Variations of SSU rDNA group I introns in different isolates of Cordyceps militaris and the loss of an intron during cross-mating

Cordyceps militaris, the type species of genus Cordyceps, is one of the most popular mushrooms and a nutraceutical in eastern Asia. It is considered a model organism for the study of Cordyceps species because it can complete its life cycle when cultured in vitro. In the present study, the occurrence and sequence variation of SSU rDNA group I introns, Cmi.S943 and Cmi.S1199, among different isolates of C. militaris were analyzed. Based on the secondary structure predictions, the Cmi.S943 intron has been placed in subgroup IC1, and the Cmi.S1199 intron has been placed in subgroup IE. No significant similarity between Cmi.S943 and Cmi.S1199 suggested different origins. Three genotypes, based on the frequency and distribution of introns, were described to discriminate the 57 surveyed C. militaris strains. It was found that the genotype was related to the stroma characteristics. The stromata of all of the genotype II strains, which possessed only Cmi.S943, could produce perithecium. In contrast, the stromata of all genotype III strains, which had both Cmi.S943 and Cmi.S1199, could not produce perithecium. Cmi.S1199 showed the lowest level of intra-specific variation among the tested strains. Group I introns can be lost during strain cross-mating. Therefore, we presumed that during cross-mating and recombination, intron loss could be driven by positive Darwinian selection due to the energetic cost of transcribing long introns.     

Preservation affects the vegetative growth and fruiting body production of <Emphasis Type="Italic">Cordyceps militaris</Emphasis>

Cordyceps militaris is a model species of Cordyceps fungi, and has been traditionally used as an edible and medicinal fungus due to its richness of bioactive and pharmacological metabolites. The fruiting bodies of this fungus are widely used as healthy food and nutrition supply. In industrial production, fruiting bodies are cultivated on artificial media, but their yield and quality are usually affected by the quality of fungal strains. In this study, the effect of colony growth rate of the fungal strains, fungal age and repeated subculturing on the fungal biomass accumulation was investigated. The results indicated that the fungal biomass was positively correlated with the colony growth rate and not affected by fungal age and the repeated subculturing. The preservation conditions for stock cultures, including choice of cultures, lyophilization, temperature and protective agents were optimized based on the mycelial formation and conidia production in artificial inoculum. The development of fruiting bodies from the fungal strains stored under the optimized preservation conditions were further analyzed to determine the ideal time period of preservation. Results indicated that storing the fungus at 4 °C could maintain the fungal vitality and fruiting body producing capacity for at least 12 months. This study established practical criteria of fungal inoculum for artificial cultivation of fruiting body and provided a simple and efficient preservation method for C. militaris. The results may shed light on preservation for other Cordyceps species and other edible fungi.     

The medicinal fungus Cordyceps militaris: research and development

Ophiocordyceps sinensis (syn. Cordyceps sinensis) is a highly valued medicinal fungus. This entomopathogen has a limited distribution, has been overharvested in the wild, and its stromata have not been artificially cultivated. Another entomopathogenic fungus, Cordyceps militaris (commonly known as orange caterpillar fungus), has chemical capacities similar to those of O. sinensis, but unlike O. sinensis, its stromata can be easily cultivated. Consequently, C. militaris is being studied as an alternative to O. sinensis, and the large-scale production of stromata is receiving substantial attention. Significant research has been conducted on the genetic resources, nutritional and environmental requirements, mating behavior, and biochemical and pharmacological properties of C. militaris. The complete genome of C. militaris has recently been sequenced. This fungus has been the subject of many reviews, but few have focused on its biology. The current paper reviews the biological aspects of the fungus including host range, mating system, cytology and genetics, insect- and non-insect nutritional requirements, environmental influence on stroma development, and commercial development.     

Optimization of culture conditions and medium components for the production of mycelial biomass and exo-polysaccharides with Cordyceps militaris in liquid culture

Both crude exo-biopolymers and mycelial biomass, produced by liquid culture of Cordyceps species, are believed to possess several potential health benefits. As a result of its known biological activities, Cordyceps militaris has been extensively characterized in regards to potential medicinal applications. However, optimized liquid culture conditions for enhanced polysaccharide productivity have yet to be developed, which is a necessary step for industrial applications. Therefore, in this study, the liquid culture conditions were optimized for maximal production of mycelial biomass and exo-polysaccharide (EPS) by C. militaris. The effects of medium composition, environmental factors, and C/N ratio were investigated. Among these variables 80 g, glucose; 10 g, yeast extract; 0.5 g, MgSO₄·7H₂O; and 0.5 g, KH₂PO₄ in 1 L distilled water were found to be the most suitable carbon, nitrogen, and mineral sources, respectively. The optimal temperature, initial pH, agitation, and aeration were determined to be 24°C, uncontrolled pH, 200 rpm, and 1.5 vvm, respectively. Under these optimal conditions, mycelial growth in shake flask cultures and 5 L jar bioreactors was 29.43 and 40.60 g/L, respectively, and polysaccharide production in shake flask cultures and 5 L jar bioreactors was 2.53 and 6.74 g/L, respectively.     

