Utilization of Methionine by Mycelial Felts of Fusarium Culmorum

When the amino-acid L-methionine was used as the sole sourceof nitrogen in the culture medium it induced appreciable inhibitionof growth, respiration, and protein synthesis by mycelial matsof Fusarium culmorum. Such inhibitory effects increased with(a) lapse of time and (b) increase in concentration of methioninein the culture medium. The results of the experiments presented below support the viewthat methionine is first oxidatively deaminated before it istaken up by the mycelial mats of the fungus being tested.     

Tea extract as an inexpensive inducer of pectin lyase in Penicillium griseoroseum cultured on sucrose

Extracts of tea, coffee, cocoa, and yeast induced pectin lyase (PL) in Penicillium griseoroseum cultured in a mineral medium with sucrose as the carbon source. PL activity and fungal growth were similar in the treatments with 0.5% tea extract, the highest concentration tested, and 0.03% yeast extract. When tea extract was added singly to the culture medium, P. griseoroseum produced 59% and 17% of the PL activity and mycelial mass, respectively, obtained in a treatment with tea extract and sucrose. These results suggest that the production of the enzyme was not proportional to mycelial growth. No PL was produced in the medium with sucrose and without inducers. The small amounts of pectic substances present in the tea extract could not be responsible for PL induction. PL activity was detected after 12 h of growth in the medium containing sucrose and tea extract added at time zero, and after 48 h of growth when tea extract was added at times 12 and 24 h. Mycelial mass in all treatments was similar after 48 h of incubation. However, the addition of tea extract at time zero increased PL activity by 20–25%. Cyclic AMP at 5 and 10 mM in the culture medium induced 20 and 30%, respectively, of the PL activity obtained with 0.03% yeast extract, suggesting that PL induction brought about by either yeast extract or tea extract might involve the intracellular metabolism of cAMP. Received 22 October 1996/ Accepted in revised form 09 January 1997     

Influence of nutritional conditions on exopolysaccharide production by submerged cultivation of the medicinal fungus <Emphasis Type="Italic">Shiraia bambusicola</Emphasis>

Maltose and yeast extract were the most favourable carbon and nitrogen sources for exopolysaccharide production by submerged culture of Shiraia bambusicola WZ-003, and initial maltose and yeast extract concentrations were at 30 and 3 g l⁻¹, respectively. Plant oils could increase the mycelial growth and exopolysaccharide production in tested concentration. K⁺ and Mg²⁺ could enhance the mycelial growth and exopolysaccharide biosynthesis. The optimal cultivation temperature and initial pH were found to be 26°C and 6.0, respectively. Exopolysaccharide concentration reached 0.53 g l⁻¹ in 15-l fermenter under optimal nutritional conditions.     

Biochemical Studies on Streptomyces sioyaensis

Non-proliferating mycelium of Streptomyces sioyaensis was shown to form siomycin in phosphate buffer without addition of an energy source or precursors. This increase of siomycin in phosphate buffer was suppressed by glucose, acetate, l-cysteine, casamino acid, metals (Fe⁺⁺, Cu⁺⁺), various metabolic inhibitors, and antibiotics (chloramphenicol, erythromycin), whereas it was promoted by yeast extract, beef extract, l-isoleucine, Mg, etc.

The mechanism of the inhibitory effect of glucose on siomycin formation was investigated. Although glucose suppressed siomycin formation, it was the best carbon source for Streptomyces sioyaensis and vigorously metabolized to keto acids and other metabolites. Glucose suppressed siomycin formation by promoting cellular metabolism and mycelial growth. Siomycin formation was not only different from but also competitive to mycelial growth (cellular protein synthesis).     

