A TaqMan(®) Assay Allows an Accurate Detection and Quantification of Fusarium spp., the Causal Agents of Tomato Wilt and Rot Diseases

SIMPLE SUMMARY: In tomato plants, Fusarium spp. have been increasingly associated with several wilt and rot diseases that are responsible for severe yield losses. In this context, a molecular-based tool was developed to increase the accuracy of detection and quantification of Fusarium spp. genomic DNA (gDNA) in tomato plants and to discriminate Fusarium spp. from other fungal species that affect tomato. This assay revealed to be highly specific and sensitive for Fusarium species. The used methodology also allowed the establishment of an absolute DNA quantification method. Finally, the effectiveness of the assay was successfully validated with the detection and quantification of Fusarium spp. in potentially infected tomato plants from an experimental field and in control plants grown under controlled conditions. The established methodology allows a reliable, sensitive, and reproducible estimation of Fusarium accumulation in infected tomato plants, gaining new insights for disease control and providing an additional tool in the screening of resistant plants. ABSTRACT: In tomato plants, Fusarium spp. have been increasingly associated with several wilt and rot diseases that are responsible for severe yield losses. Here, we present a real-time PCR TaqMan(®) MGB (Minor Groove Binder) assay to detect and discriminate Fusarium spp. from other fungal species that affect tomato plants. The methodology used is based on the selective amplification of the internal transcribed spacer (ITS) region of Fusarium spp. This assay revealed to be highly specific and sensitive for Fusarium species, targeting only the 29 Fusarium isolates from the 45 tested isolates associated to tomato diseases. Sensitivity was assessed with serial dilutions of Fusarium genomic DNA, with the limit of detection of 3.05 pg. An absolute DNA quantification method was also established, based on the determination of the absolute number of target copies. Finally, the effectiveness of the assay was successfully validated with the detection and quantification of Fusarium spp. in potentially infected tomato plants from an experimental field and in control plants grown under controlled conditions. The established methodology allows a reliable, sensitive, and reproducible estimation of Fusarium accumulation in infected tomato plants, gaining new insights for disease control and providing an additional tool in the screening of resistant plants.