Site-dependent effects of AMF–Streptomyces co-inoculation on rhizosphere functioning, maize yield, and water- and nitrogen-use efficiency in rain-fed systems

Microbial co-inoculation can improve crop performance under water limitation, but its effects often vary across environments. Single inoculations with Rhizophagus irregularis (an arbuscular mycorrhizal fungus, AMF) or Streptomyces rochei (Str) and co-inoculation with both (AMF + Str) were compared in rain-fed maize at two sites with contrasting growing-season precipitation: Heyang (HY; 135.1 mm) and Yuci (YC; 392.6 mm). At both sites, co-inoculation resulted in the highest grain yield, water-use efficiency (WUE), and nitrogen-use efficiency (NUE), with site-dependent differences in tissue N concentrations (higher grain N at YC and higher straw N at HY). At YC, co-inoculation was associated with increased homogeneous selection and reduced dispersal limitation in bacterial community assembly, whereas fungal community assembly showed the reverse pattern. Carbohydrate-active enzyme (CAZyme) analysis identified 13 responsive families at YC, compared with two at HY; glycosyltransferases GT2 and GT4 were predominantly linked to Pseudomonadota genera and positively associated with macroaggregate proportion. Analysis of Y–A–S (growth yield, resource acquisition, and stress tolerance) functional categories based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed broader pathway responses and greater resource-acquisition and stress-tolerance potential at YC. Soil enzyme responses were site-dependent, with lower urease activity at HY and higher β-glucosidase and leucine aminopeptidase activities at YC. An exploratory structural equation model indicated enzyme activity was more closely associated with crop performance than macroaggregate proportion or nutrient status at YC. Together, these results indicate that co-inoculation was associated with broader coordinated crop–soil–microbiome responses at the wetter site, suggesting that greater water availability may favor complementary inoculant functions. Microbial polysaccharide-related functional potential was associated with improved soil aggregation and sustained enzyme-mediated nutrient turnover, while a longer reproductive interval may have facilitated N allocation to grain, enhancing NUE. These site-dependent responses underscore the need to evaluate cross-kingdom inoculants under field conditions.