Unveiling Soil Microbial Diversity: Metagenomic Insights into Mineral and Hydrochar Fertilization

The global reliance on mineral fertilizers (MF) has raised significant concerns regarding their environmental and economic drawbacks, including soil degradation, water contamination, greenhouse gas emissions, and the unsustainable use of non-renewable resources. As the urgency to develop sustainable agricultural practices grows, there is increasing interest within both the scientific and agronomic communities to explore viable alternatives to conventional MF. Among these, organic amendments such as biochar and hydrochar (HC) have garnered attention due to their potential to improve soil physical and chemical properties while maintaining or enhancing crop yields. Despite the growing body of literature comparing the impacts of mineral and organic fertilizers on soil properties and crop performance, critical knowledge gaps remain with respect to their effects on the abundance and diversity of soil microorganisms. Soil microbial communities are crucial for ecosystem functioning, influencing nutrient cycling, soil health, and plant productivity. To address this gap, this study employs a metagenomic approach to evaluate both the short- and long-term impacts on microbial diversity indices and their relative abundance in soils treated with conventional MF and HC amendments. Thus, a 330-day greenhouse pot experiment was conducted in which sunflower plants were cultured on soils amended with two different application rates of MF and HC (3.25 and 6.5 t ha⁻¹), standardized based on the total applied nitrogen content. Additionally, unamended soils were included as controls. The performance under well-irrigated and water-deficit conditions was assessed.

After 77 and 330 days of cultivation, soil samples were collected from the topsoil (0-15 cm) for high-throughput ITS and 16S rRNA sequencing, followed by bioinformatic analyses. For each treatment, relative abundances and alpha-diversity were determined by calculating richness indices, including observed OTUs, Chao1, Abundance-based Coverage Estimator (ACE), and Shannon and Simpson diversity indices. Additionally, beta-diversity was assessed using Bray-Curtis distance to create the distance matrix between samples and generate the NMDS (non-metric multidimensional scaling).

Significant differences in abundance and alpha-diversity indices (richness and diversity) were observed between treatments both in the short and long term. The NMDS plots also allowed differentiation of treatments. However, the short-term results were significantly attributed to the application of HC. The results after 77 days suggested that, at high application rates and under both irrigation conditions, HC promoted greater bacterial richness and diversity than a similar nitrogen dose with MF.

Regarding fungal richness analysis, whereas HC treatments did not differ significantly from their respective nitrogen-equivalent MF treatments under well-irrigated conditions, they exhibited the lowest values under water-deficit conditions. In contrast, regardless of the irrigation condition, among the treatments, amendment of HC displayed the lowest fungal diversity. These results indicate an increased quantity, richness, and diversity of bacteria in HC-treated soils, which compete with fungal community development, leading to more uniform fungal communities dominated by a few groups.