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In general most fungi are sensitive to the respiratory inhibitors cyanide and antimycin A. However, there are a few fungi which have been reported to be at least partly insensitive to them, for example: poky mutants of Neurospora crassa Shear & Dodge (Lambowitz and Slayman, 1971), Myrothecium verrucaria (Alb. & Schw.) Ditm. (Darby and Goddard, 1950; Kidder and Goddard, 1965), anaerobically grown Mucor genevensis Lendner (Clark-Walker, 1972) and a water mold Saprolegnia sp. (Unestam and Gleason, 1968). Saprolegnia sp. is of particular interest because a large percentage of its respiration is insensitive to cyanide and antimycin A. The relationship between growth kinetics and induction of insensitivity in Saprolegnia sp. was considered in the present investigation.

A method for the estimation of erythorbic acid in the presence of ascorbic acid utilizing the unique properties of the spore ascorbase of Myrothecium verrucaria has been described. Measurements were made during partially inhibited enzyme oxidations of ratios from 10:1 to 300:1 of the substrate (ascorbic acid) to the enzyme inhibitor (erythorbic acid). Chemical analyses for reduced and oxidized ascorbic acid followed the manometric measurements. Regression lines of the amount of reduced or oxidized ascorbic acid on the logarithm of the corresponding ratio of ascorbic acid to erythorbic acid were developed from the three sets of data and were used satisfactorily for the estimation of the quantity of erythorbic acid present in “unknown” synthetic solutions. Trial measurements were made of erythorbic acid in blood and urine following test loads. Adjustments in the method must be made before definitive results can be obtained with the small amounts of the acids present in plasma. The presence of erythorbic acid was established qualitatively in urine by the method but quantitative relationships were not clearly defined due to limited data and great variation in excretion of the test load.

Spores of the fungus Myrothecium verrucaria are cryptic to maltose and isomaltose. Induction of a transport system can be effected by several sugars whose order of effectiveness is: turanose > maltulose > sucrose > d -arabinose, d -fructose, nigerose, maltotriulose, kestose > melezitose, raffinose, nystose, and stachyose. The transport system is not specific to maltose and isomaltose, and it is apparently identical to an induced trehalose permease described previously. Induction of the permease is markedly influenced by spore age—older spores being more responsive. Pure maltose is not absorbed by spores. Absorption of commercial reagent-grade maltose is due to permease induction by maltulose as an impurity. Maltulose contamination of maltose was demonstrated by charcoal column chromatography and comparison of its physical, chemical, and permease-inductive properties with those of authentic maltulose. Maltose accumulates temporarily in spores after absorption and then decreases, although no conversion to glucose can be detected. Although spores contain small quantities of maltase, metabolism of maltose may be via some nonhydrolytic pathway.

Mandels , G. R. (U.S. Army Natick Laboratories, Natick, Mass.), Rasma Vitols, and Frederick W. Parrish . Trehalose as an endogenous reserve in spores of the fungus Myrothecium verrucaria . J. Bacteriol. 90: 1589–1598. 1965.—Gross analysis of Myrothecium verrucaria spores showed approximately 3% fat, 33% carbohydrate, and 9.5% nitrogen. The water-soluble carbohydrates were trehalose, glucose, mannitol, and an unidentified phosphorylated compound. Water-soluble amino acids include leucine or norleucine (or both), valine, γ-amino- n -butyric acid, β-amino- n -butyric acid, ergothionine, glutamic acid, glutamine, glycine, aspartic acid, asparagine, cystine, and cystathionine. Ergosterol was also present. αα-Trehalose is the major reserve (20% of the dry weight), although approximately 30% of it appeared to be at the spore surface and was released by nonlethal treatment with 0.1 n HCl. Treatment with toluene or exposure to heat sufficient to kill the spores (20 min at 60 C) caused rapid liberation of all of the trehalose. Although spores could utilize exogenous trehalose with no appreciable lag, some stimulus, such as exposure to heat (10 min at 55 C), incubation with azide, or germination on exogenous substrates, was necessary to effect utilization of trehalose reserves. Spores have trehalase, but it is apparently at the spore surface, since it is inactivated by acid treatment which does not kill the spores. The metabolic pathway for utilization of trehalose is not known, but presumably it is not mediated by trehalase. The involvement of mannitol is indicated, since it tends to increase as trehalose decreases, although the changes are not quantitatively equivalent.

