Lineage-specific genes are clustered with HET-domain genes and respond to environmental and genetic manipulations regulating reproduction in Neurospora.

Lineage-specific genes (LSGs) have long been postulated to play roles in the establishment of genetic barriers to intercrossing and speciation. In the genome of Neurospora crassa, most of the 670 Neurospora LSGs that are aggregated adjacent to the telomeres are clustered with 61% of the HET-domain genes, some of which regulate self-recognition and define vegetative incompatibility groups. In contrast, the LSG-encoding proteins possess few to no domains that would help to identify potential functional roles. Possible functional roles of LSGs were further assessed by performing transcriptomic profiling in genetic mutants and in response to environmental alterations, as well as examining gene knockouts for phenotypes. Among the 342 LSGs that are dynamically expressed during both asexual and sexual phases, 64% were detectable on unusual carbon sources such as furfural, a wildfire-produced chemical that is a strong inducer of sexual development, and the structurally-related furan 5-hydroxymethyl furfural (HMF). Expression of a significant portion of the LSGs was sensitive to light and temperature, factors that also regulate the switch from asexual to sexual reproduction. Furthermore, expression of the LSGs was significantly affected in the knockouts of adv-1 and pp-1 that regulate hyphal communication, and expression of more than one quarter of the LSGs was affected by perturbation of the mating locus. These observations encouraged further investigation of the roles of clustered lineage-specific and HET-domain genes in ecology and reproduction regulation in Neurospora, especially the regulation of the switch from the asexual growth to sexual reproduction, in response to dramatic environmental conditions changes.

First Report of White Mucus Disease Caused by Fuligo gyrosa on Agrocybe chaxingu in China.

Agrocybe chaxingu is an edible and medicinal mushroom widely cultivated in China (Liu et al. 2021). Agrocybe chaxingu is extremely well-liked for the unique flavor and nutritional value. In May 2021, a serious white mucus disease was observed in the farms of A. chaxingu in the Ganxian district of Ganzhou City, Jiangxi Province, China, with an approximate disease incidence of 20%. In the years of 2022 and 2023, the same white mucus disease on A. chaxingu was observed in the farms in Nanchang City, Jiujiang City and Guangchang County, Jiangxi Province, China. The disease generally occurs on the media, stipe or pileus of A. chaxingu under condition of high humidity. The plasmodial slime molds migrated from the surface of culture media (78% hardwood sawdust, 15% wheat bran, 5% tea seed shell, 1% lime, and 1% gypsum) to the base of fruiting bodies, stipes and finally to pilei, showing as moist, sticky, and white reticulated structures. The infected fruiting bodies of A. chaxingu were completely covered by reticulated plasmodia, displaying a white or pale-yellow color. This resulted in the growth cessation, wilting and eventual death of fruiting body. Microscopic observation found that the plasmodia of slime mold enveloped the hyphae of A. chaxingu, resulting in the fragmentation of the hyphae. The disease can spread quickly, resulting in a 30% reduction in production. Slime mold cultures were isolated by transferring diseased fruiting bodies of A. chaxingu onto oat-agar medium (2% agar and 1% oatmeal) at 25 ℃. The isolates can be obtained after being subcultured for two to three generations. Purified plasmodia were placed on the semi-defined medium (1% tryptone, 1% glucose, 0.15% yeast extract, chick embryo extract and a balanced salt solution) to confirm the absence of bacteria (Daniel et al. 1964) and thus obtained the pure culture. Specimen of the voucher has been deposited in the Institute of Agricultural Applied Microbiology, Jiangxi Academy of Agricultural Sciences as number IAAM-W0002. The vegetative plasmodia have a large and well-developed scalloped structure that were white or milky white in colour. The white plasmodium became opaque pale yellow when exposed to light before fruiting. The veins merged and thickened. Fruiting bodies can be formed on the lid or side of the Petri dish under light condition. The fruiting bodies formed papillae with irregular shape, and then the color changed from translucent yellow to greyish black. Spores were usually spherical or subglobose, free, greyish brown in mass, purplish brown, 7-12 µm in diameter under light microscopy. These morphological characteristics were found to be consistent with those of Fuligo gyrosa (Synonym: Physarum gyrosum) (Kim et al. 2009; Shi et al. 2005; Jahn 1902). The identity of the isolates was further confirmed by sequence analysis of the 18S ribosomal RNA gene with primer SMNUR101/NS4 (Rusk et al. 1995; White et al. 1990). Using BLASTn searches, the sequence of 18S rRNA gene (GenBank accession number OR186216) matched the sequence of F. gyrosa (GenBank accession number LC744593) with the identity of 99.91% and coverage of 97%. A phylogenetic tree based on the 18S rRNA gene also demonstrated that the slime mold clustered with F. gyrosa. Over ten isolates have been obtained from the diseased A. chaxingu samples in different factories and identified as F. gyrosa. To test the pathogenicity of F. gyrosa, five healthy young fruiting bodies (three to five days of primordium) of A. chaxingu cultivated in mushroom-growing room were gently inoculated by a 12 mm diameter oat-agar medium with plasmodia at 24 ± 2 ℃ and then were kept with relative humidity of 90%-95%. Five fruiting bodies inoculated with a 12 mm oat-agar medium served as controls. After 5 days, white mucus characteristics and three fifths of death symptoms were observed on the fruiting bodies inoculated with the plasmodia, while the controls remained asymptomatic. The slime mold on the inoculated fruiting bodies was morphologically identical to F. gyrosa that was observed on the initial diseased fruiting bodies. It was also observed the envelopment A. chaxingu hyphae by the plasmodia of slime mold and fragmentation of the hyphae, and the fragmentation was not observed in the controls. Reisolations were prepared from the inoculated fruiting bodies and confirmed to be F. gyrosa based on morphological characteristics and 18S rRNA sequence, thus fulfilling Koch's postulates. Fuligo gyrosa has been reported to cause severe disease in oriental melon in Korea (Kim et al. 2009). This is the first report of F. gyrosa causing white mucus disease in cultivated A. chaxingu. The findings will provide important information on prevention and control of the disease, and be helpful for the development of A. chaxingu industry.

