Digging deeper to find the effect of long-term greenhouse cultivation with excessive fertilization and irrigation on the structure and assemblage of soil bacterial community

Long-term excessive fertilization and irrigation under greenhouse cultivation systems cause nitrogen leaching, while the residual content varies at different soil depths with cultivation durations. However, it remains unclear whether it changes the composition and assemblage of the soil bacterial community, especially at deeper layers (as deep as 4 m), after long-term intensified cultivation. This study selected soils from three sites in Shouguang (a typical representative intensive planting area), i.e., greenhouse monoculturing for 20 years (G20), greenhouse monoculturing for 10 years (G10), and an adjacent rotation field (F) for physicochemical property determination and high-throughput pyrosequencing. The results showed that, contrary to the vertical characterization of soil bacterial community composition, the 2-m soil was dominated by Methylomirabilota, a nitrate/nitrite-dependent anaerobic methane oxidizer, uncovered for the first time in the soil habitat. This was attributed to the high levels of dissolved organic carbon (DOC, 201.2–255.7 mg kg⁻¹), proving that applying C-rich organic fertilizers, e.g. plant residues, is effective in preventing accumulated nitrate from moving downward and threatening groundwater in greenhouse soils. Besides, greenhouse cultivation increased the inter-layer composition differences of the bacterial community, and compared with the abundant, the rare subcommunity showed higher sensitivity to environmental changes. The total nitrogen most significantly affected the bacterial community composition and assemblage. Therefore, 20 years of consecutive monocropping significantly decreased the microbial co-occurrence network complexity and species dispersal rate, yielding a low-fitted neutral community model (NCM) and more specialized ecological niches, especially for the rare subcommunity. As far as is known, this is the first study that explores the likely changes in the bacterial community composition and quantifies the responses of the rare subcommunity to long-term greenhouse cultivation at this soil depth. Discovery of Methylomirabilota broadens our understanding of micro-biodiversity in deep-soil ecosystem, and hints its application potential in soil remediation.