Isotopic and compositional evidence for carbon and nitrogen dynamics during wood decomposition by saprotrophic fungi

Highlights

• Strategies of carbon and nitrogen acquisition differ among wood decay fungi.

• Fungal taxa varied widely in chemical composition as assessed by pyrolysis GC-MS.

• Fungal carbon was from one (Mycena) to 30+ years (Fomitopsis, Hericium) old.

• Hericium preferentially assimilated ¹³C-enriched hemicellulose.

• The removal of ¹³C-depleted C₆ atoms in pentoses causes high δ¹³C in hemicellulose.

Abstract

Sporocarps of wood decay fungi contain functional information about how different taxa partition carbon and nitrogen resources from wood. We combined carbon and nitrogen concentrations, isotopic ratios (¹³C:¹²C, ¹⁵N:¹⁴N, and ¹⁴C:¹²C, expressed as δ¹³C, δ¹⁵N, and Δ¹⁴C values), and compositional patterns in wood, cellulose, and sporocarps to investigate functional and isotopic differences in six taxa of decay fungi during log decomposition. Radiocarbon (Δ¹⁴C) measurements separated fungi into heartwood colonizers (Fomitopsis and Hericium, ~30+-year-old carbon) and sapwood colonizers (Mycena, Hypholoma, and Trametes, 1-12-year-old carbon). Decay modes influenced δ¹³C, with Hericium, a selective white-rot fungus, higher in δ¹³C than nonselective white-rot fungi because Hericium preferentially assimilated ¹³C-enriched hemicellulose rather than cellulose. Fungal δ¹⁵N was lower in heartwood colonizers than in sapwood colonizers, presumably reflecting greater N turnover and ¹⁵N enrichment in sapwood than in heartwood. Sporocarp δ¹⁵N correlated with sporocarp %N and with the relative proportion of protein in N-containing pyrolysis products because fungal protein was 4–5‰ higher in δ¹⁵N (and 3–4‰ higher in δ¹³C) than non-protein. From these measurements, we improved the quantitative and conceptual understanding of how sources, composition and metabolic processing determined isotopic composition of fungi.

Introduction

Coarse woody debris in forests is important as a carbon source and nutrient sink (Harmon et al., 1986; Stevens, 1997; Siitonen, 2001). Coarse woody debris is primarily decomposed by saprotrophic fungi that have developed specialized enzyme systems to degrade the recalcitrant compounds common in wood, especially lignin. These fungi derive their energy and carbon from the decay of relatively labile substances in wood, such as cellulose and hemicellulose, and are efficient in scavenging nitrogen from decomposing wood (Rayner and Boddy, 1988). However, the difficulty of investigating fungal processes in the field, particularly in complex, impermeable substrates such as wood, has hindered efforts to determine the exact carbon and nitrogen sources assimilated by such fungi.

One approach is to use carbon and nitrogen isotope ratios (¹³C:¹²C, ¹⁵N:¹⁴N, and ¹⁴C:¹²C; expressed as δ¹³C, δ¹⁵N, and Δ¹⁴C) as natural tracers of fungal C and N dynamics in wood decay fungi. Such measurements in fungi have proven useful in investigating the cycling of these elements by saprotrophic fungi in field studies (Kohzu et al., 1999, 2005; 2007; Hobbie et al., 2001; Taylor and Fransson, 2007). For example, atmospheric radiocarbon (¹⁴C) increased from 1955 to 1963 because of ¹⁴C generated during thermonuclear testing and has declined since then after the signing of the Nuclear Test Ban treaty; radiocarbon in fungi has been used to distinguish among ectomycorrhizal, litter decay, and wood decay fungi (Hobbie et al., 2002) because of the different ages of carbon assimilated by these three functional types. This approach could presumably be used to distinguish among colonization strategies of different taxa of wood decay fungi, such as heartwood (older) versus sapwood (younger) colonization. Similarly, the decline in δ¹³C of atmospheric CO₂ because of anthropogenic addition of ¹³C-depleted fossil fuels to the atmosphere (the Suess effect, McCarroll and Loader, 2004) could lead to lower δ¹³C in fungi colonizing sapwood rather than heartwood. And finally, differences in δ¹⁵N between N sources have been used to distinguish among ectomycorrhizal fungi, wood decay fungi, and litter decay fungi (Kohzu et al., 1999). This approach may also apply to fungi colonizing different log components, such as heartwood, sapwood, or bark, since field studies indicate little or no difference in δ¹⁵N between wood decay fungi and their wood substrates (Kohzu et al., 2007).

