Fertilization measures improve agronomic traits of alfalfa by regulating root bacterial co-occurrence networks

Alfalfa (Medicago sativa; L., Fabaceae) is an important leguminous forage crop, and rational fertilization is critical for improving its productivity; however, the synergistic mechanisms by which fertilization regulates plant-soil-microorganism interactions through rhizosphere microbial networks remain poorly understood.

We examined the effects of 14 fertilization treatments-including a nonfertilized control, single-nutrient-deficient treatments, and treatments with varying or imbalanced N-P-K ratios-on soil properties, agronomic traits, and rhizosphere bacteria in alfalfa based on a two-year "3414" fertilization experiment.

All fertilization treatments significantly (p < 0.05) increased soil nutrient content, and, due to the interaction between the cumulative effects of fertilizers and associated biological processes, soil nutrient levels differed significantly between years. At the same time, fertilization significantly improved alfalfa agronomic traits (e.g., height, stem diameter, and yield), with the optimal fertilization treatment (N2P2K2: 135 kg ha^-1 urea, 175 kg ha^-1 calcium superphosphate, and 120 kg ha^-1 potassium sulfate) showing the greatest improvement. Microbial analysis revealed a lag in the response of bacterial diversity to fertilization, leading to significant interannual changes. Microbial analysis revealed that bacterial diversity varied significantly between years, while its response to fertilization was generally consistent across the two years. The co-occurrence network analysis showed that although node number did not differ significantly among treatments, the rhizosphere bacterial network under optimal fertilization (N2P2K2) exhibited markedly fewer edges and lower mean degree, density, and clustering coefficient than under the nutrient-deficient or NPK-ratio-imbalanced treatments, reflecting a "simple and efficient" topology with fewer redundant microbial interactions. In contrast, the latter treatments exhibited a "complex redundancy" pattern characterized by denser, more highly interconnected networks. Path analysis further confirmed that fertilization could improve alfalfa agronomic traits through direct and indirect pathways.

This study demonstrates that optimal NPK fertilization enhances alfalfa productivity by simplifying and stabilizing the rhizosphere bacterial co-occurrence network, providing a microbiological basis for precision fertilization management in leguminous forage production.