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Abstract: The genus Phialophora includes some of the most copper-tolerant and frequently isolated fungi causing soft rot of copper-treated wood in service. The aim of this work was to conduct a phylogenic analysis of different Phialophora species and strains isolated from treated wood, evaluate strain compatibility, and examine their copper-tolerance variability under liquid- and solid-culture conditions. Phylogenic analysis of Phialophora malorum (four strains), Phialophora mutabilis (two strains), Lecythophora mutabilis (one strain), and Phialophora sp. A. (three strains) originating from different parts of the world showed clustering into three major clades. Phialophora sp. A. refers to a number of fungal isolates identified as Phialophora spp. previously isolated by our laboratory from preservative-treated stakes and poles in Sweden. P. mutabilis strains fell into a single cluster together with Phialophora lignicola and Phialophora hoffmanii; P. malorum strains clustered in their own group (93% bootstrap value) and Phialophora sp. A. clustered together with Phialophora botulispora. Compatibility studies with the different Phialophora species/strains showed discriminative behavior. All Phialophora species showed mutual inhibition (incompatibility), as did strains of P. malorum and P. mutabilis. Phialophora sp. A. strains showed mutual intermingling but incompatibility with P. botulispora. Species/strain variability regarding copper tolerance was evaluated using in-vitro liquid and solid media containing different CuSO4 concentrations. P. malorum 211-C-15-1 showed the highest copper-tolerance growing on 6.4% CuSO4 supplemented agar and 3.2% CuSO4 in liquid cultures, followed by P. malorum ATCC 66716 (3.2% w/v CuSO4-agar). P. mutabilis strains and L. mutabilis showed growth on 0.64% CuSO4 incorporated in liquid and agar cultures and Phialophora sp. A. (25M3) at 0.32% w/v CuSO4 on agar. The results emphasize that copper tolerance varies greatly within and between species of Phialophora. [Copyright &y& Elsevier]

Abstract: At one location in central Sweden, agricultural pine (Pinus sylvestris L.) fence poles treated with a commercial copper–chromium–phosphorus preservative (CCP) formulation according to use class 4 at retention of 30 kg m−3 were prematurely degraded by fungi after only two years in-service. Light- and electron microscopy analyses showed decay to result from primarily brown rot attack. Culture studies produced on different agar and copper-containing media using small wood slivers removed from infected poles allowed establishment of a number of pure cultures of Phycomycetes, Ascomycetes, Fungi Imperfecti and Basidiomycete fungi. Using morphological characters, PCR and sequencing of isolated strains, Antrodia vaillantii was determined as the most abundant basidiomycete present and as the major causal agent of decay. Compatibility tests and comparison of the ITS nrDNA sequences of our putative A. vaillantii isolate with other A. vaillantii strains and with Antrodia radiculosa showed differences suggesting a hybrid strain. A combination of site characteristics (e.g. hot spots of A. vaillantii), the use of juvenile poles, copper tolerance and overall ineffectivity of CCP against A. vaillantii is suggested as reasons for premature decay. [Copyright &y& Elsevier]

Phialophora malorum is a well-known copper tolerant fungus causing soft rot decay of copper-treated wood in-service in ground contact situations worldwide. The aim of the present work was to develop a method that allowed studies of the mechanism(s) behind extracellular copper tolerance by examining the profile of proteins expressed by P. malorum in the cell wall/slime (subproteome) environment in mycelia grown in Cu-supplemented media. The study involves development of a novel non-destructive approach using mild extraction, concentration, separation (SDS-PAGE electrophoresis) and characterization (MALDI-TOF MS/MS) of the proteins present. Studies revealed differential protein profiles and expression in Cu-supplemented (0.1% CuSO 4) vs control shake cultures after 10 days growth. Twelve proteins including several hydrolytic (i.e. glucosidases, pectinase, polygalacturonase) enzymes were either unique or over-expressed in Cu-cultures compared to controls. The cell wall location of the proteins was consistent with a role in copper tolerance and detoxification by binding Cu-ions extracellularly. Several of the proteins (e.g. hydrolases, pectinase) are also involved in wood cell wall biomineralization. It is proposed that the concentration of metabolites (organic acids) slowly released in close vicinity of hyphal surfaces by weakly bound enzymes over-expressed through Cu-stress may function to reduce levels of free non-chelated Cu (II) ions entering the cell cytoplasm and therefore provide a first level of defense against copper toxicity. • Specific method for extracting/characterizing extracellular fungal cell wall/slime proteins developed. • Method based on SDS-PAGE protein profiling and MALDI-TOF MS/MS characterization of polypeptides. • Twelve proteins were either unique- or over expressed in Cu-grown cultures of P. malorum. • Hydrolytic enzymes in the cell wall subproteome may provide Cu-tolerance by binding Cu-ions. [ABSTRACT FROM AUTHOR]

