Search Results (217)

Pinus massoniana Lamb. is an economically important conifer native to China. However, it is highly susceptible to the pine wood nematode (Bursaphelenchus xylophilus, PWN), the causal agent of pine wilt disease (PWD), resulting in substantial ecological and economic losses. To elucidate potential molecular defense mechanisms, 50 NAC (NAM, ATAF1/2, and CUC2) transcription factors (PmNACs) were identified in the P. massoniana genome. Phylogenetic analysis divided these PmNACs into seven subfamilies, and motif analysis identified ten conserved motifs associated with stress responses. Twenty-three genes were selected for expression analysis in various tissues and under exogenous salicylic acid (SA), methyl jasmonate (MeJA), and PWN infection. Six genes (PmNAC1, PmNAC8, PmNAC9, PmNAC17, PmNAC18, and PmNAC20) were significantly up-regulated by both hormonal treatment and PWN infection, implying their involvement in JA/SA-mediated immune pathways. Functional characterization showed PmNAC8 is a nuclear-localized transcription factor with autoactivation activity. Furthermore, transient overexpression of PmNAC8 in Nicotiana benthamiana induced reactive oxygen species (ROS) accumulation and necrotic lesions. Collectively, these results elucidate NAC-mediated defense responses to PWN infection in P. massoniana and identify candidate genes for developing PWD-resistant pine varieties. Full article

Pine wilt disease, a devastating disease severely impacting pine ecosystems, is caused by the pinewood nematode Bursaphelenchus xylophilus (Steiner & Bührer, 1934) Nickle, 1970 (Nematoda: Parasitaphelenchidae). Controlling B. xylophilus is crucial for preventing and managing pine wilt disease. Recently discovered novel nematocidal lectins could provide more advantageous materials for utilizing genetically engineered bacteria to control this pathogen. Therefore, this study focuses on identifying novel nematocidal toxins within B. xylophilus lectins. Overall, we obtained twenty-one galectin, one L-type lectin (LTL), and three chitin-binding domain (CBD) genes by screening the B. xylophilus genome database; these genes were successfully expressed proteins. The bioassay results indicated that Bxgalectin2, Bxgalectin3, Bxgalectin4, Bxgalectin9, and BxLTL1 induced mortality rates exceeding 50% in B. xylophilus. Notably, Bxgalectin4 showed the strongest nematocidal activity, causing 88% mortality in the treated nematode population. The enzyme-linked immunosorbent assays further demonstrated that Bxgalectin3 (K_d = 8.992 nM) and Bxgalectin4 (K_d = 9.634 nM) had a higher binding affinity to GPI-anchored proteins from B. xylophilus. Additionally, Bxgalectin2 (K_d = 16.50 nM), Bxgalectin9 (K_d = 16.48 nM), and BxLTL1 (K_d = 24.34 nM) can bind to the GPI-anchored protein. This study reports, for the first time, that lectins endogenous to B. xylophilus exhibit nematocidal activity against their own species. These findings open up the possibility of using nematode lectins as potent control agents in the biological control of B. xylophilus. Full article

Pine wilt disease (PWD), caused by Bursaphelenchus xylophilus Steiner & Buhrer (pine wood nematodes, PWN), impacts forest carbon sequestration and climate change. However, satellite-based PWD monitoring is challenging due to the limited spatial resolution of Sentinel’s MSI sensor, which reduces its sensitivity to subtle biochemical alterations in foliage. We have, therefore, developed a slope product index (SPI) for effective detection of PWD using single-date satellite imagery based on spectral gradients in the visible and near-infrared (VNIR) range. The SPI was compared against 15 widely used vegetation indices and demonstrated superior robustness across diverse test sites. Results show that the SPI is more sensitive to changes in chlorophyll content in the PWD detection, even under potentially confounding conditions such as drought. When integrated into Random Forest (RF) and Back-Propagation Neural Network (BPNN) models, SPI significantly improved classification accuracy, with the multivariate RF model achieving the highest performance and univariate with SPI in BPNN. The generalizability of SPI was validated across test sites in distinct climate zones, including Zhejiang (accuracyZ_Mean = 88.14%) and Shandong (accuracyS_Mean = 78.45%) provinces in China, as well as Portugal. Notably, SPI derived from Sentinel-2 imagery in October enables more accurate and timely PWD detection while reducing field investigation complexity and cost. Full article

