Detection of the gummy stem blight pathogens (Stagonosporopsis spp.) in watermelon using field-adapted LAMP assays and rapid DNA extraction protocols

Gummy stem blight (GSB), caused by three Stagonosporopsis species, S. citrulli, S. cucurbitacearum and S. caricae, is one of the most economically important diseases hindering watermelon production worldwide. Since there is no commercial resistance to GSB in watermelon cultivars, its management depends on cultural practices and preventative fungicides. Therefore, efficient methods for the detection of Stagonosporopsis species that could aid management decisions are required. To help achieve this, a loop-mediated isothermal amplification (LAMP) assay specific to S. citrulli (SCIT850) was developed under two detection formats: fluorescence quantification and endpoint colorimetric detection. The SCIT850 assay was determined to be specific to its target species and exhibited a consistent sensitivity of 1 pg of genomic DNA under both formats. The assay can be combined with a previously reported LAMP assay for the collective detection of the three Stagonosporopsis spp. (STAGY), which have comparable sensitivity to SCIT850 and can aid in species discrimination. A field diagnostic system for GSB-causing Stagonosporopsis was developed by coupling the SCIT850 and STAGY assays to quick DNA extraction protocols. Two DNA extraction methods were tested: one from leaves using cellulose dipsticks, and one from steel rods (typical of spore traps) using Chelex100. With the dipstick method, we detected pathogen DNA in inoculated asymptomatic, mildly infected, and severely infected plants, while with the Chelex100 we detected pathogen DNA from rods infested with as few as 500 spores. The SCIT850 and STAGY assays coupled with these quick sample processing methods could be adapted for field deployment, which would allow growers to make efficient and timely management decisions based on detection of the actual Stagonosporopsis species present in the field.