Loop-mediated isothermal amplification (LAMP) assay for specific and rapid detection of Dickeya fangzhongdai targeting a unique genomic region

Abstract

Dickeya fangzhongdai, a bacterial pathogen of taro (Colocasia esculenta), onion (Allium sp.), and several species in the orchid family (Orchidaceae), causing soft rot and bleeding canker diseases. No field-deployable diagnostic tool is available for specific detection of this pathogen in different plant tissues. Therefore, we developed a field-deployable loop-mediated isothermal amplification (LAMP) assay using a unique genomic region, present exclusively in D. fangzhongdai. Multiple genomes of D. fangzhongdai, and other species of Dickeya, Pectobacterium and unrelated genera were used for comparative genomic analyses to identify an exclusive and conserved target sequence from the major facilitator superfamily (MFS) transporter gene region. This gene region had broad detection capability for D. fangzhongdai and thus was used to design primers for endpoint PCR and LAMP assays. In-silico validation showed high specificity with D. fangzhongdai genome sequences available in the NCBI GenBank genome database as well as the in-house sequenced genome. The specificity of the LAMP assay was determined with 96 strains that included all Dickeya species and Pectobacterium species as well as other closely related genera, and 5 hosts; no false positives or false negatives were detected. The detection limit of the assay was determined by performing four sensitivity assays with 10-fold serially diluted purified genomic DNA of D. fangzhongdai with and without the presence of crude host extract (taro, orchid, and onion). The detection limit for all sensitivity assays was 100 fg (18–20 genome copies) with no negative interference by host crude extracts. The assays were performed by five independent operators (blind test) and on three instruments (Rotor-Gene, thermocycler and dry bath); the assay results were concordant. The assay consistently detected the target pathogen from artificially inoculated and naturally infected host samples. The developed assay is highly specific for D. fangzhongdai and has applications in routine diagnostics, phytosanitary and seed certification programs, and epidemiological studies.

Dickeya fangzhongdai was prevalent and caused taro soft rot when coexisting with the Pectobacterium complex, with a preference for Araceae plants

“…Taro (Colocasia esculenta) is a world widely cultivated aroid plant featured with large starchy edible corms, and is essential for food security, especially in Asia, Africa, and the Pacific islands; in 2016, this crop had a global production value of 4.1 billion dollars (Delude et al, 2022). This crop has been widely grown in the Yangtze River and Pearl River basins in China.…”