(1) Background: A vegetable eel co-culture system is an economically efficient way of agricultural cultivation, which can have an impact on the soil microbial environment and play a pivotal role in the soil nutrient cycle, but there is little research on its impact on soil ammonia-oxidizing microorganisms. (2) Methods: NovaSeq platform sequencing was employed to investigate the richness, structure, and diversity of soil ammonia-oxidizing microbial communities, exploring the effects of a vegetable eel co-culture system on soil nitrogen cycling. Four different planting treatments were set up: unfertilized without vegetable eel (CCK), fertilized without vegetable eel (CRT), unfertilized with vegetable eel (ICK), and fertilized with vegetable eel (IRT). (3) Results: A vegetable eel co-culture system significantly increased soil pH and decreased bulk density under fertilization conditions. The soil nitrification potential rate was enhanced by a vegetable eel co-culture system to an average of 26.3%. A vegetable eel co-culture system significantly altered the community structure of all ammonia-oxidizing microorganisms, with a significant increase in the richness and diversity of ammonia-oxidizing bacteria (AOB) and comammox clade-A, while fertilization significantly increased the diversity of all ammonia-oxidizing microbial communities. Structural equation modeling (SEM) analysis showed that the main environmental factors affecting the structure of the ammonia-oxidizing microbial community were nitrate and total nitrogen. The number of amoA genes in AOB and comammox clade-B was significantly positively correlated with the soil potential N nitrification rate (PNR), which played a leading role in the nitrification of alkaline vegetable soil. The network analysis revealed that a vegetable eel co-culture system improved the modularity of AOB and comammox clade-B by 13.14% and 5.66%. (4) Conclusions: This study showed that the vegetable eel co-culture system stimulated the evolution of ammonia-oxidizing microbial communities by changing the physicochemical properties, which in turn promoted the soil nitrification reaction.
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