Effects of water glass and DMDHEU treatment on the colonisation of wood by Aureobasidium pullulans

Specimens of Scots pine sapwood (Pinus sylvestris L.) and beech (Fagus sylvatica L.) free of knots and cracks with a size of 50×25×15 mm³ (longitudinal×tangential×radial) were prepared. Modification chemicals that were used in this study are described in Table 1.

Impregnation of wood specimens was carried out by applying a vacuum of 60 mbar (60 hPa, 30 min) and subsequent pressure of 12 bar (2 h). All treatments were carried out in a laboratory scale process. After impregnation, the water glass treated specimens were stored for three weeks in a desiccator under carbon dioxide atmosphere, which was established by floating the desiccator in regular steps with CO₂ from a gas bomb. DMDHEU impregnated specimens were pre-dried at 20°C and 65% RH (168 h). Curing of DMDHEU was subsequently performed at 120°C (48 h). The weight percent gain (WPG) of the specimens was determined from the dry masses before and after treatment.

Wood specimens were ground in a disc mill (Retsch, Haan, Germany) to pass a 4 mm screen. After milling, 5 g of the wood chips were added to 150 ml cold water. The mixture was shaken for 24 h. The extract was then filtered and pH of the solution was measured with a glass electrode (Sentix 81, WTW, Weilheim, Germany). The measurements were performed in two parallel measurements of every sample. Eight samples per treatment were used to determine the pH-values.

Ten replicates of treated and untreated specimens were sterilised in an autoclave at 121°C (20 min). The sterilised specimens were dipped into a spore suspension of Aureobasidium pullulans (A. pullulans) and placed horizontally in a Kolle flask containing vermiculite which was inoculated with 15 ml of the spore suspension. The incubated specimens were stored in an incubation room at 22°C and 70% RH (8 weeks). After incubation the surface was evaluated and ranked from 0 to 5 (Table 2). Two sides of the wood specimens were rated, the side which was in contact with the vermiculite and the reverse side. The blue stained area on the sample surface was calculated using GIMP (GNU Image Manipulation Program, version 2.2.10). Furthermore, the specimens were crosscut 5 mm from each end in order to assess the penetration of fungal hyphae.

Wood specimens were ground in a disc mill (Retsch, Haan, Germany) to pass a 4 mm screen. After determination of the wood moisture content, 8 g of wood meal was weighed into folded filter paper. The grounded wood specimens were extracted with 150 ml water in a Soxhlet-extractor for 8 h. The extraction liquid was centrifuged in a laboratory centrifuge (Sigma, Osterode, Germany) at 5000 rpm for 6 min and the supernatant was decanted. These stock solutions were stored in a refrigerator at −18°C until HPLC analysis.

HPLC was carried out using a binary pump (Waters, Milford, USA) and a refractive index detector (Waters, Milford, USA). A Bio Rad HPX87P column (Bio Rad Germany, München, Germany) and an analytical guard cartridge system (Phenomenex Germany, Aschaffenburg, Germany) are used with water as the mobile phase at an operating temperature of 85°C and a flow rate of 0.6 ml/min. Arabinose, fructose, galactose, glucose, xylose and mannose were used as standards. Each treatment was replicated ten times before and after the blue stain test.

Results were statistically analysed by non-parametric one way ANOVA using the Wilcoxon two sample test, because the data were not normally distributed. The comparison between mean values was performed by the Kruskal-Wallis test with a probability level of 0.05.

The untreated specimens displayed the highest infection of blue stain on the surfaces and after cross cutting. The fungal growth started from the surface in contact with the vermiculite and penetrated through the ray cells. The staining of the reverse surface of untreated specimens was caused by the radial penetration of the hyphae from the surface in contact with the vermiculite to the reverse side. Additionally it was caused by spore germination and hyphae growth directly on the reverse surface.

The untreated specimens of beech indicated a higher amount of water soluble carbohydrates than Scots pine sapwood. Willför et al. (2005a, 2005b) showed a total amount of water soluble carbohydrates from different pine species (sapwood) between 4.6 and 6.9 mg/g dry wood and from beech 12.5 mg/g dry wood including dissolved acidic sugar units. After the blue stain test the amount of sugars decreased in untreated wood. Mannose was the most reduced monosaccharide independent of the wood species. It could not be quantified after the blue stain test (Tables 6 and 7) and appeared to be mostly utilised by A. pullulans in untreated wood specimens.

In beech specimens also glucose and fructose were reduced. Fleet et al. (2001) showed similar results for different sapstaining fungi on lodgepole pine (Pinus contorta Dougl. ex Loud.), with mannose and glucose as the most reduced sugars after fungal infection. These results indicated a preference for mannose by A. pullulans in untreated Scots pine and beech wood. The specimens showed a significantly higher level of galactose after the blue stain test. Galactose appeared not to be utilised by A. pullulans and other sapstaining fungi (Fleet et al. 2001), but glucose, glucuronic acid and also galactose and mannose were detected on cell walls of A. pullulans (Bernier 1958; Brown et al. 1973).

