The present study shows that isolates of P. vitreus have an extraordinary capacity to induce substantial permeability changes in heartwood of P. abies without causing significant losses in impact bending strength. The degradation of pit membranes by P. vitreus is an important aspect that could also have significant benefits in wood protection processes. Further studies are currently in progress with the objective of optimising the uniformity of wood colonisation and duration of incubation, so as to improve the permeability of water-borne wood preservatives or hydrophobic substances applied by brushing, dipping and impregnatio

Heeb, Markus

Landmesser, Helge

Schwarze, Francis W.M.R.

Zgraggen, Bruno

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Holzforschung, Vol. 60, pp. 450–454, 2006 • Copyright  by Walter de Gruyter • Berlin • New York. DOI 10.1515/HF.2006.071Article in press - uncorrected proofPermeability changes in heartwood of Picea abies and Abiesalba induced by incubation with Physisporinus vitreus*Francis W.M.R. Schwarze1,2,**, HelgeLandmesser1,2, Bruno Zgraggen1 and MarkusHeeb11 Wood Laboratory, Swiss Federal Laboratories forMaterials Testing and Research (EMPA), St. Gallen,Switzerland2 Institut fu¨r Forstbotanik und Baumphysiologie, Albert-Ludwigs-Universita¨t Freiburg, Freiburg i.Br., Germany**Corresponding author.Wood Laboratory, Swiss Federal Laboratories for MaterialsTesting and Research (EMPA), Lerchenfeldstr. 5, 9014 St.Gallen, SwitzerlandE-mail: francis.schwarze@empa.chAbstractThe present study shows that isolates of P. vitreus havean extraordinary capacity to induce substantial permea-bility changes in heartwood of P. abies without causingsignificant losses in impact bending strength. The deg-radation of pit membranes by P. vitreus is an importantaspect that could also have significant benefits in woodprotection processes. Further studies are currently inprogress with the objective of optimising the uniformityof wood colonisation and duration of incubation, so asto improve the permeability of water-borne wood preser-vatives or hydrophobic substances applied by brushing,dipping and impregnation.Keywords: Abies alba; heartwood; Physisporinus vitreus;Picea abies; wood permeability.IntroductionThe permeability of conifer wood is enhanced by bio-degradation of pit membranes, which usually occurs inthe course of bacterial activity in water-stored logs(Schmidt and Liese 1994). Permeability of sapwood hasbeen increased after such a treatment and as a conse-quence the uptake of preservatives was improved (Bauchet al. 1970; Johnson 1979; Schmidt and Liese 1994).Treatment of wood with enzymes that degrade the prin-cipal constituents of the pit membranes (pectin, hemi-celluloses and celluloses) is also promising (Nicholas andThomas 1968; Bauch et al. 1970; Sharma and Kumar1979). More recent findings indicate that the most effec-tive enzymes are hydrolases with a broad spectrum ofcellulolytic and hemicellulolytic activity when finelyground Picea abies wood was the substrate (Militz1993a,b). It was, however, also noted that the practical*Dedicated to Prof. Dr. (Dr. hc mult.) Walter Liese, to mark his80th birthday.importance of enzymes in this context is limited: theirdiffusion into wood is slow and the extractives adheringto aspirated pits have inhibitory effects (Militz 1993a,b).Rosner et al. (1998) demonstrated that basidiomycetesare capable of colonising sapwood and heartwood andcan improve the permeability. A major disadvantage isthat fungi also weaken the wood by degrading the sec-ondary walls as well as the pit membranes (Tucker et al.1998). The white rot fungus Physisporinus vitreus is anexception in this regard; it preferentially degrades the pitmembranes of bordered pits in the heartwood of conifersunder controlled conditions, and causes only negligiblemass losses (Schwarze and Landmesser 2000). This fun-gus is, however, capable of strongly degrading water-sat-urated timber in cooling towers (Schmidt et al. 1996,1997).The objective of the present study was to investigatewhether P. vitreus can significantly enhance the perme-ability of conifer heartwood without inducing significantstrength losses. If this approach is successful, severalprocesses for wood modification and finishing couldbe significantly improved. The improved permeability ofwaterborne solvents into conifer wood is one usefulapplication.Materials and methodsSpecimens from the