Origins of lineage-specific elements via gene duplication, relocation, and regional rearrangement in Neurospora crassa.

The origin of new genes has long been a central interest of evolutionary biologists. However, their novelty means that they evade reconstruction by the classical tools of evolutionary modelling. This evasion of deep ancestral investigation necessitates intensive study of model species within well-sampled, recently diversified, clades. One such clade is the model genus Neurospora, members of which lack recent gene duplications. Several Neurospora species are comprehensively characterized organisms apt for studying the evolution of lineage-specific genes (LSGs). Using gene synteny, we documented that 78% of Neurospora LSG clusters are located adjacent to the telomeres featuring extensive tracts of non-coding DNA and duplicated genes. Here, we report several instances of LSGs that are likely from regional rearrangements and potentially from gene rebirth. To broadly investigate the functions of LSGs, we assembled transcriptomics data from 68 experimental data points and identified co-regulatory modules using Weighted Gene Correlation Network Analysis, revealing that LSGs are widely but peripherally involved in known regulatory machinery for diverse functions. The ancestral status of the LSG mas-1, a gene with roles in cell-wall integrity and cellular sensitivity to antifungal toxins, was investigated in detail alongside its genomic neighbours, indicating that it arose from an ancient lysophospholipase precursor that is ubiquitous in lineages of the Sordariomycetes. Our discoveries illuminate a "rummage region" in the N. crassa genome that enables the formation of new genes and functions to arise via gene duplication and relocation, followed by fast mutation and recombination facilitated by sequence repeats and unconstrained non-coding sequences.

Value of CEUS features in diagnosing thyroid nodules with halo sign on B-mode ultrasound.

BACKGROUND: The results of halo sign in the differential diagnosis of thyroid nodules were conflicting, and the value of contrast-enhanced ultrasound (CEUS) in characterization of thyroid nodules with halo has not been fully evaluated. This study was therefore designed to investigate the value of contrast-enhanced ultrasound features in the differential diagnosis of thyroid nodules with halo sign on B-mode ultrasound. MATERIAL AND METHODS: Seventy-four consecutive thyroid nodules with halo sign on B-mode ultrasound were pathologically confirmed by surgery or fine needle aspiration, including 43 benign and 31 malignant lesions. All these lesions underwent pre-operative CEUS examination. The CEUS features, including enhanced time, enhanced intensity and homogeneity, and presence of enhancing ring, were compared between benign and malignant ones. RESULTS: Enhanced intensity was significant different between benign and malignant lesions with halo. Hypo-enhancement was more frequently detected in malignant nodules than that in benign ones, compared with iso-enhancement and hyper-enhancement (p = 0.013, and = 0.014, respectively). Detection rate of high-enhancing ring was significantly higher in benign nodules than that in malignant group (p = 0.001). While in nodules > 10 mm, only high-enhancing ring was the distinguishing feature between benign and malignant nodules. CONCLUSIONS: Enhanced intensity and high-enhancing ring may be helpful in the differential diagnosis of thyroid nodules with halo sign on B-mode ultrasound.

