Necromass of Diverse Root‐Associated Fungi Suppresses Decomposition of Native Soil Carbon via Impacts of Their Traits

ABSTRACT

We investigated the decomposition of diverse root‐associated fungi, their influence on native soil carbon (C) dynamics and the relationship of these processes with fungal traits. We quantified the decomposition of ¹³C‐labelled mycelium of 14 species, their priming of native soil C, impact on functional soil C pools, microbial use of C and microbial community size and composition and evaluated chemical, morphological and physiological traits of the fungi to investigate their potential to control C processes. Fungal melanin, blackness, C/N and growth rates were linked to necromass decomposability and its stabilisation. Necromass addition commonly caused suppression of native soil C decomposition (negative priming), including that of the resistant C pool, and this suppression was stronger as fungal decomposability decreased. We provide novel, clear evidence of linkages between root‐associated fungal traits, necromass decomposition, microbial C use and soil C stability which builds our mechanistic understanding of the role of dead fungi on soil C storage.

We investigated the decomposition of diverse root‐associated fungi, their influence on native soil carbon (C) dynamics and the relationship of these processes with fungal traits. Fungal melanin, blackness, C/N and growth rates were linked to necromass decomposability and its stabilisation. Necromass addition commonly caused suppression of native soil C decomposition (negative priming), including that of the resistant C pool, and this suppression was stronger as fungal decomposability decreased.

1. Introduction

As the importance of soil carbon (C) for climate regulation, nutrient and water cycles and plant productivity is better understood (Kopittke et al. 2022), the factors controlling its stocks are receiving increasing attention. Soil C results from a delicate balance of processes of inputs and their stabilisation into soil organic matter (OM) and processes that lead to destabilisation and CO₂ release. Microbes are key drivers of this balance due to their multiple roles as decomposers, regulators of plant C input (both as plant‐associated and free‐living) and sources of C themselves. Among microbes, soil fungi are demonstrating to be particularly important actors. With their enzymatic capabilities, extensive mycelial structure and high C‐use efficiency, fungi can generally acquire and retain more C compared to other microbes (de Boer et al. 2005; Hannula and Morriën 2022). Plant‐associated fungi can directly channel aboveground C into soil where it can potentially be stabilised (protected from decomposition) with fungal C potentially contributing more to mineral‐associated OM than plant C (Klink et al. 2022). Plant‐associated fungi can also interact (e.g., via competition) with soil decomposers and influence their activity or directly influence decay via their nutrient mining activity (Frey 2019). As they grow, both free‐living and plant‐associated fungi can promote aggregation by enmeshing OM with their hyphae and releasing extracellular biopolymers, which can protect C by physically constraining microbial decomposition (Lehmann et al. 2017; Liu et al. 2023). Even after death, as necromass, fungal material may persist due to chemical recalcitrance (Fernandez et al. 2019). Moreover, fungal necromass may have higher affinity for mineral surfaces promoting stabilisation (Sokol et al. 2019). While microbial (bacterial and fungal) necromass is receiving increased attention, the particularities of fungi still need more attention given their critical roles in C cycling.

Microbial necromass is known to be an important pool of soil C due to both its size (15%–80% of total soil C) (Liang et al. 2019; Angst et al. 2021) and its persistence (Kästner et al. 2021). Regarding fungal necromass, its contribution to total soil C is highly dependent on ecosystem type but is substantial (can range from 20% to 40%) and tends to be greater, above double that of bacteria (Liang et al. 2019; Wang et al. 2021). Increases in soil C in response to certain types of agricultural management have been associated with increases in fungal biomass and necromass (Yang et al. 2022; Zhou et al. 2023) suggesting the potential of fungal necromass to drive soil C changes. Necromass stocks result from the combination of inputs of fungal biomass, its turnover and decomposition (recycling) and the stabilisation of the remaining material (Buckeridge et al. 2022). Studies of fungal mycelium decomposition dynamics indicate biphasic, two‐pool decay (Gao et al. 2024; Schweigert et al. 2015; See et al. 2021) that is slowed by secondary compounds such as melanin (Fernandez and Koide 2014; Lenaers et al. 2018; Fernandez et al. 2019; Maillard et al. 2023) and accelerated by nitrogen (N) content (Fernandez and Koide 2014; Brabcová et al. 2018; Ryan et al. 2020). In turn, undecomposed fungal necromass can be stabilised via interactions with minerals and other organic surfaces, which are likely mediated by necromass chemistry (Buckeridge et al. 2020), but very little is known specifically for fungi. In a laboratory study, about half of ectomycorrhizal C remained in the non‐living OM after 7 months of incubation, suggesting significant potential for fungal necromass stabilisation (Schweigert et al. 2015). Of the available studies on fungal necromass production, decomposition and stabilisation, most have looked at necromass from ectomycorrhizal fungi and less commonly at those from arbuscular mycorrhizal, ericoid mycorrhizal or saprotrophic lifestyles. Even less is known about other root‐associated fungi such as root endophytes. However, mycelium chemistry and its relation to decomposition differ widely among (Huang et al. 2022) and within (Fernandez et al. 2016) fungal guilds, and endophytes are notoriously diverse (Rodriguez et al. 2009). Moreover, the chemical traits of root‐colonising fungi can impact the decomposition of the host roots (Fernandez et al. 2013), another source of soil C, adding relevance to the need to understand the decomposition and impacts on soil C of root‐associated fungi.

