Synthesis, characterization, and cytotoxicity analysis of selenium nanoparticles stabilized by Morchella sextelata polysaccharide.

Natural bioactive molecules have been widely used as stabilizers in the functional improvement of selenium nanoparticles (SeNPs) in recent years. In this study, Morchella sextelata polysaccharide (MSP) was introduced as a novel stabilizer for the synthesis of SeNPs based on the redox system of sodium selenite and ascorbic acid. The size, morphology, stability, and anti-cancer cell activities were respectively analyzed by various methods. The results showed that the synthesized SeNPs with MSP were 72.07 ± 0.53 nm in size, red in color, spherical in shape, and amorphous in nature. MSP-SeNPs showed high scavenging activity against DPPH and ABTS radicals. And, these MSP-SeNPs exhibited a significant anti-proliferation effect on human liver (HepG2) and cervical cancer (Hela) cells in vitro, while no significant cytotoxicity against normal human kidney cells (HK-2) was observed. Moreover, the mitochondria-dependent apoptosis pathway triggered by MSP-SeNPs in HepG2 cell was identified. The expression levels of p53, Bax, cytochrome c, caspase-3 and caspase-9 were all up-regulated in HepG2 cells after MSP-SeNPs treatment, while Bcl-2 expression was down-regulated. These results suggest that MSP-SeNPs have strong potential as the food supplement for application in cancer chemoprevention.

Prevalence and diversity of mycoviruses occurring in Chinese Lentinula edodes germplasm resource.

Incidence and banding patterns of virus-like dsRNA elements in 215 Chinese genetically diverse Lentinula edodes strains collected from wide geographic distribution (or producing areas) were first investigated, and 17 viruses were identified including eight novel viruses. The results revealed a 63.3% incidence of dsRNA elements in the cultivated strains and a 67.2% incidence in the wild strains. A total of 10 distinguishable dsRNAs ranging from 0.6 to 12 kbp and 12 different dsRNA patterns were detected in the positive strains. The molecular information of these dsRNA elements was characterized, and the molecular information of the other 12 different viral sequences with (+) ssRNA genome was revealed in four L. edodes strains with complex dsRNA banding patterns. RT-PCR was also done to verify the five dsRNA viruses and 12 (+) ssRNA ones. The results presented may enrich our understanding of L. edodes virus diversity, and will promote further research on virus-host interactions. IMPORTANCE: Viral infections involve complicated interactions including benign, harmful or possibly beneficial to hosts. Sometimes environment could lead to a transition in lifestyles from persistent to acute, resulting in a disease phenotype. The quality of spawn, such as the vulnerability to infection of viruses, is therefore important for mushroom production. Lentinula edodes, a wood rot basidiomycete fungus, was widely cultivated in the world for its edible and medicinal properties. In this study, the profile of dsRNA elements from Chinese genetically diverse L. edodes strains collected from wide geographic distribution or producing areas was first investigated. The molecular information of the dsRNA elements was characterized. Additionally, 12 different viral sequences with (+) ssRNA genome from four L. edodes strains with complex dsRNA banding patterns were identified. The results presented here will broaden our knowledge about mushroom viruses, and promote further studies of L. edodes production and the interaction between viruses and L. edodes.

Genome Sequencing Highlights the Plant Cell Wall Degrading Capacity of Edible Mushroom Stropharia rugosoannulata.

The basidiomycetous edible mushroom Stropharia rugosoannulata has excellent nutrition, medicine, bioremediation, and biocontrol properties. S. rugosoannulata has been widely and easily cultivated using agricultural by-products showing strong lignocellulose degradation capacity. However, the unavailable high-quality genome information has hindered the research on gene function and molecular breeding of S. rugosoannulata. This study provided a high-quality genome assembly and annotation from S. rugosoannulata monokaryotic strain QGU27 based on combined Illumina-Nanopore data. The genome size was about 47.97 Mb and consisted of 20 scaffolds, with an N50 of 3.73 Mb and a GC content of 47.9%. The repetitive sequences accounted for 17.41% of the genome, mostly long terminal repeats (LTRs). A total of 15,726 coding gene sequences were putatively identified with the BUSCO score of 98.7%. There are 142 genes encoding plant cell wall degrading enzymes (PCWDEs) in the genome, and 52, 39, 30, 11, 8, and 2 genes related to lignin, cellulose, hemicellulose, pectin, chitin, and cutin degradation, respectively. Comparative genomic analysis revealed that S. rugosoannulata is superior in utilizing aldehyde-containing lignins and is possible to utilize algae during the cultivation.