First Report of Bacterial Brown Rot Disease Caused by Ewingella americana on Cultivated Naematelia aurantialba in China.

Naematelia aurantialba (synonym Tremella aurantialba) is one of the jelly fungi and highly valued edible and medicinal mushrooms. It has been cultivated industrially in recent years and consumed popularly in China. In September 2022, brown rot disease of fruiting bodies was observed at the N. aurantialba factory in Tongzhou district, Beijing with a disease incidence of ~10%. Symptoms initially appeared as color changing from orange to light brown. The infected area expanded gradually until covered fully the fruiting body. Meanwhile, the interior of the fruiting body became rotten and dark brown. Finally, the whole fruiting body became wrinkled and brown, resulting in significantly reduced yield and economic loss. Isolations were made from 12 infected mushroom samples. Infected tissue within the fruiting body was mashed in sterilized 1.5 mL tubes containing 1 mL of sterile distilled water. After standing for 5-10 min, the suspensions were streaked on the Luria-Bertani (LB) medium and cultured at 37°C for 24h. The physiological and biochemical reactions of isolated strains were determined using the API 20E system (Reyes et al. 2004) according to the manufacturer's instructions. All the strains showed the same reaction results. The bacterial colonies were streaked on fresh LB medium at 37°C for 24 h, and a single pure culture was obtained with round, smooth and semitransparent. The bacterial cells were gram-negative, short-rod, (0.3) 0.8-2.0 (2.5) × (0.1) 0.6-1.0 (1.5) µm, and peri-flagellate. The isolates were further confirmed by sequence analysis of the 16S rRNA and gyrB genes with primer 27F/1492R and gyrB-UP1s/gyrB-UP2sr (Liu et al. 2018). Using EzBioCloud data searches, the 16S rRNA sequence of four strains (GenBank accession OP727593, OP727595, OP727596, OP727601) matched the sequence of E. americana type strain ATCC 33852 (accession JMPJ01000013) with identity of 99.65%~99.93 and 100% completeness. The GyrB sequence matched the E. americana in GenBank (MK460250) and showed 98.71% identity and 100% completeness. Finally, the pathogen was identified as E. americana based on morphological, physiological, biochemical, and molecular characteristics. The pathogenicity test was conducted by spreading bacterial suspensions cultured 48h onto 12 healthy cultivated fruiting bodies of N. aurantialba, with sterile distilled water as a control, and then cultured in a chamber at 23°C with 85% relative humidity. Brown symptoms, similar to natural symptoms, were observed on all inoculated fruiting bodies after 48h, whereas the controls remained symptomless. Pathogenic bacteria were isolated from the inoculated fruiting body and confirmed to be E. americana based on morphological and 16S rRNA molecular characteristics, thus fulfilling Koch's postulates. E. americana caused stipe necrosis on Agaricus bisporus in Egypt (Madbouly et al. 2014), the oak tree in Thailand, and pneumonia in Humans (Doonan et al. 2016), and brown blotch on Flammulina velutipes (Liu et al. 2018). To our knowledge, this is the first worldwide report of E. americana infecting jelly fungus N. aurantialba causing brown rot disease. E. americana is an opportunistic cross-kingdom pathogen (Liu et al. 2018). That will provide a critical alert on the prevention, effective monitoring, and control of the disease.

Hydrophobin Gene Cmhyd4 Negatively Regulates Fruiting Body Development in Edible Fungi Cordyceps militaris.

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.

Multifunctional Composite Nanosystems for Precise/Enhanced Sonodynamic Oxidative Tumor Treatment.