The induction of mycelial development inAureobasidium pullulans (IMI 45533) by yeast extract

Germ tube and subsequent mycelial development from yeast-like and swollen cells ofAureobasidium pullulans (IMI 45533) was induced by yeast extract in defined liquid medium. This morphogenetic transition was dependent on inoculum size; pH effects were not involved and once mycelial development was induced in the cells it continued even in the absence of yeast extract. The progeny of mycelium and future generations were unaffected by yeast extract. Cessation of germination was not due to any obvious medium changes but appeared to be partly due to the production of a germination inhibitor, which could also be produced by control cells grown in the absence of yeast extract.     

Growth and sporulation of Metarrhizium anisopliae and Beauveria bassiana on media containing various peptone sources

Two entomogenous fungi, Metarrhizium anisopliae and Beauveria bassiana, were cultured in liquid culture media containing various commercial peptone sources to determine the effect of the sources on growth and sporulation. Each fungus responded differently to the various peptone sources. Tryptone, Casitone, and yeast extract were effective for mycelial growth of M. anisopliae; however, yeast extract was the most effective in production of spores. Soytone Casitone, Neopeptone, and casein hydrolysate were used effectively for mycelial growth of B. bassiana, but the latter two were not as effective for production of spores. Gelatone and Peptone (Bacteriological) were not effective for production of growth or sporulation for either fungus.     

Characteristics of Protease Production by Cephalosporium sp

We investigated protease formation by Cephalosporium sp. strain KM388, which produced trypsin inhibitor in the same cultures, in medium containing polypeptone, meat extract, and glucose (natural medium) and in medium containing NaNO₃, glucose, and yeast extract (semisynthetic medium). In natural medium, protease was secreted into the culture broth after cessation of growth caused by consumption of the polypeptone, the growth-limiting substrate. Enzyme formation in the stationary growth phase was due to de novo and so-called preferential synthesis, because cycloheximide immediately inhibited enzyme formation. In semisynthetic medium, protease was produced in parallel with mycelial growth, but production was repressed by the addition of polypeptone to the medium; protease production began after the added polypeptone was consumed. On the other hand, if glucose was eliminated from natural medium, the lag period of initiation of enzyme production was reduced until the late exponential phase. The addition of phosphate up to a concentration of 1.0% to natural medium also shortened the lag period and damped the pH change of the broth during cultivation.     

The Photosynthesis and Cell Shape Rhythms can be Naturally Uncoupled from the Biological Clock in Euglena gracilis

A naturally occurring population of Euglena (Klebs Strain Z)cells, with unusual biological clock properties, has been isolated.The photosynthesis reactions, which are usually controlled bythe biological clock, are uncoupled from the clock in the newpopulation. The rate of oxygen evolution is influenced predominantlyby the environmental growth parameters instead of the biologicalclock. In addition, the rhythm in cellular shape has a differenttiming from the control population and can be temporarily uncoupledfrom the clock by lowering the light intensity used for growth Key words: Biological clock, photosynthesis, Euglena     

Exopolysaccharide production and mycelial growth in an air-lift bioreactor using Fomitopsis pinicola

For effective exopolysaccharide production and mycelial growth by a liquid culture of Fomitopsis pinicola in an air-lift bioreactor, the culture temperature, pH, carbon source, nitrogen source, and mineral source were initially investigated in a flask. The optimal temperature and pH for mycelial growth and exopolysaccharide production were 25degrees C and 6.0, respectively. Among the various carbon sources tested, glucose was found to be the most suitable carbon source. In particular, the maximum mycelial growth and exopolysaccharide production were achieved in 4% glucose. The best nitrogen sources were yeast extract and malt extract. The optimal concentrations of yeast extract and malt extract were 0.5 and 0.1%, respectively. K2HPO4 and MgSO4 x 7H2O were found to be the best mineral sources for mycelial growth and exopolysaccharide production. In order to investigate the effect of aeration on mycelial growth and exopolysaccharide production in an air-lift bioreactor, various aerations were tested for 8 days. The maximum mycelial growth and exopolysaccharide production were 7.9 g/l and 2.6 g/l, respectively, at 1.5 vvm of aeration. In addition, a batch culture in an air-lift bioreactor was carried out for 11 days under the optimal conditions. The maximum mycelial growth was 10.4 g/l, which was approximately 1.7-fold higher than that of basal medium. The exopolysaccharide production was increased with increased culture time. The maximum concentration of exopolysaccharide was 4.4 g/l, which was about 3.3-fold higher than that of basal medium. These results indicate that exopolysaccharide production increased in parallel with the growth of mycelium, and also show that product formation is associated with mycelial growth. The developed model in an air-lift bioreactor showed good agreement with experimental data and simulated results on mycelial growth and exopolysaccharide production in the culture of F pinicola.     