2. GALSTON, A. W. AND WV. S. HILLMAN. 1961. Thle degradation of auxin. In: Encyclopedia of Plant Physiology, W. Ruhland, ed. Springer-Verlag, Berlin. Vol. 14: 647-70. 3. MEIJER, G. 1959. The spectral dependence of flowering and elongation. Acta. Bot. Neerl. 8: 189-246. 4. PILET, P. E. 1960. In vitro destruction of auxin labeled with Cl4. Physiol. Plantarum 13: 766-75. 5. SEN, S. P. AND A. C. LEOPOI.D. 1954. Paper clhromatography of plant growth regulators and applied compounds. Physiol. Plantarum 7: 98-108. 6. WIGHTMAN, F. 1962. Metabolism and biosynthesis of 3-indoleacetic acid and related indole compounds in plants. Can. J. Botany 40: 689-718. 7. ZALIK, S. AND R. A. MILLER. 1960. Construction

From cultures of Myrothecium verrucaria (ALBERTINIet SCHWEINITZ) DITMARex FRIES and Myrothecium roridum TODEex FRIES four additional metabolites have been isolated: verrucarin H (C29H36O8), verrucarin J (C27H32O8), roridin D (C29H38O9) and roridin E (C29H38O8). They are closely related to verrucarin A and B, since they contain the sesquiterpenoid alcohol verrucarol as structural element.

The antifungal activity of homologous series of 2-alkenoic and 2-bromo alkanoic acids was determined against Aspergillus niger, Trichoderma viride, Myrothecium verrucaria , and Trichophyton mentagrophytes and compared with data on analogous alkanoic and 2-fluoro alkanoic acids. The fungitoxicity of all of the series of compounds was determined by chain length, pH of the medium, and presence or absence of adsorbents such as serum albumin. The order of toxicity on a molecular basis, by using a scale where the most active series is 1.0, is 2-bromo alkanoic acids (1.0) > 2-fluoro alkanoic acids (1.2) > alkanoic acids (1.4) > 2-alkenoic acids (2.5).

KOPELOFF, N., AND L. KOPELOFF. 1919. Do mold spores contain enzyms? Jour. Agric. Res. 18: 195-209. LINEWEAVER, H., AND D. BURK. 1934. The determination of enzyme dissociation constants. Jour. Amer. Chem. Soc. 56: 658-666. MANDELS, G. R., AND A. B. NORTON. 1948. Studies on the physiology of spores of the cellulolytic fungus Myrothecium verrucaria. The Quartermaster General Laboratories, Research Report, Microbiology Series No. 11: 1-50. . 1949. The synthesis and secretion of invertase by the spores of Myrothecium verrucaria. (ALb3tract) Amer. Jour. Bot. 36: 811. MICHAELIS, L., AND M. L. MENTEN. 1913. Die kinetik der invertinwirkung. Biochem. Zeitschr. 49: 333-369. MONOD, J. 1947. The phenomenon of enzymatic adaptation and its bearings on problems of genetics and cellular differentiation. Growth 11: 223-289. NEUBERG, C., AND I. S. ROBERTS. 1946. Invertase. Sugar Research Foundation (New York) Sci. Rept. Ser, No. 4: 1-62. PIGMAN, W. W. 1946. Specificity, classification, and mechanism of action of the glycosidases. Advances in Enzymology 4: 41-74. QUASTEL, J. H. 1926. Dehydrogenations produced by resting bacteria. IV. A theory of the mechanism of oxidations and reductions in vivo. Biochem. Jour. 20: 166-194.

Cellotriose, cellotetraose, cellopentaose and cellohexaose were prepared by acetolyzing cellulose and fractionating the deacetylated products on a charcoal column. A procedure is described for chromatographic separation and analysis of hydrolyzates of these sugars. Cellobiose and the above sugars, at various initial concentrations, were hydrolyzed at constant enzyme concentration by purified Myrothecium verrucaria cellulase and the hydrolyzates analyzed at various time intervals. For the conditions tested, the turnover numbers of the enzyme were calculated to be approximately as follows: cellobiose, 5–6; cellotriose, increasing directly with initial concentration from 50 to 200; cellotetraose, 400; cellopentaose and cellohexaose, at least 450. The process of chain-splitting appeared to become more random as the chain length increased. The results are discussed.

Electron microscopical studies of changes in cotton fiber microstructure, after ex posure of the fibers to the cellulase in filtrates prepared from cultures of Myrothecium verrucaria, showed evidence of the transverse, jagged cuts into the cellulose structure previously seen by cytical microscopy. The degradation appeared localized in areas along the length of the fiber which were not related to any recognized component of fiber structure. Micrographs of fragmented, degraded fibers showed etching of the macro- fibrils of the sheets of secondary wall and a sharpening of the image of the individual microfibrils. Continued enzyme attack produced smaller fragments and hydrocellulose- like particles. Measurements of changes in tensile strength, swelling in alkali, and in glucose yield were correlated with changes in microstructure. The extent of fiber degradation by cellulolytic culture filtrates was limited and could be continued only if fibers were swollen between filtrate exposures. No evidence of damage to the cellulose structure was seen which could not be explained hy hydrolysis at the β-1,4-glucosidic linkage.