Pine wilt disease (PWD), a destructive pine forest disease caused by pine wood nematode (PWN), Bursaphelenchus xylophilus, has led to huge economic losses and ecological environment damage. Thaumatin-like proteins (TLPs) are the products of a complex gene family involved in host defense and a wide range of developmental processes in fungi, plants, and animals. In this study, a tlp gene of B. xylophilus (Bxtlp) (GenBank: OQ863020.1) was amplified via PCR and cloned into the expression vector pET-15b to construct the recombinant vector PET-15b-Bxtlp, which was then transformed into Escherichia coli BL-21(DE3). The recombinant protein was successfully purified using Ni-NTA affinity chromatography. The effect of the Bxtlp gene on the vitality and pathogenicity of PWNs was elucidated through RNA interference (RNAi) and overexpression. Bxtlp dsRNA significantly reduced the feeding, motility, spawning, and reproduction abilities of PWN; shortened its lifespan; and increased the female–male ratio. In contrast, the recombinant BxTLP markedly enhanced the reproductive ability of PWN. In addition, Bxtlp dsRNA increased reactive oxygen species (ROS) content in nematodes, while the recombinant BxTLP was confirmed to have antioxidant capacity in vitro. Furthermore, the bioassays on Pinus thunbergii saplings demonstrated that Bxtlp could significantly influence PWN pathogenicity. Overall, we speculate that Bxtlp affects the pathogenicity of PWNs mainly via regulating ROS levels, the motility, and hatching of PWN. Full article

Corymbia citriodora is a eucalypt tree of significant economic value due to its essential oils (EOs), rich in citronellal, citronellol, and other oxygenated monoterpenes with diverse biological activities. Its EOs show potential for the formulation of biopesticides with a lower impact on the environment and human health. This study evaluated the in vitro nematicidal activity of C. citriodora EOs, obtained from in vivo and in vitro grown plants, and their main volatile compounds against the pinewood nematode (PWN, Bursaphelenchus xylophilus), a major phytosanitary threat. The impact of their main compounds on the environment and human health was assessed using available experimental data and predictions from specialized software. Citronellal and citronellol were the most active EO compounds and exhibited EC₅₀ values comparable to the pesticide emamectin benzoate (0.364 ± 0.009 mg/mL). They also displayed superior safety profiles, with reduced environmental persistence and toxicity to non-target organisms. Furthermore, C. citriodora shoots were efficiently propagated through an in vitro system and their volatile profile was characterized by a dominance of citronellal (64%), and citronellol (10%), which highlights their potential as a scalable and sustainable source of nematicidal compounds. Remarkably, the EO of C. citriodora in vitro shoots was strongly active against the PWN, exhibiting the lowest EC₅₀ (0.239 ± 0.002 mg/mL) obtained. These findings underline the viability of C. citriodora EOs as a promising alternative for sustainable pest management, addressing the urgent need for environmentally friendly and health-conscious biopesticides while providing a renewable approach to nematode control. Full article

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a pathogenic organism that causes pine wilt disease (PWD). To date, several molecular diagnostic methods have been developed; however, rapid, convenient, and inexpensive field diagnostic tools for detecting PWN are still limited. In this study, an enzyme-mediated duplex exponential amplification (EmDEA) method for detecting PWN from extracted nematodes or pinewood sawdust was developed and tested. This method comprised an EmDEA molecular test kit, which consisted of freeze-dried enzyme pellets that can be stored at room temperature (approximately 20–25 °C) for one year, a dry block heater, and a portable isothermal fluorescence amplification instrument. The whole procedure was completed within 30 min. The EmDEA assay could detect a single PWN at all life stages from a mixture of other nematode species or from pinewood sawdust. The detection limit was 10 copies (plasmid weight 32.66 ag) or 1/500 of that of a single adult PWN per reaction. Therefore, the EmDEA assay has potential applications in PWN detection in the field, as well as quarantine inspection in international trade. Moreover, modification of primers and probes will allow the rapid identification of other nematode species. Full article

