Metabolomic variation in wild and cultured cordyceps and mycelia of Isaria cicadae.

Isaria cicadae is one of the fungi used in traditional Chinese medicine with the longest tradition. It is used not only as a herbal medicine but also as a health food in Asia, together with cultured cordyceps and mycelia of the fungus used as substitute. However, the differences in their metabolite are unknown. Using a high-performance liquid chromatography-mass spectrometry (HPLC-MS)-based metabolomic method, we found that the fungus varies in its metabolism during growth on wild insects, artificially raised insects and artificial medium. There were 109 discriminatory metabolites detected in the samples by orthogonal projection to latent structure discriminant analysis and one-way ANOVA. High level of nonribosomal peptides (NRPs) only existed in the insect portions of the wild cordyceps (WI) and cultured cordyceps (CI), revealing that immunostimulation of the host insects enhanced the synthesis of NRPs in the fungus. The finding of a significantly higher level of sphingolipids in both the insect portions (WI, CI) and the coremia of the wild cordyceps (WC) and cultured cordyceps (CC) but not in cultured mycelia (CM) of I. cicadae implies that the immunostimulation of the live insects can induce the fungus to produce more sphingolipids, and this enhanced ability is probably heritable. Apart from NRPs and sphingolipids, the insect portions also contained higher levels of bioactive compounds such as lateritin, anisomycin, streptimidone and ustiloxins. In contrast, the coremium groups (WC, CC) and CM contained 10-fold less NRP but much higher levels of sanative metabolites such as tocotrienol, 3'-deoxy-hanasanagin, γ-aminobutyric acid and phospholipids than the insect portions. The significantly higher content of antioxidants in WC, CC and CM than in WI and CI suggests that environmental oxygen has a significant effect on the metabolites. The temperature stress which the wild cordyceps encounters during growth is responsible for the relatively high content of trehalose. These findings indicate that the immunity of the host insect and growth environment have a strong impact on the metabolomic variation in Isaria cicadae. The variation in metabolites suggests differential utilization value for the insect portions, coremia and mycelia of the fungus.

Omics data reveal the unusual asexual-fruiting nature and secondary metabolic potentials of the medicinal fungus Cordyceps cicadae.

BACKGROUND: Ascomycete Cordyceps species have been using as valued traditional Chinese medicines. Particularly, the fruiting bodies of Cordyceps cicadae (syn. Isaria cicadae) have long been utilized for the treatment of chronic kidney disease. However, the genetics and bioactive chemicals in this fungus have been largely unexplored. RESULTS: In this study, we performed comprehensive omics analyses of C. cicadae, and found that, in contrast to other Cordyceps fungi, C. cicadae produces asexual fruiting bodies with the production of conidial spores instead of the meiotic ascospores. Genome sequencing and comparative genomic analysis indicate that the protein families encoded by C. cicadae are typical of entomopathogenic fungi, including the expansion of proteases and chitinases for targeting insect hosts. Interestingly, we found that the MAT1-2 mating-type locus of the sequenced strain contains an abnormally truncated MAT1-1-1 gene. Gene deletions revealed that asexual fruiting of C. cicadae is independent of the MAT locus control. RNA-seq transcriptome data also indicate that, compared to growth in a liquid culture, the putative genes involved in mating and meiosis processes were not up-regulated during fungal fruiting, further supporting asexual reproduction in this fungus. The genome of C. cicadae encodes an array of conservative and divergent gene clusters for secondary metabolisms. Based on our analysis, the production of known carcinogenic metabolites by this fungus could be potentially precluded. However, the confirmed production of oosporein raises health concerns about the frequent consumption of fungal fruiting bodies. CONCLUSIONS: The results of this study expand our knowledge of fungal genetics that asexual fruiting can occur independent of the MAT locus control. The obtained genomic and metabolomic data will benefit future investigations of this fungus for medicinal uses.

Genetic diversity and population structure of the Chinese Fungus Metarhizium rileyi causing green muscardine in silkworm.

