Discussion

Our study for the first time assesses how the temporal dynamics of secondary components control the decomposition of the extramatrical mycelium of ectomycorrhizal fungi. We have monitored the concentrations of the main cell wall secondary components over time to evaluate how these changes affect the decomposition process. In this way, we demonstrated how the different loss of these components between species does not translate into a different overall mass loss from the decomposing necromass, which was comparable across species after six weeks. Considering that the structure of our species was very similar, since they were all cultivated in vitro in absence of external stressors, we can be sure that our results reflect the actual effects of fungal chemical composition.
Overall, the magnitude and rate of decomposition of the EM fungal species were consistent with previous findings, especially so concerning the rapid mass decline in the first seven to ten days (Drigo et al., 2012; Ryan, Schreiner, Swenson, Gagne, & Kennedy, 2020). Yet in contrast to these earlier studies, we found hardly any differences between fungal species in the decomposition of the necromass. Previous research suggested that the initial concentrations of secondary components, and especially of melanin (C. W. Fernandez, Heckman, Kolka, & Kennedy, 2019; Christopher W Fernandez & Koide, 2014), and chitin (Fernandes and Koide, 2012) underpin fungal decomposition rate. While higher concentrations of melanin in fungal litter decreased the decomposition of EM fungal necromass, a higher concentration of chitin was shown to be generally positively related to the decomposition rate . The fungal species here examined did not show a significant difference in the initial concentrations of these two components, which likely explains the similar mass loss among species. Yet, initial concentrations of glucans differed between species. However, these differences did not result in differences in mass loss during the decomposition. This happened even though the concentrations in glucans may translate into differences in nitrogen concentrations given that glucans do not contain nitrogen
Remarkably, L. deliciosus , the slowest decomposing species in our species set did not stand out in terms of initial concentration of cell-wall components. This suggests, in contrast to previous studies which did not evaluate the full dynamics in secondary compounds during decomposition, that differences in initial concentrations do not drive differences in decomposition rates. Instead, and in contrast to the initial concentration, the dynamics of loss of chitin was related to fungal species biomass loss. This is in line with the recent findings of Ryan et al. (2020), who also found that fungal species decomposition was strongly underpinned by the loss of carbohydrates from fungal biomass. However, our results indicate that this finding could not be generalized for all carbohydrates, as there was no significant relationship between necromass loss and the concentration of glucans or melanin. Moreover, our results show how the impacts of chitin on decomposition vary over time. We observed a positive relation between chitin and necromass loss during the first week of decomposition when its rate is the highest. During the remainder of the weeks, the necromass loss had a weak negative relationship to chitin concentration. This might be the result of the increased nitrogen availability in the previous stage of decomposition, which might be sufficient for decomposers to support the production of extracellular enzymes devoted to break down the organic matter (Schimel & Weintraub, 2003) later on.
The role of melanin in the decomposition process of fungi has been demonstrated previously (Koide et al 2019), and specifically to a slow-degrading necromass pool (Ryan et al, 2020). In our study, the loss of melanin was not related to the necromass loss at all. This might be a consequence of the small quantity of melanin in our samples. These consistently low concentrations of melanin among the different species might be caused by the absence of external stressors during the growth and growing conditions were optimal in terms of temperature, light and nutrients.
The patterns of loss of all cell-wall compounds examined in this study were highly species-specific (Figure 3). Yet, these differences between species in the pathways of the loss of cell-wall compounds were not reflected in the inter-specific differences in decomposition patterns. We hypothesize that the differences across fungal species in the loss of individual types of polysaccharides such as chitin and glucans, which constitute 80-90% of the fungal cell wall (Bartnicki-Garcia, 1968), may compensate each other. This likely happens due to how decomposers forage on the fungal necromass, attacking labile polysaccharides, without discriminating between these different compounds. Indeed hydrolytic enzymes (e.g. endo-chitinase, N-acetylgucosaminidase, b-glucosidase; the same enzymes that degrade plant litter compounds such as cellulose) are produced by the majority of bacteria and saprotrophic fungi (Conn & Dighton, 2000). On the other hand, the oxidative enzymes used mainly for degrading lignin and melanin, are less abundant because they are almost exclusively secreted by lignin decomposer fungi (Butler & Day, 1998). The combined enzyme actions on cell-wall compounds may also explain for the inversion of the relationship between chitin concentration and the weekly mass loss as it may impose a structural change in the cell wall or in the N availability in necromass in general. However, while these mechanisms may explain the interactions among cell-wall compounds in affecting fungal litter decomposition and the consequent absence of significant relationships for individual cell-wall compounds, they do not fully explain the species-specific decomposition patterns.
