Evaluation of decay and termite resistance of wood treated with copper in combination with boron and N′-N-(1, 8-naphthalyl) hydroxylamine (NHA-Na)

This study evaluated the relative ability of various combinations of copper sulfate with either boric acid or calcium-precipitating agent, N′-N-(1, 8-naphthalyl) hydroxylamine (NHA-Na), to inhibit fungal degradation and attack by Formosan subterranean termites (Coptotermes formosanus Shiraki). Wood specimens were treated with either 1%, 0.5%, or 0.1% concentrations of copper sulfate, boric acid, NHA-Na, copper sulfate + boric acid, or copper sulfate + NHA-Na mixtures. Treated specimens were subjected to laboratory decay-resistance tests by using petri dishes inoculated with the Basidiomycetes fungi Tyromyces palustris and Trametes versicolor for 12 weeks. Treated wood specimens were also subjected to termite-resistance tests under laboratory conditions. Increased efficacy of copper sulfate against the brown-rot fungus T. palustris was observed when either boric acid or NHA-Na was added. The most effective treatments against the fungi tested were NHA-Na only treatments at 1% and 0.5% concentration levels. Boric acid treatments were not able to protect wood against decay after leaching because of excessive leaching of boron. Similar results were obtained in termite-resistance tests in comparison with decay-resistance tests. These results indicate that the efficacy of the treatments in preventing fungal and termite attack is a function of the type of preservative.     

Mucor hiemalis: a new source for uricase production

Summary One hundred and sixty-five strains of microorganisms with the ability to grow in a medium containing uric acid as a major source of nitrogen were isolated from soil samples during a screening program. Among them, a zygomycete fungus with well-developed columellae was recognized to produce high levels of the enzyme in a short time. Classification of the isolated fungus was carried out according to the morphological and culture characteristics of the organism, and it was identified as Mucor hiemalis. The fungus was able to produce an intracellular urate oxidase in a fermentation medium mainly containing uric acid. Optimized composition of the medium consisted of (l⁻¹ of distilled water) uric acid, 7.0 g; maltose, 6.0 g; Vogel stock solution, 20 and 1 ml of 0.5 M copper sulphate. The optimum pH and temperature for uricase production in the optimized medium were pH 6 and 30 °C, respectively.     

The effect of copper on the growth of wood-rotting fungi and a blue-stain fungus

The effect of copper (II) ions on the growth of three brown-rot fungi, six white-rot fungi and one blue-stain fungus in solid medium was evaluated. The fungi were grown in malt extract agar with different concentrations of copper added, and the radial growth rate was determined. At the end of the incubation period, the mycelial biomass and the media pH were determined. The white-rot and blue-stain fungus grew up to 3 mM and 6 mM copper, respectively and the brown-rot fungi were the only ones that grew up to 10 mM, with higher growth rates than those shown by the other fungi. In general, the brown-rot fungi produced greater acidification in the culture media than the white-rot fungi and blue-stain fungus, and the acidification increased when the amount of copper was increased. The biomass production for the different species, in the absence or presence of copper, was not related to the radial growth rate, and the fungal species that produced the greatest biomass amounts did not correspond to those that presented the highest growth rates. The brown-rot fungi Wolfiporia cocos and Laetiporus sulfureus and blue-stain fungus Ophiostoma sp. demonstrated greater tolerance to high copper concentrations in solid medium than the white-rot fungi, determined as radial growth rate. On the other hand, the highest biomass producers in solid medium with copper added were the white-rot fungi Ganoderma australe and Trametes versicolor and the brown-rot fungus Gloeophyllum trabeum.     

Acid phosphatase activity in Pisolithus arrhizus mycelium treated with cadmium dust

The influence of cadmium dust (containing lead, cadmium, copper, zinc, silicium and other elements) on acid phosphatase activity of Pisolithus arrhizus was observed by means of electron microscopy. Dust-treated mycelium showed increased activity of the enzyme, especially on the surface of the cell wall. There was an increase in abundance of autophagic vacuoles marked by a strong phosphatase reaction. An increase in the number of hyphae with diffuse enzyme activity within the cytoplasm coincided with a decrease of lifespan of the fungus, rapid changes in the mictoplasm stage, earlier closing of the dolipori and presumably the earlier autolysis of cell cytoplasm. Hyphae showing strong autolytic activity were separated from other hyphae by the material deposited within the doliporus and this whole area was devoid at that stage of acid phosphatase activity. The role of the enzyme in the mechanism of resistance to toxic elements is discussed.     