Curing two predominant viruses occurring in Lentinula edodes by chemotherapy and mycelial fragmentation methods.

Previous research has established that Lentinula edodes mycovirus HKB (LeV-HKB) and L. edodes partitivirus 1(LePV1) are major mycoviruses identified in L.edodes germplasm. In this paper, two different methods for curing these two dsRNA mycoviruses, ribavirin treatment and mycelial fragmentation, were evaluated for the first time. Mycelial fragmentation was found to resulted in LeV-HKB- and LePV1-cured fungal strains, whereas ribavirin treatment could eliminate LeV-HKB only. Although no LePV1-cured strain was obtained via ribavirin treatment by the end of the experiment, the relative LePV1 concentration in the eighth successive subcultures was lower than that of the untreated control. The culture features of several virus-cured strains had faster mycelial growth rate and higher colony density than the infected ones. It was also suggested that LeV-HKB infection may affect the pigmentation in plate- and bag-cultivated mycelia of L. edodes strain L135.

Genome-wide identification of target genes for transcription factor BR-C in the silkworm, Bombyx mori.

Transcription factor Broad Complex (BR-C) is an ecdysone primary response gene in insects and participates in the regulation of insect growth and development. In this study, we performed a genome-wide identification of BR-C target genes in silkworm (Bombyx mori) using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). As a result, a total of 1006 BR-C ChIP peaks were identified, and 15% of peaks were located in the promoter regions of 133 protein-coding genes. Functional annotation revealed that these ChIP peak-associated genes, as potential BR-C targets, were enriched in pathways related to biosynthetic process, metabolic process, and development. Transcriptome analysis and quantitative real-time polymerase chain reaction (PCR) examination revealed that developmental changes in expression patterns of a portion of potential BR-C targets, including HR96 and GC-α1, were similar to those of BR-C. ChIP-PCR examination confirmed that BR-C could directly bind to the promoters of potential targets. Further, dual luciferase assays demonstrated that HR96 promoter activity was significantly upregulated following BR-C overexpression, and this upregulation was abolished when the binding motif in the promoter was truncated. This study will be helpful for deciphering the regulatory roles of BR-C during insect growth and development.

Biological Characterization and Antagonist Screening of Cladosporium anthropophilum, a Novel Pathogen Causing Stipe Black Rot on Commercial Medicinal Mushroom, Flammulina filiformis (Agaricomycetes).

We report a new fungal disease, named stipe black rot, in a cultivation factory of Flammulina filiformis (an edible mushroom cultivated worldwide) in China. The pathogen was identified as Cladosporium anthropophilum by phylogenetic analysis and morphology characterization. C. anthropophilum was characterized to mainly infect the stipe bottom and cause stipe blackening and rot, with its optimal mycelial growth conditions consisting of 25°C, pH 7, and carbon and nitrogen sources of soluble starch and sodium nitrate, respectively. Furthermore, inhibitory evaluation showed that hydrogen peroxide silver disinfectant (HPSD) can efficiently inhibit the mycelial growth of C. anthropophilum, followed by the aqueous extracts of garlic and onion. This study identified C. anthropophilum as the pathogen for the new F. filiformis black rot disease and HPSD as an effective antagonist against the pathogen, which facilitates the understanding of fungal diseases and their control in edible mushrooms.

Mycoviral diversity and characteristics of a negative-stranded RNA virus LeNSRV1 in the edible mushroom Lentinula edodes.

