This study aimed to analyze the effects of biochar on the community structure and diversity of nitrogen-fixing microorganisms (nifH gene) in soil under continuous cropping and crop rotation, and would provide a theoretical basis for the sustainable improvement of degraded soils in the black soil of Northeast China and the precise application of biochar under different cropping systems.

This study collected soil samples under continuous and rotational cropping conditions with biochar application rates of 0 (B0), 5 t·hm^-2 (B5), 15 t·hm^-2 (B15), and 25 t·hm^-2 (B25) at the soybean maturity stage based on a long-term biochar application field experiment. Real-time PCR and Illumina Miseq high-throughput sequencing were used to analyze the absolute abundance and community structure diversity of the nifH gene of nitrogen-fixing microorganisms.

Biochar application under both continuous cropping and crop rotation increased soil pH, total nitrogen (TN), total phosphorus (TP), alkaline hydrolyzable nitrogen (AN), available phosphorus (AP), and available potassium (AK), but decreased total potassium (TK) content. Biochar significantly affected the absolute abundance of the nifH gene. Under continuous cropping and crop rotation, the nifH gene abundance under the high-dose biochar treatment (B25) increased by 40.3% and 149.6%, respectively, compared with the control (B0). Moreover, the nifH gene abundance under crop rotation was significantly higher than under continuous cropping, regulated by the combined effects of total nutrients (TN, TP, and TK) and available nutrients (AN, AP, and AK). Additionally, moderate biochar application (B15) significantly enhanced the richness and diversity of the nifH gene community under both cropping systems. Redundancy analysis indicated that biochar indirectly drove changes in the nifH gene community structure by altering soil chemical properties. The dominant bacterial class in the nifH gene community composition of nitrogen-fixing microorganisms was Alphaproteobacteria, with Bradyrhizobium being the dominant genus, and its relative abundance increased with the increase of biochar application rates. Under continuous cropping conditions, the Bradyrhizobium genus showed the highest relative abundance under the B15 treatment, while in crop rotation condition, the highest relative abundance occurred under the B25 treatment. However, no significant correlation was found between its abundance and soil physicochemical properties. The second dominant genus, Skermanella, exhibited a highly significant positive correlation with AN and AP contents, and a significant positive correlation with TN content.

Biochar optimized the diversity of the nifH gene community in nitrogen-fixing microorganisms by regulating soil nutrient content, improved community structure and composition of the nifH gene, enhanced nutrient use efficiency under continuous cropping and rotation systems, and then finally promoted a virtuous cycle in the soil ecosystem.