Ultrasound-activated therapies have been regarded as the efficient strategy for tumor treatment, among which sonosensitizer-enabled sonodynamic oxidative tumor therapy features intrinsic advantages as compared to other exogenous trigger-activated dynamic therapies. Nanomedicine-based nanosonosensitizer design has been extensively explored for improving the therapeutic efficacy of sonodynamic therapy (SDT) of tumor. This review focuses on solving two specific issues, i.e., precise and enhanced sonodynamic oxidative tumor treatment, by rationally designing and engineering multifunctional composite nanosonosensitizers. This multifunctional design can augment the therapeutic efficacy of SDT against tumor by either improving the production of reactive oxygen species or inducing the synergistic effect of SDT-based combinatorial therapies. Especially, this multifunctional design is also capable of endowing the nanosonosensitizer with bioimaging functionality, which can effectively guide and monitor the therapeutic procedure of the introduced sonodynamic oxidative tumor treatment. The design principles, underlying material chemistry for constructing multifunctional composite nanosonosensitizers, intrinsic synergistic mechanism, and bioimaging guided/monitored precise SDT are summarized and discussed in detail with the most representative paradigms. Finally, the existing critical issues, available challenges, and potential future developments of this research area are also discussed for promoting the further clinical translations of these multifunctional composite nanosonosensitizers in SDT-based tumor treatment.

Homokaryotic High-Quality Genome Assembly of Medicinal Fungi Wolfiporia hoelen Reveals Auto-Regulation and High-Temperature Adaption of Probable Two-Speed Genome.

Sclerotia of Wolfiporia hoelen are one of the most important traditional Chinese medicines and are commonly used in China, Japan, Korea, and other Asian countries. In the present study, we presented the first high-quality homokaryotic genome of W. hoelen with 14 chromosomes which was evaluated with assembly index, telomere position detection, and whole-genome collinearity. A 64.44 Mb genome was assembled with a Contig N50 length of 3.76 Mb. The imbalanced distribution of transposons and chromosome characters revealed the probable two-speed genome of W. hoelen. High consistency between methylation and transposon conserved the genome stability. The expansion of the gene family about signal transduction and nutritional transport has intimate relationships with sclerotial formation. Up-regulation of expression for distinctive decomposition enzymes, ROS clearance genes, biosynthesis of unsaturated fatty acids, and change of the cell wall components maintained high-speed growth of mycelia that may be the high-temperature adaption strategy of W. hoelen. Further, the analysis of mating-control genes demonstrated that HD3 probably had no function on mating recognition, with the HD protein in a distant genetic with known species. Overall, the high-quality genome of W. hoelen provided crucial information for genome structure and stability, high-temperature adaption, and sexual and asexual process.

A Sonication-Activated Valence-Variable Sono-Sensitizer/Catalyst for Autography Inhibition/Ferroptosis-Induced Tumor Nanotherapy.

The effective deployment of reactive oxygen species (ROS)-mediated oncotherapy in practice remains challenging, mired by uncontrollable catalytic processes, stern reaction conditions and safety concerns. Herein, we develop a copper nanodot integrating sonodynamic and catalytic effects within one active center, which responds to exogenous ultrasound (US) and endogenous H2 O2 stimuli. US irradiation induces the valence conversion from CuII to CuI catalyzing H2 O2 into ⋅OH for chemodynamic therapy. Meanwhile, valence transformation results in electron-hole pairs separation, promoting ROS generation for sonodynamic therapy. Notably, copper nanodots not only block lysosome fusion and degradation leading to autophagy flux blockage, but also interfere with the glutathione peroxidase 4 and cystine-glutamate antiporter SLC7A11 function achieving ferroptosis. Furthermore, reversible valence changes, inherent hydrophilicity and renal clearance ultrasmall size guarantee biosafety.

Co-delivery of nanoparticle and molecular drug by hollow mesoporous organosilica for tumor-activated and photothermal-augmented chemotherapy of breast cancer.

