Biosynthesis of cell walls of fungi

[This corrects the article on p. 134 in vol. 43.].     

Use of confocal microscopy in examining fungi and bacteria in wood

When used in conjunction with digital image processing techniques, confocal laser scanning microscopy (CLSM) enables non-invasive optical sectioning, allowing micromorphologies of wood decay to be examined at any depth within a relatively thick (0.05-0.1 mm) wood specimen without incision. In this study, the use of specially tailored multi-fluorescent staining techniques with CLSM produced new information concerning spatial relationships between fungi and bacteria and the wood substrate, particularly in regard to their 3D characteristics. Glutaraldehyde fixation and a chitin fluorescent probe were used to locate fungal hyphae in wood. Bacteria colonising wood were examined using a fluorescent phospholipid probe. By counterstaining wood with this probe and a fluorescent dye specific for Gram-positive bacteria, it was possible to clearly distinguish Gram types through simultaneous, multichannel fluorescent CLSM imaging. The combination of glutaraldehyde fixation and phospholipid probing proved to be reliable for detecting wood-degrading bacteria in wood cell walls.     

Fermentation of plant cell walls by ruminal bacteria, protozoa and fungi and their interaction with fibre particle size

The objective of the experiment was to evaluate the contribution of various ruminal microbial groups to the fermentation of cell walls of corn stover with different particle sizes based on ruminal gas production in vitro. Physical, chemical, and antibiotical methods were used to differentiate groups of bacteria, protozoa and fungi in rumen fluid, offering following rumen microbial groups: whole rumen fluid (WRF), bacterial (B), protozoal (P), fungal (F), bacterial plus protozoal (B + P), bacterial plus fungal (B + F), protozoal plus fungal (P + F), and negative control (CON). Cell walls from corn stover were ground and ball milled to produce two different particle sizes. The results showed that digestion of the cell walls was undertaken by the interaction among ruminal bacteria, protozoa and fungi, and such co-actions seemed to fail alternation by one of three microbial groups or any combinations. However, B + P group showed a significant contribution to the degradation of milled cell walls, and B + F group revealed a great synergy effect on the ground cell walls degradation. Particle size of cell walls also had a considerable influence on their fermentation extent instead of the fermentative patterns by various rumen microbial groups.     

Fermentation of plant cell walls by ruminal bacteria,protozoa and fungi and their interaction with fibre particle size

Abstract

The objective of the experiment was to evaluate the contribution of various ruminal microbial groups to the fermentation of cell walls of corn stover with different particle sizes based on ruminal gas production in vitro. Physical, chemical, and antibiotical methods were used to differentiate groups of bacteria, protozoa and fungi in rumen fluid, offering following rumen microbial groups: whole rumen fluid (WRF), bacterial (B), protozoal (P), fungal (F), bacterial plus protozoal (B + P), bacterial plus fungal (B + F), protozoal plus fungal (P + F), and negative control (CON). Cell walls from corn stover were ground and ball milled to produce two different particle sizes. The results showed that digestion of the cell walls was undertaken by the interaction among ruminal bacteria, protozoa and fungi, and such co-actions seemed to fail alternation by one of three microbial groups or any combinations. However, B + P group showed a significant contribution to the degradation of milled cell walls, and B + F group revealed a great synergy effect on the ground cell walls degradation. Particle size of cell walls also had a considerable influence on their fermentation extent instead of the fermentative patterns by various rumen microbial groups.     

Safranine fluorescent staining of wood cell walls

Safranine is an azo dye commonly used for plant microscopy, especially as a stain for lignified tissues such as xylem. Safranine fluorescently labels the wood cell wall, producing green/yellow fluorescence in the secondary cell wall and red/orange fluorescence in the middle lamella (ML) region. We examined the fluorescence behavior of safranine under blue light excitation using a variety of wood- and fiber-based samples of known composition to interpret the observed color differentiation of different cell wall types. We also examined the basis for the differences in fluorescence emission using spectral confocal microscopy to examine lignin-rich and cellulose-rich cell walls including reaction wood and decayed wood compared to normal wood. Our results indicate that lignin-rich cell walls, such as the ML of tracheids, the secondary wall of compression wood tracheids, and wood decayed by brown rot, tend to fluoresce red or orange, while cellulose-rich cell walls such as resin canals, wood decayed by white rot, cotton fibers and the G-layer of tension wood fibers, tend to fluoresce green/yellow. This variation in fluorescence emission seems to be due to factors including an emission shift toward red wavelengths combined with dye quenching at shorter wavelengths in regions with high lignin content. Safranine fluorescence provides a useful way to differentiate lignin-rich and cellulose-rich cell walls without counterstaining as required for bright field microscopy.     

