Soil microbial activities and community structure reflect changes in soil functions in a manured Mediterranean orchard

Partial replacement of mineral fertilisers (MFs) with animal manures in Mediterranean apple orchards can maintain crop production without increasing greenhouse gas (GHG) emissions, but its effects on soil microbial properties and their value as indicators for key soil functions remain unclear. This study assessed the impacts of partial MFs replacement with cattle slurry (CS), acidified cattle slurry (ACS), cattle solid manure (CsM), and poultry manure (PM) on soil microbial properties at the end of a three-year field experiment, compared with a 100% MFs control (CTRL). Total microbial biomass (PLFA) showed limited responses to manure replacement, with significant changes mainly in Gram+ bacteria under ACS (+44%) and higher bacteria-to-fungi ratio (B/F) under CsM (+46%). Both CsM (+36%) and PM (+64%) increased β- N -acetylglucosaminidase (NAG) activity, while ACS (−55%) reduced dehydrogenase (DH) activity. β-glucosidase activity was unaffected. CsM generally showed more consistent changes in microbial structure and activity. Multivariate analysis also showed clear separation between CsM and ACS, highlighting the importance of manure composition in shaping soil microbial responses. Enzyme activities were consistently correlated with soil organic carbon and other soil chemical properties, whereas PLFA-based microbial biomass correlated with fruit nutrient uptake. Both B/F ratio ( r = −0.47) and NAG ( r = 0.58) activity were correlated with N 2 O emissions. Each indicator responded differently depending on manure type, but together, enzyme activities and PLFA-based microbial groups provided complementary information. Therefore, their combined use to assess soil functioning in Mediterranean orchard systems is recommended.