Bursaphelenchus Xylophilus Research Articles

Volume-based selection in Pinus pinaster Aiton breeding: evaluating resistance to Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle and wood properties

Key messageGenetic parameter estimates at age 12 years for trunk volume in Pinus pinaster displayed low to moderate heritability for growth and stem form, whereas wood properties showed high heritability. No correlation was detected between resistance to the nematode Bursaphelenchus xylophilus and growth, stem form, or wood properties. However, certain families stood out, showing favorable values across all traits, including resistance to the nematode.ContextThe breeding program for Pinus pinaster should advance towards a second-generation breeding population based on volume, stem form, and resistance to Bursaphelenchus xylophilus, with the challenge of incorporating novel traits of importance.AimsWe focused on assessing the genetic parameters of growth, stem form, wood properties, and resistance to B. xylophilus as well as exploring the relationship between these traits.MethodsWe evaluated 116 half-sib families at three ages (7, 12, and 17 years) in seven progeny trials established in 2005, analyzing growth, stem form, and wood traits. Resistance to B. xylophilus was assessed in 91 of the 116 half-sib families at age 2 by artificial inoculation tests under greenhouse conditions.ResultsAdvancing the selection age to 12 years proved to be feasible for volume selection in Galician P. pinaster. All traits, including resistance to B. xylophilus, showed potential for genetic improvement. Remarkably, resistance to B. xylophilus was not correlated to any other trait.ConclusionExcluding the most susceptible families to B. xylophilus from the Galician P. pinaster breeding program would not result in a significant decrease in volume, stem form, or wood properties.

Eco-friendly biocontrol of pine wilt disease: Enhancing tree defense with Bacillus subtilis JCK-1398 for sustainable forest management.

Pine wilt disease (PWD), caused by pinewood nematodes (Bursaphelenchus xylophilus), poses a significant threat to forestry worldwide. This study introduces a novel biocontrol strategy using Bacillus subtilis JCK-1398, which was selected and identified for its resistance-induction potential after extensive screening of microbial strains from pine tissues. The bacterium was found to biostimulate resistance in Pinus densiflora. Comprehensive analyses, including transcriptomics, qPCR assays, and high-performance liquid chromatography (HPLC), revealed that B. subtilis JCK-1398 significantly upregulates defense-related genes and stimulates pinoresinol production, a compound linked to resistance against nematodes. Treatment with B. subtilis JCK-1398 suppressed nematode migration and reduced nematode populations within pine tissues, effects attributed to the enhanced tree defense response. Field trials corroborated these findings, demonstrating a 72 % decrease in PWD incidence with aerial application of JCK-1398, confirming its potential for large-scale forest application. This biocontrol strategy, leveraging the stimulation of plant defense by B. subtilis JCK-1398, presents a promising and scalable, eco-friendly solution for managing PWD. Overall, this study provides new insights into the interactions between the bioagent, the host tree, and the pest nematode, offering an effective and sustainable approach to mitigate pine wilt disease.

Development of Simple Sequence Repeat of Monochamus alternatus (Coleoptera: Cerambycidae) Based on Restriction Site-Associated DNA Sequencing

Monochamus alternatus, a pest posing a serious threat to coniferous species, such as Pinus massoniana, has had devastating effects on pine forests due to its association with Bursaphelenchus xylophilus. The creation of unique simple sequence repeat (SSR) primers for M. alternatus is crucial, as there has been little study of the species’ phylogeography. The aim of this study was to identify and create polymorphic SSR primers by sequencing samples of M. alternatus obtained from three different sampling points using the restriction site-associated DNA sequencing (Red-seq) approach. Subsequently, supplementary samples were integrated, and genetic typing was performed utilizing the identified polymorphic primers. Through comprehensive analysis, a total of 95,612 SSR loci were identified. Among these, mononucleotide repeats (51.43%), dinucleotide repeats (28.79%), and trinucleotide repeats (16.74%) predominated among the SSR motif types. Ultimately, 18 pairs of SSR primers were screened out, demonstrating stable amplification and high polymorphism. Genetic typing revealed that the mean number of alleles (Na) for these primer pairs ranged from 3 to 8, observed heterozygosity (Ho) ranged from 0.133 to 0.733, polymorphic information content (PIC) ranged from 0.294 and 0.783, and Shannon’s index (I) ranged from 0.590 to 1.802. This study effectively produced 16 pairs of SSR primers that can be applied to different populations of M. alternatus. As a result, important tools for furthering studies on the phylogeography of pine wood nematodes, creating genetic maps, gene mapping, and carrying out in-depth investigations into gene function have been made available.

The impact of pine wilt disease on the endophytic microbial communities structure of Pinus koraiensis

Pine Wilt Disease (PWD) is a devastating pine tree disease characterized by rapid onset, high mortality rate, quick spread, and difficulty in control. Plant microbiome plays a significant role in the development of PWD. However, the endophytic microbial communities of Pinus koraiensis infected by pine wood nematode (PWN) Bursaphelenchus xylophilus remain largely unexplored. In this study, we analyzed the structural changes of endophytic communities of P. koraiensis after infection by the PWN using high-throughput sequencing technology. The results showed that the community structure underwent significant changes as the degree of PWN infection intensified. The diversity and abundance of endophytic fungi in P. koraiensis increased, while those of endophytic bacteria in P. koraiensis decreased during the infection process. Meanwhile, the abundance of some dominant microorganisms has also changed, including species such as Graphilbum and Pseudoalteromonas. Functional prediction analysis showed that the functional composition of endophytic fungi in P. koraiensis was significantly different across the development of PWD, while the composition of endophytic bacteria remained essentially similar. The results indicated that PWN infection had a significant impact on the structure, diversity, abundance, and functional gene composition of endophytic microbial communities in P. koraiensis, and most of the main endophytic microbial groups tended to coordinate with each other. This work provides a better understanding of the changes in endophytic community structure and function caused by PWD infection of P. koraiensis, which may benefit the exploration of potential endophytes for PWN biocontrol.

