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Effects of calcium-based materials and iron impurities on wood degradation by the brown rot lacrymans

Article in Holzforschung · January 2010 DOI: 10.1515/HF.2010.009

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Holzforschung, Vol. 64, pp. xxx-xxx, 2010 • Copyright by Walter de Gruyter • Berlin • New York. DOI 10.1515/HF.2010.009

Effects of calcium-based materials and iron impurities on wood degradation by the brown rot fungus

Jonathan S. Schilling* frequent and economically important indoor decay fungi in Department of Bioproducts and Biosystems Engineering, temperate climates, particularly in Europe (Schmidt 2000). University of Minnesota, Saint Paul, Its ability to grow long distances between carbon sources MN, USA over non-nutritional materials is distinctive and has been subject to extensive research and review (Thornton and John- *Corresponding author. son 1986; Jennings and Bravery 1991; Henry 2003). In most Department of Bioproducts and Biosystems Engineering, cases, S. lacrymans can penetrate and degrade building mate- 2004 Folwell Avenue, 108 Kaufert Lab, University of rials, such as mortar and plaster (Bech-Andersen 1987; Low Minnesota, Saint Paul, MN 55108, USA Phone: q1-612-624-1761 et al. 2000), but it is unresolved how these associations affect Fax: q1-612-625-6286 the fungus physiologically. E-mail: [email protected] A commonality among many non-woody building mate- rials is a high level of calcium (Ca). Bech-Andersen (1985) suggested first that Ca2q neutralizes oxalic acid and allows Abstract better pH control during wood decay by S. lacrymans. Fol- low-up studies showed electron micrographs of S. lacrymans Calcium-containing materials have been implicated in pro- degrading Ca-containing materials (Bech-Andersen 1987; moting wood degradation by Serpula lacrymans, but mech- Low et al. 2000) and producing Ca-oxalate away from mate- anisms remain unresolved. In this study, S. lacrymans and rial surfaces (Bech-Andersen 1991). Others observed faster Serpula himantioides degraded sapwood in agar-block linear growth rates on Ca-containing materials versus on microcosms with one of four treatments: calcium-free, 5 mM wood (Thornton and Johnson 1986). In a controlled labora- agar CaCl , high-purity gypsum (CaSO ), and gypsum 2 4 tory trial, Palfreyman et al. (1996) demonstrated that increas- amended with 1% FeSO . Calcium and iron availability were 4 ing Ca(NO ) levels in minimal nutrient medium hastened limited in minimal nutrient agar. At week 5, pine degradation 3 2 decay of pine sapwood in agar-block microcosms. The abso- was significantly higher for S. lacrymans in iron-amended lute requirement for Ca by S. lacrymans has been written gypsum treatments than other treatments, and the respective into general texts (Bech-Andersen 1991; Ridout 2000), and agar oxalate levels were also higher. Oxalate solubility was a survey written by Krzyzanowski et al. (1999) for building lowest in pure calcium microcosms. Scanning electron practitioners recommended avoiding Ca-containing materi- microscopy showed hyphae in contact with gypsum and pre- als, such as plaster, to avoid this destructive fungus. cipitation of calcium oxalate. At week 15, wood degradation However, there are some weaknesses in these conclusions by S. lacrymans was severe ()60%) in both calcium-free and recommendations. As discussed by Palfreyman et al. and iron-amended treatments, but was significantly less in f (1996), effects on degradation rates could not be conclusive- pure calcium treatments ( 45%). Cation analysis in week 2q - 15 wood revealed higher calcium and iron levels in treat- ly linked to Ca additions versus its counter ion NO3 . ments containing those element additions. Serpula himan- Nitrogen levels are very low in wood, and Watkinson et al. tioides had decayed wood equally among treatments at both (1981) have shown that nitrate, added as Na(NO3)2, signifi- harvests. Results demonstrate that calcium has an inhibiting cantly enhances hyphal growth and promotes cellulolysis in effect – and not a promoting effect as hitherto believed – S. lacrymans. Palfreyman et al. (1996) also found less decay on wood degradation by S. lacrymans. It appears that oxalate than in untreated controls when the Ca source was CaCl2. and iron play a role in stimulating wood degradation by this Steenkjær-Hastrup et al. (2005) found CaCl2 to be somewhat destructive fungus. inhibitory of wood degradation in S. lacrymans decay micro- cosms, and neither CaCl2 nor pure gypsum (CaSO4Ø2H2O) Keywords: copper tolerance; dry rot; Fenton; himantioides; have promoted wood decay by the ‘sister’ taxon S. himan- oxalic acid. tioides or by other wood degrading species (Schilling and Bissonnette 2008). Crystallization with Ca could be inhibi- tory if the soluble oxalate anion plays an active role during Introduction wood degradation by S. lacrymans, including mobilizing iron (Fe) as often theorized (e.g., Varela and Tien 2003). Serpula lacrymans is a destructive brown rot wood-degrad- Iron impurities in Ca-containing building materials might ing fungus (Kauserud et al. 2007). It is one of the most instead be responsible for any enhancement of wood degra-