BACKGROUND: In comparison with traditional therapeutics, it is highly preferable to develop a combinatorial therapeutic modality for nanomedicine and photothermal hyperthermia to achieve safe, efficient, and localized delivery of chemotherapeutic drugs into tumor tissues and exert tumor-activated nanotherapy. Biocompatible organic-inorganic hybrid hollow mesoporous organosilica nanoparticles (HMONs) have shown high performance in molecular imaging and drug delivery as compared to other inorganic nanosystems. Disulfiram (DSF), an alcohol-abuse drug, can act as a chemotherapeutic agent according to its recently reported effectiveness for cancer chemotherapy, whose activity strongly depends on copper ions. RESULTS: In this work, a therapeutic construction with high biosafety and efficiency was proposed and developed for synergistic tumor-activated and photothermal-augmented chemotherapy in breast tumor eradication both in vitro and in vivo. The proposed strategy is based on the employment of HMONs to integrate ultrasmall photothermal CuS particles onto the surface of the organosilica and the molecular drug DSF inside the mesopores and hollow interior. The ultrasmall CuS acted as both photothermal agent under near-infrared (NIR) irradiation for photonic tumor hyperthermia and Cu2+ self-supplier in an acidic tumor microenvironment to activate the nontoxic DSF drug into a highly toxic diethyldithiocarbamate (DTC)-copper complex for enhanced DSF chemotherapy, which effectively achieved a remarkable synergistic in-situ anticancer outcome with minimal side effects. CONCLUSION: This work provides a representative paradigm on the engineering of combinatorial therapeutic nanomedicine with both exogenous response for photonic tumor ablation and endogenous tumor microenvironment-responsive in-situ toxicity activation of a molecular drug (DSF) for augmented tumor chemotherapy.

Cysteine-Rich Hydrophobin Gene Family: Genome Wide Analysis, Phylogeny and Transcript Profiling in Cordyceps militaris.

Hydrophobins are a family of small secreted proteins found exclusively in fungi, and they play various roles in the life cycle. In the present study, genome wide analysis and transcript profiling of the hydrophobin family in Cordyceps militaris, a well-known edible and medicinal mushroom, were studied. The distribution of hydrophobins in ascomycetes with different lifestyles showed that pathogenic fungi had significantly more hydrophobins than saprotrophic fungi, and class II members accounted for the majority. Phylogenetic analysis of hydrophobin proteins from the species of Cordyceps s.l. indicated that there was more variability among the class II members than class I. Only a few hydrophobin-encoding genes evolved by duplication in Cordyceps s.l., which was inconsistent with the important role of gene duplication in basidiomycetes. Different transcript patterns of four hydrophobin-encoding genes during the life cycle indicated the possible different functions for each. The transcripts of Cmhyd2, 3 and 4 can respond to light and were related with the photoreceptors. CmQHYD, with four hydrophobin II domains, was first found in C. militaris, and multi-domain hydrophobins were only distributed in the species of Cordycipitaceae and Clavicipitaceae. These results could be helpful for further function research of hydrophobins and could provide valuable information for the evolution of hydrophobins.

CmVVD is involved in fruiting body development and carotenoid production and the transcriptional linkage among three blue-light receptors in edible fungus Cordyceps militaris.

Fruiting body development and carotenoid production are light-induced in Cordyceps militaris. Our previous studies have shown that two blue-light receptors, CmWC-1 and CmCRY-DASH, regulate fruiting body development and secondary metabolism. However, the photosensory system of C. militaris remains unclear. Here, gene deletion of Cmvvd, coding for another blue-light receptor, resulted in reduced conidiation level and significant promotion of carotenoid content. Cmvvd transcription levels at fruiting body stages were higher than at other stages, and fruiting bodies could not develop normally in ΔCmvvd strains, indicating that Cmvvd might play an important role in fruiting body development. Rhythm loops were not affected in ΔCmvvd strains but were regulated by Cmwc-1, and the expression of the rhythm regulator gene Cmfrq was dependent on CmWC-1. Chromatin immunoprecipitation assay confirmed that Cmvvd is the direct target of CmWC-1 in this fungus. Our results also revealed interdependent transcriptional relationships between Cmwc-1 and Cmvvd, and between Cmwc-1 and Cmcry-DASH. Cmcry-DASH expression was affected by Cmvvd, and the function-loss of Cmcry-DASH might be compensated by the high transcription of Cmvvd. This is the first report of the transcriptional linkage among the three blue-light receptors in edible fungi and will be helpful for studies of multicellular development in this fungus.

Developmental transcriptomics of Chinese cordyceps reveals gene regulatory network and expression profiles of sexual development-related genes.