In vitro inhibitory effects of rosemary and sage extracts on mycelial growth and sclerotial formation and germination of Sclerotinia sclerotiorum

The anti-fungal efficacy for two Labiate plants, rosemary (Rosmarinus officinalis L.) and Greek sage (Salvia fructicosa Mill.), against Sclerotinia sclerotiorum fungus (Lib.) de Bary has been investigated. The inhibitory effect of these plants as crude leaf ethanolic extract on the radial mycelial growth as well as on sclerotial production and germination was measured in vitro at various concentrations (stock?=?0.5?g dry leaf powder/ml ddH₂O) in the growth medium. In general, rosemary extract revealed a remarkable anti-fungal effect against the fungus, being more inhibitory than Greek sage in this respect. This was evident as total inhibition of radial mycelial growth by rosemary occurred at 10% extract concentration, while sage was half as potent producing such an effect at double the concentration (20%). Both rosemary and sage extracts were more inhibitory to sclerotial formation than to mycelial growth as the fungus ceased to produce any sclerotia at the lower concentrations of 5 and 5–10%, respectively. In addition, rosemary was highly effective in inhibiting sclerotia germination as total inhibition of germination occurred at 20% extract concentration at three?days and onward after incubation. Moreover, at this level, the survival of sclerotia was totally lost when examined after 12?days of incubation. For sage, inhibition of sclerotial germination/death was only 20% at 12th day of incubation. The results of this study indicate that the extracts of rosemary and Greek sage leaves could become natural alternatives to synthetic fungicides to manage diseases of S. sclerotiorum.     

Inhibition of Adventitious Root Growth in Tradescantia by Calmodulin Antagonists and Calcium Inhibitors

When cuttings of Tradescantia fluminensis stem were incubatedin distilled water, the buds located at the node grew into adventitiousroots. The root growth could be inhibited by calmodulin antagonists,trifluoperazine, chlorpromazine, compound 48/80 and calmidazolium,in a concentration-dependent manner. The divalent cation chelatorethyleneglycol-bis-(ß-aminoethyl ether)-N, N, N, N-tetraaceticacid had no effect, however, the intracellular chelator TMB-8completely inhibited root growth. The growth was also inhibitedby calcium ionophore A23187 [GenBank] , lanthanum, a competitive inhibitorof Ca²⁺ uptake and verapamil, a calcium channel inhibitor. AWestern blot of the adventitious root extract followed by immunostainingwith an anti-spinach calmodulin antibody clearly showed thepresence of calmodulin in this tissue. These results stronglysuggest the involvement of calmodulin and calcium in the growthof Tradescantia advenitious roots. ¹A part of this work has been published in abstract form in"Molecular and Cellular Aspects of Calcium in Plant Development"(Editedby A.J.Trewavas, Plenum Publishing Co. 1986. ³Present address: Plant Laboratory , Kirin Brewerry Co. Ltd.,Kitsuregawa-cho, Tochigi-ken 329-14 ,Japan (Received May 2, 1987; Accepted September 17, 1987)     

Optimization of submerged culture requirements for the production of mycelial growth and exopolysaccharide by Cordyceps jiangxiensis JXPJ 0109

