Extracellular amylase activities ofRhizomucor pusillus andHumicola lanuginosa at initial stages of growth

Among thermophilic fungi,Rhizomucor Pusillus andHumicola lanuginosa have been reported to be among the most prolific producers of amylase, an apparently heat stable enzyme vital to the incorporation of carbon from macromolecular sources such as starch. Yet the highest levels of extracellular amylase in starch-yeast cultures of these fungi were measured after most of the growth had occurred; pre-growth levels appeared to be very small. Since these low levels are the significant ones for growth, a procedure was devised to measure them: 1.162×10^–2 units (mg maltose/ml/min) were measured after two days of growth ofR. pusillus and 6.230×10^–3 units measured after four days of the slower-growingH. lanuginosa. Re-assays of these after dialysis to remove most of the reducing sugars gave 1.689 × 10^–2 units and 1.234 × 10^–2 units, respectively, with all correlation coefficients 0.96 or better.     

Starch-metabolic growth characteristics of Humicola grisea var. thermoidea

Very low extracellular amylase levels measured during the growth of Humicola grisea var. thermoidea in starch-yeast medium appeared to be sufficient to metabolize the starch relatively rapidly. A maximum mycelial dry weight of about 65 mg was measure after six days of incubation.     

Mycelial amylase activities of thermophilic species ofRhizomucor,Humicola andPapulaspora

Amylase activities in mycelia ofRhizomucor pusillus, Humicola grisea var.thermoidea, Humicola lanuginosa andPapulaspora thermophilia do not correspond directly with previously-measured extracellular values, and appear to decline within the time period corresponding to reduction in mycelial dry weight. The results are compared with previously-reported data on extracellular amylase.     

Amylase and growth characteristics of Papulaspora thermophilia

Mycelial dry weight of Papulaspora thermophilia reached a maximum of about 96 mg after six days of growth in a starch-yeast medium. Extracellular amylase activity was not measurable during this growth period and remained thereafter only about 0.1 unit per ml for 30 days, yet starch concentration reduced rapidly, and reducing sugar appeared in the extracellular medium within the first few days of incubation.The author wishes to thank Dr. J. Deploey for helpful discussions and constructive criticism of this work.     

The relation of extracellular amylase,mycelium, and time,in some thermophilic and mesophilic humicola species

All four fungi studied attained approximately the same dry weight of mycelium in starch-yeast extract medium. Only about one-fourth the amount of mycelia was produced in yeast extract alone (starch omitted). However, the initial growth rate ofH. grisea var.thermoidea was greater than the other three fungi. Extracellular amylase was produced by all four fungi, butH. lanuginosa produced 8 to 12 times as much as the other three. Maximum extracellular amylase was found before autolysis with these three fungi, but after autolysis withH. lanuginosa. Extracellular amylase was detected in YE medium (lacking starch), but in very low amounts (approximately one-eighth the amount observed as when starch was present). Increasing the amount of starch in the medium increased extracellular amylase. However, when the starch concentration was kept constant, increasing the concentration of yeast extract had no effect on extracellular amylase.Contribution No. 59 from the Botany Section, The Department of Biology. Portion of a thesis presented by the senior author in partial fulfillment for the M.S. degree.     

The production and activity of extracellular amylase by Rhizoctonia bataticola

Rhizoctonia bataticola produced the highest amounts of amylase in medium containing starch than that lacking starch within the 10 days of culture. Doubling the concentration of starch in the growth medium resulted in a near doubling of the amylase activity. Amylase production by the fungus is related to the type of carbon source in the medium with maximum amylase produced in medium containing starch. The maximum activity of the enzyme was detected in extracellular filtrates obtained from 4 days cultures. After this period, amylase activity decreased at first, and then increased through the 10 days incubation period. The fungus produced maximum levels of amylase prior to attainment of maximum mycelial biomass. Peak activity of the extracellular amylase was recorded at a temperature and pH range of 20–25°C and 4–5 respectively. The role of the exoenzyme in the deterioration of stored food products and its possible use in industrial fermentation processes are discussed.     

