Rapid Detection of Pythium Soft Rot in Ginger (Zingiber officinale) Caused by Pythium myriotylum Using LAMP

Pythium soft rot in ginger, caused by Pythium myriotylum, leads to significant reductions in ginger yields. In this study, a loop-mediated isothermal amplification (LAMP) detection method was established for Py. myriotylum in infected ginger plants and infested soil. The Py. myriotylum genome was compared with the genomes of seven additional oomycete species to obtain species-specific sequences. LAMP primer sets were designed and assessed at temperatures from 58 to 68°C and tested for their specificity. Furthermore, the sensitivity of the screened primer set was evaluated, and the detection limit was 1 pg of Py. myriotylum genomic DNA (gDNA) in a 25-μl reaction volume. LAMP was used to detect Py. myriotylum zoospores in solution (1 × 10²/ml), vermiculite (1 × 10²/g), and soil (1 × 10⁴/g). A rapid and portable gDNA extraction method from ginger seedlings and rhizomes using solid-phase reversible immobilization beads for purification was adopted. The LAMP assay could reliably detect Py. myriotylum in artificially inoculated diseased ginger seedlings and rhizosphere vermiculite. The portable gDNA extraction method coupled with the LAMP assay facilitates point-of-care pathogen detection, requiring 2 to 3 h for eight samples, greatly reducing the time needed for Py. myriotylum detection of Pythium soft rot in ginger.