The pine wood nematode (Bursaphelenchus xylophilus, PWN) is a globally significant quarantine pest that causes severe economic and ecological damage to coniferous forests worldwide. Additionally, PWNs continue to expand into higher latitudes. However, studies on their cold tolerance remain limited. This study investigated the overwintering environment of PWNs in epidemic areas of Liaoning Province, China. It established a protocol to induce anhydrobiosis in PWNs, evaluated their low-temperature resistance, observed morphological changes during anhydrobiosis, and explored potentially involved key genes. The results showed that (1) there was no significant difference in thermal insulation between infected and healthy wood in Liaoning Province; both effectively reduced temperature fluctuation rates, providing a protective function for PWN overwintering. (2) PWNs significantly enabled their cold tolerance through anhydrobiosis, accompanied by significant morphological changes and substantial lipid droplet depletion. (3) Eleven anhydrobiosis-related genes were identified. Among these, the collagen gene family showed consistent expression patterns throughout dehydration and rehydration. This suggests a potential role in cuticle structural changes and osmoregulation during anhydrobiosis. These findings provide a theoretical basis for understanding how PWNs survive winter conditions in high-latitude regions. Additionally, they offer valuable insights for future research into PWN anhydrobiosis and the development of effective control strategies. Full article

Pine wilt disease (PWD), caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus), poses a significant threat to global pine forests and calls for the development of innovative management strategies. Microbial control emerges as an effective, cost-efficient, and environmentally sustainable approach to eliminate the damage from PWD. This review consolidates molecular mechanisms in the microbiological control of PWD, which focus on three core strategies: microbial control activity against PWN, biological control of vector insects, and the enhancement of host tree resistance to nematode infections. The review thoroughly evaluates integrated control strategies in which microbial control is used in traditional management practices. Recent studies have pinpointed promising microbial agents for PWN control, such as nematophagous microorganisms, nematicidal metabolites, parasitic fungi that target vector insects, and microbes that boost plant resistance. In particular, the control potential of volatile organic compounds (VOCs) produced by microorganisms against PWN and the enhancement of pine resistance to PWN by microorganisms were emphasized. Moreover, we assessed the challenges and opportunities associated with the field application of microbiological control agents. We emphasized the feasibility of multi-strategy microbial integrated control, which provides a framework for future studies on microbial-based PWD control strategies. Full article

Pinus massoniana Lamb. (masson pine) is a critical species for afforestation in southern China but faces severe threats from pine wilt disease (PWD) caused by Bursaphelenchus xylophilus. To accelerate disease-resistant breeding, this study investigated the effects of cryopreservation on the embryonic capacity of the embryogenic callus as well as the effects of abscisic acid (ABA), polyethylene glycol 8000 (PEG 8000) and phytagel concentration on the somatic embryo’s maturation and germination. Furthermore, the impact of transplanting substrates on the survival and growth of regenerated plantlets were evaluated. The results showed that cryopreservation at −196 °C effectively maintained the embryogenic potential of the callus, with post-thaw tissues exhibiting superior somatic embryo maturation capacity compared to the long-term subcultured callus (38.4 vs. 13.2 embryos/mL). Key maturation parameters were systematically optimized: ABA concentration at 6 mg/L in the suspension culture maximized embryo yield of 24.1 somatic embryos/mL, while PEG 8000 at 130 g/L in solid medium achieved peak embryo production of 38.4 somatic embryos/mL, and the maximum of 26.6 somatic embryos/mL when the concentration of phytagel was 3.5 g/L. The highest germination rate of 29.8% was observed with 130 g/L PEG in the maturation medium. The highest survival rate (56.5%) and maximum plant height (22.3 cm) after 12 months of transplantation were achieved in substrates consisting of soil and vermiculite, which outperformed those containing varying proportions of mushroom residue. This study establishes a scalable protocol for the mass propagation of PWD-resistant P. massoniana, integrating cryopreservation and maturation media optimization, which offers dual benefits for disease-resistant breeding and sustainable germplasm conservation. Full article

