Age Of Mycelium Research Articles

Studies on critical analysis of factors influencing improved production of protoplasts from Trichoderma reesei mycelium

Efficient synthesis of a large number of viable protoplasts from Trichoderma longibrachiatum (Trichoderma reesei) mycelium has been found to be influenced by a number of factors. In this case, Trichoderma harzianum NCIM 1185 culture filtrate has been used to prepare protoplasts from Trichoderma longibrachiatum QM 9414. A method has been devised to isolate a large number of viable protoplasts from the mycelium. Detailed analysis of different factors affecting the synthesis of protoplasts from T. longibrachiatum has not yet been reported. The most suitable conditions for protoplasting were as follows: age of the organism in slant, 3 days; mycelium age, 20 h; volume of lytic enzymes, 190 ml; mycelial weight (dry equivalent), 1.66 g; time of contact with lytic enzymes, 2 h; temperature of protoplasting, 30°C; phosphate buffer, 25 m m , pH 6.5; KCl as osmotic stabilizer, 0.7 m . Proper spreading of the mycelium in contact with the lytic enzymes was also found to be necessary.

Influence of age of mycelium and water activity of the medium on aroma production by Trichoderma viride grown on solid substrate

The production of 2-heptanone (cheese aroma) by Trichoderma viride TS cultivated on agar media was evaluated using headspace gas chromatographic analysis. The radial growth rate of the filamentous fungi increased with high water activity values, but the mycelial density was higher for lower water activity. Maximum aroma production of a culture was obtained at a w = 0.96. An apparatus intended to measure the aroma production of different areas of a mycelial colony was set up. The study of the aroma production of these areas showed that the production values were greatly different and were evolving with time. It was shown that the mycelium aroma production was maximum when mycelia were about 3.5 to 6.5 d old.

Use of ELISA for fungal biomass measurement in standing-dead Spartina alterniflora Loisel

An ELISA was developed for Phaeosphaeria typharum (Desm.) Holm. The assay could detect 0.1 μg dry fungal organic matter·ml −1. Cross-reactivity occurred with some fungal species common to the salt-marsh but not with nonfungal materials. Mycelial age had only a moderate effect on the ELISA response. Negative interference in the ELISA of standing-dead brown leaves caused a 30% decrease in sensitivity. In a 9-month field experiment, results from the ELISA indicated that, at peak development. P. typharum biomass could account for >10% of the C in standing-dead leaves of smooth cordgrass Spartina alterniflora Loisel.

Protoplast production and regeneration from mycorrhizal fungi and their use for isolation of mutants

Methods were developed for the reproducible production of high yields of stable protoplasts from monokaryotic and dikaryotic mycelia of Hebeloma cylindrosporum. This is a prerequisite for mutation, cell fusion, and transformation of this mycorrhizal fungus. Protoplasts from Hebeloma cylindrosporum, Hebeloma edurum, Hebeloma sinapizans, and Suillus bellini were released by using cellulase Onozuka R 10 and driselase as lytic enzymes. Production was strongly influenced by the nature and molarity of the osmotic stabilizer and by the mycelial age. In spite of the high percentage of anucleate protoplasts (42%) produced, the regeneration rate averaged 15%. Fungicide-resistant mutants from monokaryotic mycelia of H. cylindrosporum were isolated following ultraviolet irradiation of protoplasts. This indicates that protoplasts will be useful to select physiological mutants from nonsporulating mycelia and to create new strains by mating complementary mycelia or by interspecific cell fusion.