Besides being a source of stable soil C, fungal necromass also has the potential to influence the cycling of existing, or ‘native’, OM C. This phenomenon, where the cycling of native organic C is altered by the addition of a fresh C substrate causing acceleration or deceleration of C loss (i.e., positive or negative priming) (Bernard et al. 2022), has been amply studied for living or dead plant tissues, but not for dead fungal mycelium. Given the considerable amount of fungal biomass production, short life spans and relatively high turnover (Godbold et al. 2006; Hendricks et al. 2016; Kästner et al. 2021), fungal necromass may be an important driver of the processing of native OM. For plant litter, its nutrient stoichiometry and C chemistry can influence native C processing. Reduced nutrient availability in litter can lead to increased soil C decomposition (Potthast et al. 2010; Wang et al. 2014). In terms of C compounds, higher concentrations of Labile C and hemicellulose in litter have been associated with increased soil C decomposition, and higher concentrations of more recalcitrant compounds (tanins, lignin) with reduced soil C decomposition (Chao et al. 2019). The direction and magnitude of the priming caused by plant litter, however, are complex, highly dependent on time since litter addition, as well as on soil attributes and on the responses and interactions with microbial communities (Fanin et al. 2020). For fungal necromass, recent studies indicate that its chemistry can shape the structure of the microbial community associated with it (the necrobiome) (Kennedy and Maillard 2023). This could, in turn, shape its activity assimilating, decomposing or stabilising native soil C. For example, a shift towards a more slow‐growing or C use efficient microbial community may promote larger proportions of more stable soil C (Tao et al. 2023). While some of the patterns observed for plant C may apply to fungal tissue, the unique biochemical features of fungi may lead to different dynamics. However, there is currently little to no information about the magnitude of this effect or the variables controlling it.

Trait‐based approaches are showing promise in helping to understand the role of microbes in soil C and ecosystem processes (Malik et al. 2019; Sokol et al. 2022). For fungi, morphological, chemical and physiological traits are being increasingly assessed and integrated to understand fungal ecological strategies and trade‐offs and their potential links with soil processes (Zanne et al. 2020; Camenzind et al. 2024), generating and supporting predictions. For example, traits associated with stress responses, such as low N content or accumulation of recalcitrant compounds, could support soil C accumulation by providing decomposition‐resistant inputs (Treseder and Lennon 2015). Dense mycelium could increase soil C by increasing soil aggregation, thus potentially stabilising native soil C (Camenzind et al. 2024). However, empirical demonstrations are still rare. Lehmann et al. (2020) identified a suite of saprotrophic fungal traits that led to increased soil aggregation. In a laboratory setting, trait‐based assemblies of arbuscular mycorrhiza were shown to determine C dynamics in controlled conditions (MAOM creation) (Horsch et al. 2023). Thus, impacts of fungal necromass on C storage could be due to direct impacts via recalcitrance of their tissues or indirect impacts on native C processing. However, there are no studies that distinguish these two mechanisms. Recently, with a suite of diverse non‐mycorrhizal root‐associated fungi and wheat plants, were observed increases in soil C and its stability under fungi with higher C/N ratios and denser mycelium (Stuart et al. 2024). However, it is not clear if the impacts occurred due to the influence of living or dead fungi. As the importance of microbial and fungal necromass C pools is recognised, it is becoming clear that the traits of both living and dead microbes can influence soil C cycling and should be considered and distinguished (Sokol et al. 2022; Camenzind et al. 2023).