An improved understanding of the events involved in cell wall polymers deposition during xylem development could provide new scientific ways for molecular regulation and biomass utilization. Axial and radial cells are spatially heterogeneous and have highly cross-correlated developmental behavior, whereas the deposition of corresponding cell wall polymers during xylem differentiation is less studied. To clarify our hypothesis that cell wall polymers of two cell types accumulated asynchronously, we performed hierarchical visualization, including label-free in situ spectral imaging of different polymer compositions during the development of Pinus bungeana. In axial tracheids, the deposition of cellulose and glucomannan was observed on earlier stages of secondary wall thickening than that of xylan and lignin, while xylan distribution was strongly related to spatial distribution of lignin during differentiation. The content of lignin and polysaccharides increased by over 130 % and 60 % respectively when the S 3 layer was formed, compared to the S 2 stage. In ray cells, the deposition of crystalline cellulose, xylan, and lignin was generally lagged compared to that in corresponding axial tracheids, although the process followed a similar order. The concentration of lignin and polysaccharides in ray cells was only approximately 50 % of that in the axial tracheids during secondary wall thickening. [Display omitted] [ABSTRACT FROM AUTHOR]

The ability of soft rot fungi possessing strong ( Phialophora malorum ), medium ( Phialophora mutabilis ) and poor copper tolerance ( Chaetomium globosum ) to degrade untreated and CuSO 4 and micronized copper treated birch- and pine wood was assessed using ENV 807 standard tests. The aim was to determine whether an ability to grow on Cu-agar and copper in liquid cultures can be transcribed into a competitive advantage to degrade Cu-treated wood. An ability to tolerate high copper levels in-vitro was not correlated with increased decay by the fungi but rather reflected the native chemistry of the wood cell walls. Both untreated and Cu-treated wood were degraded by the three fungi and showed aggressiveness in the order C. globosum > P. mutabilis > P. malorum Higher mass loss was recorded for birch than pine and decreased progressively as the copper loadings increased with statistically insignificant difference noted between Cu-treatments. Microscopy showed decay at the cell wall level to reflect degree of lignification with parenchyma cells degraded first in both untreated and Cu-treated wood. Results indicate presence of copper and its toxicity is unlikely to be the main reason for preventing soft rot decay of wood but rather the additive effect of copper binding to the wood material. [ABSTRACT FROM AUTHOR]

Degradation of lignin and non-cellulosic polysaccharides (pectins and hemicelluloses) in Norway spruce wood (softwood) by the white rot fungus Pycnoporus sanguineus was investigated using transmission electron microscopy coupled with immunocytochemistry. P. sanguineus produced selective decay in xylem cells including tracheids, ray tracheids and ray parenchyma cells. Lignin was preferentially removed first from cell walls with hemicelluloses (xylan and mannan) and cellulose remaining. Lignin in compound middle lamella (CML) regions of tracheids was also preferentially degraded prior to degradation of xyloglucan (hemicellulose). This differs from the previous concept of wood decay by selective white rot fungi demonstrating concomitant degradation of lignin and hemicelluloses. In contrast, no clear preferential degradation of lignin prior to pectins and hemicelluloses was observed in middle lamella cell corner (MLcc) regions of tracheids which were the last regions to be attacked by the fungus. Micromorphologically, prominent cell separation between tracheids by delignification of CML regions before complete degradation of secondary cell walls was observed as evidence of selective decay. Variations in resistance to decay by the fungus depending on cell wall orientation (radial vs. tangential) of tracheids and between cell types were also observed. [ABSTRACT FROM AUTHOR]