In case of the water glass treated specimens the amount of monosaccharides was low and only mannose (Scots pine sapwood) and galactose and mannose (beech) showed a significant level after the treatment. The simple sugars can be washed out during impregnation, because of the high water solubility of sugars in alkaline solutions.

A radial penetration of fungal hyphae was not visible, only a fungal infestation on the wood surfaces. The discolouration of both surfaces might be caused only by spore germination and hyphae growth directly on these surfaces. Fungal tests of water glass treated wood are difficult to carry out because of the high amounts of leaching chemicals and the high moisture content of wood specimens during fungal incubation (Furuno et al. 1991, 1992; Furuno and Imamura, 1998). The main effect which might be responsible for the reduction of blue stain is the highly alkaline pH-value and the high wood moisture content of the specimens. A secondary effect is the formation of insoluble silicates, which are mainly located in the cell lumens (Furuno et al. 1992).

The water glass treated specimens showed a highly alkaline pH-value before and after storage under carbon dioxide atmosphere. Before carbon dioxide treatment the pH-value was high due to the alkaline waterglass solution. After carbon dioxide treatment, the resulted sodium carbonate on the sample surface and the alkaline parts of the silicates caused high pH values. The pH-value for growth of A. pullulans is between 3.5 and 6.3 (McNeil et al. 1989) so that the highly alkaline pH values can influence spore germination, mycelia growth and fruit body formation (Schmidt 2006; Reiß 1997). Sodium carbonate forms an alkaline solution in the presence of water (wetted vermiculite) which could influence the spore germination and mycelia growth in the surrounding area of the sample. Investigations of Dellith (2006) also reported a high resistance of water glass treated specimens against wood destroying basidiomycetes, because of the high pH-values and the insoluble silicates in the cell lumens (Furuno et al., 1992). The moisture content of water glass treated specimens was high (mean 108%), indicating hygroscopicity of unreacted silicates and sodium carbonate on the sample surface.

The surface discolouration of DMDHEU treated specimens was reduced compared to untreated specimens and no radial penetration of fungal hyphae was visible. The discolouration of both surfaces might be caused only by spore germination and hyphal growth directly on these surfaces. The pH-values were acidic. The content and composition of monosaccharides in DMDHEU treated specimens were different from untreated wood and dependent on hard- and softwood species. Scots pine samples treated with DMDHEU showed a significantly higher amount (36.1 mg/g dry wood) of total simple sugars as compared to untreated wood (5.4 mg/g dry wood). After eight weeks of incubation with A. pullulans the amount of sugar was reduced. The percentage reduction of simple sugars in DMDHEU treated samples (65%) was comparable to the one of untreated Scots pine specimens (71%). The amount of simple sugars in DMDHEU treated Beech wood (9.8 mg/g dry wood) was significantly lower than in treated Scots pine (36.2 mg/g dry wood). Independent of the amount of simple sugars there was only a negligible difference in the percentage surface discolouration of DMDHEU treated beech and Scots pine sapwood. These results indicated that the amount of available sugar monomers does not directly influence the degree of colonisation of the sample surface of DMDHEU treated wood. Other factors caused by modification with DMDHEU might have a higher influence on the fungal colonisation, such as wood moisture content and physical blocking of the penetration pathways of fungal hyphae (Xie et al. 2008). At wood moisture contents close to fibre saturation point DMDHEU modified wood absorbs less water molecules than unmodified wood (Krause et al. 2003; Dieste et al. 2008) and the treatment reduces the speed of liquid water uptake caused by the inclusion of the molecule in the ray cells, the major penetration pathways for water in untreated wood (Xie, 2005; Xie et al., 2008). The moisture content is high enough for fungal infestation particularly on the sample surface which is in contact with the wetted vermiculite, because of the higher moisture content at the interface of wood and vermiculite. But the wood moisture content might not be adequate in the inner part of the specimen for fungal growth. The main penetration pathways for fungal hyphae are the ray cells, which are also the main penetration paths of impregnation liquids (Sell 1977). Both could be physically restricted, because of the deposition of DMDHEU molecules within the cell walls and cell lumens.

Further investigations are required to obtain more detailed information on the inhibition factors of modified wood against blue stain for further improvement of modification methods against staining fungi. The differences in the content and composition of simple sugars in DMDHEU treated wood depending on hard- and softwood species are still unclear. Therefore, more detailed studies are necessary to clarify the influence of DMDHEU treatment on the content and composition of simple sugars depending on hard- and softwood species.