We investigated the necromass decomposition dynamics of a diverse set of root‐associated fungi, their influence on native soil C dynamics and the relationship of these processes with fungal traits. For this, we used a laboratory study where dead, ¹³C‐labelled mycelium of 14 fungal species were allowed to decompose in a common soil. We quantified fungal decomposition, the priming of native soil C, functional soil C pools (Labile, Intermediate and Resistant C), microbial use of fungal C and microbial community size and composition. Separately, we evaluated chemical, morphological and physiological traits of the fungi to investigate their potential to control C processes. We hypothesised that (a) fungal necromass addition, in general, would lead to increased soil C decomposition (positive priming) as fungal C/N ratios are not particularly high, so necromass is accessible for microbial use, increasing their overall SOM decomposing activity and increasing the proportion of easily decomposable soil C; (b) fungal necromass decomposition would be higher for species with high N contents and low C/N and melanin contents; (c) necromass decomposability and associated fungal traits would be linked with soil C stability, with more decomposable fungi leading to reduced soil priming (less soil C loss) as decomposers utilise fungal C, not soil C, and to larger pools of more resistant C.

2. Methods

2.1. Fungal Growth and Labelling

Fourteen fungal isolates were originally isolated from surface‐sterilised roots of multiple species of grasses and shrubs from across diverse natural environments in southeast Australia and screened for traits that may support plant growth and soil C storage by Loam Bio Pty Ltd. (Orange, New South Wales, Australia). Isolates belonging to 10 families of Ascomycetes, comprised one species each of Acrocalymma, Beauveria, Clohesyomyces, Clonostachys, Leptodontidium, Ophiosphaerella, Thozetella and Trichoderma, two species of Phialocephala and four species of Darksidea. Fungi were cultured in a 10 At% ¹³C glucose medium following Jackson et al. (2019) (Appendix S1). Biomass was washed with sterile water, dried at 40°C, ground to powder and analysed for ¹³C (SERCON 20‐22 mass spectrometer connected with an Automated Nitrogen Carbon Analyzer (Sercon, UK)). The dried and powdered fungi were tested for viability by attempting to culture it on agar and adding it to sterile, moist sand to detect respiration. We did not detect growth or respiration, thereby confirming the material was dead.

2.2. Soil Incubations

To evaluate fungal decomposition and its impacts on soil C decomposition and C distribution across pools of different stability (Labile, Intermediate and Resistant), we assessed microbial CO₂ production in laboratory incubations of soil that had received fungal necromass. Incubations with the 14 isolates and a no‐fungal control with four replicates were maintained for 107 days in the dark at 25°C. Soil was a clay‐loam soil from an agricultural field where the past 10 years of land use history included wheat, barley, canola and sorghum (4.3% C, 0.39% N, pH 5.85). 30 g of 2 mm dried soil was mixed with 20 mg of fungal powder, brought to 42% moisture content, and placed in air‐tight jars for gas sampling. Twelve sampling events took place, and samples were analysed for CO₂ concentration and δ¹³C with a PICARRO G2201i isotopic CO₂/CH4 analyser (Picarro Inc., Santa Clara, CA, USA). The final sampling occurred at day 107, when the ¹³C enrichment of the CO₂ was approaching undetectability. See Appendix S1 for additional details about incubation conditions and sampling.

To calculate the contribution of fungal‐ and soil‐derived C to respired CO₂ at each sampling point, isotopic partitioning was used as follows:

$$\begin{array}{c}\text{Soil}\mathrm{C}\text{Fraction}=\left({\mathrm{\delta }}^{13}\mathrm{C}{\mathrm{CO}}_2\text{sample}-{\mathrm{\delta }}^{13}\mathrm{C}\text{Control}-{\mathrm{CO}}_2\right)\hfill \\ /\left({\mathrm{\delta }}^{13}\mathrm{C}\text{Fungi}-{\mathrm{\delta }}^{13}\mathrm{C}\text{Control}-{\mathrm{CO}}_2\right)\hfill \end{array}$$

where δ¹³C CO₂ sample is the C isotopic composition of CO₂ measured in each incubation jar after subtracting the mean blank value, δ¹³C Control‐CO₂ is the mean δ¹³C of CO₂ from controls that had not received fungal biomass, and δ¹³C Fungi is the δ¹³C of the fungal biomass. The Fungal C Fraction was defined as 1 minus the Soil C Fraction. These fractions were then applied to the measured CO₂ rates in each jar to calculate fungal and soil‐derived CO₂ release rates over time.

Soil‐derived CO₂ rates over time were fitted to two‐pool exponential decay curves to estimate decay kinetic parameters for each jar. Kinetic parameters derived from mid‐ to long‐term soil incubation are sensitive functional measures of the distribution of C among pools with different degrees of stability (e.g., Carney et al. 2007; Carrillo et al. 2011; Jian et al. 2020). These equations were used to calculate total soil‐derived C respired and to estimate the size of the Labile and Intermediate C pools and their mean residence time (Cheng and Dijkstra 2007; Wedin and Pastor 1993; see Appendix S1). Priming of soil C was calculated as the difference between total soil‐derived C respired in each incubation jar minus the average of the control.