Degradation of lignin and non-cellulosic polysaccharides (pectins and hemicelluloses) by the white rot fungus Pycnoporus sanguineus were investigated in European ash wood (hardwood) using transmission electron microscopy coupled with immunocytochemistry. Lignin was preferentially removed in secondary cell walls of ash xylem cells including fibers, vessel and (ray/axial) parenchyma cells prior to degradation of hemicelluloses (i.e. selective decay). Concomitantly, degradation of cell walls occurred by progressive thinning from the lumen surface outwards similar to simultaneous decay. Preferential degradation of middle lamellae before complete degradation of secondary cell walls (i.e. a typical feature of selective decay) was only observed in bi-/triseriate rays by initial attack from pit regions. Vessel and parenchyma cells showed greater resistance to decay than fibers with narrower decay zone produced. A comparison of decay between ash and spruce wood (Part I, softwood) demonstrated that P. sanguineus produces selective decay in both hard- and softwoods but more typical micromorphological features of selective decay are produced in spruce tracheids than ash fibers. This study highlights that degradation of lignin proceeds hemicelluloses in wood cell walls attacked by the selective white rot fungus P. sanguineus . [ABSTRACT FROM AUTHOR]

Aspects of the degradation of non-cellulosic polysaccharides (pectins, hemicelluloses) in spruce and ash sapwood by the brown rot fungus Postia placenta was investigated using transmission electron microscopy coupled with immunocytochemistry. Degradation of non-cellulosic polysaccharides and changes in ultrastructure were initiated in the compound middle lamella (CML)/middle lamella cell corner (MLcc) regions of xylem cells; sites that were remote from hyphal colonization in the cell lumen. Degradation of the S 3 layer was also detected in both wood species at early stages of decay. Apart from early stages of attack, the degradation patterns differed greatly between spruce and ash. Spruce showed three patterns of hemicellulose degradation in the secondary cell walls of tracheids, while ash showed a single pattern in fibers. Spruce parenchyma cells also showed lower resistance to decay than tracheids, whereas ash parenchyma cells showed higher resistance than fibers. Together, degradation of xylem cells by P. placenta differs between spruce (softwood) and ash (hardwood) even though they show similar initial degradation in CML/MLcc regions and the S 3 layer. The overall degradation patterns observed, particularly in spruce differ from previous ideas and generally accepted decay patterns produced by brown rot fungi in wood. [ABSTRACT FROM AUTHOR]

The large variability in the mechanical properties of hemp fibers is an issue in relation to their use in high-grade composites. The objective of the present study was to determine the optimal growth stage for harvesting hemp fibers for use in composites and to evaluate the effect of field retting time on mechanical performance of the fibers. Reduction in bast content and thickness of the primary bast fiber layer in stems were found to be highly significant ( P < 0.01) with plant maturity. A significant increase in the secondary fiber fraction occurred with maturity, reaching a maximum value of 10% at seed maturity. A highly significant reduction in cellulose deposition in fiber cell walls was reflected by reduced fiber wall thickness with plant maturity and was related to the development and ripening of hemp seeds. A statistically significant increase in lignin deposition and a slight decrease in pectins in hemp fiber cell walls were also noted with stem maturity. Microscopy observations and histochemical analyzes corroborated the results from the chemical analyzes and revealed variations in morphological aspects and spatial micro-distributions of carbohydrates and lignin within the cell structure of the hemp stems between early- and late growth phases. Fibers harvested at the beginning of flowering exhibited high tensile strength and strain, which decreased with plant maturity. Reduction in strength was related to the increase in proportion of secondary fibers and decrease in cellulose deposition leading to inferior properties of fibers. A negative effect of field retting occurred only after extended field retting (i.e., 70 days) which was presumably due to accelerated degradation of cellulose by the action of microorganisms. [ABSTRACT FROM AUTHOR]

