The effect of rumen chitinolytic bacteria on cellulolytic anaerobic fungi

J. KOPEČNÝ, B. HODROVÁ AND C. S. STEWART. 1996. The polycentric anaerobic fungus Orpinomyces joyonii A4 was cultivated on microcrystalline cellulose alone and in association with the rumen chitinolytic bacterium Clostridium sp. strain ChK5, which shows strong phenotypic similarity to Clostridium tertium . The presence of strain ChK5 significantly depressed the solubilization of microcrystalline cellulose, the production of short-chain fatty acids (SCFA) and the release of endoglucanase by the fungus. Co-culture of the monocentric anaerobic fungus Neocallimastix frontalis strain RE1, Neocallimastix sp. strain G-1 and Caecomyces sp. strain SC2 with strain ChK5 also resulted in depressed fungal cellulolysis. Cell-free supernatant fluids from strain ChK5 inhibited the release of reducing sugars from carboxymethylcellulose by cell-free supernatant fluids from O. joyonii strain A4. Strain 007 of the cellulolytic anaerobe Ruminococcus flavefaciens was also shown to produce small amounts of soluble products upon incubation with colloidal chitin. Mixtures of culture supernates from this bacterium and from O. joyonii strain A4 showed cellulase activity that was less than that of the component cultures. It is suggested that the ability of some rumen bacteria to hydrolyse or transform chitin may be an important factor in the interactions between bacteria and fungi in the rumen.     

A microculture hybridization technique for the detection of specific DNA sequences in filamentous fungi

A procedure that permits the successful application of the technique of colony hybridization to the filamentous fungi is described. The method involves the growth of microcultures in the wells of a microtiter tray using spore inocula. These microcultures are protoplastedin situ and transferred to a nylon filter through a device made by drilling out the wells of a second microtiter tray. The transferred protoplasts are confined to defined areas of the membrane, where they may be lysed and subjected to hybridization with a radioactive probe. The technique gives highly reproducible results and is of sufficient sensitivity to detect the integration of a single copy of a transforming plasmid into the genome ofAspergillus nidulans.     

Initial observations on the cellulolytic activities of some fungal isolates

Summary The cellulolytic activities of aTrichoderma sp.,Penicillium sp.,Cladosporium sp.,Trichoderma viride ATCC-24687,Myrothecium roredium, ATCC-28814 and unidentified fungus (F-4) were investigated. TheTrichoderma sp. and the unidentified fungus (F-4) secreted larger amounts of cellulase into the growth medium than the other strains. They also saccharified lignocellulosics more rapidly than the other strains.

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Resumen Se estudió la actividad celulolítica de: une especie deTrichoderma sp.,Penicillium sp.,Cladosporium sp.,Trichoderma viride ATCC-24687,Myrothecium roredium, ATCC-28814, y de un hongo no identificado (F-4).Trichoderma sp. y el hongo no identificado secretaron más cantidad de celulasa al medio de cultivo que las otras cepas estudiadas. Tambien saccarificaron materiales lignocelulósicos más rápidamente que los demás hongos.

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Résumé Les activités cellulolytiques d'unTrichoderma sp., d'unPenicillium sp., d'unCladosporium sp., d'unTrichoderma viride ATCC-24687, d'unMyrothecium roredium et d'une moisissure nonidentifiée (F-4) ont été examinées. La soucheTrichoderma sp et la moisissure non-identifiée (F-4) ont secrété des quantités plus importantes de cellulase dans le milieu du culture que les autres souches. Ces deux souches ont aussi saccharifié la ligno-cellulose plus rapidement que les autres souches.

     

Associative effect of cellulolytic fungi andAzospirillum lipoferum on yield and nitrogen uptake by wheat

The associative effect of cellulolytic fungi, such asAspergillus awamori andA. niger, with the nitrogen fixer,Azospirillum lipoferum was studied in a soil amended with rice straw. All the inoculants gave significantly higher grain and straw yield and nitrogen uptake by wheat crop than did the uninoculated treatment. The doubling of chemical nitrogen dose significantly increased the yield and nitrogen uptake. It was observed thatA awamori performed significantly better followed byA. niger andA. lipoferum. The maximum benefit was obtained with combined inoculation ofA. awamori andA. lipoferum. Another experiment was conducted in the subsequent year in soil amended with and without rice straw using cellulolytic culture eitherA. awamori orSclerotium rolfsii, andA. lipoferum. Application of straw in soil significantly reduced the yield and N-uptake by wheat crop as compared to the controls. All the inoculants exceptS. rolfsii gave significantly higher grain yield. However, N-uptake by grain was significantly increased only by combined inoculation ofA. lipoferum and either one of the cellulolytic fungi. Similar trends on yield and N-uptake of straw due to inoculants were observed. The maximum benefit was obtained with combined inoculation ofA. awamori andA. lipoferum followed byA. awamori alone on grain yield and only combined inoculants on N-uptake by the crop.     