Ascorbic acid oxidase activity in Myrothecium verrucaria extracts resulted in O(2) uptake exceeding 0.5 mole per mole of ascorbic acid and in CO(2) evolution. Measurement of oxidized ascorbic acid at completion of the reaction demonstrated that an average of 10% of the oxidized product disappeared. A comparison of the gas exchange data with the amount of ascorbic acid not accounted for indicated that the reaction could not be explained by independent oxidase and oxygenase systems. Chromatographic examination of the reaction mixtures identified l-threonic acid. Experiments with ascorbic acid-1-(14)C showed that C-1 was partially decarboxylated during the oxidation. Test of the fungal extracts for enzymes that might explain the deviation from expected stoichiometry showed that phenolase, glutathione reductase, cytochrome oxidase, peroxidase and oxalic decarboxylase were not involved. Addition of azide in concentrations sufficient to block catalase increased excess O(2) consumption about 65%. No enzymes were found that could directly attack oxidized ascorbic acid. H(2)O(2) accumulated during oxidation in azide-blocked systems.The O(2) excess could be explained by assuming the enzyme had peroxidative capacity on a reductant other than ascorbic acid. An intermediate of ascorbic acid oxidation appeared to function as the substrate yielding CO(2) and l-threonic acid on degradation. The increase in excess O(2) utilized in azide-blocked systems and the H(2)O(2) accumulation also were explained by the proposed scheme.Another interpretation would involve production of free radicals during ascorbic acid oxidation. Evidence for this was the ability of extracts to oxidize DPNH in the presence of ascorbic acid. Oxygen radicals formed in such reactions were considered possible agents of degradation of ascorbic acid.

Extracts from spores and mycelium of Myrothecium verrucaria contain an oxalic acid decarboxylase similar to the one Shimazono found in a wood-rotting fungus. The enzyme is highly specific for oxalic acid having no activity on 13 other carboxylic acids including formic acid. The stoichiometry approached 1 mole of oxalic acid per mole of formic acid and carbon dioxide. However, the carbon dioxide yield was about 10% low and there was an average of 0.85 μmole of oxygen consumed per 20 μmoles of oxalic acid decomposed particularly in the later stages of the reaction. The enzyme was sensitive to several heavy metal and sulfhydryl inhibitors. It was inactive under anaerobic conditions and was inhibited by certain reducing agents. The inhibition was reversed by oxygen but not by other electron acceptors. It is suggested that reductive cleavage of certain disulfide bonds inactivates the enzyme and that this process is reversible.

The structure of Verrucarin E, an antibiotic isolated from Myrothecium verrucaria, has been corrected (cf. [4]) and shown to be that of 3-acetyl-4-hydroxymethyl-pyrrole (2) by comparison with a variety of β-acetylpyrrole-derivatives, whose NMR. chemical shifts and coupling constants are reported. Verrucarin E (2) has been synthesized in low yield from 3-acetylpyrrole (12). The following previously unknown β-acetylpyrrole-derivatives are described: 3-acetyl-5-methyl-pyrrole-2-carbonic-acid (11), 4-acetyl-2-methyl-pyrrole (13), 3-acetyl-4-formyl-pyrrole (7), 3-acetyl-1-hydroxymethyl-pyrrole (14), 3-(3-hydroxypropionyl)-pyrrole (15), 3-(3-hydroxy-2-hydroxymethyl-propionyl)-pyrrole (16), and 3-(3-acetyl-pyrr-1-yl)-1-(pyrr-3-yl)-propan-1-one (17).

Endogenous respiration of spores of the fungus Myrothecium verrucaria can be stimulated up to over-10 fold by diverse chemicals or by physical treatments. Greatest effects were caused by azide (12-fold at 250 mum) and by 2,4-dinitrophenol (7-fold at 300 mum). Marked stimulation was also caused by 10 mum silver (5-fold), 30 mum pentachlorophenol (6-fold), 10 mum carbonyl cyanide m-chlorophenyl hydrazone (4.5-fold) and 10 mum merthiolate (4-fold). Physical treatments such as heat (50 C), freezing, and sonication at sublethal levels were also stimulatory. Stimulation by azide or dinitrophenol was much greater in young than in old spores, whereas response to other chemicals and to freezing was relatively unaffected by spore age. In older spores the effect of azide was no greater than some other inhibitors. During incubation with azide, the endogenous trehalose reserves decreased and changes in free amino acids occurred, both increases and decreases. Thus anabolic as well as catabolic changes occur as evidenced also by the germination of a few (up to 5%) spores. The mechanisms of stimulation must be varied and complex. Permeability changes in the membrane confining endogenous reserves are proposed as a common initial cause. Additional changes in characteristics of membranes of other subcellular particles, as well as enzymic phenomena such as uncoupling of oxidative phosphorylation, are presumably involved in instances where greater stimulation occurs. The data are consistent with the hypothesis that dormancy in these spores results from separation of substrates from metabolic enzymes and more specifically that metabolites are sequestered rather than enzymes.

