Bursaphelenchus xylophilus is a dangerous quarantine pest that causes extensive damage to pine ecosystems worldwide. Cyclobutrifluram, a succinate dehydrogenase inhibitor (SDHI), is a novel nematicide introduced by Syngenta in 2013. However, the nematocidal effect of cyclobutrifluram against plant-parasitic nematodes remains underexplored. Therefore, here, we aim to address this knowledge gap by evaluating the toxicity, effects, and mode of action of cyclobutrifluram on B. xylophilus. The result shows that cyclobutrifluram is the most effective agent, with an LC₅₀ value of 0.1078 mg·L⁻¹. At an LC₂₀ dose, it significantly reduced the population size to 10.40 × 10³ ± 737.56—approximately 1/23 that of the control group. This notable impact may stem from the agent’s ability to diminish egg-laying and hatching rates, as well as to impede the nematodes’ development. In addition, it has also performed well in the prevention of pine wilt disease, significantly reducing the incidence in greenhouses and in the field. SDH consists of a transmembrane assembly composed of four protein subunits (SDHA to SDHD). Four sdh genes were characterized and proved by RNAi to regulate the spawning capacity, locomotion ability, and body size of B. xylophilus. The mortality of nematodes treated with sdhc-dsRNA significantly decreased upon cyclobutrifluram application. Molecular docking further confirmed that SDHC, a cytochrome-binding protein, is the target. In conclusion, cyclobutrifluram has a good potential for trunk injection against B. xylophilus. This study provides valuable information for the screening and application of effective agents in controlling and preventing PWD in forests.

Bursaphelenchus xylophilu is a worldwide quarantine nematode, causing huge economic losses and ecological disasters in many countries. The sex ratio of B. xylophilus plays an important role in the nematode infestation. The laf-1-related genes are highly conserved in animals, playing crucial roles in sex determination. Therefore, we investigated the expression pattern and biological function of its orthologue, Bxy-laf-1 in B. xylophilus. Bxy-laf-1 has two typical conserved DNA-binding domains, DEAD and Helicase C. The real-time quantitative PCR data revealed that Bxy-laf-1 expression was required throughout the entire life of B. xylophilus, with the maximum expression in the J2 stage and the lowest expression in the adult stage. mRNA in situ hybridization showed that Bxy-laf-1 is mainly located in the cephalopharynx and reproductive organs of B. xylophilus. RNA interference (RNAi) indicated that the head swing frequency was dramatically decreased. The RNA interference results displayed that a significant reduction in motility was observed in the hatched larvae. The female to male sex ratio was also decreased in the F0 and F1 generations, but recovered in the F2 generation. The tail of female adults with eggs in the belly appeared deformities. This phenomenon appeared in the F0 and F1 generations, but recovered in the F2 generation. Bxy-laf-1 is a typical sex-determination gene with distinct expression patterns in males and females. As demonstrated in other species, the sex ratio was altered after knocking down Bxy-laf-1 expression. The results of this study contribute to our understanding of the molecular processes of Bxy-laf-1 in B. xylophilus, which may provide clues about how to control pine wilt disease by inhibiting ontogenic growth and reducing nematode fertility.

Bursaphelenchus xylophilus is a notorious invasive species, causing extensive losses to pine ecosystems globally. Previous studies had shown that the development of B. xylophilus was seriously suppressed by light. However, the mechanism involved in the inhibition is unknown. Here, it is the first report that Bxy-madd-4 is a light-regulated gene, plays a potential role in B. xylophilus in responding to the blue light. Transcriptome sequencing revealed that the expression level of Bxy-madd-4 declined by 86.39% under blue light. The reverse transcription quantitative real-time PCR results were in accord with the transcriptome sequencing, confirming the expression level of Bxy-madd-4 was suppressed by blue light. Bxy-madd-4 promoter::mCherry reporter constructed in Caenorhabditis elegans were utilized to mimic the spatiotemporal expression patterns of Bxy-madd-4. Bxy-madd-4A promoter activity had a strong continuity throughout all development stages in C. elegans. Further RNA interference indicated that only 36.8% of the Bxy-madd-4 dsRNA treated embryos were hatched. Moreover, 71.6% of the hatched nematodes were abnormal, such as particles on the body surface and concave tissues. Our findings contribute towards a better understanding of the mechanism of light against the destructive invasive nematode, providing a promising hint for control of the destructive invasive nematode.

Bursaphelenchus mucronatus is the sibling species of B. xylophilus, which causes pine wilt disease. Sex pheromone-mediated mating behavior underlies the development of B. xylophilus populations. The study of the molecular mechanism of sex pheromone receptor genes is vital for the control of B. xylophilus. The pivotal role of the 5-HT receptor ser-1 in Caenorhabditis elegans’s mating has been demonstrated, but there is little known in B. mucronatus and B. xylophilus. In our study, the molecular features and biological functions of Bmu-ser-1 are explored. qPCR results showed that Bmu-ser-1 was expressed at all ages, especially at J2 and J4, with significantly high expression. Notably, the expression levels in males were significantly higher than that in females. The results of in situ hybridization suggest that the Bmu-ser-1 gene was expressed at the site of the digestive system during the embryonic stage, the whole body during the J2 stage, and mainly at the end of the germinal primordium during the J3 stage, as well as centrally in the vulva of females and in the gonads and tail of males during the J4 and adult stages. The RNAi results indicate a significant decrease in hatchability and stagnation at the J1 stage after interference. Treated J2 had reduced motility and stunted growth. Males after interference showed mismatch and females showed spawning difficulties. Sexual arousal experiments further validated Bmu-ser-1 as a receptor gene for males receiving female sex pheromones. Collectively, our findings strongly suggest that the Bmu-ser-1 gene has a classical role, the control of nematode growth; and a novel role involved in mating. The study on the molecular mechanisms of growth and reproduction in B. mucronatus could provide a reference for understanding the population expansion and disease epidemics of B. xylophilus.