AIMS: The objective of the present study was to investigate the optimal culture requirements for mycelial growth and exopolysaccharide production by Cordyceps jiangxiensis JXPJ 0109 in submerged culture. METHODS AND RESULTS: The effects of medium ingredients (i.e. carbon and nitrogen sources, and growth factor) and other culture requirements (i.e. initial pH, temperature, etc.) on the production of mycelia and exopolysaccharide were observed using a one-factor-at-a-time method. More suitable culture requirements for mycelial growth and exopolysaccharide production were proved to be maltose, glycerol, tryptone, soya bean steep powder, yeast extract, medium capacity 200 ml in a 500-ml flask, agitation rate 180 rev min(-1), seed age 4-8 days, inoculum size 2.5-7.5% (v/v), etc. The optimal temperatures and initial pHs for mycelial growth and exopolysaccharide production were at 26 degrees C and pH 5 and at 28 degrees C and pH 7, respectively, and corresponding optimal culture age were observed to be 8 and 10 days respectively. According to the primary results of the one-factor-at-a-time experiments, the optimal medium for the mycelial growth and exopolysaccharide production were obtained using an orthogonal layout method to optimize further. Herein the effects of medium ingredients on the mycelial growth of C. jiangxiensis JXPJ 0109 were in the order of yeast extract > tryptone > maltose > CaCl2 > glycerol > MgSO4 > KH2PO4 and the optimal concentration of each composition was 15 g maltose (food-grade), 10 g glycerol, 10 g tryptone, 10 g yeast extract, 1 g KH2PO4, 0.2 g MgSO4, and 0.5 g CaCl2 in 1 l of distilled water, while the order of effects of those components on exopolysaccharide production was yeast extract > maltose > tryptone > glycerol > KH2PO4 > CaCl2 > MgSO4, corresponding to the optimal concentration of medium was as follows: 20 g maltose (food-grade), 8 g glycerol, 5 g tryptone, 10 g yeast extract, 1 g KH2PO4, and 0.5 g CaCl2 in 1 l of distilled water. CONCLUSIONS: Under the optimal culture requirements, the maximum exopolysaccharide production reached 3.5 g l(-1) after 10 days of fermentation, while the maximum production of mycelial growth achieved 14.5 g l(-1) after 8 days of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the submerged culture requirements for mycelial growth and exopolysaccharide in C. jiangxiensis, and this two-step optimization strategy in this study can be widely applied to other microbial fermentation processes.     

Semi-defined Medium for in vitro Cultivation of the Fastidious Insect Pathogenic Fungus Cordyceps unilateralis

Cordyceps unilateralis is a fastidious fungal pathogen affecting ants. Up to now, only the complex and expensive Grace’s insect cell culture medium has been used for in vitro cultivation (as blastospores and mycelium) of this fungus. To obtain an inexpensive and less complicated medium, the effects of carbon and nitrogen sources, salt solution and carbon-to-nitrogen (C:N) ratio on the growth of this fungus were examined. Glucose was the most important factor for blastospore formation, and yeast extract could be used as a nitrogen source for blastospore formation and mycelial growth. A suitable C:N ratio (glucose: yeast extract) was 33.3:1. As a result, a new semi-defined medium was achieved, composed of 26.68 g L⁻¹ glucose, 3.3 g L⁻¹ yeast extract and salt solution. This medium supported blastospore formation and mycelial growth of all tested C. unilateralis isolates.     

Levels of IAA, Cytokinins, ABA and Ethylene in Rice Plants as Affected by a Gibberellin Biosynthesis Inhibitor, Uniconazole-P.