SUMMARY The persistence in soil of captan and thiram was investigated by means of a technique in which the fungicidal content of soil was assayed by incubating plugs of soil containing fungicide on agar plates seeded with spores of Myrothecium verrucaria and measuring the diameter of the zone of fungal inhibition that was produced. When the fungicides were well distributed in soil they showed extremely low persistence, both fungicides having a half-life of between 1 and 2 days. In contrast, when the fungicides were added to soil in the form of dressings on the surface of glass beads they persisted well in soil, little change from their initial concentration occurring even after 21 days. These results suggest that captan and thiram persist far longer in soil when localized in high concentrations than when uniformly distributed through soil. If a glass bead is regarded as a reasonable simulation of a seed these results help to explain the effectiveness of these fungicides as seed dressings despite their apparently low persistence in soil.

AND R. T. DARBY. 1952. A rapid cell volume assay for fungitoxicity using fungus spores. Jour. Bact. 65: 16-26. , AND A. B. NORTON. 1948. Studies on the physiology of spores of the cellulolytic fungus Myrothecium verrucaria. The Quartermaster General Laboratories, Res. Report, Microbiol. Ser. 11: 1-50. ROBBINS, W. J. 1939. Thiamin and symbiosis. Bull. Torrey Bot. Club 66: 569-572. SCHOPFER, W. H. 1943. Plants and vitamins. Chronica Botanica Co. THOMPSON, R. C. 1942. Synthesis of B vitamins by bacteria in pure culture. Univ. Texas Publ. 4237: 87-96. WAKSMAN, S. A. 1941. Antagonistic relations of microorganisms. Bact. Rev. 5: 231-291. WEINDLING, R. 1938. Association effects of fungi. Bot. Rev. 4: 475-496.

1. There are certainly two, and probably three, stages in the development of B. megaterium from the spore to inception of cell division. The rapid increase in rate of respiration during the initial 10 minutes on glucose-peptone-yeast extract medium coincides with decrease in optical density and with increase in stainability. From about 10 to 100 minutes, the rate increases linearly, coinciding with swelling of the spores and ending at approximately the time of rupture of the spore case. From about 100 to 180 minutes, there is a second and steeper linear increase in respiration rate coinciding with cell elongation. These physiological and morphological phenomena are discussed as criteria for germination. 2. The rate of respiration of M. verrucaria spores also increases linearly up to about 300 minutes in sucrose-yeast extract medium. No breaks in the curves are observed during formation of the germ tubes. 3. Oxygen uptake follows the parabolic curve See PDF for Equation within the limits of experimental error for both types of spores. 4. It is postulated that metabolism during these stages of linear increase may be regulated by processes occurring at cellular or intracellular surfaces or by synthesis of a limiting enzyme at constant rate.

Ascorbic acid oxidase is widely distributed in higher plants. The enzyme from certain tissues has been highly purified, and some of its properties are well established, especially its copper protein character (6, 19, 23, 24). The function of the enzyme and the role and metabolism of ascorbic acid in plant tissues has been studied extensively (19). Although enzymatic pathways of electron transfer between dehydrogenase systems and ascorbic acid are known, the significance of these and of ascorbic acid oxidase, itself, in respiration or other metabolic phenomena remains obscure. Two atypical ascorbic acid oxidases in lower plants have been investigated, one by Mandels (17, 18) in spores of the fungus Myrothecium verrucaria, the other by Ward (29) in the slime mold Physarum polycephalum. These enzymes were termed "atypical" because they differed from the copper enzyme in response to inhibitors of metallo-enzymes, in reaction products, or in substrate specificity. Furthermore, the slime mold enzyme appeared to require an intermediate thiol compound for activity. The mycelium of M. verrucaria also contains an atypical ascorbic acid oxidase (18, 30) with certain properties distinctly different from those of the spore enzyme as well as the slime mold and higher plant oxidases. The present study deals mainly with the general properties and cellular location of the mycelial oxidase and its role as an electron transfer agent in the respiration of mycelium.

ConclusionsThe effect of actidione on the germination and the spore and mycelial respiration of Myrothecium verrucaria was measured by dosage-response curves and LD50 values and by change in per cent inhibition with time. Spore respiration was about 4 times as sensitive as germination and 10 times as sensitive as mycelial respiration. Only a small fraction of respiration seemed to be required for growth. Inhibition of spore and mycelial respiration decreased with time, in the spores because of decreasing sensitivity and possibly also because of detoxication.