Abstract: Four fields for testing wood preservatives were characterized regarding type of rot, fungi present in wood stakes by DNA-identification to genus/species level and by degradation rate. Results were compared with a previous >10 years old study of the same test fields regarding decay type. In all four test fields the major wood degrading fungi were identified to species or genus level by isolation and DNA-sequencing. Two of the test fields showed areas that could be described as “hot spots” that could be used for accelerated testing of wood protection systems. To visualize interior fungal decay throughout stakes, cross sections were made at every 2 cm. These cross sections were used in image analysis and compared with results from the traditional pick test. This provided an opportunity to identify where in the stake degradation took place and its distribution throughout the stake. Results from both methods correlated well regarding white- and brown rot decay. Combining image analysis with data concerning rot type and fungal genus/species represents a useful approach for testing new wood protection systems. [Copyright &y& Elsevier]

Abstract: Aligned epoxy-matrix composites were made from hemp fibres defibrated with the fungi Phlebia radiata Cel 26 and Ceriporiopsis subvermispora previously used for biopulping of wood. The fibres produced by cultivation of P. radiata Cel 26 were more cellulose rich (78%, w/w) than water-retted hemp due to more degradation of pectin and lignin. The defibrated hemp fibres had higher fibre stiffness (88–94GPa) than the hemp yarn (60GPa), which the fibre twisting in hemp yarn might explain. Even though mild processing was applied, the obtained fibre strength (643MPa) was similar to the strength of traditionally produced hemp yarn (677MPa). The fibre strength and stiffness properties are derived from composite data using the rule of mixtures model. The fibre tensile strength increased linearly with cellulose content to 850MPa for pure cellulose. The fibre stiffness increased also versus the cellulose content and cellulose crystallinity and reached a value of 125GPa for pure crystalline cellulose. [Copyright &y& Elsevier]

Abstract: In this paper, formation of unique carbon nanostructures via carbonization of wood in a step-wise process is reviewed. The mechanism described for the production of carbon nanotubes improves our understanding of a historic mystery related to the production of Damascus steel. [Copyright &y& Elsevier]

Microscopy was applied to explore changes in fat globules and gas chromatography was used to determine the fatty acid distribution in different lipid classes in ultra-high temperature (UHT) milk processed in a pilot plant. Four milk samples were collected at key steps during UHT-milk production. Second-stage homogenisation significantly decreased fat globule size and disrupted the original milk fat globule membrane (MFGM) organisation. Pasteurisation applied before homogenisation increased the adsorption of casein micelles to the newly formed MFGM. The final UHT treatment for 4 s at 137 °C exacerbated the damage to MFGM causing disruption of fat globules. Damaged fat globules in UHT milk were more prone to aggregation and entrapment in the protein network compared with fat globules in commercial UHT milk. The significant modifications of milk microstructure and lipid composition suggested processes applied in commercial dairy plants might contribute to destabilisation of UHT milk during shelf life. [ABSTRACT FROM AUTHOR]

Abstract In this study, we used proteomics in conjunction with microscopy to study differences in the proteome and hyphal morphology of the copper tolerant soft rot fungus Phialophora malorum grown in media containing 0.064, 0.64% Cu as CuSO 4. Unique proteins were found in the control and the copper-treated (0.064% CuSO 4) samples. Of five unique proteins found in the 0.064% CuSO 4 treated cultures, ATP synthase subunit alpha is considered to play an important role in copper tolerance as it is involved in the biosynthesis of fatty acids and steroids and may relate to morphological changes associated with hyphal cell walls of the fungus when grown in the presence of copper. ICP-AES analyses showed total mycelial Cu to increase with media Cu with 5246- and 16535 μg Cu/g dry wt mycelia respectively found in 0.064 and 0.64% Cu-cultures after 6 weeks growth. Rubeanic acid staining of 0.064% mycelia showed Cu bound in intracellular bodies while most Cu was found as extracellular precipitates on the surfaces of hyphae in 0.64% Cu. SEM showed hyphal surfaces enrobed in fibrillar polysaccharides to which Cu was bound. Highlights • The soft rot fungus Phialophora malorum was studied to understand its mechanisms of high Cu-tolerance. • P. malorum mycelia grown in 0.064% CuSO4 media bound Cu intracellularly in deposits. • In 0.64% Cu cultures, Cu adsorbed as extracellular precipitates on hyphal surfaces. • Proteomics showed up-regulation of proteins in Cu-media indicating their likely involvement in Cu-tolerance. [ABSTRACT FROM AUTHOR]