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2 J.S. Schilling

dation by S. lacrymans. Iron is theorized to play a central (‘‘CaCl2’’ treatment). The other pure Ca treatment was a gypsum role in the oxidative Fenton-based mechanisms of brown rot disc (11.5 mm diameter) (‘‘CaSO4’’ treatment) added adjacent but fungi (Goodell et al. 1997; Hyde and Wood 1997; Kerem et not touching wood blocks on top of mesh supports. Gypsum discs al. 1998). Like other brown rot fungi, S. lacrymans secretes were formed inside a cylinder of aluminum foil (shaped around a ) high levels of oxalic acid during growth (Akamatsu et al. 7 cork borer) resting on wax paper by adding 0.2 g 99% pure CaSO hemihydrate (Sigma) and then adding 200 ml deionized dis- 2004), reduces wood mechanical strength early during the 4 tilled water. After mixing, discs were allowed to solidify overnight, degradation process (Doi and Nishimoto 1986), and produces gently sanded on the edges, and autoclaved prior to adding to micro- Fe-reducing chelators linked to oxidative depolymerization q cosms. The Fe ‘impurity’ discs (‘‘Fe CaSO4’’ treatment) were cre- of cellulose (Shimowaka et al. 2004). Extracellular oxalate ated in the same manner, adding 200 ml of a fresh 1% (w/v) solution 3q coordinates well as a soluble complex with Fe (Henry of FeSO4Ø7H2O (Mallinckrodt Chemicals, Phillipsburg, NJ, USA)

