Morten Eikenes

Multiplex Real-Time PCR for Monitoring Heterobasidion annosum Colonization in Norway Spruce Clones That Differ in Disease Resistance

ABSTRACT A multiplex real-time PCR assay was developed to monitor the dynamics of the Picea abies-Heterobasidion annosum pathosystem. Tissue cultures and 32-year-old trees with low or high resistance to this pathogen were used as the host material. Probes and primers were based on a laccase gene for the pathogen and a polyubiquitin gene for the host. The real-time PCR procedure was compared to an ergosterol-based quantification method in a tissue culture experiment, and there was a strong correlation (product moment correlation coefficient, 0.908) between the data sets. The multiplex real-time PCR procedure had higher resolution and sensitivity during the early stages of colonization and also could be used to monitor the host. In the tissue culture experiment, host DNA was degraded more rapidly in the clone with low resistance than in the clone with high resistance. In the field experiment, the lesions elicited were not strictly proportional to the area colonized by the pathogen. Fungal colonization was more restricted and localized in the lesion in the clone with high resistance, whereas in the clone with low resistance, the fungus could be detected until the visible end of the lesion. Thus, the real-time PCR assay gives better resolution than does the traditionally used lesion length measurement when screening host clones for resistance.

Chemistry and ecotoxicology of furfurylated wood

Over the past decade modernized processes for furfurylation of wood have been developed. These new processes are based on completely new catalytic systems and process additives. These new systems do not add metals or halogens to the product, which is important for an environmentally acceptable product. However, little is known about the ecotoxicity of furfurylated wood or other environmental impacts that may result from the modification method. The study shows that concentrations of non-reacted furfuryl alcohol in the final products are low and do not contribute to any fungicidal effect. Environmental tests show no significant level of increased ecotoxicity, and degradation through combustion does not release any volatile organic compounds or polyaromatic hydrocarbons above normal levels for wood combustion. Hence, furfurylation of wood to enhance wood properties is not believed to be harmful to the environment.

Comparison of quantitative real-time PCR, chitin and ergosterol assays for monitoring colonization of Trametes versicolor in birch wood

Abstract This paper describes the use of quantitative real-time PCR for monitoring colonization of birch wood (Betula pubescens) by the white-rot fungus Trametes versicolor in an EN113 decay experiment. The wood samples were harvested after 4, 8, 12, 16 and 20 weeks of incubation. The mass loss was in the range of 4–40%. Chitin and ergosterol assays were conducted for comparison. Second-order polynomial fits of the mass loss of decayed wood versus chitin, ergosterol and DNA gave correlations (r2) of 0.87, 0.61 and 0.84, respectively. Compared to the other two assays employed, real-time PCR data correlated best with the relative mass loss of decayed samples 4–8 weeks after inoculation, while the saturation and decline of DNA-based estimates for fungal colonization 16–20 weeks after inoculation indicated that the DNA assay is not suited for quantification purposes in the late stages of decay. The impact of conversion factors, extraction efficiency, inhibitory compounds and background levels in relation to the three detection assays used is discussed.

Comparison of chitosans with different molecular weights as possible wood preservatives

Fungi cause serious problems in wood utilization, and environmentally benign wood protection is required as an alternative to traditional chemicals. Chitosan has shown promising antimicrobial properties against several microorganisms. In this study, we present the characterization of and antifungal properties of a commercial chitosan formulation developed for impregnation of wood. A broad range of chemical and mycological methods were used to evaluate the uptake, fixation, and antifungal properties of chitosan for wood preservation. The results show that the higher the uptake of chitosan the lower the relative recovery of chitosan in wood after leaching, and the higher the molecular weight of chitosan the higher the recovery. Chitosan with high molecular weight proved to be more efficient against decay fungi than chitosan with low molecular weight. The fungi tested on chitosan-amended nutrient agar medium were totally inhibited at 1% (w/v) concentration. In decay studies using small wood blocks, 4.8% (w/v) chitosan concentration gave the best protection against brown rot fungi.