γ-Tubulin is essential for microtubule nucleation and also plays less understood roles in nuclear and cell-cycle-related functions. High abundancy of γ-tubulin in acentrosomal Arabidopsis cells facilitated purification and biochemical characterization of large molecular species of γ-tubulin. TEM, fluorescence, and atomic force microscopy of purified high molecular γ-tubulin forms revealed the presence of linear filaments with a double protofilament substructure, filament bundles and aggregates. Filament formation from highly purified γ-tubulin free of γ-tubulin complex proteins (GCPs) was demonstrated for both plant and human γ-tubulin. Moreover, γ-tubulin associated with porcine brain microtubules formed oligomers. Experimental evidence on the intrinsic ability of γ-tubulin to oligomerize/polymerize was supported by conservation of α- and β-tubulin interfaces for longitudinal and lateral interactions for γ-tubulins. STED (stimulated emission depletion) microscopy of Arabidopsis cells revealed fine, short γ-tubulin fibrillar structures enriched on mitotic microtubular arrays that accumulated at polar regions of acentrosomal spindles and the outer nuclear envelope before mitosis, and were also present in nuclei. Fine fibrillar structures of γ-tubulin representing assemblies of higher order were localized in cell-cycle-dependent manner at sites of dispersed γ-tubulin location in acentrosomal plant cells as well as at sites of local γ-tubulin enrichment after drug treatment. Our findings that γ-tubulin preserves the capability of prokaryotic tubulins to self-organize into filaments assembling by lateral interaction into bundles/clusters help understanding of the relationship between structure and multiple cellular functions of this protein species and suggest that besides microtubule nucleation and organization, γ-tubulin may also have scaffolding or sequestration functions. [ABSTRACT FROM AUTHOR]

The durability and decay patterns of thermo-vacuum (Termovuoto process for 3–4 h at 160–220 °C) treated hardwoods (ash, beech) and softwoods (spruce, fir) TMWs exposed to three soft rot fungi ( Chaetomium globosum , Phialophora malorum , P. mutabilis ) were investigated using the soil-block test, light- and electron microscopy. Monitoring of mass loss over 1 year indicated that soft rot fungi do not attack softwood TMWs as rapidly or as extensively as hardwood TMWs. Decay resistance progressively increased in hardwood TMWs with increase in temperature but was unclear/or varied in softwood TMWs depending on fungal/wood species, particularly at lower temperatures (160–180 °C). Soft- and hardwood TMWs showed a major increase in decay resistance at 200–220 °C and 210–220 °C, respectively. Light microscopy of decayed hardwood TMWs showed formation of typical soft rot Type-I cavities in fibres at lower temperatures (190–200 °C). However, cavities were significantly inhibited or delayed at higher temperatures (210–220 °C). Cavity formation in vessels and parenchyma cells were only observed in beech TMW treated at 190 °C or references, indicating higher resistance than fibres. Transmission electron microscopy of decayed ash TMW treated at 200 °C showed a radial-like distribution of electron dense materials in cavities and lack of fibrillar-like materials within degraded fibre walls, which differed from reference. [ABSTRACT FROM AUTHOR]