The stimulation of extracellular carbohydrases of edible mushrooms by the hot water-soluble fraction from corn fiber

Corn fiber (CNF) is an abundant by-product of the wet corn milling process in the production of cornstarch. We have shown that the hot water-soluble fraction (HWSF) from CNF has a promoting effect on the mycelial growth of various edible mushrooms, including mycorrhizal fungi. To reveal the promoting mechanisms, the effect of CNF-HWSF on the stimulation of extracellular enzymes was examined. The production of extracellular carbohydrases such as amylase, CMCase, and xylanase was markedly enhanced by the addition of low molecular weight fractions (less than MW 500) prepared from CNF-HWSF. The enzymatic stimulation and enhancement of mycelial growth appeared during 3–15 days after inoculation. Furthermore, a fraction of less than MW 500 was separated by gel filtrate chromatography into five fractions (A–E), and the effect of each fraction was investigated. Promoting effects were shown from C and D fractions; mycelial growth and enzyme production of Lentinula edodes were indicated although fraction D has no sugars and amino acids in CNF-HWSF. From these results, the promoting effect of CNF-HWSF seems to be a two-step reaction. The first step could be achieved by rich nutrients such as free amino acids and monosaccharides from CNF-HWSF. The second step (during 3–15 days) is considered to be that the marked promoting effect was caused by the stimulation of extracellular enzymes.     

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.     

Changes in water content, sugars and invertase activity in developing seeds of Hibiscus esculentum

Seeds of Hibiscus esculentum were analyzed for growth, in terms of fresh and dry weights, cell size, water content, reducing and non-reducing sugars and acid invertase activity. On the basis of growth analysis seed development is divided into four distinct phases of a) cell division, d) cell elongation, c) dry matter accumulation and, d) maturation. A close parallel with water content and cell size was observed. A peak level of reducing sugars was observed during the rapid elongation growth. The role of invertase in hydrolyzing sugars and its regulation of sink development is discussed.     

Amylase synthesis inAspergillus flavus andAspergillus niger grown on cassava peel

Summary Aspergillus flavus andAspergillus niger produce extracellular amylase into the culture medium when grown on basal medium containing 2% (w/v) soluble starch or cassava peel as the sole carbon source. On soluble tarch the highest amylase activities were 1.6 and 5.2 mg of starch hydrolyzed/min per mg protein forA. flavus andA. niger, respectively. When grown on cassava peel, the highest amylase activity in the culture filtrate ofA. flavus was 170-times higher than that on soluble starch, while that ofA. niger was 16-times higher. The mycelial dry weight for both organisms was not significantly affected by the carbon sources. Maximum enzyme activity was obtained at the growth temperature of 29.0±1°C and pH 7 for both organisms. It is concluded that cassava peel might be a better substrate for the production of amylase byA. flavus andA. niger than commercial soluble starch.     

Trichoderma harzianum and its Metabolite 6-Pentyl-alpha-pyrone Suppress Fusaric Acid Produced by Fusarium moniliforme