The behaviour of the fungal‐derived CO₂ did not always show exponential decay dynamics, so estimation of total fungal‐derived C release was done by interpolation of pairs of consecutive rates to obtain average rates for each period of time, which were then applied to the duration of each period and then added together to obtain the total fungal‐derived CO₂. The fraction of fungal C that was lost was calculated as the ratio of total fungal‐derived CO₂‐C to the initial C added, based on weight and initial % C concentration.

2.3. Soil Harvest and Analysis

Following the last CO₂ sampling, soils were harvested and immediately processed for soluble and microbial C and δ¹³C, soluble N and microbial phospholipid fatty acids (PLFA). Soil microbial biomass C was assessed in fresh soils by the chloroform‐fumigation method using a 0.05 M K₂SO₄ solution in a 4:1 solution: soil ratio, 5 days of fumigation and 180 rpm shaking for an hour. Extracts were analysed for total dissolved organic C and N (Shimadzu TN, TOC‐L, Japan) and for δ¹³C to calculate microbial biomass C and its δ¹³C (See Appendix S1). For PLFA analysis, 3 g soil were extracted following the high throughput method developed and described by Buyer and Sasser (2012) (See Appendix S1).

2.4. Fungal Trait Assessment

2.4.1. Morphological and Physiological Traits

A separate in vitro plate assay was performed using 1/2 PDA plates incubated in the dark at 23°C–25°C (partial data for 10 isolates already presented in Stuart et al. (2024), 4 new isolates reported here). Radial growth rate was quantified by measuring colony areas every 2–3 days using ImageJ (National Institutes of Health, Bethesda, Maryland, US; Schneider et al. 2012) and growth parameters were calculated using the logistic function (M = 1 form of the Richards function; (Fischer and Schönfelder 2017)) and the nlshelper package in R. Hyphal density was calculated as the final fungal biomass per final colony area.

2.4.2. Fungal Chemistry

C and N content were measured on material from the liquid cultures used for incubation by Dumas combustion using an El Vario cube analyser (Elementar, Langenselbold, Germany). Melanin concentration was assessed based on Butler and Lachance (1986) with some adjustments, and tissue colour (See Appendix S1). Tissue colour was assessed as an indicator of secondary compound content, including melanin but also other compounds that may potentially influence decomposability (Velíšek and Cejpek 2011; Lenaers et al. 2018) (See Appendix S1).

2.4.3. Fungal Decomposability

We used the fungal C dynamics observed during incubation to obtain parameters related to intrinsic decomposability including (1) the fraction of fungal C that was decomposed (0%–100%), calculated based on the C concentration of the tissue, the quantity of tissue added and the total fungal C respired, (2) the initial rate of fungal C CO₂ release as a proxy for labile, accessible fungal C and (3) the final rate of decomposition, as a proxy for resistant/recalcitrant fungal C.

2.5. Statistical Analyses

We conducted ANOVA of rates, fractions, C and N pool sizes and kinetic parameters followed by Dunnett's post hoc test to determine which treatment groups were significantly different from the uninoculated control group or Tukey–Kramer post hoc test to determine significant differences between the inoculated groups. t‐tests were used to assess significant difference from zero for soil C priming. Prior to this, we tested for homogeneity of variance with the O'Brien test. Principal component analysis (PCA) and PerMANOVA were used to assess impacts on microbial communities. Redundancy analysis (RDA) of soil C priming and Resistant pools as response variables and soil and microbial community variables as explanatory variables was used to assess potential soil drivers of C responses. RDA was also used to assess relationships between fungal traits and soil C priming and Resistant C pool. RDAs were performed using the vegan package in R (Oksanen et al. 2025) and significance evaluated using the anova function in base R. Correlation analysis and Spearman's test were used to assess the significance of individual relationships between soil C responses and specific traits. Curve fitting of CO₂ rate dynamics was done using the non‐linear modelling platform in JMP 16.1.0 and the biexponential four‐parameter decay model.

3. Results

3.1. Fungal Isolates Impact the Decomposition of Native C and Its Distribution Among Pools of Different Stability

Isotopic partitioning of respiration between fungal‐ and soil‐derived C allowed the quantification of native soil C respiration. On average, 7% of original soil C was respired over the 107 days of incubation. The addition of fungal tissue influenced the amount of soil C respired depending on fungal species. Respiration decreased (Darksidea sp. 3, Leptodonditium sp) or remained unchanged compared to the control (Figure 1a), and in five cases (Beauveria sp., Darksidea sp. 3, Leptodontidium sp., Phialocephala sp. and Thozetella sp) isolates caused significant negative priming, or deceleration of decomposition (Figure 1b).