Ergosterol Content of Salt-Marsh Fungi: Effect of Growth Conditions and Mycelial Age

Species of the ascomycetc genera Phaeosphaeria, Leptosphaeria, and Buergenerula from the Spartina alterniflora decomposition system were analyzed for ergosterol content of their mycelium. Bacterial and algal members of the system were also tested. A standard laboratory liquid medium, a complex, semi-natural liquid medium, and a semi-natural solid medium were used to grow the microorganisms. Ergosterol contents of fungal mycelium ranged from 1.9-2.5 mg·g-1 dry organic mass in the standard medium, and were 3.4-16.4 mg·g 1 in the semi-natural liquid medium. For any single growth medium, ergosterol content of mycelium was less variable with mycelial age than has been previously found for glucosamine. For Leptosphaeria obiones, ergosterol content in solid medium was estimated to be similar to that in semi-natural liquid, but for Phaeosphaeria typharum, solid medium yielded much higher (X5) ergosterol estimates, possibly due to underestimated mycelial mass. Partial mycelial autolysis led to low estimates of ergosterol content (1.1 mg·g-1 dry organic). Bacteria had negligible ergosterol contents, but there was evidence that green-microalgal ergosterol content might not be negligible in some decomposition systems.

Ergosterol Content of Salt-Marsh Fungi: Effect of Growth Conditions and Mycelial Age

Ergosterol Content of Salt-Marsh Fungi: Effect of Growth Conditions and Mycelial Age

Production and Regeneration of Protoplasts from Pseudocercosporella herpotrichoides (Fron) Deighton

AbstractThis paper describes the development of a system for protoplast isolation from this pathogen using the commercially available, hydrolytic enzyme preparations Rhozyme HP150, Driselase and Cellulase CP. An analysis of the active components of these enzymes believed to be involved in protoplast release has been made. Factors affecting protoplast release including mycelial age, enzyme concentration and choice of osmotic stabilizer have been optimised, and conditions for high frequency protoplast regener, ation have been determined. The possible significance of the interaction of the several components of each enzyme is discussed. The developments described in this paper can now be exploited in genetic studies in this imperfect fungus.

Influence of fusarium oxysporum age on proportions of C, N and P mineralized after chloroform fumigation in soil

The influence of age of Fusarium oxysporum mycelia on mineralization in soil of their C, N and P contents following chloroform fumigation was determined with two soils from pasture of different productivity. The mineralization factors kc and kn could vary with mycelial age and, in prior-incubated soils, were lowest in the oldest (44-day) culture with the lowest cytoplasm and total N contents. Changes in kp with mycelial age showed no consistent trends. The use of k factors derived from physiologically young cells may lead to under-estimation of C and N in the indigenous soil biota.

Formation and regeneration of protoplasts from the ectomycorrhizal basidiomycete Laccaria bicolor

Protoplasts were released from dikaryotic mycelium of the ectomycorrhizal basidiomycete Laccaria bicolor using the lytic enzyme preparation NovoZyme 234. Protoplast release depended strongly on mycelium age, osmotic stabilizer, and temperature. The protoplasts could regenerate to form both monokaryotic and dikaryotic cultures capable of forming normal ectomycorrhizae with Pinus banksiana.

Effect of immobilization on the stability ofClaviceps purpurea protoplasts

Protoplasts released with high efficiency from vegetative and productive hyphae ofClaviceps purpurea were immobilized in 2% Ca-alginate. The yield of active immobilized protoplasts depended upon the age of the mycelium from which protoplasts were derived and was found to be 25–43% in comparison with native hyphae. During incubation in a modified production medium immobilized protoplasts were stable for at least 10–12 days. No external growth of regenerated hyphae from spherical beads of alginate gel with entrapped protoplasts was observed for 13–15 days of the batchwise incubation.

Isolation and regeneration of protoplasts from Fusarium tricinctum and F. oxysporum

A method is described for the production of protoplasts from the previously untested species Fusarium tricinctum and F. oxysporum. Factors affecting the release of protoplasts from these species were investigated using a commercial enzyme preparation Novozym 234. Maximum yields obtained were higher ( × 50) than yields previously reported for F. culmorum. A number of stabilizer solutions at a range of concentrations, were tested; the most efficient systems were 1.4 M -MgSO4 (F. tricinctum) and 1.2 M -KC1 (F. oxysporum). The effect of mycelial age, mycelial concentration in the lytic mixture and concentration of Novozym 234 were also assessed. Protoplasts liberated using this method possessed the capacity to regenerate new cell wall material and form colonies, although percentage regeneration was low. Up to 60% of protoplasts liberated were enucleate. Few contained 2 or more nuclei. Such protoplast preparations represent a useful method for the bulk production of cell-free extracts.

