Search Results (8)

The pine wood nematode (Bursaphelenchus xylophilus), the pathogen of pine wilt disease (PWD), has caused enormous economic losses in Asian forests. Whether venom allergen proteins (VAPs) are involved in the accumulation of key defense substances in pine trees during the interaction between B. xylophilus and host trees, and their specific function as putative effectors secreted through stylets, has not been fully elucidated. In this study, the role of the BxVAP2 effector protein in the infection process was analyzed through bioinformatics and phylogenetic tree construction. The expression profile of BxVAP2 during infection was analyzed using qRT-PCR, and its expression under the stress of Pinus massoniana metabolites was examined. Toxicity assays were conducted through the Agrobacterium transient expression of BxVAP2 in Nicotiana benthamiana, and its subcellular localization was investigated. The results showed that BxVAP2 contains a CAP domain and shares close evolutionary relationships with venom allergen proteins from related species, such as Bursaphelenchus mucronatus, Aphelenchoides besseyi, Aphelenchoides fujianensis, and Meloidogyne graminicola. BxVAP2 was upregulated during the infection of P. massoniana, indicating that BxVAP2 is a key effector in the infection and colonization process of B. xylophilus and may play an important role during the rapid population growth phase. BxVAP2 responds to P. massoniana metabolites, where different concentrations of α-pinene suppressed its expression, while high concentrations of β-pinene promoted its expression. Subcellular localization revealed that BxVAP2 localizes to the cell membrane and nucleus. The transient expression of BxVAP2 in N. benthamiana induced programmed cell death and regulated pattern-triggered immunity marker genes. These findings suggest that BxVAP2 plays an important role in the interaction between B. xylophilus and its host, responding to terpene stress and triggering plant defense. Full article

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. Full article

Wood- and bark-inhabiting parasitic nematodes are of great economic importance. Nematodes can cause wilt diseases in conifers and deciduous trees. In 2014–2022, during nematology surveys conducted in different regions of Russia and Belarus, adults and dauer juveniles of nematodes were collected from wood, bark and beetle vectors. Using traditional morphological taxonomic characters integrated with molecular criteria, we identified in the studied samples the following nematode species: Aphelenchoides heidelbergi, Bursaphelenchus eremus, B. fraudulentus, B. michalskii, B. mucronatus, B. willibaldi, Deladenus posteroporus, Diplogasteroides nix and Laimaphelenchus hyrcanus, several unidentified species: Aphelenchoides sp.1 and sp.2, Cryptaphelenchus sp.1, sp.2 and sp.3, Laimaphelenchus sp.1, Micoletzkya sp.1, Parasitaphelenchus sp.1, Parasitorhabditis sp.1, three unidentified tylenchid nematodes and a new species, Bursaphelenchus zvyagintsevi sp.n. Morphological descriptions and molecular characterization are provided for B. zvyagintsevi sp. n. belonging to the Abietinus group and B. michalskii belonging to the Eggersi group. Findings of Aphelenchoides heidelbergi, Bursaphelenchus eremus, B. michalskii, Deladenus posteroporus, Diplogasteroides nix and Laimaphelenchus hyrcanus are new records for Russia. Phylogenetic positions of studied species were reconstructed using D2–D3 expansion segments of 28S rRNA gene sequence analysis. The data obtained in this study may help to detect the refugia of opportunistic plant pests and find possible native biocontrol nematode agents of insect vectors causing diseases. Full article

The pinewood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine-wilt disease (PWD), is a threat to Pinus forests in Asia and Europe. Bursaphelenchus mucronatus and B. fraudulentus are closely related non-pathogenic pine-wood nematodes. In the present work, four medium-chain aliphatic alcohols (C10 to C13) were evaluated in direct-contact bioassays against B. xylophilus, B. mucronatus and B. fraudulentus. The compounds showed high nematicidal activity against the species tested. The lowest values for half-maximal effective concentrations (EC₅₀) were determined in B. xylophilus and B. fraudulentus, suggesting a higher sensibility to these compounds. Further bioassays will include compounds with different chain lengths and functional groups to explore the diversity in the activity of oxygen-containing aliphatic compounds for a more targeted sustainable-control strategy for the PWN. Full article