Study of the Kinetics of Oxidation of Monophenols by Tyrosinase. The Effect of Reducers

Kinetics of oxidation of monophenols by tyrosinase from the fungus Aspergillus flavipes 56003 and the effect of Fe²⁺, serine, and ascorbic acid on this reaction were studied. The effectors were shown to accelerate the oxidation of monophenols, decreasing the lag-time of the reaction. It is assumed that the activation of the tyrosinase in the presence of Fe²⁺ is due to a direct reduction of the active site copper ions. Serine and ascorbic acid are supposed to affect the reaction of quinone transformation. The activation of the enzyme in the presence of Fe²⁺ suggests that the oxidation of monophenols is an autocatalytic process.     

Variation in Copper Tolerance among Homokaryotic and Dikaryotic Mycelia of Polyporus palustris Berk. et Curt.

Investigations were made of the variation in copper toleranceamong homokaryons and dikaryons originating from one dikaryoticisolate of Polyporus palustris, as shown by their growth onnutrient agar containing copper sulphate, usually at 0.32 Mconcentration (c. 8 per cent of CuSO₄.5H₂O). Homokaryons obtainedby monobasidiospore isolation were crossed and further homokaryonsobtained in the same way. Selection for copper tolerance gavean increase in the second generation, but no further increasein the third; whilst selection for copper sensitivity gave substantialincreases in both second and third generations. It is concludedthat copper tolerance in this fungus is controlled by a numberof genes. It was found that, in this fungus, the copper toleranceof a dikaryon was substantially greater than that of eitherof its constituent homokaryons. It was also found that differentisolates of P. palustris varied very widely in copper tolerance.     

Natural Occurrence ofChondrostereum purpureumin Relation to Its Use as a Biological Control Agent in Canadian Forests

The incidence of natural fructification ofChondrostereum purpureumwas estimated quantitatively on southern Vancouver Island during two winter seasons in randomly located 1000-m²plots and compared with potential added fructification that might occur as a result of using the fungus to control stump-sprouting of hardwood weeds in young forest stands. Fructification was surveyed in forests as well as in urban or agricultural areas by estimating the surface areas of woody substrates covered with basidiocarps. In addition to random plots, estimates were made also in locations where the fungus would be expected to occur (woodpiles, silvicultural thinnings, and killed trees). Basidiocarps were found throughout the area in various types of forest cover as well as in urban or agricultural situations. The amount of added fructification through the use of the fungus as a biological control agent was determined from inoculated plots as well as from calculated stump-surface areas developed from published stand-density data. Added fructification was multiplied by a factor representing the maximum biological control frequency in order to compare added fructification with natural fructification values. From the various calculations, it was determined that the added fructification ofC. purpureumis of the same order of magnitude as naturally occurring levels or even lower. In addition, there is a distinct geographical separation between predominantly forestry and predominantly settled areas where fruit and ornamental trees are cultivated. Accordingly, it was concluded that using the fungus as a biological control agent in forestry is not likely to pose a significant threat to fruit growing and commercial forests.     

Role of Oxalic Acid Overexcretion in Transformations of Toxic Metal Minerals by Beauveria caledonica

The fungus Beauveria caledonica was highly tolerant to toxic metals and solubilized cadmium, copper, lead, and zinc minerals, converting them into oxalates. This fungus was found to overexcrete organic acids with strong metal-chelating properties (oxalic and citric acids), suggesting that a ligand-promoted mechanism was the main mechanism of mineral dissolution. Our data also suggested that oxalic acid was the main mineral-transforming agent. Cadmium, copper, and zinc oxalates were precipitated by the fungus in the local environment and also in association with the mycelium. The presence of toxic metal minerals often led to the formation of mycelial cords, and in the presence of copper-containing minerals, these cords exhibited enhanced excretion of oxalic acid, which resulted in considerable encrustation of the cords by copper oxalate hydrate (moolooite). It was found that B. caledonica hyphae and cords were covered by a thick hydrated mucilaginous sheath which provided a microenvironment for chemical reactions, crystal deposition, and growth. Cryo-scanning electron microscopy revealed that mycogenic metal oxalates overgrew parental fungal hyphae, leaving a labyrinth of fungal tunnels within the newly formed mineral matter. X-ray absorption spectroscopy revealed that oxygen ligands played a major role in metal coordination within the fungal biomass during the accumulation of mobilized toxic metals by B. caledonica mycelium; these ligands were carboxylic groups in copper phosphate-containing medium and phosphate groups in pyromorphite-containing medium.     