An indirect immunofluorescence study of antibodies toAspergillus fumigatusin sera from children and adults without aspergillosis

An indirect immunofluorescence (IF) method for detection of antibodies of Aspergillus fumigatus was developed: it used cryosections of formaldehyde-treated mycelium as antigen. Mycelial age was found to be of importance for titer determination, and growth in Czapek-Dox broth for 48 h at 37 degrees C was considered optimal. Fluorescein-conjugated rabbit antisera to human immunoglobulins reacted by direct IF with A. fumigatus but this staining could be abolished by absorption of conjugates with homogenized mycelium of A. fumigatus. Chessboard titrations revealed plateau end-points of conjugates in the range 0 . 05-0 . 2 antibody units ml -1, which is the same range as found in other systems. Sera from 54 adults and 69 children not suspected of having fungal disease were investigated for antibodies to A. fumigatus. IgG antibodies were present at birth and the prevalence and titers fell during the first half year. Through childhood the prevalence and titers of IgG and IgM as well as IgA antibodies increased towards adult levels. In adults IgG antibodies in titers of 40 to 5120 were present in all 54 subjects tested, IgM antibodies in titers 10 to 160 occurred in 91% and IgA antibodies in titers of 10 to 80 in 78%. These findings may be explained by continuous exposure through childhood and adult life to antigens of A. fumigatus or to cross-reacting antigens.

Colonization of unsterilized filter paper by cereal foot-rot fungi

Fusarium culmorum, Cochliobolus sativus, Curvularia ramosa, Gaeumannomyces graminis var. tritici and Phialophora graminicola were tested for their ability to produce colonies on unsterilized filter paper. Colonies of the first three species sporulated well on the paper moistened with a mineral salts solution but in no species did colonies exceed a mean diameter of 17 mm. The chief determinant of colony size seemed to be inoculum potential of the PD inocula which was determined by the nutrition of the fungus. In all species except P. graminicola , inoculum potential of the inocula declined significantly with age of mycelium. A correlation was found between linear growth rate on PD agar, taken as a parameter of general metabolic rate, and percentage reduction in diameter of colonies on filter paper, due to ageing of the mycelium. Linear growth rate has been used as the denominator of the cellulolysis adequacy index and the implications of these findings for saprophytic survival on cellulosic substrates have been discussed.

The regulation of the decay of nitrate reductase Evidence for the existence of at least two mechanisms of decay

There seem to be at least two different mechanisms of decay of nitrate reductase in Neurospora in vivo: one which is very sensitive to EDTA and cycloheximide, decreases with mycelial age and is not increased by an increase in temperature from 27 to 37°C, the other which is relatively insensitive to EDTA and cycloheximide, increases with the age of the mycelium and with the above temperature shift.

Critique of the chitin assay technique for estimation of fungal biomass

Colorimetric determination of chitin content of nine aero-aquatic hyphomycetes showed that the conversion factor (CF) values (chitin content per unit dry weight of mycelium) varied with the age of mycelium, nutrient content of the medium, and initial oxygen concentration of the water. CF values often decreased with age of mycelium and also with lowered oxygen concentrations. Consequently, for estimation of fungal biomass in tissues, this technique is of limited value.

Succinate oxidase in Neurospora.

Two kinetically distinct states of succinate oxidase have been detected in the mitochondria of Neruospora crassa. One state has a K(m) for succinate of 4.1 x 10(-3)m, and the other has a K(m) for succinate of 3.5 x 10(-4)m. The high K(m) state was found in freshly extracted mitochondria from either 20- or 72-hr mycelium. However, the succinate oxidase activity in mitochondria from 20-hr mycelium rapidly deteriorated in vitro, leaving a stable residual activity with the lower K(m) for succinate. Adenosine triphosphate (ATP) plus Mg(2+) stabilized the high K(m) state in these preparations. The high K(m) state of succinate oxidase was further characterized by a two- to threefold increase in activity over the pH range 6.6 to 8.0 and by classical competitive inhibition by fumarate and malonate. By contrast, the low K(m) state of succinate oxidase showed a relatively flat response to pH over the range 6.6 to 8.0 and a nonclassical pattern of inhibition by fumarate and malonate, as shown by nonlinear plots of reciprocal velocity versus reciprocal substrate concentration in the presence of inhibitor or reciprocal velocity versus inhibitor concentration at fixed substrate concentrations. The relationship of mycelial age to the in vitro stability of succinate oxidase is considered with reference to probable changes in the relative pool sizes of extra- and intramitochondrial ATP in response to changes in the rate of glycolysis.