We determined the mitochondrial gene sequence of Monochamus alternatus and three other mitogenomes of Lamiinae (Insect: Coleoptera: Cerambycidae) belonging to three genera (Aulaconotus, Apriona and Paraglenea) to enrich the mitochondrial genome database of Lamiinae and further explore the phylogenetic relationships within the subfamily. Phylogenetic trees of the Lamiinae were built using the Bayesian inference (BI) and maximum likelihood (ML) methods and the monophyly of Monochamus, Anoplophora, and Batocera genera was supported. Anoplophora chinensis, An. glabripennis and Aristobia reticulator were closely related, suggesting they may also be potential vectors for the transmission of the pine wood pathogenic nematode (Bursaphelenchus xylophilus) in addition to M. alternatus, a well-known vector of pine wilt disease. There is a special symbiotic relationship between M. alternatus and Bursaphelenchus xylophilus. As the native sympatric sibling species of B. xylophilus, B. mucronatus also has a specific relationship that is often overlooked. The analysis of mitochondrial gene expression aimed to explore the effect of B. mucronatus on the energy metabolism of the respiratory chain of M. alternatus adults. Using RT-qPCR, we determined and analyzed the expression of eight mitochondrial protein-coding genes (COI, COII, COIII, ND1, ND4, ND5, ATP6, and Cty b) between M. alternatus infected by B. mucronatus and M. alternatus without the nematode. Expression of all the eight mitochondrial genes were up-regulated, particularly the ND4 and ND5 gene, which were up-regulated by 4–5-fold (p < 0.01). Since longicorn beetles have immune responses to nematodes, we believe that their relationship should not be viewed as symbiotic, but classed as parasitic. Full article

The Bursaphelenchus mucronatus, which was highly similar with Bursaphelenchus xylophilus in terms of morphological characteristics and biological properties—but had weaker pathogenicity to forests—was a native species often displaced by B. xylophilus when occupying the same niche. Since the draft genome of the invasive B. xylophilus has been published, the absence of a reference genome of B. mucronatus still prevents us from understanding the molecular evidences behind competitive displacement. In this study, we employed Single Molecule, Real-Time (SMRT) sequencing and a Hi-C scaffolding approach to yield a near chromosome-level assembly of B. mucronatus, including six pseudo-chromosomes. The assembly size is 73 Mb, with scaffold N50 of 11.50 Mb and contig N50 of 1.48 Mb. Comparative genomics results showed high similarity between B. xylophilus and B. mucronatus. However, the losing of orphan genes and species-specific orthologous genes in B. mucronatus may indicate weaker adaptability to the environment. The gene family contractions of GPCRs (G Protein-Coupled Receptors) and cellulases in B. mucronatus may jointly contribute to its displacement by B. xylophilus. Overall, we introduced a valuable genomic resource for molecular and evolutionary studies of B. mucronatus, especially for studying the competitive displacement by the pinewood nematode, which could help us control the pathogenicity of pine wilt diseases. Full article

Bursaphelenchus mucronatus (B. mucronatus) isolates that originate from different regions may vary in their virulence, but their virulence-associated genes and proteins are poorly understood. Thus, we conducted an integrated study coupling RNA-Seq and isobaric tags for relative and absolute quantitation (iTRAQ) to analyse transcriptomic and proteomic data of highly and weakly virulent B. mucronatus isolates during the pathogenic processes. Approximately 40,000 annotated unigenes and 5000 proteins were gained from the isolates. When we matched all of the proteins with their detected transcripts, a low correlation coefficient of r = 0.138 was found, indicating probable post-transcriptional gene regulation involved in the pathogenic processes. A functional analysis showed that five differentially expressed proteins which were all highly expressed in the highly virulent isolate were involved in the pathogenic processes of nematodes. Peroxiredoxin, fatty acid- and retinol-binding protein, and glutathione peroxidase relate to resistance against plant defence responses, while β-1,4-endoglucanase and expansin are associated with the breakdown of plant cell walls. Thus, the pathogenesis of B. mucronatus depends on its successful survival in host plants. Our work adds to the understanding of B. mucronatus’ pathogenesis, and will aid in controlling B. mucronatus and other pinewood nematode species complexes in the future. Full article

The pine wood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease. Accurately differentiating B. xylophilus from other nematodes species, especially its related species B. mucronatus, is important for pine wood nematode detection. Thus, we attempted to identify a specific protein in the pine wood nematode using proteomics technology. Here, we compared the proteomes of B. xylophilus and B. mucronatus using Two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization -time-of-flight/time-of-flight (MALDI-TOF/TOF-MS) technologies. In total, 15 highly expressed proteins were identified in B. xylophilus compared with B. mucronatus. Subsequently, the specificity of the proteins identified was confirmed by PCR using the genomic DNA of other nematode species. Finally, a gene encoding a specific protein (Bx-Prx) was obtained. This gene was cloned and expressed in E. coli. The in situ hybridisation pattern of Bx-Prx showed that it was expressed strongly in the tail of B. xylophilus. RNAi was used to assess the function of Bx-Prx, the results indicated that the gene was associated with the reproduction and pathogenicity of B. xylophilus. This discovery provides fundamental information for identifying B. xylophilus via a molecular approach. Moreover, the purified recombinant protein has potential as a candidate diagnostic antigen of pine wilt disease, which may lead to a new immunological detection method for the pine wood nematode. Full article