heartwood of living 40–50-year-old trees ofAbies alba Mill. and Picea abies Karst were incubated with twoisolates of P. vitreus and then assessed for quantitative andspatial alterations in their water uptake and impact bendingstrength. Wood specimens of two different dimensions, but cutexactly the same in the radial and tangential planes, were inves-tigated: (1) 100 mm (longitudinal)=10 mm (tangential)=15 mm(radial) (15 cm3); and (2) 100 mm (longitudinal)=25 mm(tangential)=15 mm (radial) (37.5 cm3). A total of 448 test spe-cimens were prepared according to European standard EN 113(1997), comprising 16 specimens for each treatment and incu-bation period. Mass loss and water uptake were measured foreach specimen before and after incubation. Before water uptakemeasurements, wood specimens were dried at 1038C for18–24 h and then cooled in a desiccator and weighed. Strengthtests were performed only with the smaller specimens, andmicroscopy was carried out exclusively on the larger specimens.Selection of heartwood specimensThe water uptake of the blocks was measured (in kg my3)according to EN 113 before incubation. Based on the results,only specimens that showed the typical water uptake for heart-wood of each of the two species were selected. Wood speci-mens with atypical water uptake were rejected. This ensuredthat all specimens contained a high proportion of heartwood. Toensure that water was taken up via the tangential and radialsurfaces only, the end grain of each dried and weighed speci-men was first sealed with soft silicone grease and firmly fixedPermeability changes in wood by Physisporinus vitreus 451Article in press - uncorrected proofFigure 1 Heartwood specimens of Picea abies (top) and Abiesalba (bottom) impregnated with a 0.1% aqueous solution ofNeolan Glaucin E-A using vacuum treatment after 6-week incu-bation with Physisporinus vitreus (left, 15 cm3; right, 37.5 cm3).Numbers refer to radial and tangential uptake of water in kg my3.In comparison to weakly stained controls, cross-sections ofincubated P. abies specimens are moderately stained and thoseof A. alba are strongly stained. Arrows indicate the direction fromwhich wood samples were colonised by hyphae.between metal mounts to prevent the formation of cracks andshakes. Retention of water taken up via the tangential and radialsurfaces was then measured by immersing the blocks in distilledwater at reduced pressure (7 mbar) for 20 min in a vacuum des-iccator according to EN 113. Then the vacuum vessel wasbrought back to atmospheric pressure and the specimens wereleft for a further 120 min in water before re-weighing (Figure 1).Incubation of wood specimensBefore incubation, the blocks were sterilised with ethylene oxidefor approximately 1 h. Incubation was carried out with two iso-lates of Physisporinus vitreus (EMPA 642 and EMPA 643) in Kolleflasks according to EN 113. The flasks were incubated in a ran-dom array at 22"18C and 75"5% RH for 6, 12 and 18 weeks.After incubation, the wood blocks were dried for 48 h at 308Cand permeability changes were measured. To evaluate qualita-tive changes in uptake and distribution of the synthetic organictextile dye Neolan Glaucin E-A (Ciba Geigy), the end grain ofeach block was sealed with silicone grease as described aboveand the wood specimens were then impregnated with a 0.1%aqueous solution of the dye according to EN 113. After impreg-nation, incubated wood blocks were dried at 1038C for 18–24 hand then cooled in a desiccator and weighed for measurementof mass loss. The smaller wood specimens (15 cm3) were pre-conditioned in an incubator at 208C and 65% RH for 4 weeks.Their impact bending strength, Aw (kJ my2), was measured inthe tangential direction using a Zwick pendulum impact testingmachine in accordance with DIN standard 52 189 (1981). Forstatistical evaluation of differences between control and incu-bated blocks of each tree species for water retention and impactbending strength, t-tests (P-0.05–P-0.001) were performedusing Origin 7.5 (Microcal Software Inc.).Electron microscopyRadial longitudinal specimens were extracted from stained andunstained regions of the larger (37.5 cm3) incubated woodblocks. These were then dried in a vacuum oven at 408C and10 mbar for 12 h, glued on a specimen holder using carbon-adhesive and sputtered with a platinum layer of approximately10 nm for investigation using a field-emission SEM (Jeol 6300F)at an acceleration voltage of 5 kV and