BACKGROUND: Chinese cordyceps, also known as Chinese caterpillar fungus (Ophiocordyceps sinensis, syn. Cordyceps sinensis), is of particular interest for its cryptic life cycle and economic and ecological importance. The large-scale artificial cultivation was succeeded recently after several decades of efforts and attempts. However, the induction of primordium, sexual development of O. sinensis and the molecular basis of its lifestyle still remain cryptic. RESULTS: The developmental transcriptomes were analyzed for six stages covering the whole developmental process, including hyphae (HY), sclerotium (ST), primordium (PR), young fruiting body (YF), developed fruiting body (DF) and mature fruiting body (MF), with a focus on the expression of sexual development-related genes. Principal component analysis revealed that the gene expression profiles at the stages of primordium formation and fruiting body development are more similar than those of the undifferentiated HY stage. The PR and MF stages grouped together, suggesting that primordium differentiation and sexual maturation have similar expression patterns. Many more DEGs were identified between the ST and HY stages, covering 47.5% of the O. sinensis genome, followed by the comparisons between the ST and PR stages. Using pairwise comparisons and weighted gene coexpression network analysis, modules of coexpressed genes and candidate hub genes for each developmental stage were identified. The four mating type loci genes expressed during primordium differentiation and sexual maturation; however, spatiotemporal specificity of gene expression indicated that they also expressed during the anamorphic HY stage. The four mating type genes were not coordinately expressed, suggesting they may have divergent roles. The expression of the four mating type genes was highest in the fertile part and lowest in the sclerotium of the MF stage, indicating that there is tissue specificity. Half of genes related to mating signaling showed as the highest expression in the ST stage, indicating fruiting was initiated in the ST stage. CONCLUSIONS: These results provide a new perspective to understanding of the key pathways and hub genes, and sexual development-related gene profile in the development of Chinese cordyceps. It will be helpful for underlying sexual reproduction, and add new information to existing models of fruiting body development in edible fungi.

A breakthrough in the artificial cultivation of Chinese cordyceps on a large-scale and its impact on science, the economy, and industry.

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.

Classification, Biological Characteristics and Cultivations of Ganoderma.

Species of Ganoderma (Ling-zhi) have been widely researched and cultivated due to their highly prized medicinal value, which is famous as a traditional Chinese medicine. The aims of this chapter are to (1) review the historical taxonomy of the family Ganodermataceae, (2) provide an account of the genera and species of Ganoderma together with the distributions and habitats, (3) evaluate morphological features and phylogenetic methods to define the genera and species and (4) present two commonly used cultivated methods (wood-log cultivation and substitute cultivation) for Ganoderma.

Contrast-Enhanced Ultrasound Features of Hepatic Reactive Lymphoid Hyperplasia: Correlation With Histopathologic Findings.

OBJECTIVES: To find certain specifics of hepatic reactive lymphoid hyperplasia (HRLH) on contrast-enhanced ultrasound (CEUS) imaging as diagnostic imaging clues by retrospectively analyzing its enhancement features. METHODS: From June 2010 to June 2017, 18 histopathologically confirmed HRLH lesions in 18 patients were included in this retrospective study. The lesion's location, maximum diameter, shape, margin, echogenicity, and color flow signal on conventional ultrasound (US) imaging and enhancement pattern, presence of a feeding artery, and donutlike enhancement on CEUS imaging were observed and recorded. The lesion size on CEUS imaging at peak enhancement and that on conventional US imaging were compared and recorded. RESULTS: All of the lesions showed homogeneous hypoechogenicity with a regular well-defined margin on conventional US imaging, with a mean diameter ± SD of 14.3 ± 4.6 mm (range, 8-24 mm). On CEUS imaging, all of the lesions showed "quick-wash-in and quick-wash-out," which showed complete homogeneous hyperenhancement in the arterial phase and wash-out in the second half of the arterial phase or first half of the portal phase. In 83.3% (15 of 18) of the lesions, the lesion size that was enhanced at peak was enlarged compared with the hypoechoic area on conventional US imaging, and transient donutlike enhancement appeared when the lesion showed wash-out. In 55.6% (10 of 18) of cases, the feeding artery was detected. CONCLUSIONS: Enlarged complete homogeneous hyperenhancement in the arterial phase, consequently followed by quick wash-out of the lesion and the appearance of donutlike enhancement, may be the CEUS features of HRLH.

Artificial cultivation of true morels: current state, issues and perspectives.