The interaction of the pathogen Fusarium moniliforme and two antagonistic Trichoderma harzianum isolates was studied especially with respect to their secondary metabolites fusaric acid (FA) and 6‐pentyl‐alpha‐pyrone (6PAP). Among 10 isolates of F. moniliforme screened for FA production on maize kernels, the isolate 8 accumulated the highest amount of FA (678 μg/g). Mycelial growth and production of FA by isolate 8, determined in different liquid media revealed that the highest biomass and FA were produced in Czapek Dox Broth (CDB) followed by Richard’s solution. The amount of FA per gram mycelial dry weight reached its maximum in CDB and Richard’s solution after 14 days of incubation. Mycelial growth and conidia production of both Trichoderma isolates (T16 and T23) were retarded by increasing concentrations of FA in agar medium. At FA concentration of 300 mg/ml the radial mycelial growth of the isolates T16 and T23 were retarded by 32.5% and 45%, respectively. Conidia production was diminished in a similar extent as mycelial growth. Both T. harzianum isolates were capable to degrade FA in potato dextrose broth medium, particularly when lower doses of FA were present. In the presence of 50 mg/ml FA in the culture medium, the isolates T23 and T16 reduced FA by 51.4% and 88.4%, respectively, 9 days post‐inoculation. The antifungal metabolite 6PAP, isolated from T. harzianum T23 cultures, was introduced at different concentrations into 2‐day‐old cultures of F. moniliforme. After further 5 days of incubation of F. moniliforme in the presence of 6PAP, the FA contents per gram mycelial dry weight were significantly decreased compared to control cultures where 6PAP was absent. Dosages of 300 and 400 mg/l of 6PAP in the cultures retarded FA accumulations by 62.5% and 77.2%, respectively. The current results, however, provided the first evidence for activity of 6PAP, as a Trichoderma secondary metabolite, on degrading/synthesis suppression of the Fusarium toxin FA.     

Vigna Radiata Seed Germination under Salinity

Salinity reduced mung bean (Vigna radiata Wilczek) radicle and root elongation, delayed and inhibited hypocotyl elongation and mobilization of reserves from the cotyledons to the embryo axis. Fresh and dry masses and water content of the embryo axes were reduced. Under salinity, a net leakage of K to the media increased with time and increasing NaCl concentrations. Sugars present in the cotyledons of seeds were of primary importance for growth of the embryo axis upto 18 h after sowing whereas breakdown of starch by amylase contributed later, the contribution being delayed and reduced with increasing NaCl concentration. Even when amylase activity in the cotyledons was progressively reduced with increasing NaCl concentration, the increasing contents of soluble sugars in the cotyledons indicated that sugars were not limiting for mung bean seedling growth under salinity.     

绒毛栓孔菌液体培养过程中胞外酶活性的研究

本研究以绒毛栓孔菌为材料,采用液体培养的方法分析其在发酵过程中胞外酶的活性变化,并对其菌丝体生物量和发酵液pH值进行了测定。结果表明:胞外酶活性与菌丝体生长状况密切相关。菌丝体生物量增长呈"S"型,6～8d增长最快,第12天达到最大值,在此过程中漆酶、锰过氧化物酶、淀粉酶、羧甲基纤维素酶、果胶酶和蛋白酶活性均出现高峰。酶活性的变化表明,在液体培养过程中绒毛栓孔菌首先分解木质素,其次利用淀粉和纤维素作为碳源,蛋白质作为氮源。若要获得最大菌丝体生物量,缩短培养时间,就必须在培养过程中保证碳氮源的均衡供给。本试验说明不同的酶其分泌高峰期可以作为判断菌丝体营养利用情况和培养周期的依据,以此获取最大菌丝体生物量,为工业生产利用奠定基础。     

Enzyme Immunoassay of Herbicide Decomposition by Soil and Wood Decay Fungi

The effect of herbicide atrazine was studied on the growth and development of a number of soil and wood decay fungi: white-rot basidiomycetes (Cerrena maxima, Coriolopsis fulvocenerea, and Coriolus hirsutus), thermophilic micromycetes from self-heating grass composts (cellulolytic fungus Penicilliumsp. 13 and noncellulolytic ones Humicola lanuginosaspp. 5 and 12), and mesophilic phenol oxidase-producing micromycete Mycelia sterilia INBI 2-26. Detection of atrazine in liquid fungal cultures was performed by using the enzyme immune assay technique. Both stimulation (Humicola lanuginosa 5) and suppression (Humicola lanuginosa 12 and Penicillium sp. 13) of fungal growth with atrazine were observed on solid agar media. HyphomyceteMycelia sterilia INBI 2-26 was almost insensitive to the presence of atrazine. Neither of the thermophilic strains was capable of atrazine consumption in three-week cultivation. In contrast with that, active laccase producers Cerrena maxima, Coriolopsis fulvocenerea, and Coriolus hirsutus consumed up to 50% atrazine in 5-day cultivation in the presence of the xenobiotic and at least 80–92% in 40 days. Mycelia steriliaINBI 2-26, which also forms extracellular laccase, also consumed up to 70% atrazine in 17 days. The degree of atrazine consumption depended on the term of its addition to the fungal culture medium.     