working distance of24 mm.Results and discussionThe mass losses in heartwood of P. abies and A. albawere negligible after 6-week incubation (-1%, Table 1).After 12 and 18 weeks, there were significant differenc-es between the mass losses in P. abies and A. alba(P-0.05). Only after 6-week incubation, as the masslosses induced by both P. vitreus isolates were slight,was the wood permeability increased, as manifest by theincrease in water uptake in the test blocks to approxi-mately 300–400 kg my3 in P. abies and 400–680 kg/my3in A. alba (Table 1). The differences before and after incu-bation of the specimens were highly significant (P-0.001). The two P. vitreus isolates showed no significantdifferences in their effect on permeability. Conspicuousqualitative changes in permeability were also apparentfrom the uptake of the bluish dye Neolan Glaucin E-A.Uptake of the dye by A. alba test blocks incubated withP. vitreus was homogeneous on visual inspection, but thehomogeneity was less pronounced in corresponding P.abies blocks.FE-SEM studies revealed that uptake of Neolan Glau-cin E-A was attributable to preferential degradation of pitmembranes (Figures 2 and 3). Complete or partial hydrol-ysis of bordered pits and crossfield pits was apparent inregions stained with Neolan Glaucin E-A. Unstainedregions of pit membranes remained intact (Figure 3).Hyphae entered the pit chamber via apertures, and mem-branes were subsequently degraded (Figures 2 and 3).Degradation commenced from the thickened central partof the membrane (torus). Calcium oxalate crystals wereregularly observed on hyphae. In wood of A. alba, crys-tals accumulations were often apparent within borderedpits in close proximity to hyphae (Figure 3). Productionof polygalacturonase (PG) and hydrolysis of bordered pitmembranes during incipient decay has been describedin detail (Green et al. 1991, 1995a,b; Green and Clause1999). A key factor for pectin hydrolysis by plant patho-gens has been shown to be fungal production of oxalicacid, which lowers the pH of the substrate and chelatescalcium ions. Production of oxalic acid may serve asimilar role for P. vitreus isolates during incipient wooddecay.Crossfield pits of the xylem ray parenchyma alsoshowed signs of extensive degradation. In decayedwood specimens of A. alba, degradation of taxodioid pitmembranes was found throughout the stained woodregions. In contrast, degradation of piceoid pit mem-branes in P. abies was more localised and was restrictedto cells near the outer surfaces of the specimens. Pre-vious studies have demonstrated that most brown- andwhite-rot fungi have the capacity to hydrolyse the pectinin pit membranes during incipient decay, which facilitatescolonisation (Cowling 1961; Wilcox 1968; Green andClausen 1999; Schwarze and Landmesser 2000;Schwarze et al. 2004).The impact bending strength of incubated P. abieswood specimens was not significantly reduced in452 F.W.M.R. Schwarze et al.Article in press - uncorrected proofTable1Massloss,wateruptakeandstrengthlossinheartwoodspecimensofPiceaabiesandAbiesalbaafter6-,12-and18-weekincubationwithtwoisolatesofPhysisporinusvitreus.SampleMassloss(g)Wateruptake(kgm–3)(beforeandafterincubation)Strength(kJm–2)6weeks12weeks18weeks6weeks12weeks18weeks6weeks12weeks18weeksP.abiesControl,15cm3–25.7"4.05EMPA642,15cm30.9"0.35NS6.50"2.30***10.7"4.26***174.8"66.15170.60"69.2055.6"64.9022.7"2.00NS13.0"3.26***9.9"4.77***418.7"176.64***598.30"167.20***738.80"156.18***EMPA643,15cm30.8"0.29NS4.2"0.97***8.1"3.78*185.30"70.66328.90"188.50150.4"62.6023.2"3.57NS16.3"3.26***11.0"2.90***378.90"160.32***449.50"154.85***760.10"86.18***EMPA642,37.5cm30.8"0.24NS5.30"1.37***7.0"2.41***34.7"44.7750.40"70.7169.8"39.4–––327.0"75.22***650.3"113.78***782.20"59.87***EMPA643,37.5cm30.8"0.24NS3.9"1.24***6.3"3.19*98.90"40.9654.2"64.3053.20"58.80–––375.80"83.78***571.90"155.09***772.30"56.15***A.albaControl––25.7"4.13EMPA642,15cm30.9"0.363.41"1.255.8"2.70162.80"70.71174.10"70.71135.90"49.3017.6"2.00**18.0"3.18***14.6"2.81***660.7"64.04***672.9"174.10***819.8"70.71***EMPA643,15cm30.7"0.241.60.8"1.515.0"3.48332.60"75.48219.3"158.30157.9"76.6020.5"3.57*17.3"2.16***11.0"2.90***536.40"120.30***532.7"112.26***754.20"51.84***EMPA642,37.5cm30.7"0.241.40"1.393.2"2.70168.10"37.77168.10"37.80285.40"46.30–––647.70"88.41***654.7"75.18***758.90"47.82***EMPA643,37.5cm30.7"0.351.1"0.963.50"3.37165.60"40.89173.7"36.80303.60"43.90–––631.0"87.25***664.6"89.86***769.40"39.85***Significantdifferencesbetweencontrolandtreatment(wateruptakeandstrength)orspecies(massloss)withinthesameincubationperiodsarepresented,accordingtoat-test(ns16):NS,notsignificant;*P-0.05;**P-0.01;***P-0.001.Permeability