Morels (Morchella, Ascomycota), which are some of the most highly prized edible and medicinal mushrooms, are of great economic and scientific value. Morel cultivation has been a research focus worldwide for more than 100 years, and the outdoor cultivation of morels has succeeded and expanded to a large scale in China in recent years. In this study, we review the progress in recent research regarding the life cycle and reproductive systems in the genus Morchella and the current state of outdoor cultivation. Sclerotia formation and conidia production are two important phases during the life cycle. The morel species cultivated commercially in America is M. rufobrunnea based on molecular phylogenetic analysis. The species currently cultivated in China are black morels, including M. importuna, M. sextalata and M. eximia. The field cultivation of morels expanded in the majority of the provinces in China with a yield of fresh morels of 0-7620 kg per ha. The key techniques include spawn production, land preparation and spawning, the addition of exogenous nutrition, fruiting management and harvesting. The application of exogenous nutrition is the most important breakthrough in the field of morel cultivation, but the mechanism remains unclear. It was estimated that the total amount of field cultivated fresh morels was ∼500 t in 2015-2016. We also discuss the potential issues remaining in the current literature and suggest directions for future studies.

Reactive oxygen species induce sclerotial formation in Morchella importuna.

Morels are some of the most highly prized edible and medicinal mushrooms, and the outdoor cultivation has been achieved in China in recent years. Sclerotial formation is one of the most important phases during the morel life cycle, and the number of sclerotia indicates the spawn quality during cultivation. However, the sclerotial formation and differentiation mechanisms are poorly understood. In this study, the sclerotial formation process of Morchella importuna and the effects of reactive oxygen species on scerotial formation were studied. Scerotial formation was defined as five distinctive phases, hypha early, hyphal growth, sclerotial initiation, development, and maturation. The mycelia in the sclerotium-forming area were swollen, darkened, and dense with sclerotial formation, but hydrogen peroxide accumulated in the region lacking sclerotial formation. The expression of all six genes for superoxide dismutases tested increased with sclerotial maturation. A difference in hydrogen peroxide concentration of 20 mM could promote the sclerotial initiation and induce expression of sod genes. The MAPK signaling pathway was activated, and they passed the signal from an area of high oxidative stress to a low area to initiate sclerotial formation. An understanding of the sclerotial formation mechanisms in M. importuna may help to understand the life cycle and facilitate the fruiting body cultivation.

DASH-type cryptochromes regulate fruiting body development and secondary metabolism differently than CmWC-1 in the fungus Cordyceps militaris.

Cryptochromes (CRYs) belong to the photolyase/cryptochrome flavoprotein family, which is widely distributed in all kingdoms. A phylogenetic analysis indicated that three Cordyceps militaris proteins [i.e., cryptochrome DASH (CmCRY-DASH), (6-4) photolyase, and cyclobutane pyrimidine dimer (CPD) class I photolyase] belong to separate fungal photolyase/cryptochrome subfamilies. CmCRY-DASH consists of DNA photolyase and flavin adenine dinucleotide-binding domains, with RGG repeats in a C-terminal extension. Considerably, more carotenoids and cordycepin accumulated in the ΔCmcry-DASH strain than in the wild-type or ΔCmwc-1 strains, indicating an inhibitory role for CmCRY-DASH in these biosynthetic pathways. Fruiting body primordia could form in the ΔCmcry-DASH strain, but the fruiting bodies were unable to elongate normally, differently from the Cmwc-1 disruption strain, where primordium differentiation did not occur. Cmcry-DASH expression is induced by light in the wild-type strain, but not in the ΔCmwc-1 strain. CmCRY-DASH is also necessary for the expression of Cmwc-1, implying that Cmcry-DASH and Cmwc-1 exhibit interdependent expression. The Cmvvd expression levels in the wild-type and ΔCmcry-DASH strains increased considerably following irradiation, while Cmvvd expression in the ΔCmwc-1 strain was not induced by light. It is speculated that the photo adaptation may be faster in the Cmcry-DASH mutant based on Cmvvd transcript dynamics. These results provide new insights into the biological functions of fungal DASH CRYs. Furthermore, the DASH CRYs may regulate fruiting body development and secondary metabolism differently than WC-1.

Functional convergence and divergence of mating-type genes fulfilling in Cordyceps militaris.

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 ΔMAT1-1-1 produced barren stromata in outcrosses, whereas ΔMAT1-1-2 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.