Isolation of Fungi from Heterodera glycines and in vitro Bioassays for Their Antagonism to Eggs

Twenty fungi were assayed in vitro for antagonism to eggs of Heterodera glycines. Eight of the fungi were isolated from cysts or eggs of H. glycines during the current study, one was isolated from Panagrellus redivivus, and eleven were obtained from other researchers or collections. The bioassays were conducted on eggs from nematodes that had been grown monoxenically on excised root tips. Phoma chrysanthemicola, one strain of Verticillium chlamydosporium, and one strain of V. lecanii caused a decrease (P < 0.01, P < 0.05, P < 0.05, respectively) in the number of viable eggs, although no hyphae were observed colonizing live eggs. Trichoderma polysporum infected live eggs but enhanced (P < 0.05) egg survival. Acremonium bacillisporum, Chaetomium sp., Drechmeria coniospora (two strains), Epicoccum sp., Exophiala jeanselmei, Fusarium sp., Neocosmospora vasinfecta, Scytalidium fulvum, Trichoderma harzianum (two strains), V. chlamydosporium (one strain), V. lecanii (three strains), and an unidentified fungus did not measurably affect egg viability, even though hyphae of five of these fungi were seen in live eggs. The bioassay provides a useful step in the selection of a biological control agent for this major nematode pest.     

Regulation of cellulase production in two thermophilic fungi Melanocarpus sp. MTCC 3922 and Scytalidium thermophilum MTCC 4520

This paper reports regulation of cellulase production in two thermophilic fungi, Melanocarpus sp. MTCC 3922 and Scytalidium thermophilum MTCC 4520. The expression of endoglucanase (EG), avicel adsorbable endoglucanase (AAEG) and β-glucosidase in both fungi was inducible. Of the different carbon sources tested, rice straw induced maximal levels of cellulase in both fungi. While, the addition of fructose (1%, w/v) to the carboxymethylcellulose (CMC) medium resulted in two-fold increase in endoglucanase production in Melanocarpus sp., however, the addition of ethanol (1%, v/v) resulted in eight-fold-increased expression of endoglucanase in S. thermophilum. The expression profiles of different components of cellulase complex were shown to be co-regulated in S. thermophilum but independently regulated in Melanocarpus sp.     

Enzymatic Preparation of Aromatic Ethylamines from Aromatic L-Amino Acids

The characteristic mode for the herbicidal action of 2-amino-3-chloro-1,4-naphthoquinone (ACN) was investigated by using autotrophic green microalgae (Scenedesmus acutus). The effects of ACN on the growth, chlorophyll content, protoporphyrin-IX accumulation, and ethane production in Scenedesmus cells were measured in comparison with three reference herbicides, i.e., ioxynil, dinoseb, and chlorophthalim. ACN appeared to have a different mechanism for action from these three herbicides. S. acutus cells grown with ACN produced ethane, but the mechanism for its production is considered to have been different from that of chlorophthalim. ACN may inhibit porphyrin biosynthesis at a different stage from that by chlorophthalim.     

Ericoid mycorrhizal association: ability to adapt to a broad range of habitats

Ericoid mycorrhizal fungi are symbiotically associated with the roots of members of the Ericaceae which include genera such as Calluna, Epacris, Erica, Rhododendron and Vaccinium. These ericoid mycorrhizal associations have adapted to a broad range of habitats, from mor humus soils of the northern hemisphere to sandy soils occurring in the southern hemisphere. They also play an important part in enabling plants like Calluna vulgaris (L.) Hull in the northern hemisphere to colonize mine spoils which are inhospitable environments of toxic waste for growth of most plants. The mechanisms of utilizing complex forms of nitrogen and phosphorus and providing protection against toxic metals are described. These mechanisms carried out by ericoid mycorrhizal associations enable host plants to establish in diverse habitats.     

