Background: The pine wood nematode (PWN;Bursaphelenchus xylophilus) is the most damaging biological pest in pine forest ecosystems in China. However, the pathogenic mechanism remains unclear. Tracheid cavitation induced by excess metabolism of volatile terpenes is a typical characteristic of pine trees infected byB. xylophilus. β-pinene, one of the main volatile terpenes, influences PWN colonization and reproduction, stimulating pathogenicity during the early stages of infection. To elucidate the response mechanism of PWN to β-pinene, pathogenesis, mortality, and reproduction rate were investigated under different concentrations of β-pinene using a transcriptomics approach.Results: A low concentration of β-pinene (BL, C < 25.74 mg/ml) inhibited PWN reproduction, whereas a high concentration (BH, C > 128.7 mg/ml) promoted reproduction. Comparison of PWN expression profiles under low (BL, 21.66 mg/ml) and high (BH, 214.5 mg/ml) β-pinene concentrations at 48 h identified 659 and 418 differentially expressed genes (DEGs), respectively, compared with controls. Some key DEGs are potential regulators of β-pinene via detoxification metabolism (cytochrome P450, UDP-glucuronosyltransferases and short-chain dehydrogenases), ion channel/transporter activity (unc and ATP-binding cassette families), and nuclear receptor -related genes. Gene Ontology enrichment analysis of DEGs revealed metabolic processes as the most significant biological processes, and catalytic activity as the most significant molecular function for both BL and BH samples. Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO) analysis showed that xenobiotics biodegradation and metabolism, carbohydrate metabolism, lipid metabolism, amino acid metabolism, metabolism of cofactors and vitamins, and transport and catabolism were the dominant terms in metabolism categories.Conclusion: In addition to detoxification via reduction/oxidation (redox) activity, PWN responds to β-pinene through amino acid metabolism, carbohydrate metabolism, and other pathways including growth regulation and epidermal protein changes to overcome β-pinene stress. This study lays a foundation for further exploring the pathogenic mechanism of PWN.

Poplar are important forestry species in China, but the Botryosphaeria dothidea pathogen causes serious economic losses worldwide. To identify candidate B. dothidea resistance proteins and explore the molecular mechanisms involved in poplar–pathogen interactions, proteomic responses of stem samples from resistant and susceptible poplar ecotypes to B. dothidea were investigated using nanoflow liquid chromatography-tandem mass spectrometry with label-free quantitative analysis. We identified 588 proteins, divided into 21 biological process categories including 48 oxidoreductases, 72 hydrolytic enzymes, 80 metabolic enzymes, and 29 proteins of unknown function. Differential proteome analysis revealed large differences between resistant Populus tomentosa Carr and susceptible Populus beijingensis Hsu ecotypes before and after inoculation. Among 102 identified proteins, 22 were highly upregulated in the resistant genotype but downregulated in the susceptible genotype. Proteins induced in P. tomentosa Carr in response to B. dothidea are associated with plant defenses including oxidoreductase activity (catalase, isocitrate dehydrogenase, and superoxide dismutase), phenylpropanoid biosynthesis and phenylalanine metabolism (alcohol dehydrogenase), photosynthesis (ATP synthase subunit alpha, ATP synthase gamma chain, photosystem I P700 chlorophyll a apoprotein A2, photosystem II CP47 chlorophyll apoprotein), carbon fixation (pyruvate kinase, triosephosphate isomerase, malic enzyme, phosphoglycerate kinase, ribulose-1,5-bisphosphate carboxylase, and ribulose bisphosphate carboxylase small chain), and glycolysis/gluconeogenesis (fructose-bisphosphate aldolase). Kyoto Encyclopedia of Genes and Genomes pathway analysis identified 168 proteins related to metabolic pathways, 41 proteins related to the biosynthesis of phenylpropanoids, and 36 proteins related to the biosynthesis of plant hormones, the biosynthesis of alkaloids derived from ornithine, lysine, and nicotinic acid, and photosynthesis in response to B. dothidea. Our findings provide insight into plant–pathogen interactions in resistant and susceptible poplar ecotypes infected with B. dothidea and could assist the development of novel strategies for fighting poplar canker disease.

The pine wood nematode Bursaphelenchus xylophilus is a destructive species affecting pine trees worldwide; however, the underlying mechanism leading to pathogenesis remains unclear. In this study, a B. xylophilus gene encoding thaumatin-like protein-1 (Bx-tlp-1) was silenced by RNA interference to clarify the relationship between the Bx-tlp-1 gene and pathogenicity. The in vitro knockdown of Bx-tlp-1 with double-stranded RNA (dsRNA) decreased B. xylophilus reproduction and pathogenicity. Treatments with dsRNA targeting Bx-tlp-1 decreased expression by 90%, with the silencing effect maintained even in the F3 offspring. Pine trees inoculated with B. xylophilus treated with Bx-tlp-1 dsRNA decreased the symptom of wilting, and the disease severity index was 56.7 at 30 days after inoculation. Additionally, analyses of the cavitation of intact pine stem samples by X-ray microtomography revealed that the xylem cavitation area of pine trees inoculated with B. xylophilus treated with Bx-tlp-1 dsRNA was 0.46 mm²at 30 days after inoculation. Results from this study indicated that the silencing of Bx-tlp-1 has effects on B. xylophilus fitness. The data presented here provide the foundation for future analyses of Bx-tlp-1 functions related to B. xylophilus pathogenicity.