Bioinformatics and RT-PCR analysis of RNA from four Lentinula edodes samples identified 22 different virus-like contigs comprising 15 novel and 3 previously reported viruses. We further investigated the Lentinula edodes negative-stranded RNA virus 1 (LeNSRV1) isolated from a symptomatic sample, whose virion is a filamentous particle with a diameter of ~15 nm and a length of ~1200 nm. RT-PCR analysis detected LeNSRV1 in 10 of the 56 Chinese L. edodes core collection strains and 6 of the 22 monokaryotic strains from the L. edodes strain HNZMD. Genetic variation analysis showed that the sequences encoding the nucleocapsid protein (ORF2) from all the aforementioned LeNSRV1 positive strains are very conservative. The results presented here may enrich our understanding of L. edodes virus diversity and the characteristics of LeNSRV1, and will promote further research on virus-host interaction in L. edodes.

Identification and Characterization of the Anillin Gene in Silkworm.

Anillin is an actin binding protein and plays crucial roles during mitotic cell cycle progression in metazoan. However, the sequence and functions of the Anillin gene have not been yet characterized in the silkworm, Bombyx mori. In this study, we cloned the full-length cDNA sequence of the silkworm Anillin (BmAnillin) gene. The deduced amino acid sequence for BmAnillin protein comprises an Anillin homology region (AHR) covering an Anillin homology domain and a pleckstrin homology domain. Phylogenetic analysis and multiple alignments of the Anillin genes from silkworm and other organisms indicated evolutionary conservation in the AHR containing conserved phosphorylation sites. Reverse transcription-PCR experiments confirmed that the BmAnillin gene was highly expressed during larval development of gonads in which cells undergo mitotic cycles and exhibited an unexpected high expression in silk gland with endocycle during larval molting. RNA interference-mediated knockdown of the BmAnillin gene in silkworm BmN4-SID1 cells derived from ovary disrupted chromosome separation and resulted in a loss of the F-actin filament at cleavage furrow during anaphase, suggesting that the BmAnillin gene is essential for cytokinesis in silkworm.

Transcription factor E93 regulates wing development by directly promoting Dpp signaling in Drosophila.

Transcription factor E93 is a steroid hormone ecdysone early response gene and plays crucial roles in both the degradation of larval tissues and the formation of adult organs during insect metamorphosis with the prepupal-pupal-adult transition. However, the molecular mechanism underlying E93 regulation is poorly understood. In this study, we found that specific knockdown of the E93 gene in the Drosophila wing disrupted wing development. Analyzing ChIP-seq signals for E93 in Drosophila wing identified that the decapentaplegic (Dpp) gene was a potential downstream target of E93. ChIP-PCR analysis and dual-luciferase reporter assay confirmed that E93 could bind to the Dpp promoter and enhanced its activity. Furthermore, the expressions of Dpp and other components in the Dpp signaling pathway were upregulated following E93 overexpression in Drosophila S2 cells but were decreased after E93 knockdown in the wing. Moreover, the impairment of the Dpp signaling pathway phenocopied the defects of E93 knockdown on wing development. Taken together, our results suggest that E93 modulates the Dpp signaling pathway to regulate wing development during Drosophila metamorphosis.

Transcriptional Changes on Blight Fruiting Body of Flammulina velutipes Caused by Two New Bacterial Pathogens.