FIGURE 1.

Total soil C respired (a) and soil C priming (b) after addition of fungal necromass and its absence. C respired obtained from C isotopic partitioning of CO₂, priming calculated as difference with average of the control. Values are means with standard error n = 4. (a) Asterisks indicate significant difference to control (Dunnett's test, p < 0.05); (b) Asterisks indicate significant difference to zero (t‐test, p < 0.05).

Fitting exponential decay models to the respiration of soil C over time (Figure S1 and Table S1) allowed estimation of pools of Resistant, Intermediate and Labile C and the contribution of these to soil C. On average, the Labile pool made up less than 1% of total C, the Intermediate pool between 11% and 15% and the Resistant between 84% and 88% (Figure S2). Relative to the control, fungal mass addition reduced the fractions and corresponding pools of Labile C (three cases) and Intermediate C (10 cases) but increased the fraction and pools of Resistant C (10 cases) (Figure 2 and Figure S2).

FIGURE 2.

Pools of labile, intermediate and resistant soil‐derived C after addition of fungal tissue to soil. Pools sizes obtained from fitting exponential decay models to the respiration measured over time after addition of fungal biomass. Asterisks indicate significant difference with the control (Absent/Not inoculated) (Dunnett's test, p < 0.05). Values are means with standard error (n = 4).

3.2. Fungal Species Vary in the Dynamics and Susceptibility to Decomposition of Their Necromass

Isotopic partitioning of CO₂ between fungal‐ and soil‐derived C allowed the quantification of fungal C respiration rates over time. The initial and temporal behaviour of respiration rates varied among species, with most showing exponential decay but four showing a sharp increase before exponential decline (Figure S3). The fraction of initial fungal C that was respired over the course of the full incubation, based on cumulative respiration, also varied widely. Between 57% and 82% of the fungal C was lost over the course of the incubation (Figure 3) and thus between 18% and 43% remained in soil.

FIGURE 3.

Fraction of fungal C respired during incubation (107 days). Fraction respired calculated from cumulative fungal C respired (from isotopic partitioning of respiration) versus initial fungal C added. Values are means with standard error (n = 4). Different letters indicate significant difference (Tukey–Kramer, p < 0.05).

3.3. Fungal Necromass Impacted Soluble and Microbial C and the Proportion Derived From Soil Versus Fungal C

At harvest several fungi led to increased DOC compared to control (seven isolates, t‐test p < 0.05) (Figure 4a), although there was not marked variation among isolates (Tukey–Kramer test). For soils that received fungal mass, on average 99.8% of DOC was soil‐derived (0.2% fungal‐derived). However, this fraction varied widely with fungal isolate (Figure 4b). Microbial C only differed from the control for one isolate (t‐test) (Figure 4c) and was not observed to vary among isolates but, like DOC, the fraction of microbial C that was soil‐derived varied significantly among isolates (Figure 4d).

FIGURE 4.

Pools of DOC and Microbial C at harvest and the contribution of soil‐derived C to them based on C isotopic partitioning. Values are means with standard errors (n = 4). Asterisks indicate significant difference to control (Absent/No addition), t‐test, p < 0.05. Letters indicate Tuckey–Kramer (p < 0.05) based differences among treatments.

3.4. Fungal Necromass Impacted N Availability and Microbial Communities

Fungal necromass influenced N availability. Soluble N was significantly higher compared to the control for nine of the treatments and also varied among fungal treatments (Figure S4). While treatments did not significantly modify the size of the microbial community based on PLFA total concentration (Table S2), they impacted community structure (Figure S5, individual PLFAs in percentages, PerMANOVA p = 0.05). The greatest separation (PC1) was observed between different isolates and was related to the abundance of markers of fungi (18:1w9) and Gram‐negative bacteria (19:0 cyclo w7). Compared to several treatments, the control showed higher relative abundances of markers of the Gram‐positive bacteria (15:0 iso, 16:0 iso, PC2).