Green muscardine caused by Metarhizium rileyi affects sericulture, and is typically enzootic and occurs frequently at low incidence. We collected 152 M. rileyi isolates from silkworm cadavers in eight sericulture areas in seven provinces of China, and four strains from other Lepidoptera larvae in Qianshan(QS) County, Anhui province. Nine microsatellite inter-simple sequence repeat (ISSR) primers produced 91 distinct and reproducible bands, revealing a high level (90.11%) of DNA polymorphism. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) cluster analysis divided the populations into four groups, with isolates from Qianshan County forming a single branch. All the 156 M. rileyi isolates were heterogenic and polyphyletic and did not displayed typical regional distribution except strains from Qianshan country. PCA analysis of the nine populations of M. rileyi revealed similar phylogenies among accessions. Genetic differentiation index (Gst) among eight enzootic populations was 0.3789 and gene flow (Nm) was 0.4098, suggesting the low gene flow maintained a high degree of differentiation. Gst between the enzootic population of Qianshan County and other seven populations exceeded the threshold of severe differentiation, with moderate differentiation between the remaining seven enzootic populations. Analysis of molecular variance (AMOVA) showed most ISSR variation (61%) among isolates occurred within populations. No significant correlation was observed between geographical and genetic distance. According to cluster analysis based on single enzootic population, every enzootic population showed dominance, namely mainly constituted of strains with high genetic similarity. These data indicated that the green muscardine in each local silkworm population was predominantly caused by a native group of M. rileyi. Furthermore, Gst and Nm of M. rileyi from silkworm and other Lepidoptera larvae in Qianshan County were 0.1174 and 1.8791, respectively, suggesting strains isolated from different hosts in Qianshan County do not show obvious host specificity. This demonstrated that host transfer may take place in silkworm and other insects.

Metabolic responses of Beauveria bassiana to hydrogen peroxide-induced oxidative stress using an LC-MS-based metabolomics approach.

The entomopathogenic fungus, Beauveria bassiana, is commonly used as a biological agent for pest control. Environmental and biological factors expose the fungus to oxidative stress; as a result, B. bassiana has adopted a number of anti-oxidant mechanisms. In this study, we investigated metabolites of B. bassiana that are formed in response to oxidative stress from hydrogen peroxide (H2O2) by using a liquid chromatography mass spectrometry (LC-MS) approach. Partial least-squares discriminant analysis (PLS-DA) revealed differences between the control and the H2O2-treated groups. Hierarchical cluster analysis (HCA) showed 18 up-regulated metabolites and 25 down-regulated metabolites in the H2O2-treated fungus. Pathway analysis indicated that B. bassiana may be able to alleviate oxidative stress by enhancing lipid catabolism and glycometabolism, thus decreasing membrane polarity and preventing polar H2O2 or ROS from permeating into fungal cells and protecting cells against oxidative injury. Meanwhile, most of the unsaturated fatty acids that are derived from glycerophospholipids hydrolysis can convert into oxylipins through autoxidation, which can prevent the reactive oxygen of H2O2 from attacking important macromolecules of the fungus. Results showed also that H2O2 treatment can enhance mycotoxins production which implies that oxidative stress may be able to increase the virulence of the fungus. In comparison to the control group, citric acid and UDP-N-acetylglucosamine were down-regulated, which suggested that metabolic flux was occurring to the TCA cycle and enhancing carbohydrate metabolism. The findings from this study will contribute to the understanding of how the molecular mechanisms of fungus respond to environmental and biological stress factors as well as how the manipulation of such metabolisms may lead to selection of more effective fungal strains for pest control.

Molecular tracing of white muscardine in the silkworm, Bombyx mori (Linn.) II. Silkworm white muscardine is not caused by artificial release or natural epizootic of Beauveria bassiana in China.

