• Title/Summary/Keyword: Mycelium growth

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Studies on Protoplast Formation and Regeneration of Lyophyllum decastes (Lyophyllum decastes의 원형질체 분리와 재생에 관한 연구)

  • Bok, Jin-Woo;Kim, Jong-Pil;Jin, Mi-Rim;Choi, Eung-Chil;Kim, Byong-Kak
    • The Korean Journal of Mycology
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    • v.22 no.2
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    • pp.130-137
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    • 1994
  • This experiment was carried out to investigate proper conditions for protoplast isolation and regeneration from mycelia of Lyophyllum decastes. Novozym 234(10 mg/ml) with 0.6 M $MgSO_4$ in phosphate buffer(pH 4.0) was proper for protoplast isolation. The optimal reaction time of the mycelium with the lytic enzyme was four hours in shaking condition at 120 strokes per min. When the mycelium of L. decastes was cultured at $24^{\circ}C$ for 5 days, the formation of protoplasts was effective. The liquid medium was more effective for protoplast isolation than the solid medium. In the liquid medium, high yields of protoplasts were obtained from 0.6 M $MgSO_4$ osmotic stabilizer. Protoplasts of L. decastes were regenerated to normal hyphal growth and the regeneration frequency of the protoplasts in the complete agar medium containing Triton X-100(0.0025%) was $5.94{\sim}8.32%$. The regeneration medium stabilized with 0.6 M sucrose was the best for regeneration of the protoplasts. In contrast to protoplast formation, regeneration was inhibited by the inorganic salts used as osmotic stabilizer.

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Effect of Sterols on Phytophthora infestans and Oospore Production on detached Potato Plants (감자 역병균에 대한 스테롤류의 영향 및 감자절편에서의 난포자 형성)

  • 이왕휴;이용훈;이두구
    • Korean Journal of Plant Resources
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    • v.14 no.1
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    • pp.8-14
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    • 2001
  • The effects of media, cholesterol, $\beta$-sitosterol and lecithin on the growth and oospore production of the isolates KM10, U6, CDB6, MHB6, JD1 (A$^2$type) of Phytophthora infestans isolated in Korea and F8l7, DNC303 (A$^1$type), IB908, DN107 (A$^2$type) obtained from Japan were investigated. Mycelium of P. infestans grew better on V-8 juice agar and rye meal agar than on the other media. Oospores were produced most abundantly on V-8 juice agar. Mycelium extended more 16.6, 8.3, and 5.2% on V-8 juice agar supplemented with 5 $\mu\textrm{g}$/$m\ell$ of cholesterol, $\beta$-sitosterol and lecithin, respectively, and oospores are produced 76.0, 58.0, and 34.6 % on V-8juice agar supplemented with 5 $\mu\textrm{g}$/$m\ell$ of cholesterol, $\beta$-sitosterol and lecithin, respectively. Oospores more produced on detached potato plant disks when $A^1$ and $A^2$ type exist simultaneously which indicating that variation of population can occur in the field, but the rate of oospore formation and the number of oospores produced was low and small quantity.

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Some Factors Affecting the Protoplast Formation and Regeneration from the Mycelium of Ganoderma lucidum(Fr.) Karsten (영지(靈芝)버섯 균사(菌絲)의 원형질체(原形質體) 나출(裸出) 및 재생(再生))

  • Shin, Gwan Chull;Yeo, Un Hyung;Yoo, Young Bok;Park, Yong Hwan
    • Korean Journal of Agricultural Science
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    • v.13 no.2
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    • pp.185-192
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    • 1986
  • Some factors affecting the protoplast release from mycelia of Ganoderma lucidum and regeneration of the protoplast were investigated and the results obtained are summarized as follows; Novozym 234 as a lytic enzyme was the most effective for the protoplast release from mycelia of Ganoderma lucidu m and its optimal concentration was 10mg per ml of osmotic stabilizer. The highest number of protoplasts were released after 3 hours incubation in the reciprocal shaking bath at 120 oscillations a minute. Among six osmotic stabilizers tested, 0.6M sucrose showed the best result. SCM medium showed good mycelial growth and high yields of protoplasts. The protoplasts released from the mycelium of G. lucidum were regenerated at 0.20 to 0.27 percent on MCM, MMM and SCM. Of the cultures obtained from protoplasts regenerated, 13 to 29 percent were monokaryon.