Structure and mating system of the graminicolous fungal epibiont Echinodothis tuberiformis (Clavicipitales)

The developmental morphology and mating system of the epibiont Echinodothis tuberiformis is investigated. The structure of conidial stromata, conidia, perithecial stromata, asci, and ascospores is documented. Crosses involving transfer of conidia between stromata resulted in formation of perithecia, indicating a heterothallic mating system. Comparisons are made to other members of the Clavicipitales that are presently classified in the tribe Balansieae. It is suggested that this epibiont bears a closer relationship to the grass endophytes classified in the genus Epichloë (Fr.) Tul. than to several other members of the Balansieae.     

Local epizootics caused by teleomorphic cordycipitoid fungi (<Emphasis Type="Italic">Ascomycota: Hypocreales</Emphasis>) in populations of forest lepidopterans and sawflies of the summer-autumn complex in Siberia

Unique epizootic loci were revealed in the summer-autumn complex of the multispecies communities of forest lepidopterans and sawflies in two regions of Novosibirsk oblast and in the Baikal region at the border of the Buryat Republic and Irkutsk oblast. Mass insect mortality was caused by two species of cordycipitoid fungi: Cordyceps militaris and Cordyceps sp. At least 30 species from 7 families of millers (Macroheterocera) and sawflies from the family Cimbicidae were found to be C. militaris hosts. Lepidopterans from the family Thyatiridae are the hosts of the second species, Cordyceps sp. Total mortality was noted for the condition close to optimal for pupation, i.e., in places of mass pupa accumulation. It may be assumed on the basis of the results of the laboratory experiments that, under natural conditions, host insects may be infected by ascospores and conidia at the anamorphic stage.     

Some properties ofSaccharomyces kluyveri

Four isolates of aSaccharomyces species which differed fromS. kluyveri by their ability to use cellobiose were analyzed genetically in relation to the latter species. Isolated single spores had low viability. Spore tetrads segregated mating types 2 2, with sexual agglutination occurring between complementary mating types. All single-spore isolates assimilated cellobiose indicating that these isolates were not naturally occurring hybrids betweenS. kluyveri and a cellobiose assimilatingSaccharomyces species.Two cell types were exhibited by single-spore cultures ofS. kluyveri, one granulated (G-type) and one vacuolated (g-type). G-type cultures formed fertile hybrids with complementary mating types of both G- and g-type cultures. Hybrids between two g-type cultures were sterile. They would, however, give fertile hybrids when mixed with G-type cultures.Sporulating hybrids betweenSaccharomyces sp. andS. kluyveri were produced. However the percentage spore germination was low. Single-spore cultures examined had cell types atypical of either parent. The ability to assimilate cellobiose was dominant and appeared to segregate with mating type and cell type.Weak mating reactions occurred when the (+) and (-) mating types ofSaccharomyces sp. were mixed with (a) and () mating types ofS. cerevisiae, respectively.The species ofSaccharomyces isolated from the Pacific Coast are designated as strains ofS. kluyveri.     

Agglutinative mating types ofSaccharomyces transvaalensis

Agglutinative mating types ofSaccharomyces transvaalensis have been recovered from the type strain of this species. These mating types react with the corresponding opposite mating types obtained from two other strains of this species but do not react with one-spore cultures of the physiologically similar species,Saccharomyces dairensis. The dissimilarity of these two species is further confirmed by differences in the structure of their ascospores as observed in ultrathin sections by electron microscopy.     

Biotechnological production and applications of Cordyceps militaris,a valued traditional Chinese medicine

Cordyceps militaris is a potential harborer of biometabolites for herbal drugs. For a long time, C. militaris has gained considerable significance in several clinical and biotechnological applications. Much knowledge has been gathered with regard to the C. militaris's importance in the genetic resources, nutritional and environmental requirements, mating behavior and biochemical pharmacological properties. The complete genome of C. militaris has recently been sequenced. This fungus has been the subject of many reviews, but few have focused on its biotechnological production of bioactive constituents. This mini-review focuses on the recent advances in the biotechnological production of bioactive compositions of C. militaris and the latest advances on novel applications from this laboratory and many others.     

“Pollination” of a fungus by a fly

Summary The mechanism resulting in fertilization of Epichloë typhina, a heterothallic ascomycete that is an endophytic pathogen of grasses, has now been discovered. Conidia of one mating type are produced in stromata and are then transferred by insects to individuals of the opposite mating type. One insect, Phorbia phrenione, is a particularly important vector of conidia. Once conidia of the opposite mating type have been transferred to a stroma, the life cycle continues with the formation of perithecia.     

A new species of endophytic Balansia from Veracruz, Mexico

A new graminicolous species of Clavicipitaceae, Balansia brunnans sp. nov., has been found to infect Panicum xalapénse. Staining of living host tissues indicates the presence of intercellular endophytic mycelium. Stromata develop just below the nodes on the culms. Balansia brunnans is comparable to Balansia aristidae, B. discoidea, B. gaduae, B. nigricans, and B. strangulans in development of stromata on culms and possession of an endophytic mycelial stage. Among the differences between Balansia brunnans and other comparable species is that it possesses a brown perithecial stroma, whereas comparable species have black perithecial stromata. A key is provided to distinguish B. brunnans from similar species.     