Temporal changes in wood crystalline cellulose during degradation by brown rot fungi

The degradation of wood by brown rot fungi has been studied intensely for many years in order to facilitate the preservation of in-service wood. In this work we used X-ray diffraction to examine changes in wood cellulose crystallinity caused by the brown rot fungi Gloeophyllum trabeum, Coniophora puteana, and two isolates of Serpula lacrymans. All fungi increased apparent percent crystallinity early in the decay process while decreasing total amounts of both crystalline and amorphous material. Data also showed an apparent decrease of approximately 0.05 Å in the average spacing of the crystal planes in all degraded samples after roughly 20% weight loss, as well as a decrease in the average observed relative peak width at 2θ = 22.2°. These results may indicate a disruption of the outer most semi-crystalline cellulose chains comprising the wood microfibril. X-ray diffraction analysis of wood subjected to biological attack by fungi may provide insight into degradative processes and wood cellulose structure.     

Lignin distribution in wood cell walls determined by TEM and backscattered SEM techniques

The lignin distribution in cell walls of spruce and beech wood was determined by high-voltage transmission-electron-microscopy (TEM) in sections stained with potassium permanganate as well as by field-emission-scanning-electron-microscopy (FE-SEM) combined with a back-scattered electron detector on mercurized specimens. The latter is a new technique based on the mercurization of lignin and the concomitant visualization of mercury by back-scattered electron microscopy (BSE). Due to this combination it was possible to obtain a visualized overview of the lignin distribution across the different layers of the cell wall. To our knowledge, this combined method was used the first time to analyse the lignin distribution in cell walls. In agreement with previous work the highest lignin levels were found in the compound middle lamella and the cell corners. Back-scattered FE-SEM allows the lignin distribution in the pit membrane of bordered pits as well as in the various cell wall layers to be shown. In addition, by using TEM as well as SEM we observed that lignin closely follows the cellulose microfibril orientation in the secondary cell wall. From these observations, we conclude that the polymerisation of monolignols is affected by the arrangement of the polysaccharides which constitute the cell wall.     

Chemical imaging of poplar wood cell walls by confocal Raman microscopy

Confocal Raman microscopy was used to illustrate changes of molecular composition in secondary plant cell wall tissues of poplar (Populus nigra x Populus deltoids) wood. Two-dimensional spectral maps were acquired and chemical images calculated by integrating the intensity of characteristic spectral bands. This enabled direct visualization of the spatial variation of the lignin content without any chemical treatment or staining of the cell wall. A small (0.5 microm) lignified border toward the lumen was observed in the gelatinous layer of poplar tension wood. The variable orientation of the cellulose was also characterized, leading to visualization of the S1 layer with dimensions smaller than 0.5 mum. Scanning Raman microscopy was thus shown to be a powerful, nondestructive tool for imaging changes in molecular cell wall organization with high spatial resolution.     

Relative contributions of bacteria, protozoa, and fungi to in vitro degradation of orchard grass cell walls and their interactions

To assess the relative contributions of microbial groups (bacteria, protozoa, and fungi) in rumen fluids to the overall process of plant cell wall digestion in the rumen, representatives of these groups were selected by physical and chemical treatments of whole rumen fluid and used to construct an artificial rumen ecosystem. Physical treatments involved homogenization, centrifugation, filtration, and heat sterilization. Chemical treatments involved the addition of antibiotics and various chemicals to rumen fluid. To evaluate the potential activity and relative contribution to degradation of cell walls by specific microbial groups, the following fractions were prepared: a positive system (whole ruminal fluid), a bacterial (B) system, a protozoal (P) system, a fungal (F) system, and a negative system (cell-free rumen fluid). To assess the interactions between specific microbial fractions, mixed cultures (B+P, B+F, and P+F systems) were also assigned. Patterns of degradation due to the various treatments resulted in three distinct groups of data based on the degradation rate of cell wall material and on cell wall-degrading enzyme activities. The order of degradation was as follows: positive and F systems > B system > negative and P systems. Therefore, fungal activity was responsible for most of the cell wall degradation. Cell wall degradation by the anaerobic bacterial fraction was significantly less than by the fungal fraction, and the protozoal fraction failed to grow under the conditions used. In general, in the mixed culture systems the coculture systems demonstrated a decrease in cellulolysis compared with that of the monoculture systems. When one microbial fraction was associated with another microbial fraction, two types of results were obtained. The protozoal fraction inhibited cellulolysis of cell wall material by both the bacterial and the fungal fractions, while in the coculture between the bacterial fraction and the fungal fraction a synergistic interaction was detected.     

Bacterial degradation of lignified wood cell walls in anaerobic aquatic habitats

Test blocks of beech (Fagus sylvatica) and Scots pine (Pinus sylvestris) were buried in fresh, brackish, and seawater anaerobic muds for periods ranging between 1 and 18 months. At appropriate time intervals the test blocks were recovered and examined for changes in weight and for bacterial attack of lignified wood cell walls. Only small weight losses occurred. Scanning electron microscopy studies revealed that there was extensive superficial bacterial erosion of beech wood cell walls. The decay patterns are illustrated by micrographs and discussed in relation to other types of bacterial attack.     