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Asexual Stage and Fruit Formation of Cordyceps staphylinidaecola (유충노랑곰보동충하초(Cordyceps staphylindaecola)의 불완전세대와 자실체 형성)

  • Sung, Jae-Mo;Hong, Sung-Jun;Humber, R.A.;Spatafora, J.W.
    • The Korean Journal of Mycology
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    • v.31 no.1
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    • pp.1-7
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    • 2003
  • One hundred fifty one specimens of Beauveria spp. from 19 different locations were collected from September 1 to August 31, 2002. Most of the isolates were identified as Beauveria. bassiana. Cordyceps staphylinidaecola collected from Mt. Obong in Chunchon City covered the host with mycelia which were produced 1 to 4 stromata along with asexual spores. The size of bright yellow ununiform stromata were about 45 mm and the head about $17mm{\times}4mm$. Perithecia completely immersed were $530{\sim}550{\times}290{\sim}300{\mu}m$ in size and mainly scattered on the head. Ascospore produced in asci in the size of $400{\sim}450{\times}4{\sim}5{\mu}m$ developed thread-like secondary spores, which were directly separated into secondary conidial spores. Conidia produced at apical portion of synnemata were $2.6{\sim}3.4{\times}1.2{\sim}1.9{\mu}m$ in size. High density of mycelium was observed at $25^{\circ}C$ ranged from pH 6.5 to 8.5 after 11 days of inoculation. It took 15 to 18 days after inoculation to fully grow on the medium mixed brown rice with pupa. Mycelium developed stromata on the medium 30 days after completion of mycelial growth, where perithecia were produced in 40 days.

Antibacterial Activities of Cordyceps spp., Mugwort and Pine Needle Extracts (동충하초, 쑥 및 솔잎 추출물의 항균작용)

  • 박찬성;권충정;최미애;박금순;최경호
    • Food Science and Preservation
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    • v.9 no.1
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    • pp.102-108
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    • 2002
  • The purpose of this study was to investigate antibacterial activity of extracts of Cordyceps spp.(Paecilomyces japonica and Cordyceps militaris), mugwort and pine needle. Fruiting body and mycelium of Cordyceps spp., mugwort and pine needle were extracted with water and 70% ethanol. Antibacterial activities of each extracts against 3 kinds of Gram positive (Bacillus subtilis, Listeria monocytogenes and Staphylococcus aureus) and 3 kinds of Gram negative pathogenic bacteria(Escherichia coli O157 : H7, Shigella sonnei and Salmonella typhimurium) were tested. The yields of water and ethanol extracts of fruiting body (39∼58%) were 2.4 ∼4.4 times higher than mycellium(9∼24%) in Cordyceps sup., while those of mugwort and pine needle were less than 9%. Ethanol extract of P. japonica mycelium(JFE) had antibacterial to S. monocytogenes at 1% level and ethanol extract of C. militaris fruiting body (MFE) had antibacterial to S. aureus at 3% level. Ethanol extract of mugwort was antibacterial against L monocytogenes and S. aureus at 1% level. Water extracts of Cordyceps spp.(P. japonica and C. militaris) and mugwort had no antibacterial activity against tested bacterial strains. Water extract of pine needle had antibacterial activity against all bacterial strains except E. coli and ethanol extract had antibacterial activity against all tested bacterial strains at 1% level. Pine needle extracts had the most wide antibacterial spectrum against bacterial strains used for this experiment. Growth inhibiting activities of pine needle extracts were higher in ethanol extract than water extract for most of tested bacteria in tryptic soy broth.

Influence of Pesticides and Environmentally Friendly Agricultural Materials used in Tomato Cultivation on the Pathogenicity of the Entomopathogenic Fungus, Beauveria bassiana (토마토재배에 사용하는 농약과 친환경농자재가 곤충 병원성 곰팡이 Beauveria bassiana의 병원성에 미치는 영향)

  • Park, Jong-Ho;Hong, Sung-Jun;Han, Eun-Jung;Shim, Chang-Ki;Lee, Minho;Kim, Min-Jeong;Kim, JeongJun;Kim, Yong-Ki
    • Korean journal of applied entomology
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    • v.51 no.4
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    • pp.357-364
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    • 2012
  • This study was conducted to observe the influence of chemical pesticides and environmentally friendly agricultural materials (EFAMs) used in tomato cultivation on the pathogenicity of the entomopathogenic fungus, Beauveria bassiana. B. bassiana mycelium didn't grow on PDA media containing 13 fungicides including chlorothalonil and colonies were not formed on PDA media containing 12 fungicides. B. bassiana mycelium grew and colonies were formed on all PDA media containing insecticides and EFAMs, but mycelial growth and colony formation on most PDA media were significantly inhibited compared to the control. The insecticidal activity of B. bassiana against Trialeurodes vaporariorum was decreased when fungicides (polyoxin B, mandipropamid) and EFAMs containing sulfur were added, but insecticides (pyridaben, dinotefuran) and EFAMs originated from plant extracts did not have any influence on the insecticidal activity of B. bassiana. The pathogenicity of a mixture of B. bassiana and polyoxin B against T. vaporariorum was lower than that of B. bassiana alone under greenhouse conditions.