A blight disease of Flammulina velutipes was identified with symptoms of growth cessation of young fruiting bodies, short stipe, and brown spots on the pileus. The pathogenic bacteria were identified as Arthrobacter arilaitensis and Pseudomonas yamanorum by Koch's postulate, gram staining, morphological and 16S ribosomal RNA gene sequence analyses. Either of the pathogenic bacteria or both of them can cause the same symptoms. Transcriptome changes in blighted F. velutipes were investigated between diseased and normal samples. Compared to the control group, 1,099 differentially expressed genes (DEGs) were overlapping in the bacteria-infected groups. The DEGs were significantly enriched in pathways such as xenobiotic metabolism by cytochrome P450 and tyrosine metabolism. Based on weighted correlation network analysis (WGCNA), the module most correlated to the pathogen-treated F. velutipes samples and candidate hub genes in the co-regulatory network were identified. Furthermore, a potential diseased mechanism involved in cell wall non-extension, phenolic substrate oxidation, and stress defense response was proposed based on the up-regulation of differentially expressed genes encoding chitin deacetylase, tyrosinase, cytochrome P450, MFS transporter, and clavaminate synthase-like protein. This study provides insights into the underlying reactions of young fruiting body of F. velutipes suffering from blight disease and facilitates the understanding of the pathogenic procedure of bacteriosis in edible mushrooms.

Comparative Transcriptome Analysis Provides Novel Insight into Morphologic and Metabolic Changes in the Fat Body during Silkworm Metamorphosis.

The fat body plays key roles in energy storage and utilization as well as biosynthetic and metabolic activities in insects. During metamorphosis from larva to pupa, the fat body undergoes dramatic changes in morphology and metabolic processes. However, the genetic basis underlying these changes has not been completely understood. In this study, the authors performed a time-course transcriptome analysis of the fat body during silkworm metamorphosis using RNA-sequencing. A total of 5217 differentially expressed genes (DEGs) were identified in the fat body at different developmental time points. DEGs involved in lipid synthesis and degradation were highly expressed at the third day of the last larval instar and during the prepupal-pupal transition, respectively. DEGs involved in the ecdysone signaling and bone morphogenetic protein (BMP) signaling pathways that modulate organ development exhibited a high expression level during the fat body remodeling process from prepupa to pupa. Intriguingly, the RNA interference-mediated knockdown of either decapentaplegic (Dpp) or protein 60A (Gbb), two DEGs involved in the BMP signaling pathway, inhibited fat body dissociation but promoted lipid mobilization, suggesting that the BMP signaling pathway not only is required for fat body remodeling, but also moderately inhibits lipid mobilization to ensure an appropriate lipid supply during the pupal-adult transition. In conclusion, the comparative transcriptome analysis provides novel insight into morphologic and metabolic changes in the fat body during silkworm metamorphosis.

Genetic variation and phylogenetic analyses reveal transmission clues of Lentinula edodes partitivirus 1 (LePV1) from the Chinese L. edodes core collection.

Lentinula edodes partitivirus 1 (LePV1), a new mycovirus possibly responsible for serious morphological deformities during L. edodes cultivation, is widespread in the Chinese L. edodes germplasm. Here, LePV1 isolates from the Chinese genetically-diverse L. edodes core collection were identified to be highly conserved and devoid of codivergence between virus and its hosts. Phylogenetic analysis showed that the LePV1 isolates could be grouped into two distinct clades (subtype I and subtype II), without geographical bias in the composition of this population. Compared with the other LePV1 isolates, one non-synonymous variation was observed in the LePV1 isolate from the symptomatic strain SX12. Purifying selection appears to be the main evolutionary force acting on LePV1 and it may be subject to negative selection. Based on the aforementioned results, the domestication history of L. edodes in China and the high frequency of virus incidence in basidiospores, we postulate that LePV1 may exist in nature and have had relationship with L. edodes wild strains since early times. Moreover, wind-blown spores carrying LePV1 may play an important role for the transmission of LePV1 in nature, while artificial activities such as vegetative propagation and hybridization breeding may also transmit virus from wild strains to cultivated ones.

First report of a cross-kingdom pathogenic bacterium, Achromobacter xylosoxidans isolated from stipe-rot Coprinus comatus.

