Arbuscular Mycorrhizal Fungi and Earthworms Interact to Increase Nitrogen Sequestration in Soil Glomalin Pools of Trifoliate Orange

The individual contributions of arbuscular mycorrhizal (AM) fungi and earthworms to soil nitrogen (N) cycling are well-established; however, their combined effects on N sequestration through glomalin-related soil proteins (GRSPs) are not elucidated. This study evaluated their individual and interactive impacts on plant–soil N dynamics, with an emphasis on GRSP-mediated mechanisms. Trifoliate orange (Poncirus trifoliata) plants were treated with an AM fungus (Funneliformis mosseae), earthworms (Pheretima guillelmi), and their co-inoculation. Measurements were conducted on plant biomass and N content, soil N fractions, GRSP levels, GRSP-sequestered N (NGRSP), contribution of NGRSP to soil total N, and N-metabolizing enzymes. Co-inoculation of F. mosseae and P. guillelmi demonstrated synergistic effects, significantly increasing leaf and root N by 26% and 77%, respectively, compared to individual treatments (14–21% increases). All inoculations significantly elevated levels of total N (by 102–405%), nitrate-N (by 24–62%), soluble organic N (by 35–73%), and total dissolved N (by 31–53%), while ammonium-N decreased only with F. mosseae, with the most pronounced effect in the co-inoculation. Individual and combined inoculations significantly increased difficultly extractable (DEG) and total GRSP (TG) levels and their sequestered N content, with co-inoculation showing superior efficacy (NDEG and NTG increased by 53% and 42%). Adding F. mosseae alone and co-inoculation enhanced all NGRSP contributions to soil total N (by 17–56%), whereas P. guillelmi alone only increased NDEG and NTG contributions (by 13–17%), with co-inoculation revealing greater effects on NEEG contribution to soil total N than individual inoculations. All inoculations elevated nitrate reductase (by 72–101%) and urease (by 29–80%) activity while diminishing catalase (by 42–58%) activity, with synergistic enhancement of urease and catalase activity under co-inoculation. The synergistic interaction between earthworms and AM fungi facilitates N sequestration within the rhizosphere and promotes plant uptake.