Robust design of LAMP assays for in-field detection of major bacterial vascular diseases of banana.

Bacterial diseases of banana are a growing global threat, causing yield losses and increased management costs. Major diseases include Moko, banana blood disease (BBD), and banana Xanthomonas wilt (BXW), caused by Ralstonia solanacearum, Ralstonia syzygii subsp. celebesensis, and Xanthomonas vasicola pv. musacearum, respectively. Effective surveillance requires point-of-care diagnostics such as loop-mediated isothermal amplification (LAMP) for on-site use. We aimed to develop three LAMP assays to specifically detect the bacteria responsible for Moko, BBD and BXW, directly from banana tissues, using a simplified DNA extraction protocol. The BBD - and BXW-LAMP assays demonstrated 100% specificity, yielding negative results for a broad range of non-target bacteria, including closely related species as well as pathogenic and endophytic strains associated with banana, and positive results for all the tested target strains. For Moko disease, a duplex-LAMP assay was developed to detect all strains from the four globally most relevant sequevars: IIB-3, IIB-4, IIA-6, and IIA-24. The duplex-LAMP successfully detected all target strains, except one that was shown to be non-pathogenic to Cavendish bananas. All non-target strains tested negative, with the exception of a delayed signal for one strain belonging to Ralstonia thomasi, not associated with banana environment (hospital strain). These results were supported by an extensive in silico analysis conducted on 9,668 Burkholderiaceae and 7,483 Xanthomonadaceae genomes. Detection limits ranged from 0.1 pg/µl to 1 pg/µl DNA, and from 104 to 105 CFU/ml on banana tissues spiked with calibrated bacterial suspensions, depending on the assay. The LAMP assays prove highly effective for detecting target pathogens in both artificially inoculated banana plants and field samples, offering a promising tool for improving disease management strategies.