Strategies for the isolation of cellulolytic fungi for composting of wheat straw

It was shown that inoculation of straw with cellulolytic fungi offers potential for manipulating and improving the composting of cellulose waste, where the C:N ratio is not optimal for composting. In this paper we report on a screening strategy used to isolate novel cellulolytic fungi from field samples. The screen comprised of two phases. In phase I, 300 cellulolytic fungi were isolated to pure culture from field samples collected from Hawaii, China and the UK. Isolates were selected on the basis of high cellulolytic activities and growth rates on cellulose agar. A total of 137 lead isolates progressed to an unreplicated phase II screen to rapidly identify strains that improved quality of the resulting compost over and above that of the uninoculated control. Compost quality was assessed by measuring C:N ratio, water holding capacity, water content and potential and polysaccharide content of the resulting compost. Effect on the aggregate stability of soil and the growth of wheat seedlings was assessed when compost was added to a sandy loam soil. Performance of each isolate was quantified by allocating a utility score for each compost analysed. Utility scores were based on the sum of the logged ranked score in each assay. The 10 highest scoring isolates were subsequently processed through a replicated phase II screen and the best performing isolates identified by calculating utility scores as before. Significantly lower C:N ratios, higher water-holding capacities and improved aggregate stabilities were obtained with some inoculated treatments compared to the uninoculated control, whilst the results obtained for polysaccharide content and plant growth showed no significant differences. Isolate 304, isolated from decomposing vegetation obtained from Egham, Surrey, UK, and identifed as a Trichurus sp., appeared the most effective inoculant, significantly decreasing the C:N ratio by 36% and increasing the aggregate stability of soil by 54% compared to the uninoculated control. As a result of adopting this screening strategy, it has been possible to identify cellulolytic fungi that can, under non-sterile (laboratory) conditions, significantly improve the quality of compost. This screening approach therefore offers real possibilities for selecting microbial inoculants in low-tech agricultural practices.     

A novel PCR-based approach for the detection and classification of potential cellulolytic fungal strains isolated from museum items and surrounding indoor environment

Aims: To develop a novel PCR‐based method able to detect potential cellulolytic filamentous fungi and to classify them exploiting the amplification of the cellobiohydrolase gene (cbh‐I) and its polymorphism. Methods and Results: A mixed approach including the combination of (i) fungal cultivation and isolation, (ii) classification of fungal isolates through the amplification of the cbh gene using a fluorescently labelled primer (f‐CBH‐PCR) and (iii) final fungal identification based on amplification and sequencing of the ITS1‐5.8S rDNA‐ITS2 region of the selected fungal strains was developed. By this approach, it was possible to screen 77 fungal strains belonging to 14 genera and 26 species. Conclusions: The f‐CBH‐PCR permitted the discrimination of fungal species, producing typical f‐CBH profiles. Significance and Impact of the Study: In this study, the cbh gene was used as a preliminary classification tool able to differentiate among themselves the fungal members isolated from indoor museum items and surrounding environment. Such mixed approach consented the fast identification of all isolated fungal strains. The f‐CBH‐PCR method demonstrated its discrimination power, and it can be considered as a new molecular system suitable for the classification of fungal strains isolated from different environments.     

A new technique for the preparation of permanent reference cultures of fungi.

Fungal cultures were grown by a new microslide method and by the traditional slide-culture method. Sporulation results were similar by both methods. The microslide method has the advantages of increased safety to the user and ease of storage.     