Increased production of extracellular laccase by the white rot fungus Coriolus versicolor MTCC 138

Summary The white rot fungus Coriolus versicolor MTCC 138 has been identified as an excellent producer of the industrially important enzyme laccase. The basal medium containing glucose gave laccase activity of 155 U/ml. Screening of different media components and their effects on laccase production by Coriolus versicolor was studied using one factor at a time and L₉ (3⁴) orthogonal array method. A two-fold increase (305 U/ml) in laccase production was observed using a combination of glucose and starch as carbon source and yeast extract as nitrogen source. This activity is very high as compared to most of the reported strains. Hence this strain was explored for enhancement in laccase. The formation of extracellular laccase could be considerably stimulated by the addition of copper in the optimized medium. Addition of 1 mM copper enhanced laccase activity to 460 U/ml. Laccase production was further enhanced using different aromatic inducers. Among different inducers used 2,5-xylidine was found to be the best, and gave maximum laccase activity of 820 U/ml with 1 mM concentration. Thus, this strain could be an efficient and attractive source for laccase production.     

Enhanced production of laccase activity by <Emphasis Type="Italic">Trametes versicolor </Emphasis>immobilized into alginate beads by the addition of different inducers

In the present paper the effect of adding veratryl alcohol and copper sulphate on laccase activity production by Trametes versicolor immobilized into alginate beads has been investigated. Employing copper sulphate as laccase inducer or supplementing the culture medium with veratryl alcohol, led to maximum values of laccase activity. However, the highest laccase activity (around 4,000 U l⁻¹) was obtained in cultures simultaneously supplemented with copper sulphate (3 mM) and veratryl alcohol (20 mM). These values implied a considerable enhancement in relation to␣control cultures without any inducer (around 200 U l⁻¹). The production of laccase by immobilized T. versicolor in a 2-l airlift bioreactor with the optimized inducer has been evaluated. Laccase activities around 1,500 U l⁻¹ were attained. The bioreactor operated for 44 days without operational problems and the bioparticles (fungus grows in alginate beads) maintained their shape throughout the fermentation. Moreover, the extracellular liquid obtained was studied in terms of pH and temperature activity and stability. On the other hand, anthracene was added in two-repeated batches in order to determine the efficiency of this process to degrade pollutants. Near complete degradation was reached in both batches. Moreover, in vitro degradation of several polycyclic aromatic hydrocarbons by crude laccase was also performed.     

A saponin tolerant and glycoside producer xylariaceous fungus isolated from fruits of <Emphasis Type="Italic">Sapindus saponaria</Emphasis>

The plant Sapindus saponaria is a good producer of terpenoidal and sesquiterpenoidal saponins, which are accumulated in its fruits. Although saponins usually act as antifungal compounds, a fungus was found living on the internal part of its pericarp. Tentative identification of the fungus, based on micro and macro-morphological aspects inspection, as well as on the secondary metabolite production, suggested the fungus to be a Xylariaceous microorganism. When the fungus is placed in contact with glycosides extracted from the host plant, or exogenous glycosides and other terpenoid compounds, it showed ability to transform them in another compounds. Analysis of these products using liquid chromatography and mass spectrometry showed that these biotransformations are mainly monohydroxylation and glycosilation. These findings contrasts most of the reports found in the literature which shows that hydrolysis at sugar chain are frequently observed during glycoside biotransformation by fungi.     

Optimization of a culture medium for ligninolytic enzyme production and synthetic dye decolorization using response surface methodology

A Box-Wilson central composite design was applied to optimize copper, veratryl alcohol and l-asparagine concentrations for Trametes trogii (BAFC 212) ligninolytic enzyme production in submerged fermentation. Decolorization of different dyes (xylidine, malachite green, and anthraquinone blue) by the ligninolytic fluids from the cultures was compared. The addition of copper stimulated laccase and glyoxal oxidase production, but this response was influenced by the medium N-concentration, with improvement higher at low N-levels. The medium that supported the highest ligninolytic production (22.75 U/ml laccase, 0.34 U/ml manganese peroxidase, and 0.20 U/ml glyoxal oxidase) also showed the greatest ability to decolorize the dyes. Only glyoxal oxidase activity limited biodecoloration efficiency, suggesting the involvement of peroxidases in the process. The addition of 1-hydroxybenzotriazole (a known laccase mediator) to the ligninolytic fluids increased both their range and rate of decolorization. The cell-free supernatant did not decolorize xylidine, poly R-478, azure B, and malachite green as efficiently as the whole broth, but results were similar in the case of indigo carmine and remazol brilliant blue R. This indicates that the mycelial biomass may supply other intracellular or mycelial-bound enzymes, or factors necessary for the catalytic cycle of the enzymes. It also implies that this fungus implements different strategies to degrade dyes with diverse chemical structures.     

