Effect of Environmental Conditions on Synnema Formation and Nucleoside Production in Cicada Flower, Isaria cicadae (Ascomycetes).

Isaria cicadae (syn. Cordyceps cicadae) is one of the most valued edible and medicinal fungi and has been used in Asia as a substitute for Ophiocordyceps sinensis. Wild I. cicadae is limited and seasonal, and its cultivation is deserved. In this investigation we studied synnema formation by and nucleoside production in cicada flower under different environmental conditions. I. cicadae produced an asexual structure and mitospores instead of meiotic ascospores; this indicates that the term "synnema" is more suitable than "fruiting body" for this species. The optimal temperature was 25°C for growth of I. cicadae mycelia on potato dextrose agar plates but was 20°C for synnema formation on wheat medium. Synnemata can grow well under blue, green, and white light, and the dry weight of samples grown under these 3 light wavelengths is not significantly different. However, neither primordia nor synnemata formed under red light. Blue light promotes conidia production and white light promotes N6-(2-hydroxyethyl)-adenosine (HEA) production. Weak white light at 50 and 150 lux was more suitable for synnema production than strong-intensity light at 850 lux. The growth curve showed that HEA content has the same trend as synnema production over the entire cultivation period. The optimal harvesting time for I. cicadae cultivated on wheat medium is 35 days after inoculation. HEA content in the synnemata cultivated on wheat medium under the optimal conditions was significantly higher than that of the wild species and of synnemata cultivated on pupae, suggesting that synnemata cultivated on wheat medium may have potential as a substitute for wild resources. The results presented herein provide a new strategy for producing superior-quality synnemata of I. cicadae and further elucidate the effects of environmental conditions on metabolite accumulation in fungi.

Identification, Optimization of Culture Conditions, and Bioactive Potential of Chinese Caterpillar Mushroom Ophiocordyceps sinensis (Ascomycetes) Mycelium Isolated from Fruiting Body.

The present study deals with the challenges acquainted with in vitro culture of Ophiocordyceps sinensis. We have optimized the culture conditions for the growth of O. sinensis mycelium in semi-synthetic liquid media and determined antibacterial potential of the cultured mycelia extracts. In this study, mycelia were isolated from fruiting bodies and the isolate was identified as O. sinensis anamorph based on sequencing of internal transcribed spacer region. We investigated different culture conditions to optimize the growth of mycelia. Through this investigation, the isolated strain was observed to have its optimum growth at temperature (20°C), which yielded biomass of 12.38 g/L and pH (6.0) yielded biomass of 11.24g/L. Further to augment the production of mycelia, different carbon and nitrogen sources were optimized for mycelium growth in liquid media, out of which sucrose and corn steep powder proved to be the best carbon and nitrogen sources yielding biomass 14.01 g/L and 14.14 g/L, respectively. The evaluation of aqueous and methanolic extracts for antibacterial activity depicted that these extracts are active against all bacterial strains tested here. Aqueous extract depicted minimum inhibitory concentration (MIC) of 0.312, 0.019, 0.078, 0.312, and 0.625 mg/mL and methanolic extract depicted 1.25, 0.078, 0.009, 1.25, and 0.156 mg/mL against Pseudomonas aeruginosa, Escherichia coli, Bacillus cereus, Staphylococcus aureus, and Listeria monocytogenes, respectively. These results led to optimization of enhanced biomass production of O. sinensis, which can be a better alternative approach for further physiological studies and large-scale cultivation of this mushroom for its utilization for therapeutics and nutraceutical values.

Addition of Zeolites to Improve the Functional Characteristics of the Hen of the Wood or Maitake Medicinal Mushroom, Grifola frondosa (Agaricomycetes).

Kaolinite and the modified natural zeolite minazel plus (M+) were used as supplements in substrate used for the production of the medicinal mushroom Grifola frondosa. Growth stimulation, expressed as yield and biological efficiency, was observed when M+ (1%) was added. The production cycle was shortened by half as a result of the zeolites' ion-exchange ability, stimulation of enzyme activity, and water retain capacity. Inductively coupled plasma optical emission spectrometry of fruiting bodies showed the absence of heavy metals (arsenic, cadmium, and lead), whereas the concentration of calcium increased greatly and the concentrations of iron, magnesium, and zinc increased moderately under the influence of M+. Solid-state nuclear magnetic resonance showed a positive impact on the ß-glucan ratio, which could have been caused by the epimerization reaction stimulated by zeolites. The functionality of the mushroom was evaluated through several antioxidant activity assays, and in all cases a positive effect was established: M+ was statistically more effective in comparison with kaolinite. A strong correlation was established between the antioxidative activity of cultivated fruiting bodies and the tested compounds (total phenolic compounds, carbohydrates, and minerals).

Effects of Illumination Pattern during Cultivation of Fruiting Body and Bioactive Compound Production by the Caterpillar Medicinal Mushroom, Cordyceps militaris (Ascomycetes).

