction physiology of O. sinensis in the sexual life of natural C. sinensis and offers crucial reproductive physiology evidence, to assist in the design of the artificial cultivation of C. sinensis to supplement the increasing scarcity of natural resource.

Complete genome sequence of a novel victorivirus infecting cicada flower (Cordyceps chanhua).

Cicada flower, Cordyceps chanhua, is a precious edible and medicinal mushroom with uses in both medicine and food in China. In this study, Cordyceps chanhua strain RCEF5995 was found to be coinfected by a previously characterized alternavirus, Cordyceps chanhua alternavirus 1 (CcAV1), and a novel victorivirus, tentatively named "Cordyceps chanhua victorivirus 1" (CcV1). Molecular characterization of CcV1 showed that its complete genome is 5,232 nucleotides long with a GC content of 57.5%. Sequence analysis indicated that CcV1 contains two overlapping open reading frames (ORFs), ORF1 and ORF2, encoding a putative coat protein (CP) of 742 amino acids (aa) and a putative RNA-dependent RNA polymerase (RdRp) of 836 aa, respectively. The termination codon of the CP ORF overlaps with the initiation codon of the RdRp ORF at the tetranucleotide sequence AUGA. Homolog searches, sequence comparisons, and phylogenetic analysis based on deduced amino acid sequences of RdRp indicated that CcV1 is a new member of the genus Victorivirus, family Totiviridae.

Multiplex gene precise editing and large DNA fragment deletion by the CRISPR-Cas9-TRAMA system in edible mushroom Cordyceps militaris.

The medicinal mushroom Cordyceps militaris contains abundant valuable bioactive ingredients that have attracted a great deal of attention in the pharmaceutical and cosmetic industries. However, the development of this valuable mushroom faces the obstacle of lacking powerful genomic engineering tools. Here, by excavating the endogenous tRNA-processed element, introducing the extrachromosomal plasmid and alongside with homologous template, we develop a marker-free CRISPR-Cas9-TRAMA genomic editing system to achieve the multiplex gene precise editing and large synthetic cluster deletion in C. militaris. We further operated editing in the synthetases of cordycepin and ergothioneine to demonstrate the application of Cas9-TRAMA system in protein modification, promoter strength evaluation and 10 kb metabolic synthetic cluster deletion. The Cas9-TRAMA system provides a scalable method for excavating the valuable metabolic resource of medicinal mushrooms and constructing a mystical cellular pathway to elucidate the complex cell behaviours of the edible mushroom.

Species Diversity of Cordyceps-Like Fungi in the Tiankeng Karst Region of China.

Tiankeng acts as a refugium for biodiversity amid a changing global climate, and a previous study has shown that some ancient (Alsophila spinulosa) and unique plants (cool-adapted plants) are present in Tiankeng. However, there are few reports on Ascomycota from the Tiankeng karst region. In this research, the species diversity of Cordyceps-like fungi in Monkey-Ear Tiankeng was investigated. Seven species in the genera Akanthomyces, Beauveria, Cordyceps, and Samsoniella were identified based on internal transcribed spacer sequences and morphological characteristics. Eight new species in the genera Akanthomyces, Cordyceps, and Samsoniella were established and described according to a multilocus phylogenetic analysis and morphological characteristics. Our results revealed that Cordyceps-like fungi were abundant in Monkey-Ear Tiankeng, providing new insights into the diversity of Ascomycota in this special eco-environment. IMPORTANCE Karst Tiankeng has a special eco-environment and acts as a refugium for biodiversity. However, there are few reports on Ascomycota from the Tiankeng karst region. In this research, seven known species and eight new species in the genera Akanthomyces, Beauveria, Cordyceps, and Samsoniella were reported. The results showed that Cordyceps-like fungi are abundant in Monkey-Ear Tiankeng. Interestingly, the month of the sampling was November, which is not an active period of growth and reproduction for Cordyceps-like fungi. These results revealed that unconventional time sampling should not be ignored, especially for a special eco-environment, and provided new insights into the diversity of Ascomycota in this special eco-environment.