The fungal pathogen Beauveria bassiana causes serious economic losses in sericulture. Its origin is usually attributed to the release of B. bassiana insecticides against pine caterpillars (Dendrolimus punctuatus). In the present study, 488 B. bassiana isolates obtained from silkworm (Bombyx mori) collected from 13 Chinese provinces, and 327 B. bassiana isolates obtained from D. punctatus collected from 9 provinces, were analyzed for population genetic structure using the ISSR technique based on genetic distance. A UPGMA dendrogram clustered them into three independent clades: two B. mori clades and one D. punctatus clade. A 3-D principal component analysis further divided them into two completely independent host groups, revealing high host-specificity. This suggested that white muscardine occurring in B. mori populations throughout southern China was not caused by any B. bassiana strain either naturally prevailing in D. punctatus populations or by any strain artificially released as a fungal insecticide against D. punctatus. We further investigated the genetic differentiation coefficient Gst and gene flow between B. mori-pathogenic and D. punctatus-pathogenic B. bassiana isolates from across China and from five provinces inhabited by both B. mori and D. punctatus. The Gst value across China was computed as 0.410, while the values of the five provinces ranged from 0.508 to 0.689; all above 0.25, which is the threshold for significant genetic differentiation. This suggests that B. bassiana strains isolated from the two different hosts maintained their respective heredity without a convergent homogenization trend, and reduces the possibility that the host range of the caterpillar isolates could expand and enhance their virulence in B. mori. These findings indicate that the use of B. bassiana does not threaten the safety of sericulture.

Differential metabolic responses of Beauveria bassiana cultured in pupae extracts, root exudates and its interactions with insect and plant.

Beauveria bassiana is a kind of world-wide entomopathogenic fungus and can also colonize plant rhizosphere. Previous researches showed differential expression of genes when entomopathogenic fungi are cultured in insect or plant materials. However, so far there is no report on metabolic alterations of B. bassiana in the environments of insect or plant. The purpose of this paper is to address this problem. Herein, we first provide the metabolomic analysis of B. bassiana cultured in insect pupae extracts (derived from Euproctis pseudoconspersa and Bombyx mori, EPP and BMP), plant root exudates (derived from asparagus and carrot, ARE and CRE), distilled water and minimal media (MM), respectively. Principal components analysis (PCA) shows that mycelia cultured in pupae extracts and root exudates are evidently separated and individually separated from MM, which indicates that fungus accommodates to insect and plant environments by different metabolic regulation mechanisms. Subsequently, orthogonal projection on latent structure-discriminant analysis (OPLS-DA) identifies differential metabolites in fungus under three environments relative to MM. Hierarchical clustering analysis (HCA) is performed to cluster compounds based on biochemical relationships, showing that sphingolipids are increased in BMP but are decreased in EPP. This observation further implies that sphingolipid metabolism may be involved in the adaptation of fungus to different hosts. In the meantime, sphingolipids are significantly decreased in root exudates but they are not decreased in distilled water, suggesting that some components of the root exudates can suppress sphingolipid to down-regulate sphingolipid metabolism. Pathway analysis finds that fatty acid metabolism is maintained at high level but non-ribosomal peptides (NRP) synthesis is unaffected in mycelia cultured in pupae extracts. In contrast, fatty acid metabolism is not changed but NRP synthesis is high in mycelia cultured in root exudates and distilled water. This indicates that fungal fatty acid metabolism is enhanced when contacting insect, but when in the absence of insect hosts NRP synthesis is increased. Ornithine, arginine and GABA are decreased in mycelia cultured in pupae extracts and root exudates but remain unchanged in distilled water, which suggests that they may be associated with fungal cross-talk with insects and plants. Trehalose and mannitol are decreased while adenine is increased in three conditions, signifying carbon shortage in cells. Together, these results unveil that B. bassiana has differential metabolic responses in pupae extracts and root exudates, and metabolic similarity in root exudates and distilled water is possibly due to the lack of insect components.

Morphology and multigene phylogeny reveal three new species of Samsoniella (Cordycipitaceae, Hypocreales) from spiders in China.