Factors affecting vegetative growth and the production of perithecia in culture by Ophiobolus graminis. I. Variations in media and age of mycelium

Perithecia were readily produced in culture on a suitable solid medium under certain conditions of light and temperature, once vegetative growth was established. Investigations into the carbon and nitrogen requirements for both vegetative growth and fruiting showed that, whereas vegetative growth increased with increasing carbon supplies up to10%, reproduction occurred only within a restricted range of carbon and nitrogen concentrations. No perithecia were produced on a starvation medium. Factors involved in fruiting included concentration of nutrients and the balance between them; both were important. A suitable fruiting medium required a minimum carbon concentration of 3000 p.p.m., supplied as 0.75 % glucose or fructose, and an optimum carbon concentration of 6000 p.p.m. supplied as 1.5 % glucose or fructose. The maximum number of perithecia was produced on a medium containing 1% glucose and 0.2 % asparagine (400 p.p.m. nitrogen), which had a carbonlnitrogen ratio of 11.8. A higher than optimum nitrogen concentration was partially offset by increasing the carbon concentration, that is by keeping the carbonlnitrogen ratio approximately constant. The actual concentrations inhibiting and promoting fruiting of the fungus were influenced by the balance between carbon and nitrogen supplies. High concentrations of carbon and nitrogen increased vegetative growth but decreased the number of perithecia. There was no sudden inhibition of perithecia with increased carbon concentrations, but at 10% glucose (40,000 p.p.m. carbon) vegetative growth and pigmentation were maximal but few or no perithecia developed. No evidence was obtained that perithecial production was influenced by ageing of the mycelium, the presence of staling factors, or exhaustion of food supplies.

Autolytic enzymes in fungal cell walls.

SUMMARY: Autolytic glucanase and protease activity was detected in cell walls of some pythium species prepared by ultrasonic treatment. The glucanase activity was correlated with mycelial development. Maximal glucose release was detected in growing cultures during the linear growth period of the fungus. Assays of glucanase activity in cell walls prepared from mycelium harvested at different times showed that the enzymic activity increased with the age of mycelium from which the cell walls were prepared. The relationship between autolytic enzymes and mycelial development is discussed.

Translocation in Rhizoctonia solani

1. The mineral ions of Zn-65, S-35, Sr-89, and P-32 are readily absorbed by the hyphae of Rhizoctonia solani and are readily translocated throughout its mycelium. 2. The age of the mycelium has an effect on translocation of P-32. There was a greater movement from the hyphal tips to the old mycelium than in the reverse direction.

Alterations in the permeability of Neurospora crassa due to polyene antibiotics.

Kinsky, Stephen C. (Washington University, St. Louis, Mo.). Alterations in the permeability of Neurospora crassa due to polyene antibiotics. J. Bacteriol. 82:889-897. 1961.-Thirty-three antibiotics and synthetic fungicides were examined for their effect on the growth and morphology of Neurospora crassa. Only the polyene antibiotics (nystatin, amphotericin B, and filipin) caused a decrease in the dry weight of mycelial mats, which was accompanied by the appearance of cytoplasmic constituents in the medium. The influence of polyene concentration, incubation time, mycelial age, various metabolic inhibitors, pH, and media composition on this phenomenon are described. The present evidence indicates that the polyenes exert their primarily fungicidal effect by an alteration of permeability, probably due to direct action on the membrane of sensitive organisms.