To further understand this pattern, not only chitin lability deserves more attention, but especially glucans, as the most abundant carbohydrate fraction of the cell wall, should be further investigated. For instance, distinguishing between alpha- and beta-glucans could be an interesting line of research, as these compounds serve different functions in the cell wall compartment. Alpha-glucans contribute to the cell wall matrix (Sietsma & Wessels, 1994), while the beta-glucans play a structural role, crosslinking the chitin fibers (Wessels, Mol, Sietsma, & Vermeulen, 1990). Once we can understand the magnitude and the rate at which these components are lost from the necromass and later stored in the soil organic matter, we can more precisely predict what would be the C:N ratio of the soil organic matter, which in many cases is essential to assess its chemical ‘lability’ (Cui et al., 2020). Further insights on this C input from ectomycorrhizal fungi might provide a more comprehensive view of the contributions of these groups of microorganisms to soil organic matter. While most of the research to date has focused on their capacity to mobilize organic C by decomposing organic matter (Leake et al., 2014; D. Read & Perez‐Moreno, 2003; D. J. Read, Leake, & Perez-Moreno, 2005), we emphasize the importance of the decomposition of their necromass as a contribution to soil organic matter dynamics.
Considering that glucans and chitin are both attacked by hydrolytic enzymes, it is also worth further investigate the temporal activity of these enzymes under the influence of external factors. Such analysis of hydrolytic enzymes could provide more insights into the mechanisms that drive the ectomycorrhizal decomposition, especially concerning environmental conditions. The activity of many enzymes depend on soil abiotic parameters (pH, temperature, moisture), which drastically change between soil layers and seasonality (Wittmann, Kähkönen, Ilvesniemi, Kurola, & Salkinoja-Salonen, 2004). This can have a major effect on the ectomycorrhizal necromass decomposition, which can be modelled more easily once we can fine-tune them based on environmental conditions.
With the findings presented in this study, we encourage to follow two main paths to further understand the contribution of ectomycorrhizal necromass to SOM. First, there is a need to dig deeper into the nature of interactions between the two most abundant polymers in the cell wall (glucans and chitin), to see if those interactions may have a significant effect on the decomposition of both single components and the overall necromass. In particular, whether these interactions are prone to change under the influence of environmental conditions or that they are highly species-specific. Understanding these interactions may take a long time, in analogy to the complications that arose to understand the interaction between lignin and cellulose on plant litter decomposition (Evans, Dutton, Guillén, & Veness, 1994; Talbot & Treseder, 2012). Those interactions are particularly difficult to study in natural conditions due to the external biotic factors that are involved (Cuchietti, Marcotti, Gurvich, Cingolani, & Harguindeguy, 2014). Secondly, when it comes to developing models which aim to predict SOM dynamic, without knowing the nature of the interactions within mycorrhizal necromass – as highlighted above, we can still use the decomposition estimates from our study and others to develop descriptive models. This would demand considering mycorrhizal necromass as a separate pool next to plant litter, instead of assuming that both pools behave similarly. Either way, our results suggest that necromass decomposition does not behave similarly to plant litter, while it is a critical component to consider in estimating SOM dynamics.
Conclusions Our results show that the decomposition process of necromass of ectomycorrhizal fungal mycelium is not species-specific overall, as the rate and magnitude of decomposition do not show significant differences as for the pathway of chemical component loss from their cell walls. As evidence of this, the concentration changes of the cell wall compounds over time do not correspond to the final biomass loss. For this reason, looking at the concentration of single compounds cannot ensure an accurate prediction of the necromass loss in ectomycorrhizal fungi. Together, this brings evidence that distinct EM fungal species affect SOM through distinct pathways of cell-wall compound loss, even if these fungi show a similar overall mass loss. These pathways differ remarkably from those of plant litter and need to be further understood to be able to better predict the contribution of fungal necromass on soil organic matter dynamics.