Recombinase Polymerase Amplification Assay for Rapid Field Diagnosis of Stewart’s Wilt of Corn Pathogen Pantoea stewartii subsp. stewartii

Abstract

Stewart’s vascular wilt and leaf blight of sweet corn is caused by the Gram-negative enteric bacterium Pantoea stewartii subsp. stewartii. Stewart’s wilt results in substantial yield losses worldwide warranting rapid and accurate disease diagnosis. Recombinase polymerase amplification (RPA) is an isothermal technique that is tolerant to host plant-derived inhibitors and is, therefore, ideally suited for rapid in-field detection vis-à-vis traditional polymerase chain reaction-based molecular assays. An RPA assay coupled with a Lateral Flow Device (LFD) was developed for rapid, accurate, and sensitive real-time detection of P. stewartii subsp. stewartii directly from the infected host offering in-field pathogen detection, timely disease management, and satisfying quarantine and phytosanitary requirements. Twelve novel primer sets were designed against conserved genomic regions of P. stewartii subsp. Stewartii; however, only the primers for amplification of the intergenic spacer region between capsular polysaccharide genes cpsA and cpsB were discernibly unique and adequate for unambiguous identification of P. stewartii subsp. stewartii. The P. stewartii subsp. stewartii-specific primers were further validated in a simplex RPA assay for specificity against twenty-six bacterial species representing several Pantoea and other closely related bacterial species/subspecies/strains found in the same niche, and naturally or artificially infected plant samples. The integrated RPA/LFD assay was also optimized for rapid and sensitive on-site detection of P. stewartii subsp. stewartii with an empirical detection limit of 0.0005 pg μL−1 bacterial DNA and 1 × 102 CFU mL−1 (app. two bacterial cells used per RPA reaction) in minimally processed samples for accurate, low-cost, and point-of-need diagnosis of the quarantine pathogen P. stewartii subsp. stewartii.