Hyperspectral signatures reveal hidden stress in soybean caused by residual nicosulfuron

Abstract Residual nicosulfuron in soil can induce persistent phytotoxic effects on subsequent soybean ( Glycine max L.) crops, yet the physiological trajectory from early stress to photosynthetic collapse remains unclear. Here, we established a multimodal phenotyping framework that integrates hyperspectral reflectance, ultraviolet‐excited multichannel fluorescence, and chlorophyll fluorescence quenching imaging to capture stage‐specific soybean responses under nicosulfuron stress. Early pigment disruption was marked by a 12.3% decrease in the chlorophyll index 3, while metabolic activation was indicated by an increase in fluorescence at 450 nm to 3.26 ± 0.16 at 100 μg/kg, compared to 2.05 ± 0.05 in the control group. These were followed by photosystem II (PSII) dysfunction, including a decline in the maximum quantum efficiency of PSII under light adaptation (Fv/Fm_Lss) from 0.71 to 0.37, a 102% increase in non‐photochemical quenching under light adaptation (NPQ_Lss), and a 38% reduction in maximum fluorescence under light adaptation (Fm_Lss), reflecting photosystem disintegration. Such impairments culminated in a marked elevation of the Integrated Biomarker Response version 2. This study identifies a distinct injury–regulation–collapse pathway and phase‐specific markers, while the integrated imaging approach enables earlier, non‐invasive detection and dynamic monitoring, providing a mechanistic basis for risk assessment in herbicide‐impacted rotation systems.