Effects of uniconazole-P, a triazole-type growth retardant,on endogenous levels of IAA, cytokinins, ABA and ethylene inrice seedlings were investigated. Endogenous levels of IAA andABA were similar between control and uniconazole-P-treated riceshoots. Evolution of ethylene was promoted slightly, being 1.8times greater under 0.3 ppm uniconazole-P treatment than thatof control. The most obvious effect was the increase of trans-Zand trans-RZ in shoots. Shoots treated with uniconazole-P (10mg/m² nursery box) contained 3.4 times and 3 times more trans-Zand trans-RZ than control, respectively. No significant differencesof cytokinin levels were recognized in roots except for cis-RZ.The increase of ethylene and active forms of cytokinins, andthe decrease of gibberellin in the shoots may be the basis forphysiological phenomena caused by uniconazole-P, namely thepromotion of flowering in woody plants and the enhancement offemaleness in cucumber. (Received September 9, 1987; Accepted October 20, 1987)     

Defective threonine-linked glycosylation of human insulin-like growth factor in mutants of the yeast Saccharomyces cerevisiae

Mutants of the yeast Saccharomyces cerevisiae were identified,in which O-glycosylation at threonine 29 of a heterologous protein,human insulin-like growth factor (hIGF-1), is defective. Inmutant M195, O-glycosylation of hIGF-1, but not of yeast proteinschitinase and a-agglutinin, was reduced; in mutant M577 yeastproteins were affected besides hIGF-1. The mutations of M195and M577 did not affect viability and could not be complementedby the PMT1 or PMT2 genes. The mutant phenorype of strain M195was reconstituted in an in vitro system, in which a hIGF-1-derivedpeptide encompassing residues 24–34 was not used as acceptorfor mannosylation, while unrelated peptides were glycosylatedat wild-type levels. hIGF-1 glycosylation was drastically reducedin pmt1 disruptants and to a lesser extent in pmt2 disruptants,suggesting interaction between the PMT gene products and componentsmutated in M195 and M577 cells. The results suggest that mutationsmay only affect O-glycosylation of a specific subset of secretedproteins in yeast. insulin-like growth factor O-glycosylation protein mannosyltransferase Saccharomyces cerevisiae     

Effect of light on sexual flocculation in Schizosaccharomyces japonicus

A fission yeast, Schizosaccharomyces japonicus can sporulateabundantly under conditions of vegetative growth. Prior to sporulation,strong flocculation was observed. When the culture medium containeda sufficient amount of yeast extract, sporulation was completelyinhibited. When the medium was cultured under light, flocculationoccurred, but not zygote formation. Neither flocculation norzygote formation was observed in the culture under completedarkness. The effect of light occurred even with short-periodillumination at the early to mid logarithmic growth phase. Thepresence of a definite amount of yeast extract was essentialfor the phenomenon in question. (Received July 20, 1976; )     

Sporulation in Rhizopus sexualis and Some Other Fungi Following a Period of Intense Respiration

Rhizopus sexualis grown at 20° C. on liquid I per cent.malt or glucose-asparagine medium showed a peak of respiratoryactivity between 40 and 55 hours-after inoculation. Rate ofrespiration then fell until it reached a steady low level whichcoincided with maximum mycelial growth. Zygospore initiationoccurred at or just after the peak of respiration. At a low temperature (9° C.) or with high concentrationsof glucose the respiration peak was less marked and no zygosporesdeveloped. Single ‘plus’ or ‘minus’strains of the heterothallic species Mucor hiemalis and Phycomycesblakesleeanus showed a pattern of respiration and mycelial growthsimilar to that of R. sexualis but no zygospores were formed.Zygospores did not develop without a preliminary period of intenserespiration, but such a peak period could occur without beingfollowed by zygospore formation. A strain of Sordaria fimicola was grown at 25° C. on a syntheticmedium with 5.0 per cent. sucrose or glucose as source of carbon.Respiration reached a peak at approximately 4 and 5 days respectively,the actual peak value being twice as large on sucrose as onglucose. Dry weight of mycelium was greater on glucose thanon sucrose. Perithecia were formed only on the sucrose medium.Visible peri-thecial initials were first seen shortly afterthe occurrence of the respiratory peak. Mature perithecia werepresent 3 days later. The possible significance of these results is discussed.     

