A panel of qPCR assays to detect and quantify soybean soil-borne pathogens

Fusarium oxysporum,F. graminearum,F. acuminatum,F. equiseti,F. proliferatum,F. solani, and Rhizoctonia solani are soil-borne fungal pathogens that cause substantial yield loss in a widespread list of crops worldwide. The objective of this study was to develop a panel of TaqMan assays for the detection and quantification of these six widespread soil-borne fungal species using real-time polymerase chain reaction (qPCR). The primers and probes were designed based on the intergenic spacer ribosomal RNA and translation elongation factor 1-alpha gene (tef1). These assays, although not multiplexed, can be performed simultaneously as they have similar reaction conditions, allowing more efficiency when targeting multiple pathogens in a sample. The assays presented high efficiency (94.3%-108.9%) and sensitivity, with a limit of detection of 0.05 picograms (50 femtograms) of target DNA. Results from an assay targeting 19 non-target and closely related species confirmed the specificity of the developed assays. The assays were also evaluated to detect the target species in different matrices, such as soil and plant material. This panel of qPCR assays is an additional tool that can be used by plant pathologists, microbiologists, plant breeders, diagnostic clinics, and other researchers interested in these fungal species.