Fourier transform infrared (FT-IR) attenuated total reflection (ATR) spectroscopy was used to discriminate five commonly encountered soil-borne fungi that cause severe economic damage to agriculture: <i>Colletotrichum, Fusarium, Pythium, Rhizoctonia</i>, and <i>Verticillium</i>. Contrary to previous studies related to microorganism discrimination using FT-IR-ATR spectroscopy, the pathogen samples were not dried on the ATR crystal, which is a time-consuming operation. Rather, after removing some pathogen filaments from the solution using tweezers, these were placed directly on a flat ATR crystal and pressure was applied using a pressure clamp. Following water subtraction, baseline correction, and normalization of the spectra, principal component analysis was used as a data-reduction step and canonical variate analysis was used for discrimination. Discrimination was performed at the genus level and at the strain level for <i>Colletotrichum</i>. For discrimination between the five fungi at the genus level, the success rate for the validation samples ranged from 75% to 89%. For discrimination between the two <i>Colletotrichum</i> strains, the success rate was 78%. Comparison with spectra of similar fungi dried on the ATR crystal showed that both types of spectra were very similar, indicating that drying the samples on the ATR crystal is not required and can be replaced by mathematical post-processing of the spectra. For routine analyses that involve rapid screening of very large amounts of samples, this approach allows for increasing significantly the number of samples that can be analyzed daily.

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