We investigated the effects of light intensity in the 3 cultivation stages separately-the mycelium colonization stage, the primordial initiation stage, and the fruiting stage (in order)-on fruiting body and bioactive compound production by Cordyceps militaris. In the mycelium colonization stage, rice substrates were incubated in a spawn running room at 23°C. During the primordial initiation stage, C. militaris was grown at 18°C and illuminated 12 hours/day. In the fruiting stage the temperature was 23°C, with illumination provided 12 hours/day. The highest fruiting body yield and biological efficiency were 4.06 g dry weight/bottle and 86.83%, respectively, under 1750 ± 250 lux during the second and third stages. The cordycepin content was highest during the second and third stages under 1250 ± 250 lux. The mannitol and polysaccharide contents were highest under 1250 ± 250 and 1750 ± 250 lux during the primordial initiation stage and the fruiting stage, respectively. Thus, with controlled lighting, C. militaris can be cultivated in rice-water medium to increase fruiting body yield and bioactive compound production.

Various grain substrates for the production of fruiting bodies and bioactive compounds of the medicinal caterpillar mushroom, Cordyceps militaris (Ascomycetes).

In this study, several grains such as brown rice (Br), plumule rice (Pr), wheat (W) and pearl barley (Pb) supplemented with 1% (w/w) peptone (P), yeast extract (Ye), ammonia sulfate (As), and monosodium glutamate (Mg) as a nitrogen source, respectively, were used to produce fruiting bodies and bioactive compounds of two strains of Cordyceps militaris. Among these grain substrates, the substrate most suitable to mycelial growth was Pb+Ye for C. militaris H and L. The mushroom strains colonized this substrate in 12.8 and 12.6 days, respectively. For C. militaris L, the fewest days were required for primordial initiation on Br+Ye and Pr+P substrates. The highest yield and biological efficiency was observed with Pb substrate (25.16 g/bottle and 87.36%) and Br+P substrate (21.84 g/bottle and 75.83%) for C. militaris H and L, respectively. In the fruiting bodies of C. militaris H, the highest cordycepin content was cultivated on W+Mg substrate (25.07 mg/g), the highest mannitol content was cultivated with Pr+Mg (153.21 mg/g) and Pr (151.65 mg/g) substrates, and the highest adenosine content was cultivated with Pr+Ye (0.94 mg/g) and Pb+Ye (0.90 mg/g) substrates. In the fruiting bodies of C. militaris L, the highest cordycepin content was cultivated with W+Mg substrate (22.14 mg/g); the highest mannitol content was cultivated with Pb substrate (189.33 mg/g); and the highest adenosine content was cultivated with Pb+Ye substrate (0.71 mg/g).

Elucidating the inhibitory mechanisms of the ethanolic extract of the fruiting body of the mushroom Antrodia cinnamomea on the proliferation and migration of murine leukemia WEHI-3 cells and their tumorigenicity in a BALB/c allograft tumor model.

The aim of this study was to explore whether the ethanolic extract of Antrodia cinnamomea (EEAC), a medical mushroom form Taiwan, could affect the proliferation and migration of WEHI-3 cells in vitro and to explore the antitumor effects of EEAC in BALB/c mice engrafted with WEHI-3 cells. The results showed that EEAC inhibited the proliferation of WEHI-3 cells, resulting in the accumulation of cell in G0/G1 and G2/M phases, as determined by flow cytometry. Moreover, EEAC markedly reduced the migration of WEHI-3 cells, as determined by a transwell assay. Treatment of WEHI-3 cells with EEAC also decreased MMP-9 protein expression and enzyme activity. The protein levels of p-Akt, p-ERK1/2 were also decreased, whereas the expression of p21 and p27 was increased. Furthermore, in an in vivo model, EEAC treatment reduced the infiltration of WEHI-3 cells into the liver and spleens and decreased tumor growth. Other bioactive compounds, such as cordycepin and zhankuic acid A, have been demonstrated to reduce the expression of MMP-9, cyclin E, cyclin D1 and to increase the expression of p21, p27. This is the first study to investigate that the mechanisms by which EEAC reduce the proliferation and migration of WEHI-3 cells in vitro, as well as the ability of EEAC to reduced infiltration of WEHI-3 cells into the liver and spleen in vivo. The results suggest that EEAC may prove to be useful in future antileukemic therapies.

Association of various fruiting body macromorphological traits with spore yield in Ganoderma Lingzhi (higher Basidiomycetes), a new medicinal mushroom from China.

To study the correlations and relationship between spore yield and various macromorphological traits of Ganoderma lingzhi, a field experiment was conducted in a randomized block design with 3 replications. Ten macromorphological traits, including pileus diameter, pileus crust thickness, context thickness, tube thickness, pileus thickness, stipe length, stipe diameter, stipe weight, pileus weight, and spore yield, were recorded for all of the tested strains. There was significant variation among the strains for all of the traits studied. The results indicated that the highest variation was observed in spore yield and pileus weight. Correlation studies revealed that among 9 macromorphological traits, only the pileus weight of the fruiting body was significantly positively correlated with spore yield (r2 = 0.674*). Pileus diameter showed significant positive association with pileus weight of fruiting body (r2 = 0.838*). Stepwise multiple regression analysis showed that pileus weight and spore yield had a linear relationship (spore yield = -21.95 + 1.51 * pileus weight). The coefficient of determination of stepwise regression analysis (r2 = 0.4543) revealed 45.43% variation in the spore yield because of its relationship with pileus weight. Regression coefficient (b = 1.51) showed that a unit (1 g) increase in the pileus weight of the fruiting body resulted in a proportional increase of 1.51 g in spore yield. The derived information would be very useful when selecting potentially breeding strains for future G. lingzhi improvement programs.