Pichia angophorae,sp.n. from the exudate of an Australian Red Gum tree

A new species ofPichia has been described.Pichia angophorae sp.n. was the only yeast present in an exudate ofAngophora costata collected near North Ryde, N. S. W., Australia. The new species exists in both the homo- and heterothallic state. Heterothallic mating types have been isolated as segregants from originally homothallic cultures.     

A strain of Chaetomium olivaceum

A strain ofChaetomium isolated from the aerial plant parts of Polish-type rape has been tentatively classified asC. olivaceum. Frequentin situ germination of perithecial hair and their occasional branching was shown. A re-evaluation of the use-fulness of characters of perithecial hair and development of other taxonomic concepts in the genusChaetomium is advocated.Contribution, Alberta Department of Agriculture, Plant Industry Laboratory, Edmonton, Alberta.     

野生蛹虫草菌株培养特性及子实体多糖含量比较分析

蛹虫草(Cordyceps militaris)是一种药食两用真菌,为获得高产优质的菌种资源,以采集分离的5株蛹虫草野生菌株(XY002、XY008、XY011、XY029、XY032)为研究对象,通过对5株菌株的分子鉴定、交配型基因分子检测、培养特性及子实体多糖含量测定,确定5株菌株皆为蛹虫草菌株,除XY008只含有MAT1-1-1交配型基因外,其他菌株都含有两种交配型基因MAT1-1-1和MAT1-2-1,5株菌株在菌丝生长速度、分生孢子数量、子实体形态、产量及子实体多糖含量均存在较大差异。综合培养特性及多糖含量分析的结果表明,菌株XY011子实体产量较高达29.60 g/瓶,多糖含量最高达100.79 mg/g,子实体长度最长达11.41 cm,而且子实体粗壮,发育周期短,确定为优势菌株,具有较好的开发价值。     

Hybridization between Japanese and North American Chlamydomonas reinhardtii (Volvocales,Chlorophyceae)

The microalga Chlamydomonas reinhardtii is a model organism whose whole genome has been sequenced. Although considered a cosmopolitan species, only eastern North American isolates of C. reinhardtii were available before 2010, when new Japanese isolates were reported. In the study describing the new Japanese isolates, zygote formation between Japanese and North American strains was shown, but germination was not demonstrated. In this study, the germination of intercontinental hybrid zygotes was examined using wild‐type Japanese strains and mutant American strains that cannot utilize nitrate. Several clonal progeny strains were established, and the progeny strains were screened based on mating type and nitrate utilization to confirm their hybrid nature. The establishment of four intercontinental hybrid strains with different phenotypic combinations was confirmed by sequencing mating type‐specific and nitrate reductase‐related genes. The potential for hybrid formation between Japanese and North American strains suggests the existence of a worldwide mating population of C. reinhardtii.     

<Emphasis Type="Italic">In vitro</Emphasis> biosynthesis of antioxidants from <Emphasis Type="Italic">Hemidesmus indicus</Emphasis> R. Br. cultures

Summary Shoot cultures and callus cultures from roots and leaves of Hemidesmus indicus R. Br (Asclepiadaceae) were established on Murashige and Skoog medium with various hormonal combinations. The production of antioxidants (lupeol, vanillin, and rutin) in shoot cultures, callus cultures derived from leaf cells and root cells, was compared with root and aerial portions of the parent plant. Shoot cultures and leaf callus cultures produced more antioxidants than root callus cultures. In vitro culture of this species might ofter an alternative method for production of these important pharmaccuticals, which would reduce the collection pressure on this rare plant.     

Chaunopycnis pustulata sp. nov., a new clavicipitalean anamorph producing metabolites that modulate potassium ion channels

Two morphologically similar strains of hyphomycetous fungi, MF5785 and MF5638, produced potent indole diterpene antagonists of the calcium-gated potassium ion channel, Maxi-K, and a diterpene blocker (nalanthalide) of the voltage-gated potassium channel, Kv 1.3, respectively. The two strains were tentatively identified in the literature as Nalanthamala species. Analyses of their secondary metabolite profiles by HPLC and mass spectroscopy demonstrated that both produced a series of indole diterpenes, of which at least one was produced by both strains. Another strain, Chaunopycnis alba (MF6799), that produced the diterpene, nalanthalide, also produced indole diterpene metabolites. Morphological comparisons and phylogenetic studies based on 28S and ITS rDNA sequences indicated that MF5785 and MF5368, and another soil-derived strain GB6597, belonged to a monophyletic clade of the Clavicipitaceae that included the anamorph Chaunopycnis alba and several Cordyceps and Tolypocladium species. A new species of Chaunopycnis is therefore proposed to accommodate MF5785, MF5368, and GB6597. The possible synonymy of Albophoma yamanashiensis with C. alba also is discussed. Within the Clavicipitaceae, indole diterpenoid compounds have only been known from the graminicolous species (subfamily Clavicipitoideae); therefore delineation of a Chaunopycnis clade has revealed a previously unrecognized lineage of indole diterpene-producing fungi among the subfamily Cordycipitoideae.