Bacterial and Fungal Communities Respond Differently to Changing Soil Properties Along Afforestation Dynamic

Spontaneous afforestation following land abandonment has been increasingly recognized as a nature-based solution to mitigate climate change and provide measurable benefits to biodiversity. However, afforestation effects on biodiversity, particularly on soil microbial communities, are still poorly characterized, with most previous studies focusing on artificial plantations rather than forest rewilding dynamics. Here, we assessed changes in topsoil physical-chemical properties and related dynamics of bacterial and fungal community composition and structure following spontaneous afforestation of abandoned grasslands in Northeast Italy over the last 70 years. With a space-for-time approach, we selected four chronosequences representing different successional stages: grassland, early (2000-2020), intermediate (1978-2000), and late (1954-1978). Results showed that spontaneous afforestation progressively reduced topsoil pH and total phosphorus (P), while soil organic carbon (SOC), nitrogen (N), and C:N ratio increased. Correspondingly, the overall α-diversity of the fungal community, assessed by ITS DNA metabarcoding, progressively decreased after an initial increase from grassland conditions, following substrate acidification and trophic specialization. Bacterial diversity, assessed by 16S DNA metabarcoding, was highest at the initial stages, then progressively decreased at later stages, likely limited by lower organic matter quality. Shifts of fungal community composition included an increase of ectomycorrhizal Basidiomycota linked to topsoil's higher SOC, N, and C:N ratio. Differently, bacterial community composition responded substantially to pH, with topsoil acidity favoring Proteobacteria (Pseudomonadota) and Acidobacteria (Acidobacteriota) at the late afforestation stages. Our findings provide a first contribution to clarify how fungi and bacteria respond to spontaneous afforestation. This is particularly relevant in the context of climate change mitigation, considering the fundamental role of microorganisms in shaping soil carbon storage dynamics.