2003), potentially facilitating Fe availability for Fenton and deionized water to CaSO4 powder, matching the sulfate counter chemistry in early stages of the brown rot mechanism (Goo- ion to isolate effects of a high Fe impurity level. A 1% level is dell et al. 1997). Iron has been reported to promote S. lacry- relevant to real-world levels, and was chosen to approximate levels mans wood degradation (Paajanen 1993), and Low et al. in materials tested by Low et al. (2000) that ranged from 0.3% to (2000) showed Fe translocation by S. lacrymans from plaster 4.2% Fe. Microcosms (ns6 per treatment) were inoculated with two 1 cm2 and stone into wood and observed ‘rusty’ hyphae on high- blocks from 2-week-old fungal cultures and incubated in the dark iron materials. at constant temperature (208C). Five replicates were used to com- The hypothesis for this work was that Ca would not pare wood weight loss. The sixth replicate was used for electron enhance S. lacrymans wood decay and that by immobilizing microscopy. Inoculum, blocks, and gypsum discs were arranged in oxalate, Ca might inhibit Fe sequestration and interfere with an ‘X’ configuration, with two adjacent blocks on mesh, a center the brown rot wood degradation mechanism. The aim of this gypsum disc (if applicable) on mesh, and two adjacent sources of work was to isolate effects of Ca and to test contributing inoculum on agar, with no contact among components. Control effects of Fe as an impurity in Ca-containing materials. microcosms with no fungal inoculum (‘‘control’’ treatment) served to monitor any contamination (none was detected), to monitor wood moisture contents, and to serve as ‘time zero’ material. Materials and methods Harvesting and microscopy preparation Fungal strains Out of five of the six replicate treatment microcosms, one of the Serpula lacrymans (Wulfen: Fries) Schroeter strain EMPA 65 two blocks was aseptically removed at week 5, with the final block (ATCC 32750) and Serpula himantioides (Fr: Fr) P. Karst (ATCC harvested at week 15. Blocks were oven-dried for 48 h at 1038C 36335) were used in this study. The latter was originally deposited and weighed, as before. Decay progress was calculated as a percent as S. lacrymans, but has twice been positively identified as S. of the original mass lost during degradation (weight loss). Blocks himantioides by amplification of the internal transcribed spacer were then ground to 20-mesh in a Wiley mill and powder was stored (ITS) region and BLAST searches of sequences in GenBank over dessicant. Non-inoculated control blocks were treated similarly, (Steenkjær-Hastrup et al. 2005; Kauserud et al. 2006). Both isolates except fresh weights were also recorded to allow calculation of w were maintained on 20 ml yeast malt agar (2% agar, ATCC medium moisture content on a standard dry-weight basis (water weight/oven = x No. 200) at 208C to standardize inoculum input volume. dry weight) 100 . Wood blocks from the sixth replicate, used for scanning electron microscopy, were removed, transferred to a clean glass slide, and Agar-block microcosms cut into segments with a razor. Wood segments were added to 2% Petri dishes (25 mm height) containing 20 ml of a minimal nutrient glutaraldehyde in phosphate buffer (pH 7) and stored at 48C until medium in an agar type (Type A) with a low Ca concentration all harvests were complete. Wood segments were fixed in OsO4, (Schilling and Bissonnette 2008) were used as decay microcosms. dehydrated through an ethanol series and freeze-fractured in liquid nitrogen with a razor prior to sputter coating with gold to approx- No Ca or Fe was added to basal salts in agar, except in CaCl2 treatments amended to 5 mM agar concentration. Thick (2 mm) imately 500 A˚ . Gypsum discs were removed and cut, as above, but plastic mesh supports (35=50 mm) were used to keep wood blocks were vapor-fixed in OsO4, similar to Schilling and Bissonnette and gypsum treatments above agar contact. Southern pine sapwood (2008). blocks (1.8 cm each face) were split in half longitudinally but added to the same microcosm to allow two harvests from paired material. Scanning electron microscopy (SEM) The split blocks were dried (48 h, 1038C), weighed, autoclaved, and and microanalysis then added to the microcosms with the split, non-planed face down. This design achieved weight-loss in excess of 60% in this experi- Microanalysis was performed by SEM to verify hyphal contact with ment. This is nearly complete wood degradation for a brown rot the gypsum treatments and to show any production of calcium oxa- fungus on a wood source which we previously measured with 62% late crystals, distinct from sulfur-containing gypsum crystals, during polysaccharide (glucanqhemicellulose) content (Schilling et al. translocation from gypsum treatments. A Hitachi S3500N SEM 2009). (Pleasanton, CA, USA) with a digital image collection system was Treatments included microcosms with no added Ca or Fe (‘‘Ca- used with a 5 kV accelerating voltage for imaging. Accelerating free’’ treatment), two ‘pure’ Ca treatments, and one Ca treatment voltage of 12 kV was used for microanalysis by electron dispersive containing a controlled Fe ‘impurity’. One of the pure Ca treatments spectroscopy (EDXA), using a Genesis EDXA system (Mahway, was 5 mM CaCl2 (Sigma, St. Louis, MO, USA) added to the agar NJ, USA). Spot probing was done to collect EDXA data over a Article in press - uncorrected proof