Coprinus comatus is an edible mushroom widely cultivated in China as a delicious food. Various diseases have occurred on C. comatus with the cultivated area increasing. In this study, the pathogenic bacterium JTG-B1, identified as Achromobacter xylosoxidans by 16S rDNA and nrdA gene sequencing, was isolated from edible mushroom Coprinus comatus with serious rot disease on its stipe. A. xylosoxidans has been confirmed as an important opportunistic human pathogenic bacterium and has been isolated from respiratory samples from cystic fibrosis. It is widely distributed in the environment. Here, we first report that fungi can also serve as a host for A. xylosoxidans. We confirmed that it can cross-kingdom infect between animals (mice) and fungi (C. comatus). The results of pathogenicity tests, physiological, biochemical and genotyping analysis of A. xylosoxidans from different hosts suggested that different strain of A. xylosoxidans may have pathogenicity differentiation. A. xylosoxidans not only is pathogenic to C. comatus but also may threaten human health.

Biological and Molecular Characteristics of a Novel Partitivirus Infecting the Edible Fungus Lentinula edodes.

A new partitivirus named Lentinula edodes partitivirus 1 (LePV1) was isolated from a diseased L. edodes strain with severe degeneration of the mycelium and imperfect browning in bag cultures. The nucleotide sequences of LePV1 dsRNA-1 and dsRNA-2 were determined; they were 2,382 bp and 2,245 bp in length, and each contained a single ORF encoding RNA-dependent RNA polymerase (RdRp) and coat protein (CP), respectively. The purified virus preparation contained isometric particles 34 nm in diameter encapsidating these dsRNAs. Phylogenetic analyses showed LePV1 to be a new member of Betapartitivirus, with the RdRp sequence most closely related to Grapevine partitivirus. RT-PCR analysis showed that 27 of the 56 Chinese L. edodes core collection strains carry LePV1, with the virus being more common in wild strains than cultivated strains. In addition, qPCR analysis suggested that coinfection with L. edodes mycovirus HKB (LeV-HKB) could increase replication of the RdRp gene of LePV1. This study may be essential for the development of more accurate disease diagnostics and the formulation of control strategies for viral diseases in L. edodes.

Genome Sequence of the Edible Cultivated Mushroom Lentinula edodes (Shiitake) Reveals Insights into Lignocellulose Degradation.

Lentinula edodes, one of the most popular, edible mushroom species with a high content of proteins and polysaccharides as well as unique aroma, is widely cultivated in many Asian countries, especially in China, Japan and Korea. As a white rot fungus with lignocellulose degradation ability, L. edodes has the potential for application in the utilization of agriculture straw resources. Here, we report its 41.8-Mb genome, encoding 14,889 predicted genes. Through a phylogenetic analysis with model species of fungi, the evolutionary divergence time of L. edodes and Gymnopus luxurians was estimated to be 39 MYA. The carbohydrate-active enzyme genes in L. edodes were compared with those of the other 25 fungal species, and 101 lignocellulolytic enzymes were identified in L. edodes, similar to other white rot fungi. Transcriptome analysis showed that the expression of genes encoding two cellulases and 16 transcription factor was up-regulated when mycelia were cultivated for 120 minutes in cellulose medium versus glucose medium. Our results will foster a better understanding of the molecular mechanism of lignocellulose degradation and provide the basis for partial replacement of wood sawdust with agricultural wastes in L. edodes cultivation.

Diversity and effect of Trichoderma spp. associated with green mold disease on Lentinula edodes in China.

Lentinula edodes, one of the most important edible mushrooms in China, is affected heavily by the infection of green mold that overgrows mushroom mycelia. We collected the diseased samples from main L. edodes cultivation regions in China to characterize the pathogen and to study the effect of Trichoderma spp. on L. edodes species. We identified six Trichoderma species, that is, T. harzianum, T. atroviride, T. viride, T. pleuroticola, T. longibrachiatum, and T. oblongisporum based on the internal transcribed spacer or tef1-α sequences and morphology characteristics. In confrontation cultures on Petri plates or in tubes, and in L. edodes cultures in a medium containing Trichoderma metabolites, L. edodes mycelia were not only distorted and swollen, but also inhibited by Trichoderma isolates. It is not possible that adjusting pH value or temperature is used for controlling L. edodes green disease, because the growth of most of Trichoderma isolates and L. edodes shared similar pH and temperature conditions.