The wide variation of mechanical properties of natural fibers limits their applications in matrix composites. The aim of this study is to evaluate the properties of hemp fibers from different stem sections (top, middle and bottom) and to assess fungal retting pretreatment of hemp from different stem sections with the white rot fungi Phlebia radiata Cel 26 and Ceriporiopsis subvermispora . For the untreated hemp fibers, no apparent difference in tensile behavior for fiber bundles from different stem sections was observed, and more than 90% tested samples demonstrated plastic flow behavior. Fiber strength and stiffness were highest for the fibers from the top and middle stem sections. These properties were related to the compositional make up and morphological properties of hemp fibers, notably the secondary fiber cell contents. In fungal retting, there was a strong dependence of depectinization selectivity on stem section, which decreased from bottom to top presumably due to the significantly higher lignin content in the bottom section than in the top section (middle section was in between). Consequently, the fungal retting caused a lower reduction in strength of fibers from the bottom section than in those from the top stem section, and essentially reversed the influence of stem section on fiber tensile strength through depectinization selectivity. At whole hemp stem level, the fungal retting with P. radiata Cel 26 exhibited better mechanical properties with an ultimate tensile strength, strain and stiffness of 736 MPa, 2.3% and 42 GPa, respectively, while fibers treated with C. subvermispora exhibited lower mechanical properties of 573 MPa, 1.9% and 40 GPa, respectively. The study thus also showed that less variable and high strength fibers may be produced using the dependence of depectinization selectivity on stem section for composite application. [ABSTRACT FROM AUTHOR]

High-amylose potato starches were produced through genetic modification resulting in changed granule morphology and composition, with higher amylose content and increased chain length of amylopectin. The increased amylose content and structural changes in amylopectin enhanced film-forming behavior and improved barrier and tensile properties in starch films. The molecular structure in these starches was related to film-forming properties. Solution-cast films of high-amylose starch revealed a homogeneous structure with increasing surface roughness at higher amylose content, possibly due to amylose aggregation. Films exhibited significantly higher stress and strain at break compared with films of wild-type starch, which could be attributable to the longer chains of amylopectin being involved in the interconnected network and more interaction between chains, as shown using transmission electron microscopy. The oxygen permeability of high-amylose starch films was significantly decreased compared with wild-type starch. The nature of the modified starches makes them an interesting candidate for replacement of non-renewable oxygen and grease barrier polymers used today. [ABSTRACT FROM AUTHOR]

Abstract: An investigation was undertaken to understand the mechanism(s) by which chitosan exerts its antifungal effects against the wood-degrading fungi Sphaeropsis sapinea and Trichoderma harzianum. Exposure to increasing concentrations of chitosan caused an increase in the amount of hydrogen peroxide accumulation in cultures of S. sapinea, which was accompanied by a decrease in superoxide formation. The same effect was not observed in T. harzianum. Potassium ion leakage was an early event for both test fungi, leakage being more pronounced for S. sapinea than T. harzianum for the first 5min, particularly at higher concentrations of chitosan treatment. Fluorescence microscopy provided evidence that the effect of chitosan on fungal hyphae was mediated through alterations in the plasma membrane properties. Chitosan also severely affected fungal morphology. Increasing concentrations of chitosan induced excessive branching, vacuolation, and a reduction in hyphal diameter. Transmission electron microscopy, which showed more severe ultrastructural changes in S. sapinea hyphae from chitosan treatment as compared to T. harzianum, provided valuable complementary information. The data suggest that the plasma membrane may be the primary target of chitosan action, and that the two fungi differ in the extent to which they are affected. [Copyright &y& Elsevier]

Abstract: The effect of chitosan on cell wall deposition was investigated in the two wood-inhabiting fungal species Trichoderma harzianum (CBS 597.91) and Sphaeropsis sapinea (NZFS 2725). The study used three independent analytical techniques to quantify chitin in the fungal mycelium. A colorimetric method for the detection of d-glucosamine was compared with two gas chromatography–mass spectroscopy (GC-MS) methods employing alditol acetates analysis and pyrolysis. The latter used a stable-isotope-labelled internal standard, d3-N-acetyl glucosamine. At least in the case of S. sapinea, the study provided evidence of an increase in the chitin content in the mycelium due to chitosan treatment, indicating that chitosan treatment affected cell wall deposition. Electron microscopy techniques showed alteration in surface morphology and cell wall texture due to chitosan treatment. The implications of these results are discussed with a view to analysing possible mechanisms for growth inhibitory effects of chitosan on fungal hyphae. [Copyright &y& Elsevier]