Serpula, calcium, and iron 3

60 s collection period, and valence shell peaks were layered using using one-way analysis of variance (ANOVA) and, when protected probe data within the same image field when applicable. at as0.05, Tukey’s means comparison tests were performed among treatments. Wood cation analysis

Wood powders from time zero and week 15 blocks were ashed Results (4808C) and dissolved in HCl (10% w/v). A Thermo Scientific ARL 3560 (Waltham, MA, USA) inductively coupled plasma optical Growth patterns emission spectrometer (ICP-OES) was used to determine Ca and Fe in wood powders. Similar to previous work, weight-based cation Both test fungi grew in direct contact with gypsum treat- data were normalized to account for the loss in wood density during ments, but S. lacrymans mycelia were different in appearance fungal biodegradation (Schilling and Jellison 2007). Instead of among treatments. In the Ca-free treatment, growth was expressing cation content per wood volume, cations are expressed sparse and white, whereas appearing yellow and dense on -1 per wood original mass by multiplying final mmol g by the per- both CaCl and CaSO treatments, and rust-colored (similar cent-of-original weight for each block. This eliminates variability in 2 4 to that described by Low et al. 2000) and dense on the callipering volumes of severely decayed wood. q Fe CaSO4 treatments. At week 15, pine blocks were more fragile and clearly more decayed in the Ca-free and Oxalate and pH q Fe CaSO4 treatments than in the CaCl2 and CaSO4 In S. lacrymans microcosms only, oxalate (solubleqextractable) treatments. was measured in week 5 agars, when the fungus was likely actively translocating elements to the reaction front. Agar/water slurries in Weight loss (WL) a ratio of 1:1 (v/v) were sampled for oxalate after an equilibration period of 2 h and after measuring slurry pH. Aliquots for soluble Pine blocks degraded by S. lacrymans for 5 weeks were sig- q oxalate were removed and filtered to 0.22 mm. Remaining slurry nificantly (p-0.001) more degraded in Fe CaSO4 (19.3%) was acidified to 0.1 N with HCl, buffered in pH 1.4 phosphate versus other treatments (overall mean 9.0%). Wood degraded buffer (acidqmonobasic), pH-adjusted with 1 N NaOH, and filtered during 15 weeks had significantly (p-0.001) higher WL in q to 0.22 mm. Soluble and extracted oxalate were measured by high- the Ca-free (61.9%) and Fe CaSO4 (60.0%) treatments than performance liquid chromatography (HPLC) under conditions out- in CaCl2 (46.0%) or CaSO4 (44.8%) treatments (Figure 1a). lined in Schilling and Jellison (2006); analytical column: Aminex WL)60% approaches complete removal of glucan and HPX-87H from Bio-Rad, Hercules, CA, USA. hemicelluloses by a brown rot fungus, so it is unclear if S. lacrymans might have reached complete polysaccharide re- Statistics q moval in Fe CaSO4 treatments before Ca-free treatments, or All data are expressed as means ("standard deviation). Percentage vice versa. data were log-transformed and pH data were converted to absolute In contrast, treatment did not affect wood degradation by Hq concentration to normalize prior to statistics using SYSTAT S. himantioides, reaching an average of 25.9% WL in week (Systat Software, Inc., Chicago, IL, USA), comparisons among the 15 (Figure 1b). To verify decay progress, six Ca-free micro- q four treatments (Ca-free, CaCl2, CaSO4,Fe CaSO4) were made cosms were inoculated with S. himantioides and incubated

Figure 1 Weight loss in southern yellow pine degraded by S. lacrymans (a) or S. himantioides (b) in microcosms with or without Ca and Ca/Fe amendments. Means"standard deviations (SD) with the same letter harvested from a fungal species on the same week are not significantly different. Article in press - uncorrected proof

4 J.S. Schilling for 24 weeks (not included in Materials and methods sec- tion), with a final WL of 43.4% ("8.3), demonstrating that wood degradation in week 15 was still in progress. Although S. himantioides has a growth optimum of 278C, not 208Cas applied here, inhibition of decay by the agar-block design has been noted in past work at higher incubation tempera- tures for S. himantioides along with other isolates (Schilling and Bissonnette 2008). Week 15 wood block moisture contents in control blocks were favorable for biodegradation, averaging 40.4% ("11.0), above the average fiber saturation point (28.6%) reported by Yao (1969) for second-growth southern yellow pine.

