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Applied and Environmental Microbiology, August 2006, p. 5662-5665, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.00281-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Metal Accumulation without Enhanced Oxalate Secretion in Wood Degraded by Brown Rot Fungi

Jonathan S. Schilling^* and Jody Jellison

Department of Biological Sciences, University of Maine, Orono, Maine 04469

Received 3 February 2006/ Accepted 30 May 2006

Top Abstract Introduction References

 
Brown rot fungi were incubated in agar and agar-wood microcosms containing metallic or hydroxide forms of Al, Cu, and Fe. Metal dissolution was associated with elevated oxalate concentrations in agar, but metals translocated into wood did not affect oxalate accumulation, crystal production, or decay rate, demonstrating a substrate-dependent oxalate dynamic.

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Brown rot fungi initiate wood decay by demethylating lignin (9) and oxidatively depolymerizing holocellulose via Fe-dependent Fenton chemistry (12, 17). These fungi characteristically produce oxalic acid during metabolism (20). Secreted oxalate may promote brown rot by weathering soil minerals (2), mobilizing Fe³⁺ (3, 11), and detoxifying metals (21).

Oxalate secretion in response to metals has been demonstrated for brown rot fungi in artificial media (26), and Cu tolerance in wood has been associated with Cu oxalate crystallization (24). Similarly, oxalate has been theorized to chelate excess Fe³⁺ near hyphae, minimizing radical formation (16), although excessive Fe chelation in wood could impede Fenton-based depolymerization (15, 25). It remains uncertain whether metal detoxification in wood is attributable to increased oxalate secretion (resistance) (5), incidental to oxalate production (tolerance) (6, 13), or related to other mechanisms (4, 10). Oxalate dynamics in artificial media often do not reflect those in the wood matrix, where brown rot fungi may decarboxylate (18) and control surplus oxalate (7, 23).

This in situ experiment tests the hypotheses that Al, Cu, and Fe translocated into wood from metallic and soil-relevant hydroxide forms during brown rot enhance oxalate secretion and metal-oxalate crystallization, that Fe enrichment accelerates decay, and that enhanced wood oxalate results in elevated Ca²⁺ accumulation.

The brown rot fungi Gloeophyllum trabeum (strain ATCC 11539) and Fomitopsis pinicola (strain FP-105877R) were grown for 10 weeks in agar-wood microcosms. Microcosm setup, processing, and analysis were as in previous reports (23), except as follows: two birch strips supported two spruce blocks (cut on the tangential plane), a separate strip between inoculum and blocks supported metal treatments, inoculum and microcosm plates contained 20 ml agar to standardize volume, only acid-extractable oxalate was measured in agar-only microcosms, and the ergosterol analysis column temperature was 35°C.

Treatments were metallic Al shot (99% pure), Cu wire (99.9% pure), and Fe chips (99.98% pure) (Sigma), plus two hydroxide treatments: Fe(OH)₃, precipitated from equimolar NaOH and FeCl₃ at pH 7, and 100 mM Al(OH)₃ in type A agar (Sigma). Metal treatments and their birch supports had no contact with other strips or with spruce.

Positive controls contained no metal treatments, negative controls contained no inoculum, and agar-only controls contained metals on glass coverslips and no wood. Corrosion controls were with or without metallic Fe in direct contact with spruce, with weight loss assessed at week 6. All treatments and controls, except corrosion controls (n = 4), included five replicates. Statistical analyses were analysis of variance with protected Tukey's tests and t tests ( = 0.05).

Crystals in wood were evaluated with scanning electron microscopy and energy-dispersive spectrometry. Wood was dehydrated, freeze fractured, critical point dried, and Au coated to 300 nm. A Cameca SX 100 microscope at 25 kV, 150 pA beam, and 25 s per frame was used for imaging. X-ray spectra were collected with a Rontec flash EDS detector probing for 100 s.

By week 10, both fungi often produced colored exudates (e.g., green droplets in Cu treatments) where mycelia contacted metals. In agar-wood microcosms, blocks decayed with some treatment metals were darkened.

Metal enrichment in wood blocks was confirmed by cation analysis of milled wood (Table 1). For both fungi, wood incubated with a particular metal treatment was significantly enriched in that metal versus the controls with no added metals, with the exception of G. trabeum cultures with metal hydroxides.

