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Notes: Summary Cell-free extracts of mycelia and sclerotia of Sclerotinia sclerotiorum (Lib.) D By. grown on synthetic liquid medium with various carbon sources contained trehalase (α,α′-glucoside 1-glucohydrolase; EC3.2.1.28) activity. The enzyme was not usually detected in the culture filtrate. Treatment with ammonium sulfate or MnSO4 and alumina resulted in a 2- to 3-fold purification. The optimum pH (5.0), K m with trehalose (1.7×10-3 M) and other properties are within the range reported for trehalase from other fungi.

Notes: Summary Cell-free extracts of mycelia and sclerotia ofSclerotinia sclerotiorum (Lib.) D By. grown on synthetic liquid medium with various carbon sources containedd-mannitol-1-phosphate: NAD oxidoreductase (EC 1.1.1.17) and weakd-mannitol: NADP oxidoreductase (EC 1.1.1.67) activity. The difference in specific activity of the two enzymes coupled with the presence ofd-mannitol-1-phosphatase activity indicates the major pathway for mannitol synthesis. $$\begin{gathered} D - Fructose - 6 - P + NADH + H^ + \mathop \rightleftharpoons \limits_2^1 D - Mannitol - 1 - P + NAD^ + \hfill \\ D - Mannitol - 1 - P\xrightarrow{{ 3 }}D - Mannitol + P_i \hfill \\ \end{gathered} $$ . Both enzymes were present in mycelia and sclerotia at all times, but highest activity occurred during early stages of growth. The optimum pH for reactions 1,2 and 3 was 7.5, 10.5 and 6.5 respectively. The oxidoreductase was quite specific in substrate requirements. Both enzymes were inhibited by sulfhydryl reagents and the phosphatase was activated by Mg2+.

Notes: Summary Sclerotia of Sclerotinia sclerotiorum (Lib.) D By. were obtained from commercial pea-and bean-cleaning operations or grown on potato-dextrose agar and synthetic glucose-and sucrose-salts agar media. The crude fat (ether extract) content of sclerotia varied from 0.8 to 1.5%. Extraction and fractionation of the lipids followed by gas chromatographic analysis showed that sclerotia from pea cleanings contained one predominant hydrocarbon which was absent from sclerotia produced in the laboratory. Sclerotia from natural sources and grown in the laboratory contained a similar distribution of C18 unsaturated free fatty acids, however, quantitative differences were noted. Palmitic, oleic and linoleic were the major free fatty acids of the laboratory-grown sclerotia while a high proportion of linoleic acid was also found in sclerotia from natural sources. Sclerotia were fractionated into water-soluble and water-insoluble fractions. After acid hydrolysis of the waterinsoluble fraction, both fractions were analyzed for amino acids. Twenty-one compounds, including 2 unknowns, were detected in the soluble fraction. The hydrolyzates contained 19 amino acids, including the same 2 unknowns. Two compounds tentatively identified as ornithine and γ-aminobutyric acid were found only in the water-soluble fraction. The relative amino acid composition of the water-insoluble fraction of sclerotia from various sources was fairly constant but the arginine content decreased on the synthetic media.

Notes: Summary Sclerotinia sclerotiorum (Lib.) D By. was grown in stationary liquid mineral-salts medium, pH 4.3, containing various carbon sources and the weight of mycelia and sclerotia was determined at regular intervals. When grown on various glucose concentrations (0–24 g of C/l), more sclerotia were produced at 8–12 g of C/l. Sclerotia were not usually formed in shake cultures. The ability of the fungus to use other carbon sources for growth and sclerotium formation was tested at 12 g of C/l in the stationary mineral-salts medium. The highest weights of mycelia and sclerotia occurred with raffinose, sucrose, maltose, lactose, d-mannose, d-glucose, d-fructose or l-arabinose. Good growth but decreased sclerotium production were found on cellobiose and d-xylose. Reduced or poor growth, a long lag period and few or no sclerotia occurred on trehalose, melibiose, l-sorbose, l-rhamnose, d-ribose, d-arabinose, l-xylose or 8 polyols. No growth was observed with erythritol or i-inositol. A combination of glucose plus trehalose or polyols resulted in increased growth and the formation of sclerotia. Organic acids supported little or no growth and no sclerotia were produced. Generally culture filtrates which supported growth and formation of sclerotia became acid (about pH 3.5). The pH of the culture filtrate usually increased slowly during the growth period when the fungus grew poorly and no sclerotia were formed. The alcoholsoluble sugars and polyols present in culture filtrates, mycelia and sclerotia were determined by paper and thin-layer chromatography. Regardless of the carbon source, mannitol was usually present in culture filtrates. The occurrence of other compounds in the filtrates depended on the carbon source. Trehalose, mannitol and usually small quantities of glucose or fructose were present in mycelia and sclerotia from all carbon sources. Galactitol or pentitols occurred in mycelia and sclerotia when the fungus grew on galactose and oligosaccharides containing galactose or the corresponding pentose, sugars. Acid hydrolyzates of the alcohol-insoluble fraction of mycelia or sclerotia contained glucose, smaller amounts of galactose and mannose and traces of ribose and rhamnose.

