Pine wilt disease (PWD), which poses a significant risk to pine plantations across the globe, is caused by the pathogenic agent Bursaphelenchus xylophilus, also referred to as the pine wood nematode (PWN). A droplet digital PCR (ddPCR) assay was developed for the quick identification of the PWN in order to improve detection sensitivity. The research findings indicate that the ddPCR assay demonstrated significantly higher analysis sensitivity and detection sensitivity in comparison to traditional quantitative PCR (qPCR). However, it had a more limited dynamic range. High specificity was shown by both the ddPCR and qPCR techniques in the diagnosis of the PWN. Assessments of reproducibility revealed that ddPCR had lower coefficients of variation at every template concentration. Inhibition tests showed that ddPCR was less susceptible to inhibitors. There was a strong linear association between standard template measurements obtained using ddPCR and qPCR (Pearson correlation = 0.9317; p < 0.001). Likewise, there was strong agreement (Pearson correlation = 0.9348; p < 0.001) between ddPCR and qPCR measurements in the evaluation of pine wood samples. Additionally, wood samples from symptomatic (100% versus 86.67%) and asymptomatic (31.43% versus 2.9%) pine trees were diagnosed with greater detection rates using ddPCR. This study’s conclusions highlight the advantages of the ddPCR assay over qPCR for the quantitative detection of the PWN. This method has a lot of potential for ecological research on PWD and use in quarantines.

Catalases play a crucial role in the metabolism of reactive oxygen species (ROS) by converting H₂O₂ into molecular oxygen and water. They also contribute to virulence and fungal responses to various stresses. Previously, the MrCat1-deletion mutant (ΔMrCat1) was generated using the split-marker method in Metarhizium rileyi. In this study, the Cat1 gene was identified, and its function was evaluated. Under normal culture conditions, there were no significant differences in colony growth or dimorphic switching between ΔMrCat1 and the wild-type (WT) strains. However, under oxidative stress, the colony growth was inhibited, and the yeast–hyphal transition was suppressed in the ΔMrCat1 strain. Hyperosmotic stress did not differ significantly between the two strains. In the ΔMrCat1 strain, microsclerotia (MS) formation was delayed, resulting in less uniform MS size and a 76% decrease in MS yield compared to the WT strain. Moreover, the ΔMrCat1 strain exhibited diminished virulence. Gene expression analysis revealed up-regulation of ΔMrCat1, MrCat2, MrCat4, and MrAox in the ΔMrCat1 strain. These findings indicate that the MrCat1 gene in M. rileyi is essential for oxidative stress tolerance, MS formation, and virulence.