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.

Recently, due to rapid industrialization and urbanization, many studies have focused on the assessment of soil metal contamination. The present research aimed to investigate the concentration of potentially hazardous elements (Al, Cr, Cu, Fe, Mg, Mn, Ni, Pb, V and Zn) in three different areas (forest, wildfire forest and urban) of the Mediterranean region. Contamination levels were further assessed calculating different indices: contamination factor (CF), geo-accumulation index (GI) and ecological risk (E). The results showed high concentrations of V, Mg and Mn in forest and wildfire forest soils, as well as high concentrations of Al, Fe, Ni, Pb and Zn in urban soils. According to the contamination indices, Pb exceeded the background level in both wildfire forest and urban soils. According to human risk assessment, inhalation appeared the main route of exposure of metals in soils, especially for children. The overall cancer risk was higher than the safe level, especially for Pb. Different relationships were found between the contamination indices and the potential risk of carcinogenic effects according to the diverse metal concentrations. Particularly, wildfire soils showed human health risks mainly linked to Pb, Cu and Cr contaminations, due to human activities, and Ni, due to both anthropogenic and pedogenetic input. Instead, the urban soils showed that Zn contamination, mainly related to urban traffic, influenced the potential carcinogenetic risk in this area. The carcinogenic risk was higher than acceptable values for all the metals assessed. These findings highlighted the need to develop further management practices to protect soils from metal pollution and reduce human health risks.

Background: Previous evidence demonstrated DNA methylation changes in response to stress in plants, showing rapid changes within a limited time frame. Exposure to self-DNA inhibits seedling root elongation, and it was shown that it causes changes in CG DNA methylation in Lactuca sativa. We assessed cytosine methylation changes and associated gene expression patterns in roots of Arabidopsis thaliana Col-0 seedlings exposed to self-DNA for 6 and 24 h. Methods: We used whole genome bisulfite sequencing (WGBS) and RNA-seq analyses to assess genomic cytosine methylation and corresponding gene expression, respectively, on DNA and RNA extracted with commercial kits from roots exposed to self-DNA by an original setup. Fifteen hundred roots replicates, including the control in distilled water, were collected after exposure. Sequencing was performed on a NovaSeq 6000 platform and Ultralow Methyl-Seq System for RNA and DNA WGBS, respectively. Results: Gene expression in roots exposed to self-DNA differed from that of untreated controls, with a total of 305 genes differentially expressed and 87 ontologies enriched in at least one treatment vs. control comparison, and particularly after 24 h of exposure. DNA methylation, particularly in CHG and CHH contexts, was also different, with hyper- and hypomethylation prevailing in treatments vs. controls at 6 h and 24 h, respectively. Differentially expressed genes (DEGs) analysis, Gene Ontology (GO) enrichment analysis, and differentially methylated regions (DMRs) analysis, provided an integrated understanding of the changes associated with self-DNA exposure. Our results suggest differential gene expression associated with DNA methylation in response to self-DNA exposure in A. thaliana roots, enhanced after prolonged exposure. Conclusions: Main functional indications of association between DNA methylation and gene expression involved hypomethylation and downregulation of genes related to nucleotide/nucleoside metabolism (ATP synthase subunit) and cell wall structure (XyG synthase), consistent with previous observations from metabolomics and physiological studies. Further confirmation of these findings will contribute to improving our understanding of the plant molecular response to self-DNA and its implications in stress responses.

This study investigates the response of soil microbial community to spontaneous afforestation—a natural rewilding process that has been progressing for decades across national and European landscapes following land abandonment. The research was conducted at two Italian sites: the Foreste Casentinesi National Park (CF) and Julian Prealps (JP). In both areas, five successional stages (I–V) of afforestation were identified based on historical orthophotos (1954–2020) and replicated across four chronosequences. Topsoil samples (0–10 cm) were analyzed for soil properties, including pH, bulk density, organic carbon, total nitrogen, and water content. Soil microbial communities were characterized through environmental DNA extracted from fine soil fractions, followed by DNA metabarcoding using ITS and 16S rRNA gene markers to target fungi and bacteria, respectively. We hypothesized that: (i) topsoil properties change along the afforestation dynamics, thereby controlling the pattern of soil microbiota α‐diversity; (ii) microbial β‐diversity differs between the two study areas as a result of the interplay between afforestation dynamics and soil properties. Results showed that topsoil became increasingly acidic and less compact (i.e., lower bulk density), accompanied by a rise in organic matter content. However, these trends were modulated by site‐specific variability. Microbial α‐diversity generally peaked at intermediate successional stages and declined in late forests. Bacterial communities were dominated by Verrucomicrobiota and Pseudomonadota , while fungal communities were dominated by Ascomycota and Basidiomycota , the latter increasing as afforestation progressed. β‐diversity differed markedly between the two areas. In CF, but not in JP, a significant successional trajectory was evident, particularly for fungi. Bacterial community composition in CF was most associated with soil pH, and fungal composition with soil water content, while in JP multiple soil properties explained community variation. In conclusion, successional effects on soil microbiota were most evident in the more homogeneous environment of CF, whereas in the heterogeneous JP area, site‐specific filtering driven by high variability in soil properties predominated.