Abstract
Bacterial soft rot caused by Pectobacterium species is a serious disease in konjac (Amorphophallus konjac), a healthy source of starch particularly in East Asia. An effective diagnostic method is crucial to control the disease and reduce losses in konjac production. In this study, we evaluated a loop-mediated isothermal amplification (LAMP) assay with a specific primer set for the rapid and accurate detection of P. aroidearum. A comparative genomics approach was used to identify the specific genes suitable for the design of LAMP primers. The candidate target genes were determined through a first-round comparison with a 50-genome nucleotide database, and subjected to a second-round screening with the GenBank NR database. As a result, nine specific genes of P. aroidearum were selected for LAMP primer design. After screening of the primers, the primer set 1675-1 was chosen for LAMP detection owing to its high specificity and sensitivity. The LAMP assay could detect the presence of P. aroidearum genomic DNA at a concentration as low as 50 fg and 1.2 × 104 CFU/g artificially infected soil within 40 min at 65 °C. Subsequently, this primer set was successfully used to specifically detect P. aroidearum in naturally infected and non-symptomatic plant samples or soil samples from the field. This study indicates that a comparative genomic approach may facilitate the development of highly specific primers for LAMP assays, and a LAMP diagnostic assay with the specific primer set 1675-1 should contribute to the rapid and accurate detection of soft-rot disease in konjac at an early stage.
Highlights
Konjac (Amorphophallus konjac) is a perennial herbaceous species that mainly grows in Southeast Asia and Africa [1]
We developed a loop-mediated isothermal amplification (LAMP) method for the specific detection of P. aroidearum based on primers derived from comparative genomics
We first used 16S ribosomal DNA (rDNA) sequencing and the 35 strains were placed as Pectobacterium, while the 15 strains were placed as Dickeya (D. fangzhongdai)
Summary
Konjac (Amorphophallus konjac) is a perennial herbaceous species that mainly grows in Southeast Asia and Africa [1]. Bacterial soft rot of konjac was mainly caused by Pectobacterium carotovorum subsp. We confirmed that the main pathogen causing bacterial soft rot on konjac from Yichang, China is P. aroidearum using multilocus sequence typing (MLST) analysis (Figure S1). Once the konjac corms are infected, there is no effective method to control bacterial soft rot. We developed a loop-mediated isothermal amplification (LAMP) method for the specific detection of P. aroidearum based on primers derived from comparative genomics. LAMP assays have been applied to the diagnosis of some Pectobacterium species [28] These assays are not capable of detecting P. aroidearum. We developed a novel gene target specific to P. aroidearum for LAMP detection using a comparative genomics approach. We validated the applicability of the LAMP assay in naturally infected konjac and soil samples from the field
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