The genus Samsoniella was erected based on orange cylindrical to clavate stromata, superficial perithecia and conidiophores with Isaria-like phialides and to segregate them from the Akanthomyces group. In this study, based on morphological features and multigene (SSU, LSU, TEF, RPB1 and RPB2) phylogenetic analysis six Samsoniella species parasitizing spiders were collected in China. Three of them belong to known species S.alpina, S.erucae and S.hepiali. Three new species S.anhuiensissp. nov., S.araneasp. nov. and S.fusiformisporasp. nov. are illustrated and described. They are clearly distinct from other species in Samsoniella occurring in independent subclades. Furthermore, among the four insect-pathogenic fungi specimens collected from similar sites, three of them were identified as the new species described below. Our study significantly broadens the host range of Samsoniella from Insecta to Arachnida, marking a noteworthy expansion in understanding the ecological associations of these fungi. Additionally, the identification of both mononematous and synnematous conidiophores in our study not only expands the knowledge of Samsoniella species but also provides a basis for future research by comparing the ecological significance between these conidiophore types. In conclusion, our study enhances the understanding of Samsoniella diversity, presenting a refined phylogenetic framework and shedding light on the ecological roles of these fungi in spider parasitism.

Genetic diversity of the fungal pathogen Metarhizium spp., causing epizootics in Chinese burrower bugs in the Jingting Mountains, eastern China.

Based on the internal transcribed spacer and inter-simple sequence repeats (ISSR), the phylogenetic relationship and genetic diversity of Metarhizium spp., pathogens found in Chinese burrower bugs, Schiodtella formosana, were analyzed. The results showed that the causative agents of the epizootic green muscardine disease in populations of S. formosana were actually composed of M. anisopliae and its sister species, M. robertsii. The genetic structure of Metarhizium spp. populations were assessed using ten ISSR. A 3D principal component analysis of 51 isolates sampled on different occasions revealed that the Metarhizium spp. populations were temporally heterogeneous. They differentiated into two main clades including over 71 % of all strains causing epizootics, with a similarity of 83 %. The population differentiation was relatively low (G ( ST ), 0.2080), reflecting a large proportion of gene differentiation (79.2 %) within the populations. Further knowledge of the complex species and heterogeneous populations of Metarhizium spp. may be necessary for sustainable control methods of S. formosana.

Integration of Untargeted Metabolomics with Transcriptomics Provides Insights into Beauvericin Biosynthesis in Cordyceps chanhua under H2O2-Induced Oxidative Stress.

Cordyceps chanhua is an important cordycipitoid mushroom widely used in Asia and beyond. Beauvericin (BEA), one of the bioactive compounds of C. chanhua, has attracted much attention because of its medicinal value and food safety risk. In order to clear up the relationship between oxidative stress and BEA synthesis, we investigated the impact of H2O2-induced oxidative stress on the secondary metabolism of C. chanhua using untargeted metabolomics and a transcript profiling approach. Metabolic profiling of C. chanhua mycelia found that in total, 73 differential metabolites were identified, including organic acids, phospholipids, and non-ribosomal peptides (NRPs), especially the content of BEA, increasing 13-fold under oxidative stress treatment. Combining transcriptomic and metabolomic analyses, we found that the genes and metabolites associated with the NRP metabolism, especially the BEA biosynthesis, were highly significantly enriched under H2O2-induced stress, which indicated that the BEA metabolism might be positive in the resistance of C. chanhua to oxidative stress. These results not only aid in better understanding of the resistance mechanisms of C. chanhua against oxidative stress but also might be helpful for molecular breeding of C. chanhua with low BEA content.

Identification of the molecular origin and development of a panzootic caused by Beauveria bassiana in praying mantis populations in eastern China.

A panzootic in praying mantid species Tenodera sinensis and Statilia maculate, caused by Beauveria bassiana, occurred in north, southwest and southeast regions of Anhui Province, eastern China in Autumn, 2009. A 3-d principal component analysis (PCA) of 123 isolates from three sites revealed that the B. bassiana populations were heterogeneous with obvious dominance. Furthermore, the causal source of the panzootic in Anhui was shown to be polyphyletic. The populations were homogenized into homogenous subunits for investigation of genetic structure by inter-simple sequence repeat (ISSR) markers. Variance was greater than 70%, largely due to genetic differences within populations and subpopulations. Genetic distances and genetic differentiation were negatively associated with geographic distances and it was speculated that this was due to the effects of monsoons and topography. Mantid isolates were divided into five pathotypes based on a two-way cluster analysis of genetic distance. Pathotype I consisted of the predominant subpopulations of Huangcangyu and Chashui populations, with a genetic distance of 0.120 and gene flow up to 1.833. This pathotype caused a widespread epizootic in north and southwest Anhui, and Pathotype III caused enzootic at Site A in September and then epizootic in October, while the other three pathotypes caused enzootics at all three investigation sites. The widespread epizootics and isolated enzootics composed the polyphyletic panzootic in Anhui. A strong gene flow between isolates from the two mantid species was identified, resulting in negligible gene differentiation. This indicated a lack of host specificity in mantid isolates of B. bassiana.