Pine wilt disease, caused by the nematode Bursaphelenchus xylophilus, poses a significant threat to pine forests worldwide. Understanding the dynamics of its spread is crucial for effective disease management. In this study, we investigated the involvement of asymptomatic carrier trees in the expansion of pine wilt disease through a series of experiments. Cage-releasing experiments revealed that sexually immature Japanese pine sawyer beetles, Monochamus alternatus, feeding on healthy pine branches drops only a minimal number of nematodes (primary infection). However, sexually mature beetles, still harboring numerous nematodes, fly to weakened trees for breeding and extend their feeding activities to healthy pines around weakened trees, infecting them with nematodes and thus spreading the disease further. Inoculation experiments on field-planted black pine seedlings demonstrated that even a small number of nematodes can lead to a high occurrence of asymptomatic carrier trees. Our findings suggest that nematode infections transmitted by sexually mature Monochamus beetles significantly contribute to the expansion of pine wilt damage and play a crucial role in the persistence of asymptomatic carrier trees. This conclusion is based on cage-release experiments demonstrating nematode transmission by mature beetles and inoculation experiments highlighting the conditions leading to asymptomatic carrier trees. Full article

To understand the spread of the pine wood nematode Bursaphelenchus xylophilus (Steiner and Buhrer, 1934; Nickle, 1970) and identify infected trees, the dispersal ability of insects was examined. In this study, the effects of sex, adult age, and morphological traits on the flight performance (such as flight distance, time, and speed) of Monochamus alternatus (Hope, 1843) were investigated using flight mills. Over a period of 8 weeks, a total of 42 newly emerged adult beetles were tethered to flight mills once a week. Flight distance and time were recorded for 2 h during each session. The total flight distance of individuals that flew more than 100 m during each session was calculated. The results showed that females flew an average of 6.65 km, for 59.6 min, while males flew an average of 9.89 km, for 85.8 min. In a single flight experimental session, both sexes could fly up to approximately 2 km and were found to fly at an average speed of 1.9 m s⁻¹. In our principal component analysis (PCA), the first and second principal components were found to be highly correlated with the sex, morphological traits, and flight performances of M. alternatus. This study demonstrates that the flight ability of M. alternatus varies significantly according to body size and sex, which is fundamental and crucial for understanding the dispersal patterns of pine wilt disease. Full article

Bursaphelenchus xylophilus is a pine wood nematode capable of destroying pine forests. Exploring the genes providing resistance to this pathogen and understanding their resistance mechanisms is thus necessary and constitutes an effective way to tackle this problem. We used Pinus tabuliformis Carriere to dissect its response to B. xylophilus strain BxFC. The 30 d inoculation results showed that the P. tabuliformis germplasms exhibited a wide resistance spectrum. Some lines were sensitive with the needles fully wilted and the MDA content and the relative conductivity of needles greatly increased, while some lines demonstrated strong resistance with good needle vigor and better physiological conditions. Moreover, the transcriptome analysis revealed 7928 differentially expressed genes (DEGs) between the resistant and sensitive germplasm pools, including 3754 upregulated and 4174 downregulated genes in the resistant lines. These DEGs were specially enriched in the pathways of plant–pathogen interaction (318 genes), phenylpropanoid biosynthesis (108 genes), ubiquitin-mediated proteolysis (47 genes), carotenoid biosynthesis (18 genes), and monoterpenoid biosynthesis (9 genes). Accordingly, P. tabuliformis utilized multiple ways to control the proliferation and activity of B. xylophilus, such as immune response, ubiquitination, thickening plant cell walls, and increasing its terpenoid and antioxidant contents. Our results could thus help in better understanding the resistance process of P. tabuliformis against B. xylophilus and offer some new strategies and gene resources for a molecular breeding program of resistant P. tabuliformis. Full article

