Arbuscular mycorrhizal hyphal networks and glomalin-related soil protein jointly promote soil aggregation and alter aggregate hierarchy in Calcaric Regosol

•Aggregate hierarchy was analyzed by fitting soil aggregate disruption and dispersion characteristic curves.•Hyphal length and glomalin-related soil protein strongly contributed to soil aggregate stability.•AM fungal inoculation decreased the disruption rate of macroaggregates.•No evident aggregate hierarchy was detected in Calcaric Regosol. The Loess Plateau of China (LPC) is one of the most severely eroded areas in the arid and semi-arid regions of northern China. Improving soil aggregate stability and hierarchy in Calcaric Regosol is vital for mitigating soil erosion. However, Calcaric Regosol exhibits weak aggregate hierarchy, and there is limited correlation between its aggregate stability and soil organic matter (SOM). Arbuscular mycorrhizal (AM) fungi, known for their soil-structuring capabilities, may hold potential for improving aggregate stability, yet their specific impact on calcareous soil remains unclear. In this study, a three-compartment growth system was used to separate the root and AM fungi, and the impact of AM fungi on soil aggregate stability and hierarchy was quantified. The AM fungi, Rhizophagus intraradices and Funneliformis mosseae, were separately inoculated into mycorrhizal compartments under well-watered and drought stress conditions. Aggregate stability was measured through the wet sieving method and ultrasonic dispersive technology, while aggregate hierarchy was assessed by characteristic disruption and dispersion curves. The results revealed that AM fungi significantly increased the water-stable aggregate stability, and the inoculation reduced the rate of macroaggregate disruption and microaggregate dispersion rate mediated by hyphal network and glomalin. Despite these improvements, the characteristic curves indicated no strong aggregate hierarchy. A permutation test identified hyphal length and glomalin-related soil protein (GRSP) as critical factors contributing to soil aggregate stability. These results suggest that increases in hyphae and GRSP, which are important components of SOM, promote soil aggregation and modify aggregate hierarchy in calcareous soils. This study introduces an energy-based approach to investigate the soil aggregate hierarchy, proposing AM fungi as an effective ecological strategy to restore aggregate stability and mitigate soil erosion on the LPC.