Long-term preservation, regeneration, and cultivation of Paecilomyces tenuipes (Peck) Samson (Ascomycetes), an entomopathogenic fungus inoculated into the silkworm larva of Bombyx mori.

Paecilomyces tenuipes reportedly have anticancer and immune activities, along with various other medicinal uses. Cultured products with P. tenuipes are certified for use in food in South Korea, and processed goods containing this fungus have been developed in many countries, particularly South Korea, Japan, and China. Research on mass production technology-procured raw materials for the manufacture of P. tenuipes is very important; however, cultures of the fungus have been unstable. This study identified stable cultivation conditions, focusing on growth inhibition and revitalization. Moisture regulation and preservation of pupae inoculated with P. tenuipes were used to control growth inhibition and revitalization. When inoculated silkworm pupae were dehydrated to 4% moisture and preserved freeze-dried or at -70 degrees C, -20 degrees C, or 4 degrees C, the mycelia in their bodies were able to survive for 14 d. Inoculated silkworm pupae were rehydrated for 3 h and the mycelia within their bodies were recovered at 94.3-96.3%. Silkworm pupae at 4% moisture were able to survive for 135 d at temperatures < 4 degrees C and for 1 y after freeze-drying. Optimal conditions for synnemata induction were 25 degrees C and 100-300 1x.

Population genomics and evolution of a fungal pathogen after releasing exotic strains to control insect pests for 20 years.

Entomopathogenic fungi are one of the key regulators of insect populations in nature. Some species such as Beauveria bassiana with a wide host range have been developed as promising alternatives to chemical insecticides for the biocontrol of insect pests. However, the long-term persistence of the released strains, the effect on non-target hosts and local fungal populations remains elusive, but they are considerable concerns with respect to environmental safety. Here we report the temporal features of the Beauveria population genomics and evolution over 20 years after releasing exotic strains to control pine caterpillar pests. We found that the isolates within the biocontrol site were mostly of clonal origins. The released strains could persist in the environment for a long time but with low recovery rates. Similar to the reoccurrence of host jumping by local isolates, the infection of non-target insects by the released strains was evident to endemically occur in association with host seasonality. No obvious dilution effect on local population structure was evident by the releases. However, the population was largely replaced by genetically divergent isolates once per decade but evolved with a pattern of balancing selection and towards expansion through adaptation, non-random outcrossing and isolate migration. This study not only unveils the real-time features of entomopathogenic fungal population genomics and evolution but also provides added values to alleviate the concerns of environmental safety regarding the biocontrol application of mycoinsecticides.

[Anti-tumor compounds from submerger fermentation products of a Paecilomyces militaris strain].

OBJECTIVE: The aim was to identify the antitumor compounds of the fermentation broth of Paecilomyces militaris (strain RCEF0927). METHODS: Anti-tumor activity was tested with a resazurin cytotoxicity assay model with Chinese hamster ovary (CHO) cells. Bioactive compounds were analysed with combined method of High Performance Liquid Chromatography (HPLC), High Resolution Mass Spectrometer (HRMS) and antitumor assay. RESULTS: Antitumor test showed that the fermentation broth had strong cytotoxicity against CHO cells. Extraction tests showed that ethylacetate was the best solvent for the bioactive constituents extracting. HPLC-DAD- HRMS-Cytotoxicity assay revealed that the molecular formula of the active compounds in the extract were possibly C10 H13 N5 O3, C22 H22 O9, C23 H24 O10, C22 H22 O10, C15 H10 O4, C15 H10O, C36 H70 O11, C36 H68 O12, C38 H74 O11, C40 H76 O13 and C44 H82 O14. CONCLUSION: The possible structures of the bioactive compounds were deduced with bioassay, HRMS, Uv and database inquiry as cordycepin, daizein, genistein 7,4'-dihydroxyisoflavone-7-O-(4"-O-methyl)-b-D-glucopyranoside, 7, 4'-dihydroxy-6- methoxyisoflavone-7-O-( 4"-O-methyl)-b-D-glucopyranoside, 5, 7, 4'-trihydroxy-isoflavone-7-O-(4"-O-methyl)-b-D-glucopyranoside, polyoxyethylene ether oleate, dehydro products of polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, polyoxypropylene or polyoxybutene contained derivants of polyoxyethylene sorbitan fatty acid ester. Cytotoxicity of the compounds was revealed for the first time.