双间柱顶孢的研究进展

双间柱顶孢是一种暗色真菌,能感染植物和人类。主要存在于热带、亚热带地区。世界各地均有散在报道,泰国的感染最高,但我国很少有报道。为进一步认识该菌,现就双间柱顶孢的分类和命名学的研究进展、流行病学现状、形态特点、生物化学、致病性及治疗等情况进行综述。     

Purification and biochemical properties of a thermostable xylose-tolerant β-<Emphasis Type="SmallCaps">D</Emphasis>-xylosidase from <Emphasis Type="Italic">Scytalidium thermophilum</Emphasis>

The thermophilic fungus Scytalidium thermophilum produced large amounts of periplasmic -D-xylosidase activity when grown on xylan as carbon source. The presence of glucose in the fresh culture medium drastically reduced the level of -D-xylosidase activity, while cycloheximide prevented induction of the enzyme by xylan. The mycelial -xylosidase induced by xylan was purified using a procedure that included heating at 50°C, ammonium sulfate fractioning (30–75%), and chromatography on Sephadex G-100 and DEAE-Sephadex A-50. The purified -D-xylosidase is a monomer with an estimated molecular mass of 45 kDa (SDS-PAGE) or 38 kDa (gel filtration). The enzyme is a neutral protein (pI 7.1), with a carbohydrate content of 12% and optima of temperature and pH of 60°C and 5.0, respectively. -D-Xylosidase activity is strongly stimulated and protected against heat inactivation by calcium ions. In the absence of substrate, the enzyme is stable for 1 h at 60°C and has half-lives of 11 and 30 min at 65°C in the absence or presence of calcium, respectively. The purified -D-xylosidase hydrolyzed p-nitrophenol--D-xylopyranoside and p-nitrophenol--D-glucopyranoside, exhibiting apparent K_m and V_max values of 1.3 mM, 88 mol min^–1 protein^–1 and 0.5 mM, 20 mol min^–1 protein^–1, respectively. The purified enzyme hydrolyzed xylobiose, xylotriose, and xylotetraose, and is therefore a true -D-xylosidase. Enzyme activity was completely insensitive to xylose, which inhibits most -xylosidases, at concentrations up to 200 mM. Its thermal stability and high xylose tolerance qualify this enzyme for industrial applications. The high tolerance of S. thermophilum -xylosidase to xylose inhibition is a positive characteristic that distinguishes this enzyme from all others described in the literature.     

Thermostable conidial and mycelial alkaline phosphatases from the thermophilic fungus Scytalidium thermophilum

An extracellular (conidial) and an intracellular (mycelial) alkaline phosphatase from the thermophilic fungus Scytalidium thermophilum were purified by DEAE-cellulose and Concanavalin A-Sepharose chromatography. These enzymes showed allosteric behavior either in the presence or absence of MgCl₂, BaCl₂, CuCl₂, and ZnCl₂. All of these ions increased the maximal velocity of both enzymes. The molecular masses of the conidial and mycelial enzymes, estimated by gel filtration, were 162 and 132 kDa, respectively. Both proteins migrated on SDS-PAGE as a single polypeptide of 63 and 58.5 kDa, respectively, suggesting that these enzymes were dimers of identical subunits. The best substrate for the conidial and mycelial phosphatases was p-nitrophenylphosphate, but β-glycerophosphate and other phosphorylated compounds also served as substrates. The optimum pH for the conidial and mycelial alkaline phosphatases was 10.0 and 9.5 in the presence of AMPOL buffer, and their carbohydrate contents were about 54% and 63%, respectively. The optimum temperature was 70–75°C for both activities. The enzymes were fully stable up to 1 h at 60°C. These and other properties suggested that the alkaline phosphatases of S. thermophilum might be suitable for biotechnological applications. Journal of Industrial Microbiology & Biotechnology (2001) 27, 265–270. Received 10 January 2001/ Accepted in revised form 10 July 2001     

Optimization of culture conditions for production of cellulases and xylanases by Scytalidium thermophilum using Response Surface Methodology