Sources of carbon and their isotopic fractionation during biosynthesis can influence fungal isotopic patterns (Hobbie et al., 2012). For example, lignin is 3–4‰ depleted in ¹³C relative to cellulose (Benner et al., 1987). Hemicellulose is somewhat higher in δ¹³C than cellulose (Deines, 1980) and the pentose monomers of hemicellulose, xylose and arabinose, are higher in δ¹³C than hexose monomers such as the glucose of cellulose (Teece and Fogel, 2007; Dungait et al., 2008, 2011). The dominant wood decomposers in forests, white-rot fungi, have good abilities to degrade lignin but do not incorporate lignin-derived carbon (Hobbie, 2005). Since wood decay fungi selectively assimilate wood carbohydrates and lose ¹³C-depleted CO₂ during metabolism (Kohzu et al., 2005), sporocarps of wood decay fungi are generally enriched in ¹³C by 3–4‰ relative to bulk wood and by ~2‰ relative to wood cellulose (Gleixner et al., 1993; Kohzu et al., 1999; Hobbie et al., 2001). White-rot fungi differ in their preferences for the two primary wood carbohydrates, hemicellulose and cellulose (Blanchette, 1991), with fungi preferentially attacking hemicellulose and lignin known as selective white-rot fungi and those attacking hemicellulose, cellulose, and lignin simultaneously known as nonselective white-rot fungi. These decay modes have not yet been linked to δ¹³C patterns in wood decay fungi.

The chemical composition of fungi can also influence their isotopic patterns (Hobbie et al., 2012). For example, compounds are enriched in ¹³C in the order protein > carbohydrates > chitin > lipids and enriched in ¹⁵N in the order protein > chitin (Taylor et al., 1997). Thus, compositional information that provides the relative abundance of different compound classes may help to interpret isotopic patterns. One common technique to assess composition in environmental samples is pyrolysis coupled to gas chromatography-mass spectrometry (pyr-GC-MS) (Grandy et al., 2009; Wickings et al., 2012; Haddix et al., 2016). This technique, suitable for solid samples such as fungal and wood biomass, can be used to quantify the relative abundance of hundreds of individual compounds which can be then grouped into broad compound classes (e.g. proteins, carbohydrates, lipids), thus providing a ‘fingerprint’ of the chemical composition.

One opportunity to study resource use of wood decay fungi during decomposition began in 1985 at the H.J. Andrews Experimental Forest of Oregon, USA (Harmon et al., 1994). Experimental logs were of four dominant tree species of the Pacific Northwest, Tsuga heterophylla, Pseudotsuga menziesii, Abies amabilis, and Thuja plicata. Wood samples were collected from Year 0 (1985) and Year 10 (1995) and sporocarp samples of different fungal species were collected between Year 3 and Year 7. This provided a multi-year opportunity to study wood decay fungi and carbon and nitrogen dynamics during log decomposition by comparing isotopic patterns in wood and different fungal taxa.

Our hypotheses included:

• Radiocarbon can distinguish between sapwood (young) and heartwood (old) colonizers.

• Because atmospheric CO₂ has declined over time in δ¹³C (the Suess effect), heartwood colonizers will have higher δ¹³C signatures than sapwood colonizers.

• ¹³C partitioning among different source compounds can alter δ¹³C patterns. Fungi selectively targeting hemicellulose will be higher in δ¹³C than those targeting cellulose or targeting both hemicellulose and cellulose.

• Fungal δ¹⁵N will reflect the δ¹⁵N of the colonized wood.

• Because different chemical classes differ in their isotopic values (e.g., protein is higher in δ¹³C and δ¹⁵N than chitin), sporocarp chemical composition will also influence δ¹³C and δ¹⁵N patterns.

To assess these hypotheses, we: (1) measured isotopic patterns in wood, bark, and wood decay fungi from the study; (2) assessed how fungi differed in chemical composition (from pyrolysis) and in age (from radiocarbon) of assimilated carbon; (3) tested how fungal composition, age of assimilated carbon, or fungal life history characteristics, such as colonization patterns (e.g., sapwood versus heartwood colonizers) or carbohydrate preference (e.g., hemicellulose versus cellulose), affected fungal δ¹³C or δ¹⁵N.