Utilization of some monosaccharides by two species of Colletotrichum

Summary Utilization of 8 monosaccharides, viz., glucose, fructose, galactose, mannose, sorbose, arabinose, xylose and rhamnose, by some plant pathogenic isolates ofColletotrichum gloeosphorioides andC. dematium has been studied with the help of paper chromatography. Among hexoses, the rate of utilization of glucose, fructose and mannose was fast, whereas, that of galactose was comparatively slow. The rate of assimilation of sorbose was very slow at early stages of incubation, although at later stages this rate showed marked enhancement. The pentoses were utilized readily. The dry weight of mycelial mats showed an increase up to the end of final incubation period (15 days), on sugars which were slowly assimilated. In cases where the sugars were consumed up rapidly, the dry weight at later stages of incubation either became nearly stationary or recorded slight fall.     

The effects of fungicides on the growth rates of thermophilous fungi

The effects of the fungicides Captan, Dicloran, Thiram, Verdasan and mercuric chloride (HgCl₂) on the radial and mycelial growth rates of thermophilous fungi isolated from living green leaves were studied. On controlplates Thermoascus aurantiacus andTorula thermophila had the highest radial growth rates whileMalbranchea pulchella var.sulfurea andTalaromyces duponti had the lowest growth rates. Good sporulation was seen on all control plates.With increasing concentrations of fungicides the growth rates were correspondingly retarded and sporulation was reduced. At het higher fungicide concentrations the hyphal tips and young hyphae were thicker and warty or granulated.Of the fungi studies,Aspergillus andMucor pusillus were the most tolerant species; they were capable of growth in more concentrations of fungicides compared with the other species.Talaromyces duponti andThermomyces lanuginosus emerged as the most sensitive species.Most marked reductions in the growth rates were produced by Thiram and Verdasan than by the other fungicides. All the ten thermophilous species studied were capable of growth with more concentrations of HgCl₂ than with the organo mercurial Verdasan.Six of the ten thermophilous species studied showed higher radial growth rates compared with the two fastest growing mesophilic species studied;Trlchoderma viride andZygorhynchus moelleri. While M. pusillus was capable of growth in dry weight at 0.05 ppm of all fine fungicides,Sporotrichum thermophile did not show mycelial growth with Verdasan at 0.05 ppm.Thermomyces lanuginosus showed reduced growth rates with Thiram and no mycelial growth with both HgCl₂ and Verdasan.     

Factors affecting growth and cellulose hydrolysis by the thermotolerant aspergillus nidulans from composts

The ability of Aspergillus nidulans (EIDAM) WINT to grow and sporulate at various temperatures and to degrade soluble and insoluble forms of cellulose were studied. A. nidulans was found to grow and sporulate best at 37°C in continuous light and alternating light-darkness respectively. The fungus was able to cause losses in the dry weights of filter papers on incubation and made appreciable growth on CMC and hemicellulose. The culture filtrates contained cellulases which hydrolysed filter papers and CMC to reducing sugars, and were only able to produce these enzymes in the presence of cellulose or its derivatives in the growth medium. The CM-cellulases had peak activity at pH 5.2 and at 50°C while optimal FP-activity occurred at a pH of 5.5 and at 45°C. The participatory role of A. nidulans in composting is discussed.     