Effects of glycerol on the growth,adhesion, and cellulolytic activity of rumen cellulolytic bacteria and anaerobic fungi

The effect of glycerol on the growth, adhesion, and cellulolytic activity of two rumen cellulolytic bacterial species,Ruminococcus flavefaciens andFibrobacter succinogenes subsp.succinogenes, and of an anaerobic fungal species,Neocallimastix frontalis, was studied. At low concentrations (0.1–1%), glycerol had no effect on the growth, adhesion, and cellulolytic activity of the two bacterial species. However, at a concentration of 5%, it greatly inhibited their growth and cellulolytic activity. Glycerol did not affect the adhesion of bacteria to cellulose. The growth and cellulolytic activity ofN. frontalis were inhibited by glycerol, increasingly so at higher concentrations. At a concentration of 5%, glycerol totally inhibited the cellulolytic activity of the fungus. Thus, glycerol can be added to animal feed at low concentrations.     

A spectrophotometric assay for the detection of fungal peroxygenases

Rapid and simple spectrophotometric methods are required for the unambiguous detection of recently discovered fungal peroxygenases in vivo and in vitro. This paper describes a peroxygenase-specific assay using 5-nitro-1,3-benzodioxole as substrate. The product, 4-nitrocatechol, produces a yellow color at pH 7, which can be followed over time at 425 nm (ε(425)=9,700 M(-1) cm(-1)), and a red color when adjusted to pH >12, which can be measured in form of an end-point determination at 514 nm (ε(514)=11,400 M(-1) cm(-1)). The assay is suitable for detecting peroxygenase activities in complex growth media and environmental samples as well as for high-throughput screenings.     

Effects of an organophosphorus insecticide on the growth and cellulolytic activity of fungi

The organophosphorus insecticide Selecron [O-(4-bromo-2-chlorophenyl) O-ethyl S-n-propyl-phosphorotioate] at 10 and 50 ppm significantly decreased respiration, mycelial protein, extracellular protein and mycelial dry weight of Aspergillus fumigatus, A. terreus and Myceliophthora thermophila when grown at 45°C. C_x and C₁ cellulases of tested fungi were significantly decreased. However, C₁ cellulase of A. fumigatus was slightly increased.     

A plate assay method for the detection of fungal polygalacturonase secretion

Abstract A method for the detection of polygalacturonase activity has been developed using ruthenium red staining of fungal colonies on polygalacturonate- agarose plates. Ruthenium red was shown to penetrate beneath the surface layers of the gel, in the regions surrounding a fungal colony where degradation of polygalacturonate had occurred. Without degradation of polygalacturonate ruthenium red did not penetrate the medium, was restricted to binding to the surface layers and was easily washed off. The medium containing undegraded polygalacturonate was a colourless clear background and areas of polygalacturonate degradation around the colonies were visualised as an intense purple-red halo. The method has been used to screen yeasts and filamentous fungi for polygalacturonase secretion.     

An assessment of the efficiency of fungal DNA extraction methods for maximizing the detection of medically important fungi using PCR

To date, no single reported DNA extraction method is suitable for the efficient extraction of DNA from all fungal species. The efficiency of extraction is of particular importance in PCR-based medical diagnostic applications where the quantity of fungus in a tissue biopsy may be limited. We subjected 16 medically relevant fungi to physical, chemical and enzymatic cell wall disruption methods which constitutes the first step in extracting DNA. Examination by light microscopy showed that grinding with mortar and pestle was the most efficient means of disrupting the rigid fungal cell walls of hyphae and conidia. We then trialled several published DNA isolation protocols to ascertain the most efficient method of extraction. Optimal extraction was achieved by incorporating a lyticase and proteinase K enzymatic digestion step and adapting a DNA extraction procedure from a commercial kit (MO BIO) to generate high yields of high quality DNA from all 16 species. DNA quality was confirmed by the successful PCR amplification of the conserved region of the fungal 18S small-subunit rRNA multicopy gene.     

A microfuge tube technique for disrupting microquantities of fungal spores

A practical apparatus was assembled and through its use a technique was developed to disrupt microquantities of Ceratocystis ulmi spores. spores were placed in a microfuge tube with buffer and glass beads. The microfuge tube was sealed and placed inside a holding tube and then subjected to rapid vibrations over a Vortex mixer. By this technique 80–90% of the spores were disrupted and it was possible to extract a high-quality ribosomal RNA fraction from the supernatant fluid of the disrupted spore sample. The technique has also proved applicable to spores of fungi other than C. ulmi.