Objectives The aim of this study was to provide a theoretical basis and data support for replacing chemical fertilizer with green manure in karst areas. We studied the effects of applying green manure on soil nutrient status and soil bacterial community.

Methods A green manure–rice rotation field experiment was conducted for three years in karst paddy field with Chinese milk vetch. There were three treatments: chemical fertilizer (CK), Chinese milk vetch green manure (MV), and the combination of Chinese milk vetch and chemical fertilizer (MF). The soil bacterial community diversity, characteristics and co-occurrence network of soil (0–20 cm) were analyzed using Illumina Novaseq PE250 high-throughput sequencing technology.

Results Compared with CK, MV and MF increased soil organic carbon (SOC), total nitrogen (TN), and available nitrogen (AN), but significantly decreased soil pH, readily potassium (AK), and C/N ratio. There was no significant difference (P>0.05) in soil bacterial diversity index among the fertilization treatments. The dominant bacteria flora of karst paddy soil were Chloroflexi, Proteobacteria, Nitrospirae, Bacteroidetes and Acidobacteria, regardless of the treatment. However, at the genus level, Geobacter, Anaerolinea, and RBG-16-58-14 were identified as the key genera of karst calcareous paddy soil. Novosphingobium, Syntrophorhabdus, and Phenylobacterium were (P<0.05) higher in MF than CK. Desulfatiglans was higher in CK than MF. The co-occurrence network analysis indicated that CK and MV had similar co-occurrence networks, while MF increased the complexity of the soil bacteria network and the relative abundance of eutrophic bacteria such as Proteobacteria and Bacteroidetes. Meanwhile, the RDA analysis results revealed that soil exchangeable readily available potassium, exchange calcium ions, and total N were the key environmental factors affecting the composition of soil bacterial communities.

Conclusions The combined use of Chinese milk vetch and chemical fertilizer could improve soil nutrient content and the relative abundance of eutrophic bacteria than a single use of chemical fertilizer and Chinese milk vetch. Our findings are important for maintaining the sustainable development of the karst paddy ecosystem.