# Herbicide Bioassay Using a Multi-Well Plate and Plant Spectral Image Analysis
## 4 Discussion  
### Spectral Image Responses to Herbicides  
Plant imaging is becoming a core technique for plant phenotyping and diagnoses in plant and agricultural sciences [19]. One of the benefits of a plant image analysis is that it allows for a non-destructive and high-throughput screening of plant responses [19; 20; 21; 22]. In this study, we developed a multi-well plate assay by combining it with a plant spectral image analysis for the rapid diagnosis of herbicide activity and modes of action. Three different sensors, RGB, CF and IR thermal sensors, were used to acquire spectral responses at different time points after herbicide treatment and showed differential spectral responses depending on the herbicide mode of action and the time when the spectral images were acquired, suggesting a potential diagnosis of herbicide modes of action based on spectral image data. Among the spectral image parameters, those related to CF images seemed to be the most responsive to herbicides, particularly \(\Phi\)PSII (Figure 4). Other image parameters derived from RGB and IR thermal images were also informative, although they appeared to be relatively slow and less responsive than the CF images (Figures 3 and 5). An RGB image is most commonly used for analyzing vegetation indices associated with plant growth and canopy development and has a broad range of applications, because it is easily accessible and provides informative and high-resolution results, even with commercially available cameras installed in smartphones [23]. In this study, an RGB color change in response to the herbicides was analyzed by an RGB image analysis, particularly the level of greenness in the leaves, as leaf discoloration such as yellowing and bleaching is the most common symptom after herbicide treatment [24]. The mNDI and ExG values showed significant evidence for changes in leaf color, particularly greenness, and a suppression of plant development resulting from herbicide action (Figures 2A and 3). Moreover, the observed changes varied depending on the herbicide mode of action. The herbicides related to reactive oxygen species (ROS), including paraquat (PSI), difenacil (PPO) and glufosinate (GS), exhibited rapid changes in these RGB-based parameters. Glyphosate (EPSPSPS) showed  
Figure 7: PCA results of six spectral parameters of crabgrass by aggregating all of the data, including 3 h, 6 h, 24 h, 48 h, 72 h, and 120 h after treatment of herbicides with different modes of action. Each symbol represents a replication. a rather mild and slow response, while isoxaflutole (HPPD) and penosvalum (ALS) showed very little changes. These parameters allowed for the explanation of plants' responses to herbicides depending on the mode of action, suggesting that mNDI and ExG are highly useful to quantify plant damage caused by herbicides and to diagnose herbicide modes of action. Given that photosynthesis requires plant pigments, antioxidant systems, and proteins linked to several enzymes and carbohydrates, any stress experienced by plants is likely to impact photosynthesis [25; 26; 27; 28]. Herbicides act on distinct targets depending on the mode of action and consequently impact photosynthesis in various ways. In our study, paraquat exhibited the most rapid and significant reduction in the CF parameters within 3 HAT, despite the absence of any apparent visual impact, while no discernible difference in the CF images was observed in the penosvalum (ALS) treatment (Figures 2B and 4). Other herbicides, such as tiafenacil (PPO), glufosinate (GS), glyphosate (EPSPS), and isoxaflutole (HPPD), exhibited distinct herbicide effects in the CF images and parameters based on their respective modes of action. Among these herbicides, tiafenacil (PPO) was the fastest, followed by glufosinate (GS), glyphosate (EPSPS), and isoxaflutole (HPPD). Interestingly, isoxaflutole (HPPD) showed a sudden decrease in the CF parameters at 120 HAT.