• We evaluated effects of fungi on N2O emission in Chinese milk vetch-containing soils.
• Fungi to contributed to soil N2O production in CMV-amended soils.
• Fungi accounted for 56% of N2O emission in CMV-amended soils.
• Fungi may be important contributors to N2O production in CMV-amended soils.
Fungi play an important role in soil nitrous oxide (N2O) emission in many agricultural soil systems. However, the effect of fungi on N2O emission in Chinese milk vetch (CMV)-containing soils has not been examined sufficiently. This study investigated the contribution of bacteria and fungi to soil N2O emission in CMV-amended soils. We compared soils from an experimental field in the Fujian Academy of Agricultural Sciences that had been treated with 30 000 kg of CMV per 667 m2 per year with one that was not treated with CMV. We incubated soil using cycloheximide and streptomycin to differentiate fungal and bacterial N2O emissions, respectively. Quantitative PCR (qPCR) was performed to investigate bacterial and fungal abundances in the two agricultural soil ecosystems. The contribution of fungi to soil N2O emission in CMV-amended soils was greater than that in non-CMV-amended paddy soils, with fungi accounting for more than 56% of the emissions in CMV-amended soils. Quantitative PCR showed that the ratio of the internal transcribed spacer to 16S rDNA was significantly higher in CMV-amended soils than in non-CMV-amended paddy soils. Furthermore, soil properties, such as pH (P<0.05) and NH4+ concentration (P<0.05), significantly and negatively affected N2O emission by fungi in soil, whereas the total organic carbon (P<0.05) and NO3- concentration (P<0.05) showed significant positive effects. Fungi may be important contributors to N2O production in CMV-amended soils, which may create challenges for mitigating N2O production.