Development of Effective Screening Method for Resistance of oyster mushroom to Trichoderma disease in vitro (느타리버섯 푸른곰팡이병 저항성 실내검정 방법의 개발)

  • Jhune, Chang-Sung;Yun, Hyung-Sik;Lee, Chan-Jung;Kong, Won-Sik;Cheong, Jong-Chun;Jang, Kab-Yel
    • Journal of Mushroom
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    • v.9 no.3
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    • pp.110-115
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    • 2011
  • Trichoderma disease of oyster mushroom has not been effectively detected in the field for testing its resistance against the disease with its varieties. In this study, we investigated the methods to detect its resistance in the laboratory by using media, which enables us to understand the relevant characteristics (e.g., lysis, toxin enzyme, mycelial growth rate). In coculturing with strains of Trichoderma and oyster mushroom, it is possible to observe the difference in the resistance of oyster mushroom against Trichoderma with the phenomena of barrage reaction, overgrowth and lysis. We also observed the inhibition of mycelial growth of oyster mushroom using the dilution method with 48-well plate, but could not observed the inhibition of mycelial growth using the filter paper method of cultural supernatant. In simultaneously culturing both Trichoderma and oyster mushroom, it was possible to detect the inhibition of the mycelial growth of oyster mushroom, but Trichoderma mycelium did not overgrow against oyster mushroom. We found that the pathogenicity was efficient in using solid medium with the phenomena of overgrowth and lysis by inoculating Trichoderma on top of mycelia of oyster mushroom. In conclusion, the methods (e.g., coculture method, dilution method with 48-well plate, post-inoculation method) are recommended to detect the resistance of oyster mushroom against Trichoderma disease.

Characteristics of fruiting body growth according to alternative substrates of king oyster mushroom (Pleurotus eryngii) (큰느타리 대체배지 종류에 따른 자실체 생육 특성)

  • Hye-Sung, Park;Gyong-Jin, Min;Eun-Ji, Lee;Tai Moon, Ha
    • Journal of Mushroom
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    • v.20 no.4
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    • pp.274-278
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    • 2022
  • The present study aimed at selecting a cost-effective substrate for king oyster mushroom based on the growth characteristics of fruiting body for use as a basic resource to ensure stable production on farms. Compositional analysis of substrates manufactured with different materials in each process revealed that the total carbon content was 42.24-48.22% higher and the total nitrogen content was 1.7-2.29% higher in the processed lot than in the control (i.e., substrate used by the farmhouse; 40.86% and 1.39%, respectively). Meanwhile, the carbon-to-nitrogen ratio was the highest in the control (27.9% vs. 19.12-27.88% in the processed lot). When cultured for 28 days, the mycelium growth was 11.5 and 11.3 mm in substrates 1 and 6, respectively, indicating the fastest growth; meanwhile, the values were 10.1-10.3 mm in the control and substrate 11, showing a similar tendency. Mycelial density did not differ significantly among the processed lots. Yield per bottle was higher in substrates 8 (205.95 g/bottle), 7 (178.51 g/bottle), and 11 (170.63 g/bottle) than in the control (152.2 g/bottle). Fruiting body quality was comparable to controls in all processed lots. Overall, economic effects, such as substrate material prices, should be analyzed, and stability evaluations, such as residual pesticide and harmful microorganisms, should be undertaken along with further detailed examination to ensure safe and stable production on farms.

Growth-promoting effect on Tricholoma matsutake mycelium by Terrabacteria isolated from pine mushroom habitats in Korea (국내 송이 자생지에서 분리된 Terrabacteria에 의한 송이균사체 생장촉진 효과)

  • Doo-Ho Choi;Jae-Gu Han;Kang-Hyo Lee;Gi-Hong An
    • Journal of Mushroom
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    • v.21 no.3
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    • pp.190-193
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    • 2023
  • To cultivate pine mushroom (Tricholoma matsutake) artificially, co-cultivation with microorganisms has been introduced. Here, experiments were performed to assess the growth-promoting effect of bacteria on T. matsutake mycelia. Bacteria were isolated from soil samples collected in Yangyang County, Korea. Four of the bacterial isolates (Y22_B06, Y22_B11, Y22_B18, and Y22_B22) exhibited a growth-promoting effect on T. matsutake mycelia (154.67%, 125.91%, 134.06%, and 158.28%, respectively). To analyze the characteristics of the bacteria, especially the antifungal activity, 𝛼-amylase and cellulase activity assays were performed. In comparison with the controls, the isolated bacteria exhibited low 𝛼-amylase and cellulase activity. 16S rRNA gene sequencing was performed to identify the four bacterial isolates. The isolates belonged to the Terrabacteria group and were identified as Microbacterium paraoxydans, Paenibacillus castaneae, Peribacillus frigoritolerans, and P. butanolivorans. These bacterial isolates are expected to have contributed to the growth promotion of T. matsutake mycelia and the artificial cultivation of T. matsutake.

Sesquiterpenoids Bioconversion Analysis by Wood Rot Fungi

  • Lee, Su-Yeon;Ryu, Sun-Hwa;Choi, In-Gyu;Kim, Myungkil
    • 한국균학회소식:학술대회논문집
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    • 2016.05a
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    • pp.19-20
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    • 2016
  • Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

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