Bursaphelenchus xylophilus, the causative agent of pine wilt disease (PWD), poses a severe global threat to coniferous forests. Esteya vermicola, an endoparasitic nematophagous fungus, exhibits promising biocontrol potential against this pinewood nematode. The vesicular transport system, evolutionarily conserved in eukaryotes, is essential for fungal pathogenicity. Based on our genome sequence of E. vermicola CBS115803, we identified EvSec22, a gene encoding a SNARE protein implicated in vesicular transport process. This study investigates the role of EvSec22 in E. vermicola during nematode infection, utilizing our optimized gene knockout methodology. Infection assays revealed that EvSec22 deletion significantly impaired the pathogenicity of E. vermicola against B. xylophilus. Phenotypic analyses revealed that the ΔEvSec22 mutant exhibited suppressed hyphal growth, reduced conidiation, and abnormal septal spacing. Furthermore, the mutant showed significantly diminished tolerance to osmotic stress (sorbitol) and oxidative stress (hydrogen peroxide). Overall, the EvSec22 gene is associated with the virulence of E. vermicola CBS115803 against B. xylophilus, and its deletion also affects the normal growth of E. vermicola and its tolerance to abiotic stress. This study providing new insights into SNARE protein functions in fungal biocontrol agents. Full article

Glutamate-gated chloride channels (GluCls), a class of ion channels found in the nerve and muscle cells of invertebrates, are involved in vital life processes. Bursaphelenchus xylophilus, the pathogen of pine wilt disease, has induced major economic and ecological losses in invaded areas of Asia and Europe. We identified 33 GluCls family members by sequence alignment analysis. A subsequent bioinformatic analysis revealed the physicochemical properties, protein structure, and gene expression patterns in different developmental stages. The results showed that GluCls genes are distributed across all six chromosomes of B. xylophilus. These proteins indicated a relatively conserved structure by NCBI-conserved domains and InterPro analysis. A gene structure analysis revealed that GluCls genes consist of 5 to 14 exons. Expression pattern analysis revealed BxGluCls were extensively involved in the development of second instar larvae of B. xylophilus. Furthermore, BxGluCls15, BxGluCls25, and BxGluCls28 were mainly associated with the development of eggs of B. xylophilus. BxGluCls12, BxGluCls18, and BxGluCls32 were predominantly linked to nematode resistance and adaptation. Investigation the structure and expression patterns of BxGluCls is crucial to understand the developmental trends of B. xylophilus. It also helps identify molecular targets for the development of biopesticides or drugs designed to control this nematode. Full article

Pine wilt disease, caused by Bursaphelenchus xylophilus, poses severe ecological and economic threats to coniferous forests. This study isolated two fungal (Arthropsis hispanica, Penicillium sclerotiorum) and two bacterial (Bacillus amyloliquefaciens, Enterobacter hormaechei) strains from Pinus massoniana rhizospheres, evaluating their biocontrol potential against pine wood nematodes. Molecular characterization confirmed strain identities. In vitro assays demonstrated that combined fermentation filtrates of CSX134+CSZ71 and CSX60+CSZ71 significantly enhanced plant growth parameters (height, biomass) and root-associated soil enzyme activities (urease, acid phosphatase) in P. massoniana. Treated plants exhibited elevated defense enzyme activities and upregulated defense-related gene expression. The treatments achieved 75.07% and 69.65% nematode control efficacy, respectively, compared to controls. These findings highlight the potential of microbial consortia in activating systemic resistance and suppressing pine wilt disease through the dual mechanisms of growth promotion and defense induction. Full article