Poplar canker, mainly caused by Botryosphaeria species, is a serious disease that has resulted in the reduced productivity and death of poplar worldwide. Different Populus species have varied resistance levels to poplar canker; however, whether phenolic compounds in poplar are involved in this resistance remains uncertain. Here, we determined the concentrations of phenolic compounds and their antifungal activities in canker-resistant P. tomentosa and canker-susceptible P. beijingensis. We identified 29 phenolic compounds with significantly different concentrations between the two species. Salicylic acid (SA), tremuloidin, salicin, and poplin were dominant in P. tomentosa, while benzoic acid (BA) and catechol were dominant in P. beijingensis. These six phenolic compounds were further tested for antifungal activities. SA, BA, and catechol showed significant antifungal activities against Botryosphaeria dothidea, while the other three compounds showed no activity. As BA or catechol can be converted in plants to SA, which is a remote signaling molecule that plays an important role in plant defenses, we presumed that the resistance of P. tomentosa was initiated by SA, while the susceptibility of P. beijingensis resulted from the absence of SA. Further studies are required to confirm this hypothesis. These results provide a foundation for future research on the disease-resistance mechanisms of poplar.

Pine wilt disease is a major forest disease worldwide, including in China, where it has severely damaged pine forest ecosystems, and the pathogen is pine wood nematode (Bursaphelenchus xylophilus). The thaumatin-like protein-1 gene (Bx-tlp-1) is a key gene associated with B. xylophilus pathogenicity, which is also responsive to α-pinene. In this study, an examination of Pinus massoniana seedlings infected by B. xylophilus revealed that monoterpene (sesquiterpene) levels peaked on days 15 and 27 (days 18 and 27). Meanwhile, P. massoniana Pm-tlp expression levels were high on days 3, 12, and 27, which were consistent with the expression of key enzymes genes in the terpene biosynthesis pathway. The functional similarity of B. xylophilus Bx-TLP-1 and P. massoniana Pm-TLP suggests Bx-TLP-1 and Pm-TLP may have similar roles in P. massoniana. There was also no secondary accumulation of terpenes in P. massoniana seedlings during B. xylophilus treated with dsRNA targeting Bx-tlp-1 (dsTLP1) infections, reflecting the decreased pathogenicity of B. xylophilus and the delayed disease progression in pine trees. And the results of micro-CT showed that the degree of cavitation for the trees inoculated with Bx-TLP-1 (0.3811 mm³) was greater than that for the trees inoculated with dsTLP1 PWNs (0.1204 mm³) on day 15 after inoculation. Results from this study indicated that B. xylophilus Bx-tlp-1 gene may induce the upregulated expression of related genes encoding enzymes in the terpene synthesis pathway of P. massoniana, resulting in the accumulation of terpenes, which also provided an insight to investigate the B. xylophilus pathogenicity in the future.

Pine wood nematode, Bursaphelenchus xylophilus, is a worldwide pest of pine trees, spreading at an alarming rate and with great ecological adaptability. In the process of causing disease, the nematode causes metabolic disorders and changes in the endophytic microbial community of the pine tree. However, the changes at the pine nidus during early nematode invasion have not been well studied, especially the differential metabolites, in Pinus massoniana, the main host of B. xylophilus in China. In this study, we analyzed the endophytic bacterial and fungal communities associated with healthy and B. xylophilus-caused wilted pine trees. The results show that 1333 bacterial OTUs and 502 fungal OTUs were annotated from P. massoniana stem samples. The abundance of bacterial communities in pine trees varies more following infection by B. xylophilus, but the abundance changes of fungal communities are less visible. There were significant differences in endophytic microbial diversity between wilted and healthy P. massoniana. In wilted pine trees, Actinobacteria and Bacteroidia were differential indicators of bacterial communities, whereas, in healthy pine trees, Rhizobiales in the Proteobacteria phylum were the major markers of bacterial communities. Meanwhile, the differential markers of fungal communities in healthy pines are Malasseziales, Tremellales, Sordariales, and Fusarium, whereas Pleosporaceae is the key marker of fungal communities in wilted pines. Our study examines the effect of changes in the endophytic microbial community on the health of pine trees that may be caused by B. xylophilus infection. In parallel, a non-targeted metabolomic study based on liquid mass spectrometry (LC-MS) technology was conducted on pine trees inoculated with pine nematodes and healthy pine trees with a view to identifying key compounds affecting early pine lesions. Ultimately, 307 distinctly different metabolites were identified. Among them, the riboflavin metabolic pathway in pine trees may play a key role in the early pathogenesis of pine wood nematode disease.