Two-fold Red Excess (TREx): A simple and novel digital color index that enables non-invasive real-time health assessment of green-leaved as well as anthocyanin-rich crops

Background Digital color indices provide a reliable means for assessing plant health status by enabling real-time estimation of chlorophyll (Chl) content, and are thus adopted widely for crop monitoring. However, as all prevalent leaf color indices used for this purpose have been developed using green-leaved plants, they do not perform reliably for anthocyanin (Anth)-rich red-leaved varieties. Hence, the present study investigates digital color indices for six types of leafy vegetables with different levels of Anth to identify congruent trends that could be implemented universally for non-invasive crop monitoring irrespective of species and leaf Anth content. For this, datasets from three digital color spaces, viz., RGB (Red, Green, Blue), HSV (Hue, Saturation, Value), and L*a*b* (Lightness, Redness-greenness, Yellowness-blueness), as well as various derived plant color indices were compared with SPAD Chl meter readings and Anth/Chl ratio of n = 320 leaf samples. Results While most digital color features and indices presented abrupt shifts between Anth-rich and green-leaved samples, the newly-developed color index Two-fold Red Excess (TREx) as well as the color feature R showed very strong correlation with SPAD readings ( R 2 > 0.84), and did not exhibit any deviation due to leaf Anth content. Moreover, both parameters could predict SPAD values reliably ( R 2 > 0.75). Further, logarithmic decline of G/R and Augmented Green-Red Index (AGRI) with increasing Anth/Chl ratio ( R 2 > 0.82) revealed that relative Anth content affected digital color indices markedly by shifting the R:G balance until the Anth/Chl ratio reached a certain threshold. Conclusion The present study provides the first in-depth assessment of variations in RGB-based digital color indices due to high leaf Anth contents, and uses the data for Anth-rich as well as green-leaved crops belonging to different species to develop a universal digital color index TREx that can be used as a reliable alternative to handheld Chl meters for rapid high-throughput monitoring of green-leaved as well as red-leaved crop varieties.