Cytological and chemical changes in cell walls ofRhodotorula gracilis

The phenotype and genotype of six strains of the genusRhodotorula Harrison and of one strain of the genusCryptococcus Phaff et Fell, with anomalous thickening of cell walls were investigated. The present studies showed that the strains investigated represent different stages of the life cycles of the genusRhodosporidium Banno. The anomalous thickening of the cell walls can be explained by extreme conditions resulting in the formation of surviving forms (teliospores, chlamydospores).     

Ultrastructural appearance of embedded and polished wood cell walls as revealed by Atomic Force Microscopy

Atomic Force Microscopy (AFM) was used to investigate the ultrastructural appearance of transverse wood cell wall surfaces in embedded and polished Norway spruce wood blocks. The prepared surfaces showed only little height differences, suitable for high resolution AFM phase contrast imaging. Our results revealed randomly arranged wood cell wall components in the thick secondary 2 (S2) layers of the tracheid cell walls. It is concluded that the observed distribution pattern of the cellulose fibril/matrix structure is close to the original cell wall structure. In this context, the plasticity of wood cell wall components to re-arrange and adjust to different conditions resulting in diverse structural pattern is discussed.     

Polysaccharide changes in cell walls of ripening apples

Changes in the polysaccharide composition of apple fruits ripening on and off the tree were compared. Polysaccharide fractions defined by their method of extraction were analysed colorimetrically, and the monosaccharide composition of total acetone insoluble material was analysed. Neutral carbohydrate associated with pectic extractives decreased; correspondingly galactose residues were lost in detached fruit, while galactose and arabinose residues were lost in fruit on the tree. Decreases in hemicellulose were correlated with losses of wall glucan; xylose contents did not change. Soluble polyuronide increased especially in detached fruit. DEAE-cellulose chromatography showed that this polyuronide was free from neutral sugar residues. Amounts of soluble neutral polysaccharides and glycoproteins did not change during fruit ripening.     

Biodegradation of eucalyptus urograndis wood by fungi

We focused in selecting four fungi, naturally living in Eucalyptus sp. fields, for application in accelerating stump decay. The wood-rot fungi Pycnoporus sanguineus (Ps), Lentinus bertieri (Lb) and Xylaria sp. (Xa) were isolated from Eucalyptus sp. field and the fungus Lentinula edodes (Led) was obtained from a commercial strain. All fungi were studied according to their capacity to degrade eucalyptus urograndis wood. In order to evaluate mass losses of seven years old eucalyptus urograndis' wood test blocks from heartwood were prepared added to glass flasks with red clay soil. The humidity of the soil was adjusted with 50 and 100% of its water retention capacity. Mass loss evaluations occurred at 30 until 120 days after eucalyptus wood degradation. Chemical analysis and soil pH were measured only in the last evaluation. Mycelial growth assays with potato-dextrose-agar, malt-agar and sawdust-dextrose-agar at three temperatures was carried out in order to get information about the best conditions of fungi growth. On the 120^th day, Ps and Lb showed good capacity of wood degradation by leading to a high mass loss in soil with highest humidity. These fungi were the best consumers of lignin, hemicellulose, cellulose and extractives, caused acidification in the soil. Ps and Lb had faster mycelial growth in sawdust-dextrose-agar, especially in high temperature, comparing to Xa and Led. Xa and Led are not good eucalyptus urograndis heartwood degraders, because they consume preferentially hemicellulose.     

Short-term changes in the biomass of fungi and bacteria in soddy-podzolic soil

Short-term (diurnal or daily) changes in fungal and bacterial biomass were studied using direct count. Diurnal changes in microbial biomass were found to be unreliable. Daily observations have shown that the length of fungal mycelium can change 3--4-fold within 10--30 days, and the number of bacteria, 2--3-fold, the frequency and amplitude of changes depending on the season. Fungal biomass was found to be greater than bacterial biomass by a factor of dozens; that is why, while estimating the biomass of soil fungi, other indices of the biological activity of soil (DNA content, enzymic activity, respiration, etc.) must be taken into account.     

L-form bacteria,cell walls and the origins of life

The peptidoglycan wall is a defining feature of bacterial cells and was probably already present in their last common ancestor. L-forms are bacterial variants that lack a cell wall and divide by a variety of processes involving membrane blebbing, tubulation, vesiculation and fission. Their unusual mode of proliferation provides a model for primitive cells and is reminiscent of recently developed in vitro vesicle reproduction processes. Invention of the cell wall may have underpinned the explosion of bacterial life on the Earth. Later innovations in cell envelope structure, particularly the emergence of the outer membrane of Gram-negative bacteria, possibly in an early endospore former, seem to have spurned further major evolutionary radiations. Comparative studies of bacterial cell envelope structure may help to resolve the early key steps in evolutionary development of the bacterial domain of life.