Soil organic matter (SOM) is increasingly recognized as a key component of terrestrial carbon cycling, yet the relative contribution of microbial necromass—and especially fungal necromass—remains poorly understood. We produced fungal necromass of different biochemical quality (low vs. high melanin content) from Hyaloscypha bicolor and decomposed it in forest topsoil to study (i) how microbial decomposer and predator communities differ between soil and necromass, (ii) whether necromass-associated communities are subsets of bulk soil communities or contain additional “unseen” colonizers, and (iii) if microbial predators (protists, nematodes) exert top-down control on the necromass decomposers and necromass decomposition rates. Over two sampling times (4 and 12 weeks), necromass consistently exhibited rapid early mass loss, followed by reduced decay, and more strongly stabilized mass in high melanin residues. Quantitative PCR revealed substantially higher bacterial and fungal abundances in necromass relative to soil, especially for low melanin necromass. Metabarcoding showed distinct decomposer communities in necromass as compared to soil, with half or more of the observed necromass taxa absent in bulk soils at the site level. Necromass also supported differentiated predator communities, including a high nematode richness at early decay stages and a predominance of protist bacterivores. Structural equation modeling indicated that predator community composition influenced both bacterial and fungal abundance, and marginally significantly affected necromass decomposition rates. We conclude that fungal necromass acts as a microbial “hotspot” enriched in specialized decomposers and predators, and that predator–decomposer interactions can potentially regulate necromass decay. Our findings highlight the need to integrate top-down control and colonization processes into models of fungal necromass turnover and its ultimate contribution to soil organic carbon.

We conducted the decomposition experiment in a mixed-age white pine (Pinus strobus) forest at the Cedar Creek Ecosystem Science Reserve (Cedar Creek; 45°25' N, 93°10' W) in eastern Minnesota, USA. Cedar Creek is a 2300-ha site and National Science Foundation Long-Term Ecological Research site located on the Anoka Sand Plain, which is characterized by excessively drained soils containing up to 90% sand. In early August 2020, we buried mycobags containing either low- or high-melanin necromass within the top 5 cm of mineral soil in twelve plots, placing two mycobags of each necromass type per plot (48 bags in total). We retrieved them after one and three months (i.e., 4 and 12 weeks) on September 3 and November 5, 2020, respectively. These times were chosen based on previous necromass experiments, including at Cedar Creek, to capture the earlier and later stages of decomposition. Each retrieved bag was individually packaged, stored at 4°C, and promptly transported to the lab. One bag was lost during the course of the experiment and thus excluded from all analyses. In the lab, the remaining necromass was transferred to sterile 2 ml plastic tubes using a clean spatula. It was then freeze-dried for 3 days at -50degrees C, under vacuum. The final dry weight (mg) of necromass for each sample was then measured using an analytical microbalance and mass remaining was calculated as percentage of initial dry weight added to each bag.