3.5. Priming and Resistant C Pools Were Associated With Shifts in Microbial Communities and Their Use of Soil C and Dissolved C and N

We ran RDA analysis to assess factors potentially mediating treatment‐driven shifts in priming of native soil C and Resistant C pool, as measures of C permanence. RDA (Figure 5. ANOVA: F = 3.3471, p = 0.001) indicated that reduced priming (deceleration of native C decomposition) was associated with low levels of microbial C and low relative abundances of Gram‐negative bacteria and fungi. Larger pools of Resistant C were associated with larger pools of DOC and dissolved N, and lower abundances of Gram‐positive bacteria. The fungal treatments that showed a combination of increased priming and low Resistant C (lowest C permanence) were linked with greater microbial use of soil‐derived C and its incorporation into DOC (Fraction of soil‐derived C) and with higher relative abundance of actinobacteria.

FIGURE 5.

Redundancy analysis of microbial community size, composition, microbial use of soil C and soil N and dissolved C (blue text) as predictors of soil C responses (priming and Resistant C; red text) under various fungal isolates (symbols). Significance: ANOVA: F = 3.3471, p = 0.001 and adjusted R ² = 0.439. Microbial C, DOC and Dissolved nitrogen based on K₂SO₄ extractions and chloroform fumigation. Fractions of soil‐derived C in microbial C and DOC based on isotopic partitioning of those pools. Microbial groups (as relative abundance) and Total PLFA based on PFLA analysis. Symbols are averages with standard error n = 4.

3.6. Priming and Resistant C Pools Were Associated With Fungal Traits

RDA analysis indicated that fungal traits (including their decomposability and chemical and physiological traits, Table S3, Figure S3) significantly explained treatment‐driven shifts in priming of native C and Resistant C, as measures of C permanence (Figure 6a. ANOVA: F = 2.68, p = 0.002, Adjusted R ² = 0.34). RDA (Figure 6a) and further individual correlation analysis (Figure 6b) indicated that priming was negatively related to melanin, blackness, fungal C/N ratio and time to maximum growth (low growth rate), which in turn were negatively related to the fraction of added fungal necromass that was decomposed and respired during incubation. Larger pools of Resistant C were negatively related to the initial rate of fungal C decomposition.

FIGURE 6.

Association between fungal traits and soil C responses. (a) Redundancy analysis of fungal traits (blue text) as predictors of soil C responses (priming and Resistant C; red text) under various fungal isolates (symbols). Symbols are averages with standard error n = 4. Significance: ANOVA: F = 2.68, p = 0.002 and adjusted R ² = 0.349. (b) Heatmap of Spearman correlations coefficients (−1 to 1) and their significance (***p < 0.001, **p < 0.01, *p < 0.05, ^. p < 0.1). For fungal trait data see Table S3.

4. Discussion

With the broad aim of understanding the role of fungal necromass on soil C, we investigated the decomposition dynamics of a diverse set of root‐associated fungi, their influence on native soil C dynamics, and the relationship of these processes with fungal traits. Combining C isotopic tracing into C pools and fluxes and assessment of a suite of fungal traits, we found clear evidence that the necromass of diverse root‐associated fungi often suppressed the decomposition of soil C and increased the pool of Resistant C, and that these impacts were linked with specific fungal traits and their impacts on soil microbial communities and microbial use of C sources.

We hypothesised that fungal necromass addition would generally accelerate SOM decomposing activity (positive priming) with an associated increase in easily decomposable Labile‐C. This was expected because the C/N of fungi, an important controller of priming, is low (12–25 in this case) and similar to that of easily decomposable plant litter (Brabcová et al. 2018), which prompts positive priming, and because of observations for ectomycorrhizal fungi (Zhang et al. 2018). In contrast, we observed suppression of decomposition in most cases and a reduction of the pool of Labile C. Suppression of soil C decomposition may have resulted from preferential C and nutrient utilisation of readily available fungal necromass by microbes, thereby reducing the demand for SOM (Michel et al. 2023). However, the species used included several fungi with high melanin content and dark in colour, variables related to recalcitrance (Fernandez and Koide 2014; Lenaers et al. 2018) and suppression of SOM decomposition also occurred in these cases, taking away support from preferential substrate utilisation as a cause for suppression of decomposition. An alternative explanation is that the addition of fungal necromass contributed to the physical protection of native soil C and/or reduced microbial assimilation and respiration of Resistant soil C. This is consistent with the commonly observed increases in the native pool of Resistant C (Figure 2), which, in turn, were associated with reduced microbial use of soil C (Figure 5). Fungal necromass addition may have caused shifts in the microbial community (Kennedy and Maillard 2023) that could have led to reduced use of Resistant soil C. Supporting this explanation, several treatments reduced the abundance of Gram‐positive bacteria relative to the control, a group known to rely on more complex C sources (Fanin et al. 2019). The addition of fungal necromass may have also increased physical protection of native C, for example, by enhancing soil aggregation; however, this parameter was not evaluated. Our findings demonstrate that suppression of native soil C decomposition was common across the diverse genera studied and point to shifts in microbial use of Resistant C via microbial community composition changes and/or physical protection as potential causes.