Insecticidal evaluation of Beauveria bassiana engineered to express a scorpion neurotoxin and a cuticle degrading protease.

To improve the insecticidal efficacy of the entomopathogen Beauveria bassiana, the fungus was genetically modified with an insect-specific scorpion neurotoxin AAIT and an insect cuticle degrading protease PR1A from another insect pathogen (Metarhizium anisopliae). The wild-type and the transformants were bioassayed against the larvae of Masson's pine caterpillar Dendrolimus punctatus and the wax moth Galleria mellonella. In comparison to the wild-type strain, engineered isolates took fewer spores to kill 50% of pine caterpillars, 15-fold less for the aaIT single transformant Bb13T and eightfold less for the double transformant Bb13TPR1A, respectively. The median lethal times for Bb13T and Bb13TPR1A were reduced by 40% and 36.7%, respectively against D. punctatus and 24.4% and 20.9%, respectively against G. mellonella. Our data showed that the cotransformation of these two genes produced no synergistic effects on virulence improvement. It is evident from this study that AAIT could be degraded by the protease PR1A when they are expressed together, emphasizing that protein interactions need to be evaluated when working with multiple genes, particularly if they include proteases.

[Analysis and isolation of radical scavengers from metabolites of Hirsutella sp].

OBJECTIVE: During a screening for free radical scavengers from metabolites of entomogenous fungi, we fond a fermentation broth of the strain RCEF 0881 of Hirsutella sp. exhibited strong radical scavenging activity. To make clear of the constituents of the active compounds, and prepare some pure active compound for further structure identification we launched this study. METHODS: We used organic solvent for active compounds extraction. DPPH-TLC and DPPH- Microplate assay were used for activity analysis. Components analysis was carried out on a HPLC-DAD-HRMS, and bioactive compound preparation on a preparative RP-HPLC. RESULTS: Our extraction tests showed that ethylacetate was the best solvent for the bioactive constituents extracting. HPLC-DAD-HRMS-DPPH assay revealed that the molecular formular of the radical scavengers existed in the extract were possibly C7H6O4, C8H8O3 and C12H14N2O. From the chromatographic and Uv properties, and the MS fragments, and database consulting, the compounds could be deduced as dihydroxybenzoic acid, methyl-hydroxyl benzoic acid, and an alkaloid, however, the structures are still needed to be confirmed. The pick area of HPLC and MS showed that the compound C12H14N2O was the main component of the extract. It was isolated via activity directed fractionation. The activity of the prepared compound was confirmed with DPPH-TLC assay and its purity was confirmed with HPLC-DAD-HR-ESIMS. The occurance of the three active compounds in entomogenous fungi was revealed for the first time.

[Genetic diversity of Beauveria bassiana (Bals.) Vuill. in forest ecosystem assessed by inter-simple sequence repeat (ISSR) markers].

In the present paper, the genetic diversity of 48 Beauveria bassiana strains from different altitudes and at different seasons in Dabie Mountains of western Anhui was estimated using inter-simple sequence repeat (ISSR) markers. Twelve among 33 ISSR primers were chosen for their reproducibility and high polymorphism. Seven (2 - 11) markers per primer were scored, and a total of 84 fragments were amplified, in which 73 (81%) were polymorphic. Genetic diversity analysis revealed a relatively high level of intraspecific genetic diversity of B. bassiana in Dabie Mountains of western Anhui: the percentage of polymorphic loci (PPL) was 81%, Nei's genetic diversity (He) was 0.3187 and Shannon's genetic diversity index (I) was 0.4782. The genetic differentiation, Gst was 0.1028, indicating that a low degree of genetic differentiation occurred in the B. Bassiana among populations.

