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Charcoal rot caused by the fungal pathogen Macrophomina phaseolina is an important disease of soybean and the use of resistant cultivars is recommended to manage the disease. Since symptoms, including leaf wilt, typically occur as soybeans reach maturity, screening varieties for tolerance to charcoal rot can be time-consuming, requiring nearly an entire growing season. In this study, soybean seedlings (V1) were exposed to a culture filtrate of M. phaseolina containing toxin(s) produced by the fungal pathogen. The effect on the seedlings was measured with hyperspectral spectroscopy on leaves. Two spectrometers integrated with a fiber optic light source and a 6.35 mm diameter probe yielding 480–850 nm and 900–2400 nm ranges after preprocessing were used. The spectra of the untreated plants measured at 0 h, 4 h, 8 h, 12 h, and 24 h post-exposure to the fungal extract were nearly indistinguishable. In contrast, the toxin-treated plants had significantly different spectra from the untreated plants at each of the 4 h, 8 h, 12 h, and 24 h measurements. Reflectance increased in the NIR (900–2400 nm) region with extended exposure to the fungal extract. This change was most noticeable in the 1450 nm and 1940 nm wavebands. Across the spectra, the 24 h reflectance was significantly higher than that of 12 h, which was significantly higher than those of 8 h, 4 h, and 0 h. Jeffries-Matusita (JM) distance, quantifying class separability, was used as a feature selection method and the 24 h measurement had the highest JM distance values, which indicated good separability. Based on JM Distance the most sensitive wavebands were in the regions of 1370–2400 nm. A ratio of the reflectance at 0 h to reflectance at the other times for each of the wavebands was calculated. The ratio curves had two noticeable troughs centered on 1450 nm and 1940 nm, with respective ratios of 0.47 and 0.32 for the 24 h measurement. The 1940 nm ratio at 24 h was proposed as a relative measure of charcoal rot susceptibility of soybean varieties. A ratio of 1.0 indicated no susceptibility with lower ratios indicating greater susceptibility to charcoal rot toxin(s). This study has implications in terms of developing tools to screen for soybean varieties tolerant to charcoal rot and potentially for other biotic or abiotic factors that induce foliar wilting. [ABSTRACT FROM AUTHOR]