Natural farming (NF), an environmentally friendly agricultural practice similar to organic farming, was developed in Japan. Unlike conventional farming, little is known about the influence of NF on soil microbial communities, especially the surface soil. We therefore compared the effect of seven years’ conventional practice (CP), conventional practice without chemicals (CF), and NF on soil properties and microbial community structure at two soil depths (0–10, 10–20 cm) in an experimental cabbage field. Both soil depth and agricultural practice significantly influenced edaphic measures and microbial community structure. NF improved bulk density, pH, electrical conductivity, urease activity, and nitrate reductase activity in topsoil; similar trends were observed in deeper soil. Pyrosequencing demonstrated that the use of pesticides in conventional farming (CP) led to lower microbial abundance and diversity in topsoil than CF. Similarly, NF increased microbial abundance compared to CP. However, distinct taxa were present in the topsoil, but not deeper soil, in each treatment. CP-enriched microbial genera may be related to plant pathogens (e.g., Erwinia
) and xenobiotic degraders (e.g., Sphingobacterium
). The microbial community structure of NF was distinct to CP/CF, with enrichment of Pedomicrobium
, which may prefer stable soil conditions. Network analysis of dominant genera confirmed the more stable, complex microbial network structure of the 0–10 cm than 10–20 cm layer. Flavisolibacter
and Candidatus Nitrososphaera
are potentially fundamental taxa in the 0–10 cm and 10–20 cm layer networks, respectively. Overall, we show that NF positively affects soil quality and microbial community composition within sustainable farming systems.
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