Militarinone A, a neurotrophic pyridone alkaloid from Paecilomyces militaris.

[structure: see text] A new pyridone alkaloid, militarinone A (1), was isolated by bioassay-guided fractionation from the mycelium of the entomogenous fungus Paecilomyces militaris. Its structure was established by extensive spectroscopic analysis. The compound features an unprecedented side chain and a 1,4-substituted cyclohexyl moiety not previously encountered in microbial metabolites. Militarinone A had a pronounced neurotrophic effect in PC-12 cells at 10 microM concentrations.

New tyrosinase inhibitors from Paecilomyces gunnii.

Through screening 50 strains of entomopathogenic fungi and rescreening of 7 strains of Paecilomyces gunnii, a methanol extract of liquid-cultivated mycelia of P. gunnii was found to have the strongest tyrosinase inhibitory activity. Preparative high-speed counter-current chromatography (HSCCC) guided by high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-high-resolution mass spectrometry (HRMS) was employed for the isolation and purification of the active components, and three new compounds with half inhibition concentration (IC50) of 0.11, 0.17, and 0.14 mM against diphenolase were obtained from the extract, respectively. Their chemical structures were identified by HRMS, one- and two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy as paecilomycones A, B, and C. Structure and activity studies showed that the tyrosinase inhibition activities are positively related to the number of hydroxyl groups on the paecilomycones.

Comparison of cytotoxic extracts from fruiting bodies, infected insects and cultured mycelia of Cordyceps formosana.

A resazurin method was employed to test and compare cytotoxicity of extracts from fruiting bodies, insects and cultured mycelia of Cordyceps formosana against Chinese hamster ovary (CHO) cells. Results showed that the cultured mycelia had much stronger cytotoxicity than that of the fruiting bodies and infected insects. This suggests that using cultured mycelia to substitute a natural Cordyceps may result in poisoning. A combined method of HPLC-PAD-HRMS and cytotoxic analysis revealed that the most toxic compound (Compound 1) was found mainly in the cultured mycelia and also a small amount in the infected insect body of the Cordyceps, but not in the fruiting body. The second toxic compound (Compound 2) was found in all structures of Cordyceps and in cultured mycelia. Different contents of the toxic compounds resulted in the different cytotoxicity of the extracts. Compound 1 and Compound 2 were prepared with preparative HPLC as yellow and orange powders, respectively. Cytotoxic tests showed that the median lethal dose (LD50) against CHO cells of Compound 1 was 18.3 ± 0.2 and 103.7 ± 5.9 µg/mL for Compound 2. Compound 1 and Compound 2 were identified as rugulosin and skyrin by HRMS, UV and NMR data. The two compounds were never previously isolated from the genera Cordyceps and Hirsutella and their cytotoxicity against CHO cells was also reported for the first time.

Phylogenetic-based nomenclatural proposals for Ophiocordycipitaceae (Hypocreales) with new combinations in Tolypocladium.

Ophiocordycipitaceae is a diverse family comprising ecologically, economically, medicinally, and culturally important fungi. The family was recognized due to the polyphyly of the genus Cordyceps and the broad diversity of the mostly arthropod-pathogenic lineages of Hypocreales. The other two cordyceps-like families, Cordycipitaceae and Clavicipitaceae, will be revised taxonomically elsewhere. Historically, many species were placed in Cordyceps, but other genera have been described in this family as well, including several based on anamorphic features. Currently there are 24 generic names in use across both asexual and sexual life stages for species of Ophiocordycipitaceae. To reflect changes in Art. 59 in the International Code of Nomenclature for algae, fungi, and plants (ICN), we propose to protect and to suppress names within Ophiocordycipitaceae, and to present taxonomic revisions in the genus Tolypocladium, based on rigorous and extensively sampled molecular phylogenetic analyses. When approaching this task, we considered the principles of priority, monophyly, minimizing taxonomic revisions, and the practical utility of these fungi within the wider biological research community.