Identification of microRNAs associated with defense against Bursaphelenchus xylophilus in Pinus koraiensis and their potential as early diagnostic targets for pine wilt disease

BACKGROUND Pine Wilt Disease (PWD), a devastating pine disease caused by Bursaphelenchus xylophilus , severely threatens forest ecosystems. microRNAs (miRNAs) are crucial in host‐pathogen interactions, but pine miRNA research is limited and their function in PWD remains unclear. Additionally, PWD lacks effective early diagnostic methods, hindering timely spread control. This study focuses on Pinus koraiensis to explore its miRNAs' role in PWD defense and potential as early diagnostic targets. RESULTS We identified 1026 miRNAs via small RNA high‐throughput sequencing, 149 of which were differentially expressed between infected and control groups. Functional enrichment analysis showed that these miRNAs regulate genes related to defense pathways, lipid metabolism, and organic acid metabolism. To overcome PWD diagnostic limitations, we developed the CRISPR/Cas12a‐assisted ligation‐initiated loop‐mediated isothermal amplification (CAL‐LAMP) method, targeting novel‐miR574 and novel‐miR544 with high sensitivity (detection limits as low as 1 amol L −1 ) and specificity. CONCLUSIONS This study developed a novel and rapid diagnostic tool specifically for early‐stage PWD—a critical advancement given the long‐standing lack of effective early detection methods for the disease. By targeting specific miRNAs, the CAL‐LAMP method was capable of detecting target miRNAs in asymptomatic P. koraiensis as early as 10 days post‐inoculation under experimental conditions, with high sensitivity and specificity. This early diagnostic capability may provide a useful tool for the timely initiation of disease management measures, although further validation in field settings is necessary to confirm its practical utility.