We found that fungal C loss to respiration (fraction C decomposed) and thus retention of fungal C in soil (fraction of C not respired) was highly variable and linked to fungal traits. Overall, decomposition was faster for species with lower C/N and melanin contents (fraction C decomposed negatively related to C/N, melanin, Figure 6), agreeing with studies of mass loss for ectomycorrhizal and saprotrophic fungi (Fernandez and Koide 2014; Brabcová et al. 2018; Fernandez et al. 2019). However, we also observed a case where a species with high melanin content (Darksidea sp. 4) had a high rate of decomposition. This species also had among the lowest C/N, and higher initial and final decomposition rates, indicating distinct chemistry and demonstrating that high melanin and other pigments may co‐occur with high N contents and high levels of more accessible C compounds and that decomposition is co‐regulated. Additionally, melanin, blackness and C/N were also related to ‘Time to maximum growth area’, an inverse measure of in vitro growth rate, demonstrating association between chemical and physiological traits, and specifically, that slow growing fungi were also less decomposable and their C more likely to persist in soil. Furthermore, as our study tracked fungal C in soil over time, it extends beyond measures of mass loss and brings insight into the fate of fungal C in soil. At the end of the incubation, when in most cases close to 70% of fungal C had been respired, fungal C was being decomposed at very slow rates (final rate of decomposition) suggesting fungal C was becoming stabilised and that this was more marked in fungi that combined high C/N and high melanin and blackness. Together, our observations of fungal C dynamics and their relationships with fungal traits indicate that C/N, melanin and growth rates are linked to necromass loss and potential fungal C stabilisation.

We hypothesised that necromass decomposability and associated fungal traits would be linked with soil C stability. We found that the fraction of fungal C decomposed was positively related to priming, so that the more decomposable fungi caused the greatest amount of soil C loss and, conversely, given most fungi suppressed decomposition, the most recalcitrant fungi (particularly those with high C/N, melanin and blackness) suppressed decomposition the most. In other words, the degree of decomposition suppression was linked to fungal traits. A link between C chemistry and priming has been observed for plant litter, where in later stages of decomposition (more recalcitrant) reduced C losses (Chao et al. 2019). In their case, they attributed this to lower amounts of decomposition by‐products that could act as easily accessible C to prompt microbial activity and soil C decomposition. Agreeing with this, in our study, the more recalcitrant fungi would have contained low levels of accessible C, thus not prompting microbial activity and modifying microbial communities. Indeed, more fungal recalcitrance and suppression of C decomposition were associated with changes in microbial community size and composition, specifically low microbial biomass C, fungi and, importantly, low abundances of Gram‐negative bacteria, which are thought to rely on accessible C (Fanin et al. 2019). Besides impacting microbes and decomposition by impacting C availability, a reduction in microbial biomass and change in composition may also have been related to the potential antibiotic properties of melanin (Michael et al. 2023). These changes suggest that low fungal decomposability and compounds associated with it can influence the microbial biomass, reducing its size and reliance on accessible C, and thus reducing C decomposition (reducing priming).

While the overall loss of soil C over the course of incubation (priming) was linked to melanin, blackness and C/N, we found that larger pools of Resistant soil C after fungal necromass addition and incubation were more closely related to Initial rates of fungal C decomposition and to maximum growth rate. This indicates that in addition to influencing overall decomposition, fungal traits also impacted the partitioning of soil C into functional pools. This agrees with our previous observations in soils planted with wheat (Stuart et al. 2024) and suggests that one of the contributing mechanisms in live plant–soil systems is the input of fungal necromass to soil. The negative relationship of Resistant C with initial rate of fungal decomposition is likely reflecting effects of fungal C chemistry. A high initial rate of decomposition denotes a highly accessible pool of fungal C, so fungal necromass with this feature reduced the pool of Resistant C. This may be explained by this accessible fungal C causing an early and strong stimulation of microbial use of more Resistant soil C, thus depleting this pool. This could occur if this highly accessible C led to high microbial demand for nutrients (Fanin et al. 2020) and need to mine SOM to access them. This is consistent with our observations of the negative relationship between Resistant C and the use of soil C by microbes as well as Gram‐positive bacteria, who rely on complex C (Fanin et al. 2019). Accordingly, the strong positive relationship of Resistant C with soluble DOC and N is likely reflecting greater retention of these in the Resistant pool (due to reduced mining), that were then solubilised during the extraction procedure. Our findings suggest that fungal necromass with low initial decomposition rates and thus small pools of highly accessible C may favour the persistence of Resistant C in soil.

