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Accelerating crop improvement for increased yield and better adaptation to changing climatic conditions is an issue of increasing urgency in order to satisfy the ever-increasing global food demand. However, the major bottleneck is the absence of high-throughput plant phenotyping methods for rapid and cost-effective data-driven variety selection and release in plant breeding. Traditional phenotyping methods that rely on trained experts are slow, costly, labor-intensive, subjective, and often require destructive sampling. We explore ways to improve the efficiency of crop phenotyping through the use of unmanned aerial vehicle (UAV)-based multispectral remotely sensed data in maize (Zea mays L.) varietal response to maize streak virus (MSV) disease. Twenty-five maize varieties grown in a trial with three replications were evaluated under artificial MSV inoculation. Ground scoring for MSV infection was carried out at mid-vegetative, flowering, and mid-grain filling on a scale of 1 (resistant) to 9 (susceptible). UAV-derived spectral data were acquired at these three different phenological stages in multispectral bands corresponding to Green (0.53–0.57 μm), Red (0.64–0.68 μm), Rededge (0.73–0.74 μm), and Near-Infrared (0.77–0.81 μm). The imagery captured was stitched together in Pix4Dmapper, which generates two types of multispectral orthomosaics: the NoAlpha and the transparent mosaics for each band. The NoAlpha imagery was used as input into QGIS to extract reflectance data. Six vegetation indices were derived for each variety: normalized difference vegetation index (NDVI), green normalized difference vegetation index (GNDVI), Rededge NDVI (NDVIrededge), Simple Ratio (SR), green Chlorophyll Index (CIgreen), and Rededge Chlorophyll Index (CIrededge). The Random Forest (RF) classifier was used to evaluate UAV-derived spectral and VIs with and without variable optimization. Correlations between the UAV-derived data and manual MSV scores were significant (R = 0.74–0.84). Varieties were classified into resistant, moderately resistant, and susceptible with overall classification accuracies of 77.3% (Kappa = 0.64) with optimized and 68.2% (Kappa = 0.51) without optimized variables, representing an improvement of ~13.3% due to variable optimization. The RF model selected GNDVI, CIgreen, CIrededge, and the Red band as the most important variables for classification. Mid-vegetative was the most ideal phenological stage for accurate varietal phenotyping and discrimination using UAV-derived multispectral data with RF under artificial MSV inoculation. The results provide a rapid UAV-based remote sensing solution that offers a step-change towards data availability at high spatial (submeter) and temporal (daily/weekly) resolution in varietal analysis for quick and robust high-throughput plant phenotyping, important for timely and unbiased data-driven variety selection and release in plant breeding programs, especially as climate change accelerates.

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