Summary Scytalidium thermophilum type culture Humicola insolens MTCC 4520 isolated from composting soil was optimized for production of cellulolytic and hemicellulolytic enzymes (endoglucanase, Avicel-adsorbable endoglucanase, FPase, β-glucosidase, xylanase and mannanase) by solid-state fermentation (SSF). Initial experiments showed that culture medium containing rice straw and wheat bran (1:3) as carbon source prepared in a synthetic basal medium supported maximal enzyme production at 45 °C. Further optimization of enzyme production was carried out using Box-Behnken design of experiments to study the influence of process variables (inoculum level, (NH₄)₂SO₄ and pH) on enzyme production. The response surface plots revealed the conditions for obtaining optimal enzyme levels. The models computed for R ² value ranged between 95% and 98.7% indicating they are appropriate and can be useful to predict the effect of inoculum level, (NH₄)₂SO₄ and pH on enzyme production. Under optimized conditions 62.5 ± 0.50, 23.0 ± 0.58, 3.0 ± 0.50, 151.00 ± 8.194, 196 ± 5.033 and 4.9 ± 0.32 (units/g substrate) of endoglucanase (EG), Avicel-adsorbable endoglucanase (AAEG), FPase, β-glucosidase, xylanase and mannanase were produced, respectively. Isoelectric focusing (IEF) of the crude extract showed that S. thermophilum produced six different EG isoforms, of which the EG corresponding to pI values of 8.4, 7.9 and 6.5 showed affinity for Avicel, thereby indicating the presence of a cellulose-binding domain (CBD). Furthermore, seven isoforms of β-glucosidase and ten multiple forms of xylanase distributed over a wide range of pI were also detected.     

Xylanase XYL1p from Scytalidium acidophilum: Site-directed mutagenesis and acidophilic adaptation

The role of residues Asp60, Tyr35 and Glu141 in the pH-dependent activity of xylanase XYL1p from Scytalidium acidophilum was investigated by site-directed mutagenesis. These amino acids are highly conserved among the acidophilic family 11 xylanases and located near the catalytic site. XYL1p and its single mutants D60N, Y35W and E141A and three combined mutants DN/YW, DN/EA and YW/EA were over-expressed in Pichia pastoris and purified. Xylanase activities at different pH’s and temperatures were determined. All mutations increased the pH optimum by 0.5–1.5 pH units. All mutants have lower specific activities except the E141A mutant that exhibited a 50% increase in specific activity at pH 4.0 and had an overall catalytic efficiency higher than the wild-type enzyme. Thermal unfolding experiments show that both the wild-type and E141A mutant proteins have a T_m maximum at pH 3.5, the E141A mutant being slightly less stable than the wild-type enzyme. These mutations confirm the importance of these amino acids in the pH adaptation. Mutant E141A with its enhanced specific activity at pH 4.0 and improved overall catalytic efficiency is of possible interest for biotechnological applications.     

Purification,characterization, and identification of a novel bifunctional catalase-phenol oxidase from <Emphasis Type="Italic">Scytalidium thermophilum</Emphasis>

A novel bifunctional catalase with an additional phenol oxidase activity was isolated from a thermophilic fungus, Scytalidium thermophilum. This extracellular enzyme was purified ca. 10-fold with 46% yield and was biochemically characterized. The enzyme contains heme and has a molecular weight of 320 kDa with four 80 kDa subunits and an isoelectric point of 5.0. Catalase and phenol oxidase activities were most stable at pH 7.0. The activation energies of catalase and phenol oxidase activities of the enzyme were found to be 2.7 +/- 0.2 and 10.1 +/- 0.4 kcal/mol, respectively. The pure enzyme can oxidize o-diphenols such as catechol, caffeic acid, and L-DOPA in the absence of hydrogen peroxide and the highest oxidase activity is observed against catechol. No activity is detected against tyrosine and common laccase substrates such as ABTS and syringaldazine with the exception of weak activity with p-hydroquinone. Common catechol oxidase inhibitors, salicylhydroxamic acid and p-coumaric acid, inhibit the oxidase activity. Catechol oxidation activity was also detected in three other catalases tested, from Aspergillus niger, human erythrocyte, and bovine liver, suggesting that this dual catalase-phenol oxidase activity may be a common feature of catalases.