Systemic Uptake of Rhodamine Tracers Quantified by Fluorescence Imaging: Applications for Enhanced Crop–Weed Detection

Systemic fluorescence tracers introduced into crop plants provide an active signal for crop–weed differentiation that can be exploited for precision weed management. Rhodamine B (RB), a widely used tracer for seeds and seedlings, possesses desirable properties; however, its application as a seed treatment has been limited due to potential phytotoxic effects on seedling growth. Therefore, investigating mitigation strategies or alternative systemic tracers is necessary to fully leverage active signaling for crop–weed differentiation. This study aimed to identify and address the phytotoxicity concerns associated with Rhodamine B and evaluate Rhodamine WT and Sulforhodamine B as potential alternatives. A custom 2D fluorescence imaging system, along with analytical methods, was developed to optimize fluorescence imaging quality and facilitate quantitative characterization of fluorescence intensity and patterns in plant seedlings, individual leaves, and leaf disc samples. Rhodamine compounds were applied as seed treatments or in-furrow (soil application). Rhodamine B phytotoxicity was mitigated by growing in a sand and perlite media due to the adsorption of RB to perlite. Additionally, in-furrow and seed treatment methods were tested for Rhodamine WT and Sulforhodamine B to evaluate their efficacy as non-phytotoxic alternatives. Experimental results demonstrated that Rhodamine B applied via seed pelleting and Rhodamine WT used as a direct seed treatment were the most effective approaches. A case study was conducted to assess fluorescence signal intensity for crop–weed differentiation at a crop–weed seed distance of 2.5 cm (1 inch). Results indicated that fluorescence from both Rhodamine B via seed pelleting and Rhodamine WT as seed treatment was clearly detected in plant tissues and was ~10× higher than that from neighboring weed plant tissues. These findings suggest that RB ap-plied via seed pelleting effectively differentiates plant seedlings from weeds with reduced phytotoxicity, while Rhodamine WT as seed treatment offers a viable, non-phytotoxic alternative. In conclusion, the combination of the developed fluorescence imaging system and RB seed pelleting presents a promising technology for crop–weed differentiation and precision weed management. Additionally, Rhodamine WT, when used as a seed treatment, provides satisfactory efficacy as a non-phytotoxic alternative, further expanding the options for fluorescence-based crop–weed differentiation in weed management.