Screening of chitosan against wood-deteriorating fungi

Chitosan, a derivative of the natural amino polysaccharide chitin, has proven effective as a potential environmentally benign antimicrobial component. Few studies have focused on chitosan applied to wood against wood-inhabiting and decaying fungi. In these screening studies several mycological experiments were performed to screen chitosan as a potential wood-protecting agent. Growth studies on chitosan-amended media showed total inhibition of Poria placenta, Coriolus versicolor and Aspergillus niger using 1% w/v concentration. Chitosan with high average molecular weight (MW) was more efficient against mould and staining fungi than chitosan with low MW. Agar plate leaching tests showed only a small leaching effect using a 5% concentration on A. niger and P. placenta. Decay testing with P. placenta demonstrated efficacy using 5% and 2.5% concentrations in unleached samples. Leaching decreased the efficacy of chitosan and further investigations are needed to improve the fixation in wood.

Detection of chlorine-labelled chitosan in Scots pine by energy-dispersive X-ray spectroscopy

The aim of this study was to use energy-dispersive X-ray spectroscopy (EDX) to localize chitosan in the cell wall of chitosan-impregnated Scots pine. It was of interest to investigate the concentration of chitosan in wood to gain further knowledge and understanding of the distribution of chitosan in the wooden matrix. After deacetylation, chitosan was re-acetylated with chloroacetic anhydride to achieve a covalent bonding of chloride to the chitosan polymer. Chloride-labelled chitosan was measured by EDX using a scanning electron microscope and described as chloride intensity. Analysis of free chloride anions was performed by dialysis and inductively coupled plasma atomic emission spectroscopy. There was a significant correlation between the molecular weight of chitosan and the intensity of covalent-bonded chloride to the chitosan polymer. High molecular weight chitosan showed a better interaction with the cell wall structure than low molecular chitosan.

Evaluation of factors that have an influence on the fixation of chitosan in wood

Abstract When using chitosan as an antifungal agent in wood it is important to understand which factors contribute to a higher fixation ratio to optimize the utilization of chitosan, the active component. Small pine samples were impregnated with chitosan solutions varying in molecular weight, concentration, pH, polymerization agent, acid and degree of deacetylation. Different post-treatments such as time, temperature, moisture content and the effect of present air were applied to the samples to evaluate the effect on the relative retention. After impregnation, the samples, with a volume of 1.5 cm3, were leached in separate test-tubes according to EN-84. The samples were prepared in a paired design where both samples were impregnated, but only one was leached. Both leached and unleached samples were analysed for their chitosan content, and the relative ratio was used as a measure for the relative retention of chitosan during leaching. The results from these trials show that pH in the range of 5.1–5.9 is favourable. The molecular weight should be as high as possible yet able to penetrate the wood structure, and the use of acetic acid gives far better fixation than the use of hydrochloric acid.

Screening of properties of modified chitosan-treated wood

Abstract Chitosan is a biopolymer derived from chitin in crustacean shells. Over the past decade it has been studied as an environmentally benign wood-protecting agent. It is assumed to act as a fungi-stat against a wide range of fungi and even as a fungicide at higher concentrations. This study investigated the properties of wood treated with modified chitosan of different molecular weights. Scots pine (Pinus sylvestris L.) and beech (Fagus sylvatica L.) samples were impregnated with two chitosan solutions differing in their average molecular weights. The chitosan solutions were depolymerized by nitrous acid to one solution of high molecular weight and one solution of low molecular weight with a concentration of 5% (w/v). The results show changes in sorption properties, antifungal properties, fire-retardant properties and mechanical properties of modified chitosan-treated wood. Heat-modified, chitosan-treated wood showed similar properties to chitosan-treated wood, except for brownish coloration, enhanced hydrophobation, and slightly reduced antifungal and fire-retardant properties. The modulus of rupture and hardness showed little or no change. The modulus of elasticity of the heat-modified, chitosan-treated wood increased by 27% compared with untreated wood.