Scanning electron microscopy (SEM) and microanalysis

Both fungi grew on gypsum, extracted Ca, and precipitated Ca oxalate. SEM of the surface of gypsum discs showed hyphae of both fungi grew in direct contact with gypsum (Figure 2a,b). Fungi produced bipyramid crystals, typical of Ca-oxalate morphology, away from the gypsum disc surface and on the wood surface (Figure 2c). No crystals were observed within wood cells. To verify Ca-oxalate in the same image frame with gypsum, bipyramid crystals near the gyp- sum surface were probed using EDXA. Calcium oxalate was discerned from CaSO4 by confirming the lack of sulfur (Figure 3a,b).

Wood cation flux

Calcium levels in wood degraded by test fungi were elevated in treatments containing Ca amendments. Wood Ca at week 15 for S. himantioides and S. lacrymans showed statistical significance among treatments (ps0.026, ps0.033, respec- tively) (Table 1). Wood had more Ca accumulation in CaSO4 s q treatments for S. himantioides (p 0.016) and in Fe CaSO4 for S. lacrymans (ps0.027) than in the Ca-free treatment wood. Wood Fe levels were higher when fungi had access to Fe- amended gypsum. Wood Fe was lower in pure Ca treatments and the Ca-free treatment than in controls for both S. lacry- mans (p-0.001) and S. himantioides (p-0.001). Wood q degraded 15 weeks in the presence of Fe CaSO4 by S. himantioides contained 50% more Fe than controls, and by S. lacrymans contained 250% more Fe than controls. Figure 2 Scanning electron micrographs showing the gypsum sur- Oxalate and pH face (a), fungal hyphae of S. himantioides growing in direct contact with the gypsum surface (b), and S. lacrymans depositing bipyramid The amount of oxalate and its solubility in the agar matrix Ca oxalate crystals on the surface of an adjacent pine wood block was significantly affected by the treatments for S. lacrymans after contact with the gypsum surface (c). (Table 2). Solubility was lowest in pure Ca treatments. Sol- uble oxalate was statistically equal among treatments Ca- percentage was in Ca-free treatments, followed by Fe- q free, with CaCl2, and CaSO4, but much higher in the CaSO4, CaCl2, and CaSO4 treatments, respectively. q Fe CaSO4 treatment (p-0.001 for each fungus). Extracta- Agar pH in cultures of S. lacrymans was influenced by q ble oxalate was higher in the Fe CaSO4 treatment than treatment (p-0.001). pH in pure Ca treatments was lower q other treatments, with the exception of the CaCl2 treatment than in the Ca-free or Fe CaSO4 treatments (Table 2). (ps0.108). Of the extractable oxalate, the highest soluble Although these pH values mirror the oxalate solubility pat- Article in press - uncorrected proof

Serpula, calcium, and iron 5

Figure 3 SEM with EDXA of S. lacrymans in contact with the gypsum surface (a) and S. himantioides in contact with the gypsum surface (b). As denoted in the legend for the spectral overlays, gray-filled spectra are EDXA data from the gypsum surface (probe point x), whereas black lines are EDXA data from Ca oxalate crystals (probe point q). terns, it is important to note that observed pH might reflect related taxon S. himantioides in this study. Variability was decay phase and growth stage among the same treatments. notably low, and greater than 60% wood WL was achieved by week 15 by S. lacrymans, demonstrating a controlled and favorable environment for wood decay. The absence of a Discussion positive Ca effect, in either early or late stages of wood decay, is an important result. It places S. lacrymans alongside Calcium from different ‘pure’ treatment sources did not other white and brown rot species observed growing in direct enhance degradation of pine sapwood by S. lacrymans or the contact with Ca and extracting Ca from non-woody sources

Table 1 Week 15 mean wood calcium (Ca) and iron (Fe) contents ("SD) in agar- block microcosms containing type A low-calcium agar, a test fungus, southern pine blocks resting on plastic mesh, and one of four treatments.