Two methods are described for purification of the cellulase of Myrothecium verrucaria from concentrated culture filtrates. The steps of method I are (1) fractionation with ammonium sulphate, (2) elution through Sephadex G25, (3) elution through Sephadex G75, (4) precipitation with polymethacrylic acid, and (5) elution through Amberlite CG50 with citrate buffer containing a gradient of urea concentration. The steps of method II are precipitation by saturated ammonium sulphate, (2) and (4) as above, elution through DEAE-cellulose with phosphate buffer containing 7 M urea, followed by (2) and (3) as above. The two methods gave final products with identical specific activities toward carboxymethyl cellulose; the increase in specific activity was approximately 12-fold. Starch-gel electrophoresis at pH 6.8 showed no major indications of heterogeneity. The purified enzyme was unstable but could be stored frozen in dilute salt solution.Enzyme passed through DEAE-cellulose without an inhibitor of cellulase activity was contaminated by DEAE-substituted oligoglucosides and subject to proteolysis. Urea could be replaced by cellobiose as an inhibitor but the latter gave enzyme contaminated by products of transfer reactions.Binding of urea by the resin is shown to influence significantly the resolution attainable in chromatographic fractionations on Amberlite CG-50 with buffers containing gradients of urea concentration.Procedures for dialysis and desalting of cellulases and a compact reaction vessel for pH stats are described.

An improved procedure for the production of cellulase by Myrothecium verrucaria is described. The mold is grown in two stages in the same culture vessel: firstly on a semisolid medium of ground extracted cotton linters moistened with a solution of inorganic salts, and then, by addition of further salt solution, in submerged culture.

1. Cell-free enzymes from Myrothecium verrucaria and Trichoderma koningii hydrolyse native undegraded cellulose, as found in cotton fibres, in a random manner to short insoluble fibres and to minor amounts of soluble products. 2. Enzyme preparations from M. verrucaria fail to attack the short fibres whereas preparations from T. koningii solubilize them completely to sugars at an optimum pH4·2–4·6. 3. The mode of hydrolysis of cotton cellulose by preparations from T. koningii involves from the earliest stages the formation of reducing sugars, followed closely by the appearance of short fibres, until the insoluble and soluble products each constitute about 40–50% of the weight of the initial substrate. After this stage the quantity of sugars increases at the expense of the insoluble short fibres. 4. Depending upon the method of preparation, derived forms of cellulose may be hydrolysed more slowly, much more rapidly, or at the same rate as cotton fibres by enzyme preparations from T. koningii.

The incorporation of nine 14C-labelled, assumed biosynthetic precursors of verrucarin E (1) formed by Myrothecium verrucaria have been measured. The antibiotic and 3-acetyl-4-methyl-pyrrole (3) derived from it were oxidized, and the radioactivity of four of the seven C-atoms determined. It has been found that the carbon skeleton of verrucarin E does not originate from proline, glutamic acid and its biogentic equivalents nor from 5-amino-levulinic acid, but is built up from four acetate units with loss of one carboxyl C-atom. These results are rationalized by proposing a biogenetic pathway for verrucarin E.

A severe petal blight was found affecting the canna ’Pfitzers Dwarf’ at Ottawa in 1959. The principal cause proved to be a pathogenic strain of an Alternarai of the tenuis type. Petal infection readily occurred from 5° to 30 °C. but lesions developed most rapidly at 25 °C The fungus also infected wounded leaves of canna, but it caused little decay. Myrothecium verrucaria (Alb. & Schw.) Ditmar ex Fr., isolated once from a diseased petal, could infect petals but it caused less rapid decay than the Alternaria.

Muconomycin A (M-A) and muconomycin B (M-B), two nonnitrogenous antibiotics derived from culture broths of Myrothecium verrucaria, possess unique structural features and high mammalian toxicity. The ld 50 in mice (i.p.) was found to be 0.50 to 0.75 mg/kg for both antibiotics. M-A induced a severe creatinuria in rats, and this effect was attenuated by pre-administration of vitamin E. Both antibiotics were found to inhibit creatine phosphokinase (CPK) in vitro; I50 values were 29.8 × 10−4 M for M-A and 7.0 × 10−4 M for M-B. CPK was then assayed, at various concentrations of substrate, with M-A. A Hunter-Downs plot of the data showed that M-A was a noncompetitive inhibitor of CPK; the Ki was 3.5 × 10−4 M. It is suggested that the high toxicity of these antibiotics, as well as their ability to cause creatinuria, may be related to their inhibitory effect on CPK.

