Development of loop-mediated isothermal amplification assays for the rapid and accurate diagnosis of Exserohilum turcicum for field applications.

Abstract

Northern corn leaf blight (NCLB), caused by Exserohilum turcicum, is one of the most devastating foliar diseases of maize. Rapid and accurate diagnosis for this disease is urgently needed but still limited. Here, we establish a field-deployable diagnostic method to detect E. turcicum based on loop-mediated isothermal amplification (LAMP) assays. A software application called K-mer Elimination by Cross-reference (KEC) was utilized to search for the specific sequences belonging to E. turcicum by comparing the whole genome sequence between E. turcicum and other known maize pathogens. Five LAMP primer sets were designed based on specific and single-copy fragments of E. turcicum. Post-LAMP analyses indicated that only the primer set, Et9468_set1, was the most suitable, producing a ladder-like amplification pattern in the agarose gel electrophoresis and a strong fluorescence signal in the presence of SYBR Green I. The LAMP assay using Et9468_set1 primers demonstrated a high level of specificity in distinguishing E. turcicum from six other common fungal pathogens of maize, as well as 12 more fungal and oomycete strains including the epiphytic fungi from maize leaves and other crop pathogens. Moreover, it exhibited remarkable sensitivity by detecting 5 copies per reaction which was approximately 104 times more sensitive compared to conventional PCR. The LAMP assay successfully detected E. turcicum in field maize leaves without DNA extraction, demonstrating its suitability for rapid on-spot detection of NCLB. Our study provides a direct LAMP diagnostic method to detect E. turcicum, which enables on-site pathogen detection in the field and the development of preventive strategies for NCLB management.