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TABLE 1. Cation content in spruce blocks decayed for 10 weeks by brown rot fungi in agar-wood microcosms containing metal treatments

Accumulation of treatment metals in wood during decay did not influence wood weight loss at week 10 for either test fungus (Table 2). Ergosterol, a proxy for fungal biomass (22), was similarly unaffected by metals. Wood pH was characteristically lowered during decay by both species and was lower in metallic Fe treatments than in other treatments or positive controls. For each week 10 variable, the overall mean for one test fungus was significantly different than that of the other. For both fungi, wood in direct contact with metallic Fe had higher weight loss at week 6 than did wood with no added Fe.

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TABLE 2. Weight loss, ergosterol (fungal biomass), and pH in spruce decayed by brown rot fungi in agar-wood microcosms containing metal treatments in contact with (week 6) or separate from (week 10) wood blocks

Soluble and acid-extractable oxalate levels in metal-enriched wood were not different from each other or from positive controls (Fig. 1A and D). For both soluble and acid-extractable wood oxalate, the pooled mean (n = 30) in G. trabeum cultures was significantly higher than in F. pinicola cultures. This pattern was reversed in the agar of agar-wood (Fig. 1B and E) and agar-only microcosms (Fig. 1C and F), where F. pinicola produced substantially more oxalate. Oxalate production in agar was enhanced by some metals, particularly Fe in agar-only microcosms. Soluble oxalate fractions were generally higher in agar than in wood, although soluble agar oxalate in agar-wood G. trabeum microcosms was below detection.

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FIG. 1. Oxalate at week 10 in microcosms containing either F. pinicola (A to C) or G. trabeum (D to F). Oxalate in agar-wood microcosms containing spruce blocks is shown from both the wood component (A and D) and the agar component (B and E), and oxalate (acid extractable only) from agar-only microcosms is shown from agar (C and F). Wood oxalate data are adjusted for mass loss as oxalate per wood volume. In each graph, bars with the same fill pattern and same letter are not significantly different. Letters are included only when treatment effects occur.

Microscopy and X-ray analysis revealed copious Ca oxalate crystals in wood degraded by F. pinicola, while crystals were not observed in wood degraded by G. trabeum. Import of Ca²⁺ consequent to wood oxalate secretion was not observed for either fungus, and wood Ca²⁺ concentrations were not affected by treatments (Table 1). No Cu oxalate was observed, and crystal microanalysis generated only Ca and Au peaks, with no associated treatment metals.

Reduced oxalate solubility in wood may be due to esterification with wood fibers (14) instead of crystallization with Ca²⁺ or Cu²⁺. Because metal enrichment did not affect the decay rate or fungal biomass, these fungi may have detoxified metals via intracellular sequestration or perhaps by pH modulation (10), evident in this study during degradation of Fe-enriched wood.

Only F. pinicola, a more common forest floor decomposer than G. trabeum (8), mobilized metals from soil-relevant hydroxides. For both fungi, enhanced decay with Fe in contact with wood suggests electrolytic corrosion (1) and encourages caution when interpreting Fe-promoted brown rot (19).

The difference in agar oxalate patterns between microcosms with and without wood blocks suggests that wood may influence oxalate secretion in soil mycelia. Although this could influence nutrient acquisition and forest biogeochemistry, Ca²⁺ translocation was not necessarily dependent on oxalate concentrations, as hypothesized, and was against the wood-agar Ca gradient.

This work demonstrates a capacity for brown rot fungi to control the extracellular environment differently inside and outside the wood matrix. Metal tolerance among brown rot fungi, including Cu intolerance in G. trabeum, has been associated with oxalate production patterns in artificial media (13). These correlations have been inconsistent (4), and differences observed in this study suggest that in situ trials are valuable for evaluating the role of oxalate both in metal tolerance/resistance and in oxidative brown rot.

 
This work has been supported by USDA grant 2002-34158-12783 and the Maine Agricultural and Forestry Experiment Station (MAFES publication number 2876).

We acknowledge Kelly Edwards, Barry Goodell, and Martin Yates for providing facilities, and we thank Aria Amirbahman and Andrea Ostrofsky for experimental and editorial assistance.

 
* Corresponding author. Mailing address: 311 Hitchner Hall, Department of Biological Sciences, University of Maine, Orono, ME 04469. Phone: (207) 581-3032. Fax: (207) 581-2537. E-mail: jonathan{at}maine.edu.

Top Abstract Introduction References

 

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Applied and Environmental Microbiology, August 2006, p. 5662-5665, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.00281-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Schilling, J. S. Articles by Jellison, J. Search for Related Content PubMed PubMed Citation Articles by Schilling, J. S. Articles by Jellison, J. Agricola Articles by Schilling, J. S. Articles by Jellison, J.