SUMMARY: A study has been made of the degradation of different forms of cellulose (‘cellulolysis’), comprising soluble substituted derivatives (carboxymethyl-cellulose), insoluble cellulose powder, swollen cellulose, hydrocellulose and native cotton fibres, by concentrated suspensions of mixed rumen micro-organisms and by the aerobic fungus Myrothecium verrucaria. Mixed rumen micro-organisms are shown to be one of the most powerful sources of cellulolytic enzyme, in that they produce almost complete solubilization of all the above forms of cellulose in a relatively short period (3 days). Enrichment cultures of rumen micro-organisms were prepared by growing concentrates of mixed micro-organisms on cellulose powder. Cellulolysis was followed by determining the cellulose disappearance, formation of cellulolytic activity and gas evolution. Freeze-dried powders and their derived acetone powders obtained from washed concentrated supensions (non-enrichment cultures) of mixed rumen micro-organisms solubilized up to 80% of insoluble cellulose powder. Cell-free extracts were isolated: (a) from concentrated suspensions and from freeze-dried powders of rumen micro-organisms by extraction with butanol; (b) from concentrated suspensions of enrichment cultures by grinding with alumina at low temperature. The butanol extracts solubilized cellulose powder to a small extent (10% solubilization) and were more effective against the soluble carboxymethylcellulose. In contrast, the alumina extracts were more active against insoluble cellulose (30 % solubilization) but were only weakly active against carboxymethyl-cellulose. Cell-free filtrates from Myrothecium verrucaria, an aerobic fungus much used in work on cellulose metabolism, were shown to possess cellulolytic properties very similar to those of alumina extracts from enrichment cultures of rumen microorganisms. The significance of the results is discussed in relation to the mode of breakdown of cellulose.

Carbon nutrition of Chaetomium sp., Myrothecium verrucaria, Pestalotia gracilis, and Pleurotus ostreatus was studied. Nitrogen nutrition was also examined with Nigrospora oryzae included. The effect of C/N ratio on fruiting was measured for the first four fungi. Several carbon sources including alcohols, carbohydrates, and organic acids were tested; in general, dextrin and L-arabinose were the most favorable for growth, starch was less so, whereas sodium acetate and sodium citrate were the least favorable. Maltose supported good sporulation of M. verrucaria, P. gracilis, and P. ostreatus. Organic nitrogen was superior to inorganic nitrogen for growth of M. verrucaria, N. oryzae, and P. ostreatus. The opposite was noticed for Chaetomium sp. and P. gracilis. Not all could grow on sodium nitrite at the concentration used nor sporulate on DL-methionine or ammonium sulfate. The best sporulation of P. gracilis was obtained with the maximum glucose and nitrate concentrations used (4% and 1% respectively), whereas the minimum concentrations (0.1 and 0.05% respectively) were best for the sporulation of P. ostreatus. Chaetomium sp. and M. verrucaria fruited best on intermediate concentrations of glucose and nitrate.

SUMMARY: When Alternaria solani is grown on any one of a variety of defined culture media, an antibiotic metabolic product, alternaric acid, accumulates in the medium. The quantity of alternaric acid produced is directly related to the amount of mycelium formed; good yields of alternaric acid are obtained on any medium which supports good growth. Optimal media contain high concentrations (7·5% (w/v) or more) of sucrose, which is better than any other carbon source tested. Nitrogen may be supplied as nitrate or casein hydrolysate; ammonia as nitrogen source is equally good when supplied in conjunction with a suitable organic acid, 0·25% (w/v) acetic acid being particularly favourable. The antibiotic is isolated from optimal media by extraction with chloroform after adjustment to pH 3·5; the solvent is then evaporated and the residue recrystallized from benzene. Yields of the order of 150 mg./l. are obtained. Alternaric acid is not antibacterial. Germination of spores of some fungi (e.g. Absidia glauca, Myrothecium verrucaria) is prevented by 1 μg./ml. or less of alternaric acid. Germination of spores of other fungi (e.g. Botrytis allii) is unaffected by concentrations as high as 100 μg./ml., but extension of the germ-tubes is markedly retarded shortly after germination. This secondary retarding effect may be produced by very low concentrations; 0·01 μg./ml. produced an obvious effect with B. allii.

