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One strategy presented as an alternative to avoid using chemical substances in soil disinfestation consists in the technique of biosolarization. The aim of the present study was to analyze the effect of seasonality on the effectiveness of biosolarization with different organic amendments for the control of Fusarium oxysporum f. sp. lactucae (FOLAC) on lettuce plants, and to compare the results obtained using a classical soil infectivity bioassay and a qPCR-based molecular technique. None of the plants subjected to biosolarization in the summer season (469–700 and 0–463 h with temperature > 42 °C at 15 and 30 cm soil depth, respectively) showed damage by the pathogen except the untreated control. Conversely, in autumn (3–5 and 0–0 h at temperature = 38–40 °C at 15 and 30 cm soil depth, respectively), only two biosolarization treatments (wheat + semi-composted manure, sunflower pellets) that reduced FOLAC inoculum in soil and plants did not show any disease at the lowest depth (15 cm) in the soil infectivity bioassay. This same result was only obtained at 30 cm soil depth in the biosolarization treatment with sunflower pellets. The number of FOLAC sequences per gram of soil determined with qPCR was null in the biosolarization treatments in summer at both soil depths and corresponded to the absence of disease in the soil infectivity bioassay. A threshold of 145 sequences per gram of soil determined by the qPCR-based molecular technique corresponded to the presence of 10% of diseased lettuce plants infected by FOLAC. Therefore, this molecular technique has been shown to be useful for establishing the soil inoculum thresholds required for crop infection by pathogens, while reducing the time and execution tasks necessary to perform soil infectivity bioassays.