Notes: Summary Pentitol: NADP and pentitol: NAD oxidoreductase were detected in cell-free extracts of mycelia and sclerotia ofSclerotinia sclerotiorum (Lib.) D By. grown on synthetic liquid medium containingd-xtlose,l-arabinose ord-ribose. Both enzymes were similar to those reported from other fungi. The enzymes were not detected when the fungus was grown on a medium containingd-glucose.

Notes: Summary Citrate synthase (EC 4.1.3.7), aconitate hydratase (EC 4.2.1.3), NADP specific isocitrate dehydrogenase (EC 1.1.1.42), fumarate hydratase (EC 4.2.1.2) and malate dehydrogenase (EC 1.1.1.37) were detected in cell-free preparations of Sclerotinia sclerotiorum (Lib.) D By. grown on liquid glucose-salts medium in stationary culture. Isocitrate lyase (EC 4.1.3.1) was present when the fungus grew on a carbohydrate-free medium but was not detected when the cultures grew on the glucose-salts medium. The amount of oxalate in the culture filtrate declined as the specific activity of citrate synthase and malate dehydrogenase in the mycelium declined. Increasing the initial pH of the medium resulted in an increase of the dicarboxylic acids in the culture filtrate and the specific activity of malate dehydrogenase in the mycelium. The specific reaction(s) leading to oxalic acid formation were not identified.

Notes: Cell-free extracts of mycelial mats of Pgrenochaeta terrestris grown in stationary culture on synthetic glucose or sucrose - salts liquid media contained D-mannitol-1-Phosphate:NAD oxidoreductase (EC 1.1.1.17) activity. Greatest activity occurred early in the growth period. The optimum pH for the reduction of NAD+ in the presence of Fru-6-P was 7.4–7.5 while the optimum pH for the oxidation of NADH in the presence of Mtl-1-P was 8.1–8.2. The enzyme was stabilized to some extent in Tris-maleate buffer, pH 7.5, and by the addition of 10% (NH4)2SO4, to this buffer. A 10- to 16-fold purification was attained by a combination of (NH4)2SO4 fractionation and gel filtration on Sephadex G-100. The enzyme was relatively specific in its substrate and coenzyme requirements. The Km values were determined as: Fru-6-P - 3 × 10−4 M, Mtl-1-P - 1 × 10−4 M, and NAD+ and NADH - 3 × 10−5 M.

Notes: Pyrenochaeta terrestris (Hansen) Gorenz, J. C. Walker and Larson produces D-mannitol in the mycelium but not in the cutture filtrates when grown in a sucrose salts liquid medium.In the present study, P. terrestris was grown in stilt culture on a synthetic salts medium containing 30 g of sucrose per liter. After inoculation with a myceliat suspension, the mycelial mats were harvested and the dry weight and the amount of mannitol were determined. Maximum mycelial mat production occurred at 15 days after inoculation while the amount of mannitol was greatest at about 7 days after inoculation. The percentage of mannitot on a dry weight basis was maximal (20–25 per cent) within a few days after inoculation and decreased rapidly to 3–4 per cent at the time mycelial mat production was greatest. The same percentage of mannitot was produced when the fungus was grown in shake culture or when the sucrose was replaced by equivalent amounts of D-fructose, D-glucose, D-mannose, maltose, trehalose, and raffinose. Increasing the amount of sucrose or decreasing the amount of sodium nitrate increased the amount of mycelium produced but the percentage of mannitol in the mycelium remained about the same.Mannitot was reutilized when mycelial mats were transferred to a mineral medium without a carbon source. It was concluded that mannitot probably serves as a reserve carbohydrate in P. terrestris.