Structure 1 has been established for roridin H (C29H36O8), an antibiotic isolated from Myrothecium verrucaria and formerly namend verrucarin H [3]. Base catalysed hydrolysis of 1 gave the known terpene alcohol verrucarol (8; C15H22O4) and myrothecinic acid (3; C14H18O6), a previously undescribed dicarboxylic acid. The structure of 3 was determined by spectral methods and by chemical cleavage of the unusual acetal group of dimethyl hexahydromyrothecinoate (6) by hydrolysis with HCl in the presence of 2, 4-dinitrophenylhydrazine, producing the 2, 4-dinitrophenylhydrazone 13 of 3-methylglutaric acid semialdehyde 10 and methyl 6, 7-dihydroxy-octanoate (11). 13 was transformed in to dimethyl 3-methylglutarate (15). Cleavage of 11 with HIO4 gave acetaldehyde and adipic acid semialdehyde (17) which was oxidized to adipic acid (19). The 220 MHz NMR spectra of roridin H (1) and dimethyl hexahydromyrothecinoate (6) allowed unequivocal assignment of the signals to the majority of the protons in the antibiotic.

A combination of Fickian diffusion and Michaelis–Menten kinetics is proposed to describe the rate of diffusion-coupled biochemical reactions. This postulate leads to a nonlinear mathematical model which is solved by a perturbation technique. The result is a relation which permits identification of zones of relative diffusion or reaction influence. The conversion of cellulose to protein by Myrothecium verrucaria is a heterogeneous process that is well-suited to this type of analysis, although the data requirements are severe.

Exposure of cotton fiber to cell-free filtrates obtained from certain culture media after the growth of microorganisms increases the degree of subsequent swelling of the fiber in NaOH solutions. The active agency in the filtrates is here termed " S factor." Its action is measured by a previously described alkali-centrifuge test [12]. With the fungus Myrothecium verrucaria, S factor was produced during growth in media which included any of several cellulose-containing materials but not in any of several media lacking in cellulose. This finding and related results suggest that S factor consists at least in part of an enzyme or enzymes of the cellulase complex. A simple and reliable method of preparing Myrothecium filtrates of uniform and high S activity is reported. Several properties of S factor in Myrothecium filtrates have been observed. It is operative over a wide pH range. A very considerable uptake of the factor from solution by cotton fiber and a major increase in alkali-centrifuge value of the fiber occur in 10 min. Strength losses take place much more slowly. S factor is moderately stable in sterile filtrates at room tempera ture. Filtrates may be stored in frozen condition or may be dried at 40°C and stored dry with little loss in activity. The active factor is precipitated by acetone and is inactivated by crystal line pepsin. Although partial inactivation of S factor by heat occurred in 1/2 hr. at temperatures of 60°C and higher, complete inactivation did not take place even at much higher temperatures, and easily measurable activity remained even after heating at 100°C for 1/2 hr. Autoclaving at 15 lbs. steam pressure for 15 min. caused complete inactivation. The same filtrates also exhibited resistance to complete cellulase inactivation by heat when tested by a carboxymethylcellulose- viscosity test for cellulase.

Summary 1. Initially the growth of Myrothecium verrucaria was found to be inhibited through cyanamide. A concentration of 10-4 M of cyanamide caused a slight inhibition. 2. The addition of cyanamide to the culture medium brought about an induction of a cyanamide hydratase. This enabled the fungi to grow, with cyanamide as the only source of nitrogen. Urea was formed first, and then decomposed into ammonia and carbon dioxide. 3. Cyanamide hydratase was isolated from the fungi mycelium and concentrated approximately seventy times. 4. The characteristics of cyanamide hydratase were examined, i. e., to show that the reaction NH = C = NH + H2O → (NH2)2CO could be proved quantitatively, and that there was an absence of inhibitors in the preparations. In addition, it was found that the temperature optimum ranged between 40–55° C, that the pH optimum was pH 7.7 and that the Km of the reaction was 3,7 · 10-6 Mol/l. The cyanamide hydratase seemed to be crucial for the reaction. The velocity of the reaction was greatly enhanced through additional Mn2+. The enzyme was classified as one of the E. C. group 4.2.1. 5. The question of an active secretion of the enzyme into the culture medium was discussed.

Activities of several enzymes associated with cellulolysis were compared using as substrates cell-walls of Lolium multiflorum and cotton cellulose. Purified enzymes C 1 (see Ref. 1 for definition), C. x (CM-cellulase) and β-glucosidase were employed as well as culture filtrates containing C x . Activities were determined by ability to digest the substrates and to release H 2 O-soluble phenolic compounds from the grass cell-walls. The culture filtrates most active on cotton cellulose were obtained using the fungi Trichoderma viride and Fusarium solani ; with grass cell-walls the most active were from T. viride , Gliocladium roseum , a species of Basidiomycetes, and one strain of Myrothecium verrucaria (IMI Strain 25 291). For the crude enzyme preparations tested, there were highly significant correlations between the digestibility of grass cell-walls and the UV-absorption of the filtrate at λ max 290 nm and at λ max 324 nm but there was no significant correlation between the digestibility of grass cell-walls and that of cotton cellulose. Partially purified C 1 and C x from two different fungal sources showed activity on both substrates. Differences in MW of the H 2 O-soluble phenolic compounds obtained by treatment of grass cell-walls with C 1 and C x components suggest that these enzymes could have different modes of action. Synergism between C 1 and C x from T. koningii occurred with both substrates but with C 1 and C x from F. solani synergism only occurred with cotton cellulose.