Factors affecting mycelial biomass and exopolysaccharide production in submerged cultivation of Antrodia cinnamomea using complex media

Submerged cultures were used to identify growth-limiting nutrients by Antrodia cinnamomea strains. The mycelial biomass and EPS production by A. cinnamomea BCRC 35396 were markedly higher than other A. cinnamomea strains. A relatively high C/N ratio was favorable for both the mycelial growth (5.41 g/l) and EPS production (0.55 g/l); the optimum ratio was 40. The glucose was available utilized preferentially for mycelial growth, rather than for EPS production. Flushing the culture medium with nitrogen had a stimulating effect on both mycelial growth and EPS production. In addition, peptone, yeast extract and malt extract appeared to be important and significant component for EPS production. Phosphate ion, magnesium ion and thiamine were probably not essential for mycelial growth. By optimizing the effects of additional nutrition, the results showed that 5% (w/v) glucose, 0.8% (w/v) peptone, 0.8% (w/v) yeast extract, 0.8% (w/v) malt extract, 0.03% (w/v) KH2PO4, 0.1% (w/v) MgSO4 .7H2O and 0.1% (w/v) thiamine could lead to the maximum production of EPS (1.36 g/l).     

Selection of strains of Lentinula edodes and Lentinula boryana adapted for efficient mycelial growth on wheat straw

Mycelial growth rates are presented for 11 strains of Lentinula edodes and six strains of Lentinula boryana cultivated on solid media: derived from malt extract (MEA); malt yeast extract (YMEA); and, YMEA plus soluble lignin derivatives (YMEA+WSLD). The results were compared with data for mycelial growth rates, of the same strains cultivated on substrates derived from wheat straw treated at different temperatures (50, 65, 75 and autoclaving at 121 degrees C). In general, the addition of WSLD significantly reduced mycelial growth rates in both species. The greatest mycelial growth rate was obtained on sterilized straw at 121 degrees C for the majority of strains. However, this growth was not significantly different from that obtained at 75 degrees C. L. edodes showed greater growth rates than L. boryana. The feasibility of using estimates of mycelial growth rate on YMEA and YMEA+WSLD are discussed as possible indicators of a strain's potential for mycelial growth on substrates derived from wheat straw.     

Nutritional requirements of mycelial growth of Cordyceps sinensis in submerged culture

AIMS: The nutritional requirements for mycelial growth of Cordyceps sinensis in semi-synthetic liquid media were investigated. The results provide a basis for further physiological study and industrial fermentation of the fungus. METHODS AND RESULTS: Nutritional requirements, including 17 carbohydrates, 16 nitrogen compounds, nine vitamins, four macro-elements, four trace-elements and eight ratios of carbon to nitrogen, were studied for their effects on the mycelial growth in submerged cultures of C. sinensis by using one-factor-at-a-time and orthogonal matrix methods. Among these variables, sucrose, peptone, folic acid, calcium, zinc and a carbon to nitrogen ratio 12 : 1 were identified as the requirements for the optimum mycelial growth. The concentrations of sucrose, peptone and yeast extract were optimized and the effects of medium composition on mycelial growth were found to be in the order sucrose > yeast extract > peptone. The optimal concentration for mycelial growth was determined as 50 g l(-1) sucrose, 10 g l(-1) peptone and 3 g l(-1) yeast extract. CONCLUSIONS: Under optimal culture conditions, over 22 g l(-1) of mycelial biomass could be obtained after 40 days in submerged cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Cordyceps sinensis, one of the most valued medicinal fungi, is shown to grow in axenic culture. This is the first report on nutritional requirements and design of a simplified semi-synthetic medium for mycelial growth of this psychrophilic species, which grows slowly below 20 degrees C. The results of this study will facilitate research on mass production of the fungus under defined culture conditions.