Genes Encoding Potential Molecular Mimicry Proteins as the Specific Targets for Detecting Bursaphelenchus xylophilus in PCR and Loop-Mediated Isothermal Amplification Assays

Abstract

The introduction of the pine wood nematode (Bursaphelenchus xylophilus) to new areas has affected the international forestry industry because this pathogen causes pine wilt disease (PWD). Therefore, methods for the accurate and reliable detection of B. xylophilus are essential for controlling and managing this pest. The PCR and Loop-Mediated Isothermal Amplification (LAMP) techniques developed in this study involve species-specific primer sets targeting B. xylophilus genes encoding potential molecular mimicry proteins (Bx-tlp-1, Bx-tlp-2, and Bx-cpi), which are associated with pathogenicity. The PCR and LAMP results revealed that the primers were specific for B. xylophilus Bx-tlp-1, Bx-tlp-2, and Bx-cpi. Moreover, our LAMP assay targeting Bx-tlp-1 conducted at 63°C detected B. xylophilus within 20 min and B. xylophilus from Monochamus alternatus or M. saltuarius within 30 min. The lower limits of detection for the LAMP and PCR assays were 10 pg and 10 ng genomic DNA, respectively, implying these assays may be useful for the rapid detection of B. xylophilus in pine forests. Designing primers specific for Bx-tlp-1, Bx-tlp-2, and Bx-cpi enabled the relatively rapid detection of B. xylophilus isolates as well as M. alternatus or M. saltuarius carrying B. xylophilus. These primers, which were designed following a thorough functional analysis of key B. xylophilus pathogenicity-related genes, may be useful for developing improved assays for the early diagnosis and prevention of PWD.

Evolution of parasitism genes in the plant parasitic nematodes

“…These nematodes secrete proteins that contain a conserved 12-amino acid C-terminal motif, which is highly similar to plant CLAVATA3/ESR (CLE) ligand peptides 48 . Similarly, migratory endoparasitic nematodes like B. xylophilus have been found to produce thaumatin-like proteins and a cystain-like peptidase inhibitor 12 , 49 , which agreed with the present study. Recent studies have identified several proteins in plant-parasitic nematodes that could mimic host plant peptidase and peptidase inhibitors.…”

Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

“…LAMP ( Notomi et al., 2000 ) is a highly efficient nucleic acid amplification technology that can potentially improve disease diagnosis in plant protection. LAMP technology has been applied for detecting B. xylophilus and the methods have constantly been improved ( Kikuchi et al., 2009 ; Leal et al., 2015 ; Meng et al., 2022 ). LAMP assays can detect B. xylophilus with high sensitivity and efficiency; however, interpretation of LAMP reaction results has been a critical issue.…”

“…Isothermal amplification approaches, such as loop-mediated isothermal amplification (LAMP) and recombinant polymerase amplification (RPA), have been successfully applied to detect B. xylophilus . These approaches have eliminated the need for thermocycling instruments and can be used for real-time detection, thereby supporting field and point-of-care testing ( Zhou et al., 2022 ; Meng et al., 2022 ). However, these technologies generally are unable to distinguish nucleotide sequences that differ in only one, or even several, bases; they need to be optimized, and may result in false positives because of non-specific amplification, cross-contamination, and/or primer dimerization ( Wang et al., 2018 ; Joung et al., 2020 ).…”