Growth and glucoamylase production by the thermophilic fungusThermomyces lanuginosus in a synthetic medium

Summary The production of glucogenic amylase from the thermophilic fungus Thermomyces lanuginosus was studied in shake flasks and laboratory fermentors. As conidia were not able to germinate in media without yeast extract, pregerminated conidia were applied as inoculum. By this procedure it was possible to use different NH _inf4 ^sup+ salts as the sole source of nitrogen for growth and amylase formation in a synthetic medium. In pH-controlled fermentors a fourfold increase in the extracellular glucogenic amylase activity was obtained with (NH₄)H₂PO₄ as the nitrogen source as compared with yeast extract. However, by fractionation of these activities, comparable yields of partially purified glucoamylases were obtained. The glucoamylase preparation from fermentations with either of the nitrogen sources had a temperature optimum at 70° C and showed similar thermal stability. By incubation without substrate at 60° C. 90% of the activity was still present after 5 h. At 70° C, 50% of the activity was retained after 30 min incubation. Offprint requests to: I. Hassum     

Effect of environmental factors and precursors on oxalic acid production,mycelial biomass and virulence of a potential bioherbicide isolate of Sclerotium rolfsii SC64 produced in liquid culture

The fungus Sclerotium rolfsii is presently under development as a bioherbicide for broadleaf weed species using fungus-infested substrates as application material in this laboratory. The effect of environmental factors and three precursors (citric acid, ascorbic acid, and sodium succinate) on mycelial growth, oxalic acid production, and virulence by SC64 in liquid culture were investigated. The results showed that for mycelia growth the optimum liquid medium was Modified Richard's solution (MRS) among the five tested media, but potato dextrose broth (PDB) produced the maximum oxalic acid production and virulence on detached Solidago canadensis leaves. When PDB was used as the basic medium, the oxalic acid/mycelial dry weight (mg g^–1) ratio reached the peak 4 days after inoculation. The optimum temperature for oxalic acid production was at 27°C, but increased mycelial dry weight and virulence were observed at 30°C. The optimum range of initial pH value for oxalic acid accumulation was 4.0–6.0, with the optimal pH 5.0; highest mycelial growth was with an initial pH 3.5–6.0 (optimum pH 5.0) and subsequently pH 3.5–5.5 (maximum at pH 3.5). Both mycelial dry weight and oxalic acid production showed a decreasing trend as a result of the precursor of oxalic acid being added to PDB. Among the three precursors, the greatest decrease in mycelial dry weight, and oxalic acid production was caused by sodium succinate. This clarification of optimal conditions for production of mycelial biomass while insuring high concentrations of oxalic acid and high virulence should be useful for further development of this fungus as biocontrol agent.     

An Improved Growth Medium to Assess Mycelial Compatibility Groups in Sclerotium rolfsii

Mycelial compatibility is assayed mainly by pairing mycelial plugs of field isolates on Petri dishes with agar media. Although methodologically simple, mycelial compatibility testing requires an artificial growth medium that permits the identification of compatible and incompatible interactions. In this work, several growth media were studied to assess consistently mycelial interactions between Sclerotium rolfsii isolates. A modification of Patterson’s medium with an increment of 25% glucose from the original concentration at a rate of 23.4 g/l and amended with 180 μl/l of red food colouring was the most effective combination for enhancing the size, density and distinctiveness of the aversion zone between incompatible isolates. This medium allowed the unequivocal identification of compatible and incompatible reactions of a set of five S. rolfsii isolates, which could be determined quickly after 5 days of incubation in the dark at 25°C. This new formulation improved significantly and consistently the assessment of the aversion zone reaction that was visible as a red line on the colony reverse as compared to that assessed using previous media formulations, for which the visualization of aversion zones was scarcely discernible. The utility of the improved growth medium was validated by microscopic observations of the contact area of hyphal pairings between isolates of S. rolfsii in microscope slide cultures.