Mycopappus quercus sp. nov., causing frosty mildew inQuercus acutissima

The causal fungus of frosty mildew inQuercus acutissima was taxonomically studied. Brown spots with conical white to yellowish tufts of the causal, fungus developed on the leaves, followed by successive defoliation. The fungus was regarded as a new species ofMycopappus, M. quercus, from the morphology of the multicellular propagules. The ascogenous stage and true conidia of the fungus were not observed, although sclerotia were formed on the fallen diseased leaves.     

Phosphatase production and activity in copper (II) accumulating Rhizopus delemar

The fungus Rhizopus delemar produced extracellular and cellular acid phosphatase during the growth in starch-supplemented medium in the presence or absence of copper ions. The levels of both AP-ase activities were maximal at the end of exponential growth phase and were dependent on copper concentrations. Copper ions in the medium provoked slight decrease of specific AP-ase activities and significant increase of the values of secreted enzyme per gram dry cells. On the other hand, an increase of copper ions in the reaction mixture leads to considerable increase of the values of cellular enzyme activity. Total uptake of copper (II) was highest at the highest copper (II) concentration, when resting cells were used. Between 27 and 30% copper (II) was not removed by acid washing, suggested that this copper was bound intracellularly by mycelium. Determination of the Michaelis constant for the cellular AP-ase gave value of 0.325 mM. The pH optimum of the enzyme was determined to be in the range of 3.5–4.5 using p-nitrophenyl phosphate (pNPP) as a substrate. The data obtained indicated a possible participation of AP-ases in the processes of heavy metal resistance and heavy metal uptake of this fungus.     

Comparative studies on metal biosorption by two strains of Cladosporium cladosporioides

Two strains of a fungus, Cladosporium cladosporioides 1 and C. cladosporioides 2 showed different metal biosorption properties. Strain 1 showed preferential sorption of gold and silver, while strain 2 could bind metals such as copper and cadmium in addition to gold and silver. Strain 1 had a cell-wall hexosamine content of 0.1%. X-ray photoelectron spectroscopy (XPS) and Fourier transform infra-red spectroscopy (FTIR) analyses indicated that nitrogen was not involved in metal biosorption by the strain. In strain 2 the cell-wall hexosamine content was 150 times that of strain 1. These results indicated that hexosamine was responsible for non-specific metal binding while cell-wall polymers other than hexosamines had a role in conferring selectivity in precious-metal binding.     

Exserohilum israeli, a new species isolated from soil from Timna Park (Israel), and its physiological properties

Fungi isolated from soil in Timna Park (Israel) were found to belong to a new species of Exserohilum for which the name Exserohilum israeli sp. nov. is proposed. The main physiological properties of members of this species are discussed and the influence of temperature and copper concentrations on the growth and morphology of the fungus were investigated.     

Ultrastructural analysis of the behavior of the dimorphic fungus Microbotryum violaceum in fungus-induced anthers of female Silene latifolia flowers

Summary. The development of male organs is induced in female flowers of the dioecious plant Silene latifolia by infection with the fungus Microbotryum violaceum. Stamens in a healthy female flower grow only to stage 6, whereas those in an infected female flower develop to the mature stage (stage 12), at which the stamens are filled with fungal teliospores instead of pollen grains. To investigate these host–parasite interactions, young floral buds and fungus-induced anthers of infected female flowers were examined by electron microscopy following fixation by a high-pressure freezing method. Using this approach, we found that parasitic hyphae of this fungus contain several extracellular vesicles and have a consistent appearance up to stage 8. At that stage, parasitic hyphae are observed adjacent to dying sporogenous cells in the infected female anther. At stage 9, an increased number of dead and dying sporogenous cells is observed, among which the sporogenous hyphae of the fungus develop and form initial teliospores. Several types of electron-dense material are present in proximity to some fungi at this stage. The initial teliospores contain two types of vacuoles, and the fungus cell wall contains abundant carbohydrate, as revealed by silver protein staining. The sporogenous cell is probably sensitive to infection by the fungus, resulting in disruption. In addition, the fungus accelerates cell death in the anther and utilizes constituents of the dead host cell to form the mature teliospore. Correspondence and reprints (present address): Molecular Membrane Biology Laboratory, RIKEN, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan.