Root nodule senescence is a major factor limiting the symbiotic nitrogen fixation efficiency of leguminous crops. Delaying nodule senescence and prolonging its functional period is considered an effective strategy to improve crop yield and nutritional quality. To address the challenges of low nodulation efficiency, poor nitrogen fixation, and susceptibility to aflatoxin contamination in peanuts, our team has developed the ARC microbial agent, which effectively enhances nodulation while improving yield and quality. This study aims to elucidate the alleviating effects of the ARC microbial agent on peanut root nodules under dark stress, and to investigate its role in extending nodule lifespan and maintaining nitrogenase activity and functional stability.

A rapid senescence model was established by applying dark stress to peanut plants at the flowering and pegging stage. Root nodule phenotypic traits were dynamically extracted at different stress time points (0, 6, 12, 24, 36, 48, and 72 h) using the YOLOv8s algorithm, while nitrogenase activity was determined via the acetylene reduction assay. Based on both phenotypic and functional parameters, the regulatory effects of the ARC microbial agent on the rapid senescence of root nodules were comprehensively evaluated.

Compared with the control group, treatment with the ARC microbial agent significantly improved both the structural and functional traits of root nodules: the average diameter increased by 6.34%, and nitrogenase activity per gram increased by 117.11%. In addition, the average surface brightness and chroma of nodules increased by 6.32% and 3.05%, respectively. RGB color analysis further showed that nodules in the treatment group exhibited significantly higher color intensity across all channels, with mean increases of 6.32%, 8.06%, and 9.35% in the red, green, and blue channels, respectively, indicating a more vivid and brighter color appearance. During the dark stress-induced rapid senescence process, the ARC microbial agent effectively mitigated the decline in both nodule color phenotype and nitrogen fixation activity across different time points. In the early stress stage (0-6 h), the average brightness and chroma of control group nodules decreased by 8.36% and 16.85%, respectively, and nitrogenase activity dropped by 82.65%, indicating rapid onset of senescence. In contrast, nodules in the ARC treatment group showed only 1.26% and 11.31% decreases in brightness and chroma, respectively, with nitrogenase activity decreasing by 63.99%, reflecting a clear delay in early senescence and a strong protective effect. By the late stage of stress (72 h), the ARC treatment group exhibited brightness and chroma reductions of only 4.85% and 20.96%, significantly lower than the 13.82% and 23.15% declines observed in the control group, effectively alleviating the trend of color deterioration. At the same time, nitrogenase activity in the treatment group remained at 22.36% of its initial level, while nodules in the control group had already lost activity by 48 h, further confirming the sustained regulatory effect of ARC on nodule senescence. In addition, Pearson correlation analysis revealed that the red channel intensity was significantly positively correlated with nitrogenase activity (r=0.573, P=0.0003), while the green channel showed a weaker correlation and the blue channel showed no significant correlation. Notably, chroma, as a composite indicator of color variation, exhibited the strongest correlation with nitrogenase activity (r=0.736, P<0.001). Furthermore, with the progressive decline in nitrogenase activity, root nodules showed a gradual color transition from bright red to dark red and brown.

The ARC microbial agent not only promotes peanut nodulation and nitrogen fixation, but also effectively delays the rapid senescence of root nodules induced by dark stress, thereby maintaining structural stability and sustaining nitrogen-fixing function. This contributes to enhanced symbiotic nitrogen fixation efficiency in leguminous crops. Moreover, nodule chroma was found to reflect changes in physiological activity and can serve as an early warning indicator of functional decline. These findings provide new perspectives and methodological support for advancing the study of nodulation and nitrogen fixation in legumes.