changes in wood by Physisporinus vitreus 453Article in press - uncorrected proofFigure 2 Scanning electron micrographs (5 kV) showing bordered pits (a–d) in Picea abies and Abies alba after 6-week incubationwith Physisporinus vitreus. Hyphae entered the bordered pits via the aperture and degraded the torus; lysis of the warty layer (pointer)and calcium oxalate crystals (arrows) occurred in close proximity to hyphae within bordered pit chambers (b). After 12 weeks, mostbordered pits showed partial to complete dissolution (c,d) of pit membranes, so that both pit apertures in pit pairs were occasionallyexposed (arrow).Figure 3 Scanning electron micrographs showing crossfield pits in sound and degraded heartwood of Picea abies (a,b) and Abiesalba (c,d), respectively. After 6-week incubation with Physisporinus vitreus, some crossfield pits showed partial dissolution (b; arrows)in P. abies, whereas most pit membranes of A. alba were completely degraded (d; arrows).454 F.W.M.R. Schwarze et al.Article in press - uncorrected proofcomparison to controls after 6 weeks, whereas there wasa significant reduction in A. alba specimens (EMPA 642,P-0.01; EMPA 643, P-0.05; Table 1). After 12 and18 weeks, wood strength was significantly lower in allcases in comparison to the controls (P-0.001).Colonisation of wood specimens of A. alba and P.abies by P. vitreus was more homogeneous when theywere incubated with their tangential surfaces, rather thantheir radial surfaces, in contact with the mycelium. Thelatter orientation was used for the smaller specimens, toallow them to rest on their broader (radial) surfaces. Theradial surfaces of wood specimens are relatively resistantto colonisation by fungal hyphae (Kleist and Seehann1997; Schwarze et al. 2004). Thus, a more rapid hyphalgrowth in the radial than tangential direction resulted ina higher increase of water uptake in larger wood speci-mens irrespective of the wood species. In wood speci-mens of A. alba, water uptake was also facilitated byhomogenous degradation of the large taxodioid pit mem-branes in the xylem ray parenchyma. The results appearto suggest that P. vitreus colonises wood of A. alba morerapidly, but on the basis of losses in mass, it seems toshow a preference for degrading heartwood of P. abies.Although permeability increased with incubation time,significant strength losses were recorded after 12 and18 weeks. The potentially disadvantageous strength lossin A. alba appears to be correlated with a more intensecolonisation by P. vitreus, which could be mitigated sim-ply by using shorter incubation periods and highergrowth temperatures.AcknowledgementsWe would like to thank the Fond zur Fo¨rderung der Wald- undHolzforschung (FFWH), Bern, for partly funding these studies(Projektnumbers 2005.01 and 2004.16). A German patent hasbeen issued (No. 10 2005 002 366.5) and a European application(No. 05027812.6) has been submitted regarding the use of P.vitreus to increase the permeability of conifer heartwood. Theauthors wish to thank E. Strub for technical assistance with FE-REM and Dr. D. Lonsdale for comments on the paper.ReferencesBauch, J., Liese, W., Berndt, H. (1970) Biological investigationsfor the improvement of the permeability of softwoods. Holz-forschung 24:199–205.Cowling, E.B. (1961) Comparative biochemistry of the decay ofsweetgum sapwood by white-rot and brown-rot fungi. WO-TB-1258. USDA Forest Service, Washington, DC.DIN standard 52189 (1981) Testing of wood; Impact bendingtest; Determination of impact bending strength.European standard (1997) EN 113. Wood preservatives – testmethod for determining the protective effectiveness againstwood destroying basidiomycetes. Determination of toxicvalues.Green, F., Clausen, C.A. (1999) Production of polygalacturonaseand increase of longitudinal gas permeability in southern pineby brown-rot and white-rot fungi. Holzforschung 53:563–568.Green, F., Larsen, M.J., Winandy, J.E., Highley, T.L. (1991) Roleof oxalic acid in incipient brown-rot decay. Mater. 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Permeability changes in heartwood of Picea abies and Abies alba induced by incubation with Physisporinus vitreus

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