SUMMARY The mycelia of 45 species of fungi were examined using a direct-vision spectroscope and in every instance absorption bands of cytochromes were seen. The relative positions of the bands varied, but this variation was not considered sufficient to suggest the presence of cytochromes additional to those normally found in yeast. Since Keilin's original paper entitled 'On cytochrome, a respiratory pigment common to animals, yeast and higher plants' (Keilin, 1925), there have been few observations on the occurrence of cytochromes in fungi. Cytochromes have been recorded in Neurospora crassa and Penicillium, notatum (Keilin and Tissieres, 1953); certain strains of N. crassa show variations from the normal cytochrome system of the wild type (Tissieres and Mitchell, 1954). Neilands (1952) isolated cytochrome c from Ustilago sp. whilst cytochrome oxidase has been shown to occur in Myrothecium verrucaria (Darby and Goddard, 1950), in Aspergillus spp. (Tamiya, 1942), and in Blastocladiella emersonii (Cantino and Horenstein, 1955). In the latter organism the enzyme was apparently not present in the thick-walled resistant sporangia. This paper records the results of direct spectroscopic examinations for the presence of cytochromes within the mycelium of a series of fungi. MATERIAL AND METHODS

Cellulase production by strains of Myrothecium verrucaria, Stachybotrys atra and Trichoderma viride was examined. Myrothecium verrucaria was found to give the greatest yields. A variety of media were examined as potential substrates for the industrial production of cellulase. The salts content of the medium was varied and was found to affect cellulase production. Glucose, carboxymethylcellulose (CMC), filter paper and three industrial wastes were examined as possible cellulase inducers. Filter paper was found to be the most effective, followed by sugar cane bagasse and CMC.

There is relatively little knowledge concerning the respiration of filamentous fungi. This paper describes some of the features of the tespiration,of a strain of Myrothecium verrucaria (Alb. and Schw.) Dit. ex Fr. and methods developed in handling such a septate fungus in respirometry. A review of the few published papers dealing with fungus respiration shows that wide differences exist among the results of various workers, and it is possible that much of the variability can be ascribed to differences in methodology rather than to real differences in physiology of the organisms concerned. The difficulty encountered in evaluating such differences is aggravated by the lack of supporting information such as development curves, pH data, environmental effects, etc. Some of the criticisms leveled against the older work have been stated by Wassink (1934). This literature based on analytical methods is not considered in any detail here, but attention is given in this short summary only to those papers using manometric or volumetric methods. None of the papers has undertaken a unified evaluation of fungus respiration with particular attention to the effects of technique. The need for the standardization of methods and the integration of the data concerning age, physiological condition, nutrition, etc. is emphasized. Various methods have been used by the several investigators in preparing fungi for respirometry. Usually pieces of mycelial mats taken from unshaken cultures have been used directly in the respirometers. In these cases it is often impossible to ascertain the homogeneity of the material with respect to vegetative and sporulating types. Dry weights, if determined, were taken at the end of the experiment. Although most of the papers were aimed at more specialized problems, some information about "normal" respiration can be extracted from them. Giese and Tatum (1946) reported a Qo2 ([tl.O2 consumed /mg. dry wt./hr.) from 10 to 55 for endogenous respiration of Neurospora. They were of the opinion that the rather wide variation was due to the age of the culture or its state of nutrition. Wolf (1947) using washed 6?/2-day-old mycelial pellets of Penicillium notatum suspended in buffer reported Qo2 values between 3.11 and 8.97 in fifty-five runs. The oxygen uptake of pellets of P. chrysogenum and P. notatum reported by Koffler and co-workers (1945) taken in the propagation medium reached a peak on the third to fifth day with Qo2 values around 30; the Qo2 then rapidly fell off to about 2 on the seventh day. This latter value is of about the same magnitude as that of Sordaria fimicola as deter

SUMMARY: Myrothecium verrucaria can produce cellulase when grown on media containing glucose or glycerol as sole carbohydrate source. The enzyme complex can depolymerize cellulosic substrates ranging from carboxymethylcellulose to cotton fibres. Hemicellulase is also produced. These depolymerases are first detected intracellularly near the time of the deceleration phase of growth and appear extracellularly a few hours later. Since cellulase can be produced by reducing the organism's growth rate in the absence of cellulose or any of its constituent sugars, it is suggested that this enzyme is constitutive and that production may be partly controlled by catabolite repression.