Abstract
Pectobacterium parmentieri (formerly Pectobacterium wasabiae), which causes soft rot disease in potatoes, is a newly established species of pectinolytic bacteria within the family Pectobacteriaceae. Despite serious damage caused to the potato industry worldwide, no field-deployable diagnostic tests are available to detect the pathogen in plant samples. In this study, we aimed to develop a reliable, rapid, field-deployable loop-mediated isothermal amplification (LAMP) assay for the specific detection of P. parmentieri. Specific LAMP primers targeting the petF1 gene region, found in P. parmentieri but no other Pectobacterium spp., were designed and validated in silico and in vitro using extensive inclusivity (15 strains of P. parmentieri) and exclusivity (94 strains including all other species in the genus Pectobacterium and host DNA) panels. No false positives or negatives were detected when the assay was tested directly with bacterial colonies, and with infected plant and soil samples. Sensitivity (analytical) assays using serially diluted bacterial cell lysate and purified genomic DNA established the detection limit at 10 CFU/mL and 100 fg (18–20 genome copies), respectively, even in the presence of host crude DNA. Consistent results obtained by multiple users/operators and field tests suggest the assay’s applicability to routine diagnostics, seed certification programs, biosecurity, and epidemiological studies.
Highlights
Pectobacterium parmentieri, which causes soft rot disease in potatoes, is a newly established species of pectinolytic bacteria within the family Pectobacteriaceae
Since soft rot diseases are caused by a complex of bacterial pathogens of different species and phenotypes, detection of an individual bacterial species requires a robust, accurate diagnostic tool[13]
The loop-mediated isothermal amplification (LAMP) assay has gained popularity for pathogen detection and point-of-need application[18,19]. This isothermal nucleic acid amplification technique is based on auto-cyclic amplification and a high DNA strand displacement activity facilitated by a Bacillus stearothermophilus (Bst) polymerase[18,20]
Summary
Pectobacterium parmentieri (formerly Pectobacterium wasabiae), which causes soft rot disease in potatoes, is a newly established species of pectinolytic bacteria within the family Pectobacteriaceae. Despite serious damage caused to the potato industry worldwide, no field-deployable diagnostic tests are available to detect the pathogen in plant samples. We aimed to develop a reliable, rapid, field-deployable loop-mediated isothermal amplification (LAMP) assay for the specific detection of P. parmentieri. The LAMP assay has gained popularity for pathogen detection and point-of-need application[18,19] This isothermal nucleic acid amplification technique is based on auto-cyclic amplification and a high DNA strand displacement activity facilitated by a Bacillus stearothermophilus (Bst) polymerase[18,20]. Several closely related Pectobacterium species cause soft rot and blackleg diseases in potatoes and shared high pairwise homology in their genomic regions. Primer specificity for P. parmentieri is important since it occurs in highly heterogeneous populations in different geographic locations[28] This selectivity eliminates cross-reactivity with non-target pathogens
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