Our controlled environment incubation study allowed us to isolate the impacts of fungal biomass inputs and functionally assess impacts on soil C dynamics. Under natural conditions, however, the presence of live roots and fluctuating abiotic environment would likely modify fungal C decomposition, for example by modifying nutrient availability (Gao et al. 2024). Living roots and their exudation could also influence the impact of fungal C on native C dynamics, for example by making mineral associated C more available for microbial access (Keiluweit et al. 2015), thus potentially counteracting the suppression of decomposition brought about by fungal C addition. Importantly, under natural conditions, additional fungal traits, such as morphology and architecture (Certano et al. 2018) would be at play and they could possibly interact with the traits considered here. While our study shed light on potential mechanisms by which necromass addition suppressed Resistant C decomposition, further insight may be possible via direct assessment of soil versus plant and fungal‐derived C in soil aggregates and size fractions as well as soil versus fungal/plant C use by microbial taxa at higher resolution in plant–soil systems.

Combining C isotopic tracing into C pools and fluxes and assessment of a suite of fungal traits, we brought clear and highly novel insights into the potential impact of fungal necromass and its traits on soil C stability and potential underlying mechanisms. Our findings demonstrate that, in the absence of living roots influence, suppression of native soil C decomposition was common across the diverse genera studied and point to shifts in microbial use of Resistant C via shifts in microbial community composition or physical protection as potential causes. Fungal C/N, melanin and growth rates were linked to necromass decomposability and the potential for its stabilisation. Moreover, we show that as fungal decomposability decreases, this may influence the microbial community, reducing its size and reliance on accessible C, and thus reducing C decomposition (reducing priming) and that fungi with low initial decomposition rates, that is, lacking a substantial pool of highly accessible C, could prevent mining of resistant soil C pools. This study makes empirical linkages between root‐associated fungal traits, necromass decomposition and soil C stability, which builds our understanding of the role of dead fungi in soil C storage and may inform selection of fungal traits with the potential to support it.

Author Contributions

E.K.S. and C.Y. carried out the incubation work. E.K.S. carried out the trait assessment work with support from J.R.P., E.K.S., C.Y. and J.R.P. carried out the analyses. C.Y. wrote the paper with feedback from coauthors.

Supporting information

Appendix S1: ele70216‐sup‐0001‐AppendixS1.docx.

Figure S1: Soil‐derived CO₂‐C release rates over time after addition of fungal biomass. Soil‐derived CO₂ obtained with isotopic partitioning of CO₂. Values are means with standard error (n = 4).

Figure S2: Fractions of labile, intermediate and resistant soil C after addition of fungal tissue to soil. Fractions calculated from pools sizes obtained from fitting exponential decay models to the respiration measured over time after addition of fungal biomass (See Section 2). Asterisks indicate significant difference with the control (Absent/Not inoculated) (Dunnet's test, p < 0.05). Values are means with standard error (n = 4).

Figure S3: Fungal‐derived CO₂‐C release rates over time after addition of fungal biomass. Soil‐derived CO₂ obtained with isotopic partitioning of CO₂ (See Section 2). Values are means with standard error (n = 4). Note different scale of y axes.

Figure S4: Soluble N in soils at harvest time. Values are means with standard errors (n = 4). Asterisks indicate significant difference to control (Absent/No addition), Dunnet's‐test, p < 0.05. Letters indicate Tuckey‐Kramer (p < 0.05) based differences among treatments.

Figure S5: Principal component analysis of phospholipid fatty acids (PLFA) at the time of harvest. Analysis of percentage of total PLFA. Permanova p = 0.05.

Table S1: Soil‐derived C decay parameters from two pool exponential decay fits used for calculations of labile and intermediate pool sizes (See Section 2). n = 4.

Table S2:. Total concentration of PLFA and relative abundance of PLFA markers at harvest. Values are means followed by standard error (n = 4).

Table S3: Traits of fungal isolates used for incubation. Traits assessed in in vitro assays (see Section 2).

Yolima, C. , Stuart E. K., and Powell J. R.. 2025. “Necromass of Diverse Root‐Associated Fungi Suppresses Decomposition of Native Soil Carbon via Impacts of Their Traits.” Ecology Letters 28, no. 9: e70216. 10.1111/ele.70216.

Data Availability Statement

The data has been archived on the WSU institutional repository and openly published to Research Data Australia for discovery. A permanent and unique DOI has been assigned and all required metadata included. The location of the published dataset on RDA is https://doi.org/10.26183/swsa‐nx87.