Microscopic and LF-RPA assay approaches to the detection of the fungal peanut pathogen Cercospora arachidicola

Abstract

Peanut brown spot, caused by the fungus Cercospora arachidicola, represents a serious threat to global peanut production and is associated food security. The mechanisms whereby this fungus can penetrate the epidermis of infected plants, however, remain poorly understood. In this study, we found that C. arachidicola is able to enter pores and extend hyphae into leaves, thereby infecting peanut plants. Developing a tool that is well-suited to diagnose peanut brown spot in its early stages is vital to manage this disease effectively in the field. To that end, we combined a simplified DNA extraction method with a newly developed lateral flow strip-based recombinase polymerase amplification (LF-RPA) assay in order to facilitate the rapid and equipment-free detection of C. arachidicola. This assay recognizes highly conserved internal transcribed spacer (ITS) sequences associated with this pathogen and functions across a wide temperature range (25–45 °C). This approach positively identified all 12 tested C. arachidicola, without any cross-reactivity with other tested Cercospora species or fungal species. It was also able to detect DNA concentrations as low as 10−4 ng, making it 10 times more sensitive than traditional PCR-based approaches. By utilizing this LF-RPA assay in combination with a simplified PEG-NaOH-based approach to extracting DNA from plant samples, we were able to execute the entire protocol in just 30 min without the need for special equipment, thus allowing us to detect C. arachidicola in artificially infected peanut plants. The LF-RPA assay established herein is simple, rapid, and cost-effective, and is potentially amenable to further development as a diagnostic kit for the diagnosis of peanut brown spot in resource-limited settings or the field.