Recombinase Polymerase Amplification–Lateral Flow Dipstick Assay for Rapid Detection of Fusarium circinatum Based on a Newly Identified Unique Target Gene

Abstract

Pitch canker caused by the fungus Fusarium circinatum is an important disease affecting pine trees in Europe and South Africa. Several countries, including China, have listed F. circinatum as a quarantine pathogen. Therefore, timely detection of F. circinatum could efficiently prevent its introduction into new areas or facilitate spread management in already infected sites. In this study, a recombinase polymerase amplification–lateral flow dipstick (RPA-LFD) assay was developed for rapid detection of F. circinatum based on a new target gene, Fcir2067, identified from whole-genome sequences. The assay was highly specific to F. circinatum. In fact, it exclusively detected F. circinatum isolates; 53 isolates of fungal and oomycete species and 2 nematodes of Bursaphelenchus xylophilus and B. mucronatus were not detected. By detecting as little as 10 pg of F. circinatum genomic DNA in a 50-µl reaction, the RPA-LFD assay was 10 times more sensitive than conventional PCR assays. F. circinatum was also detected in artificially inoculated pine needles of Cedrus deodara. These results demonstrated that the developed RPA-LFD assay has the potential for rapid detection of F. circinatum in regions at high risk of infection. The RPA-LFD assay might serve as an alternative method for the early detection of F. circinatum.