Serpula lacrymans Serpula himantioides Ca Fe Ca Fe Treatment (mmol g-1)* (mmol g-1)* (mmol g-1)* (mmol g-1)* Control 18.18 (2.79) 0.50 (0.23) 18.18 (2.79) 0.50 (0.23) Ca-free 18.12 (1.89)a 0.15 (0.03)a 18.54 (2.31)a 0.21 (0.09)a ab a ab a CaCl2 19.95 (1.65) 0.12 (0.02) 22.02 (2.79) 0.27 (0.09) ab a b a CaSO4 21.86 (3.27) 0.20 (0.10) 24.78 (4.59) 0.33 (0.12) q b b ab b Fe CaSO4 26.41 (6.82) 1.25 (0.59) 21.40 (2.18) 0.74 (0.06)

CaSO4 treatments were solid gypsum discs, whereas CaCl2 treatment was 5 mM final concentration in agar. *Mass-based ICP-OES data were converted to account for mass loss by multiplying by ratio of final/original mass remaining after biodegradation (e.g., 35% weight losss0.65 conversion factor). Treatment data followed by the same letter in a column denote not significantly different (as0.05), after protected Tukey’s tests. Article in press - uncorrected proof

6 J.S. Schilling

Table 2 Week 5 agar pH, soluble (Sol) and extractable (Ext) oxalate in agar- block microcosms containing type A low-calcium agar, Serpula lacrymans (ATCC 32750), southern pine blocks resting on plastic mesh, and one of four treatments.

Soluble Extractable oxalate oxalate Sol/Ext Treatment pH (mM) (mM) (%) Control 5.90 (5.87, 5.93) 0.06 (0.00) 0.13 (0.01) 43.1 Ca-free 3.83 (3.61, 4.28)a 0.24 (0.18)a 0.25 (0.15)a 97.9 b a b CaCl2 2.69 (2.53, 2.93) 0.22 (0.05) 0.68 (0.19) 34.7 b a ab CaSO4 2.62 (2.57, 2.67) 0.12 (0.02) 0.51 (0.19) 26.2 q a b b Fe CaSO4 3.07 (2.94, 3.26) 0.71 (0.13) 1.14 (0.50) 69.5

CaSO4 treatments were solid gypsum discs, whereas CaCl2 treatment was 5 mM final concentration in agar. *Standard deviation (SD) in parentheses. Mean pH"SD is calculated based on absolute Hq concentration, so the upper and lower range is indicated. Data fol- lowed by the same letter within a column are not significantly different (as0.05), after protected Tukey’s tests.

