Bursaphelenchus xylophilus is fatal to the pine trees around the world. The production of the pine tree secondary metabolite gradually increases in response to a B. xylophilus infestation, via a stress reaction mechanism(s). α-pinene is needed to combat the early stages of B. xylophilus infection and colonization, and to counter its pathogenesis. Therefore, research is needed to characterize the underlying molecular response(s) of B. xylophilus to resist α-pinene. We examined the effects of different concentrations of α-pinene on the mortality and reproduction rate of B. xylophilus in vitro. The molecular response by which B. xylophilus resists α-pinene was examined via comparative transcriptomics of the nematode. Notably, B. xylophilus genes involved in detoxification, transport, and receptor activities were differentially expressed in response to two different concentrations of α-pinene compared with control. Our results contribute to our understanding of the molecular mechanisms by which B. xylophilus responds to monoterpenes in general, and the pathogenesis of B. xylophilus.

Bursaphelenchus xylophilus is a nematode species that has damaged pine trees worldwide, but its pathogenesis has not been fully characterized. α-pinene helps protect host species during the early B. xylophilus infection and colonization stages. In this study, we identified potential molecular mimicry proteins based on a comparative transcriptomic analysis of B. xylophilus. The expression levels of three genes encoding secreted B. xylophilus proteins were influenced by α-pinene. We cloned one gene encoding a thaumatin-like protein, Bx-tlp-2 (accession number MK000287), and another gene encoding a cysteine proteinase inhibitor, Bx-cpi (accession number MK000288). Additionally, α-pinene appeared to induce Bx-tlp-1 expression, but had the opposite effect on Bx-cpi expression. An analysis of the expression of the potential molecular mimicry proteins in B. xylophilus infecting pine trees revealed that the α-pinene content was consistent with the expression levels of Bx-tlp-1 (Bx-cpi) and Pm-tlp (Pm-cpi) over time. Thus, these genes likely have important roles contributing to the infection of pine species by B. xylophilus. The results of this study may be relevant for future investigations of the functions of Bx-tlp-1, Bx-tlp-2 and Bx-cpi, which may provide a point to explore the relationship between B. xylophilus and host pines.

Studying sex communication is necessary to develop new methods to control the population expansion of gonochoristic species Bursaphelenchus xylophilus, the pathogen of pine wilt disease (PWD). Small chemical signals called ascarosides have been reported to attract potential mates. However, they have not been studied in the sex attraction of B. xylophilus. Here, we confirmed the sex attraction of B. xylophilus using a chemotaxis assay. Then, we cloned the downstream ascaroside biosynthetic gene Bx-daf-22 and explored its function in the sex attraction of B. xylophilus through bioinformatics analysis and RNA interference. The secretions of females and males were the sources of sex attraction in B. xylophilus, and the attractiveness of females to males was stronger than that of males to females. Compared with daf-22 of Caenorhabditis elegans, Bx-daf-22 underwent gene duplication events, resulting in Bx-daf-22.1, Bx-daf-22.2, and Bx-daf-22.3. RNA interference revealed that the attractiveness of female secretions to males increased after all three Bx-daf-22 genes or Bx-daf-22.3 had been interfered. However, the reciprocal experiments had no effect on the attractiveness of male secretions to females. Thus, Bx-daf-22 genes, especially Bx-daf-22.3, may be crucial for the effectiveness of female sex attractants. Our studies provide fundamental information to help identify the specific components and signal pathways of sex attractants in B. xylophilus.

Pinewood nematode, Bursaphelenchus xylophilus, a pine-parasitic nematode, poses a serious threat to pine trees globally, causing pine wilt disease. When dispersal-stage juvenile 4 (dauer, J_IV, a durable stage) of B. xylophilus enters the new pine, it transforms into a propagative adult (dauer moulting) and reproduces quickly. Our previous studies have found that pine-volatile β-pinene promotes dauer moulting of B. xylophilus; however, this mechanism is not clear. Here, this study is attempting to unravel the molecular process underlying dauer moulting of B. xylophilus through signal chemical tests and transcriptome analysis. The results showed that β-pinene could promote dauer moulting of B. xylophilus, while other common dauer moulting signals, such as dafachronic acid (DA), part of the TGF/insulin signal pathway, were inoperative. Moreover, the J_IV soaked in 1% β-pinene for only 6 h could transform into adults at a significant rate. Therefore, the transcriptomes of J_IV soaked in 1% β-pinene for 6 h were sequenced. It was found that 15,556 genes were expressed; however, only 156 genes were expressed differentially and enriched in the metabolism of xenobiotics, peroxisome, fatty acid metabolism, and carbon metabolism, indicating that energy metabolism was active at the early stage of dauer moulting. With a stricter parameter, the number of differential genes fell to 19, including 4 sterol hydroxylase, 5 dehydrogenase, 2 glucuronosyltransferase, 5 nuclear-related factor, 1 calcium-binding protein, 1 nitrogen metabolic regulation protein, and 1 cystathionine gamma-lyase. These results indicated that dauer moulting of B. xylophilus into adults might not be regulated by the TGF-β/insulin signal pathway but by another new signal pathway related to the 19 differential genes which need more exploration. Our results contribute to the understanding of the molecular mechanisms behind dauer moulting and may be useful in reducing pine wilt disease by suppressing this moulting to cut the life cycle of B. xylophilus.

Pine wilt disease (PWD), caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus), is one of the most serious threats to pine forests worldwide. The fungus Esteya vermicola, with its lunate conidia capable of parasitizing the PWN, has shown promise as an efficient biological control agent against PWD. Solid-state fermentation (SSF) is preferred for large-scale applications in the field, as it facilitates microbial agent transport and ensures a long shelf life. However, research on enhancing the yield of lunate conidia from E. vermicola through SSF is limited. In this study, we initially achieved a yield of 3.04 × 10⁸ conidia/g using a basic SSF medium composed of wheat bran, corn flour, and soybean flour. To improve this yield, we employed an orthogonal experimental design (OED) to identify the optimal medium composition, which required a wheat bran-to-corn flour-to soybean flour ratio of 7:2:1 (w/w/w), a substrate-to-water ratio of 1:0.7 (w/v), and the addition of 1.33% (w/w) glucose, 1.33% (w/w) yeast extract fermentation, and 1.33% (w/w) MgSO₄. Using the response surface methodology (RSM), we calculated the optimal fermentation conditions, which were 24.9 °C, 78.0% relative humidity (RH), an inoculation volume of 16.3% (v/w), and a fermentation time of 7.1 days. Under these conditions, the yield of lunate conidia reached a maximum of 16.58 × 10⁸ conidia/g, a 4.45-fold increase after optimization. This study improved the yield of E. vermicola lunate conidia and provides insights for developing biopesticides based on this strain.