q without consequent enhancement in wood degradation ment of wood degradation by Fe CaSO4 in week 5, when (Steenkjær-Hastrup et al. 2005; Schilling and Jellison 2006, compared to the Ca-free treatment, supports this supposition. 2007). The data refute the supposition that Ca necessarily Because Fe levels in wood degraded by S. lacrymans in Ca- promotes wood degradation by S. lacrymans, although cor- free treatments were similar to those in pure Ca treatments, roborating this observation would be useful by means of oth- it will be important to compare the chemical state of Fe in er S. lacrymans isolates. This has significant implications on these treatments. Iron hydroxides versus mobile Fe oxalate cultural management of this destructive pest. complexes could affect the Fenton-based capacity of S. Inhibition of S. lacrymans wood degradation by Ca, with lacrymans without an observable difference in total wood Fe Fe relieving this inhibition, is also an important observation concentration. with mechanistic implications. It supports the concept that There are also comparisons between calcium oxalate oxalate helps mobilize Fe during brown rot. If the role for dynamics observed here and a role for oxalate in detoxifi- oxalate during brown rot is to sequester and supply Fe3q for cation of copper (Cu) in wood preservatives, also often Fenton chemistry (Goodell et al. 1997; Varela and Tien attributed to crystallization and immobilization. Tolerance of 2003), crystallization with Ca could indirectly limit Fe avail- Cu has been shown to be diminished by Ca additions, prob- ability. This might inhibit brown rot in these Fe-limited ably as a consequence of immobilizing oxalate in non- microcosms and would explain why addition of Fe-amended exchangeable crystals (Steenkjær-Hastrup et al. 2005). gypsum helped the fungus overcome apparent decay inhibi- Tolerance of Cu has also been enhanced in the presence of tion by Ca. As supportive data, rust-colored hyphae were Fe (Ruddick and Morris 1991; Morris and Ingram 1998). q observed in Fe CaSO4 treatments, as was reported by Low This can be interpreted in the following way: Cu toxicity in et al. (2000) when S. lacrymans was in contact with natural brown rot fungi might relate to immobilization of oxalate sandstones and plaster. Calcium-oxalate crystals were also and subsequent reduction of available Fe, although there are produced in copious amounts and there was less soluble oxa- many Cu detoxification routes possible (Gadd 1993). late in pure Ca treatments, as is logical and was shown pre- The inference that Fe, not Ca, in non-woody materials viously in liquid culture (Schilling and Jellison 2004). Fe might exacerbate brown rot in structural timber has signifi- might have stimulated oxalic acid secretion. Oxalic acid cant implications on indoor wood decay. Many of the non- secreted by S. lacrymans might have been immobilized non- woody materials used in structures are Ca-based, such as productively by Ca2q, with any associated pH drop too weak plaster and mortar. These materials inherently must contain to mobilize Fe and overcome decay inhibition. It will be Ca. By contrast, Fe can be viewed as a contributing element important to test the effects of Fe separately and to probe or an impurity and therefore could be limited through mate- early and more thoroughly, when strength loss issues are rial selection and processing. In the broad array of scenarios critical. where S. lacrymans is a significant issue, this suggests a The difference between S. lacrymans and S. himantioides, potential management tool for avoidance and requires more as well as other brown rot fungi tested in the past, might research. relate to efficient use of nutrients, such as nitrogen. Decay capacity of S. lacrymans in these element-limited conditions is impressive, compared to our past work. If S. lacrymans Acknowledgements has superior efficiency utilizing nitrogen when it is limiting for other fungi, including S. himantioides, Fe might by This work was supported, in part, by the McIntire Stennis Fund default be the limiting element for S. lacrymans. Enhance- (Project No. MIN-12-074) and the University of Minnesota. I wish Article in press - uncorrected proof

Serpula, calcium, and iron 7

to acknowledge Dr. Robert Blanchette and Dr. Shona Duncan for Krzyzanowski, N., Oduyemi, K., Jack, N., Ross, N.M., Palfreman, editorial assistance, and Gilbert Ahlstrand for electron microscopy J.W. (1999) The management and control of dry rot: a survey assistance. I wish to acknowledge past collaboration with Dr. Jody of practitioners’ views and experiences. J. Environ. Manage. 57: Jellison, Dr. Andrea Ostrofsky, and Kaitlyn Bissonnette whose input 143–154. was integral. Low, G.A., Young, M.E., Martin, P., Palfreyman, J.W. (2000) Assessing the relationship between the dry rot fungus Serpula lacrymans and selected forms of masonry. Int. Bio-deterior. Bio- References degr. 46:141–150. Morris, P.I., Ingram, J.K. (1998) The effect of high iron loadings on brown rot decay of treated wood by copper- and arsenic- Akamatsu, Y., Takahashi, M., Shimada, M. (2004) Production of tolerant fungi. Wood Protection 3:55–59. oxalic acid by wood rotting basidiomycetes grown on low- and Paajanen, L.M. 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