Optimized emamectin benzoate trunk injection: addressing temperature limitations for pine wilt disease control.

Pine wilt disease (PWD), caused by the pinewood nematode Bursaphelenchus xylophilus, poses a significant threat to global forestry, resulting in extensive economic and ecological damage. Traditional trunk injection agents (TIAs) are limited by environmental factors, such as high winter temperatures and vigorous resin flow, particularly in southern China. This study aimed to develop an ordinary temperature trunk injection agent (OTTIA) for year-round application and to evaluate its performance under high-temperature conditions in comparison to commercial TIAs. Extensive laboratory and field tests identified N,N-Dimethylformamide (DMF) and benzyl acetate as optimal solvents and Tween-40 as an effective emulsifier for the OTTIA formulation. The new agent was fully absorbed by Pinus massoniana within 3 h at temperatures exceeding 30 °C, outperforming existing commercial agents. Treated trees maintained comparable emamectin benzoate (EB) levels but achieved lower LC90 lethal concentration values (27.65 mg L-1), indicating greater efficacy. The OTTIA provided protection against PWD for ≤360 days postinjection. The development of OTTIA marks a significant advancement in PWD management, offering an effective solution for high-temperature regions and potentially transforming year-round treatment strategies. The results highlight the critical role of optimizing solvent and emulsifier combinations to enhance the efficacy and application of trunk injection agents, contributing to sustainable forest management and improved protection against PWD. © 2024 Society of Chemical Industry.

Detection Methods for Pine Wilt Disease: A Comprehensive Review.

Pine wilt disease (PWD), caused by the nematode Bursaphelenchus xylophilus, is a highly destructive forest disease that necessitates rapid and precise identification for effective management and control. This study evaluates various detection methods for PWD, including morphological diagnosis, molecular techniques, and remote sensing. While traditional methods are economical, they are limited by their inability to detect subtle or early changes and require considerable time and expertise. To overcome these challenges, this study emphasizes advanced molecular approaches such as real-time polymerase chain reaction (RT-PCR), droplet digital PCR (ddPCR), and loop-mediated isothermal amplification (LAMP) coupled with CRISPR/Cas12a, which offer fast and accurate pathogen detection. Additionally, DNA barcoding and microarrays facilitate species identification, and proteomics can provide insights into infection-specific protein signatures. The study also highlights remote sensing technologies, including satellite imagery and unmanned aerial vehicle (UAV)-based hyperspectral analysis, for their capability to monitor PWD by detecting asymptomatic diseases through changes in the spectral signatures of trees. Future research should focus on combining traditional and innovative techniques, refining visual inspection processes, developing rapid and portable diagnostic tools for field application, and exploring the potential of volatile organic compound analysis and machine learning algorithms for early disease detection. Integrating diverse methods and adopting innovative technologies are crucial to effectively control this lethal forest disease.

Climate change may make pine wilt disease more prevalent

Abstract Pine wilt disease is one of the most severe and devastating diseases affecting pine forests worldwide, resulting in huge economic losses in many countries. The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease and is obligately vectored by pine sawyer beetles, of the genus Monochamus. For the disease to be present, the habitat must be suitable for the PWN, and include at least one vector species, and at least one host species. To predict its potential distribution, a model must consider all three components. However, no comprehensive study has examined the influence of climatic suitability on the distribution of this “biological complex”. This study addresses this gap by incorporating biotic interactions, specifically involving 13 vectors and 61 host plants, into projections based on the PWN model. We predicted the global potential distribution of pine wilt disease and compared it with the PWN model to highlight the importance of including biotic interactions in species distribution models under climate change. We found that the model revealed an overall trend of increasing suitability scores for both the PWN and pine wilt disease models under future climate scenarios. Furthermore, compared to the PWN model, the biotic model results in an apparent increase in suitability worldwide in the future as the climate will be more suitable to vector and host complexes, suggesting that pine wilt disease could potentially spread to other places via available hosts and vectors. Synthesis and applications. By incorporating biotic interactions, we projected a more accurate suitable area for pine wilt disease, offering valuable insights into regions at high risk for future invasions by the disease and its vectors. This information supports the development of management and early detection strategies in areas of high suitability, helping to mitigate potential economic and ecological losses. Additionally, this study introduces a novel approach for integrating biotic factors into species distribution models.

Relationship between the distribution of the pinewood nematode (Bursaphelenchus xylophilus) and the development of xylem embolism in the stems of Japanese black pine (Pinus thunbergii) seedlings monitored by magnetic resonance imaging.

The development of xylem embolism in 1-year-old stems of Japanese black pine (Pinus thunbergii) seedlings was monitored by compact magnetic resonance imaging (MRI) after inoculation with the pinewood nematode (Bursaphelenchus xylophilus). In parallel, the nematode distribution and population structure in the stems were examined by isolating the nematodes using the Baermann funnel technique. The vertical length and volume of massive embolisms in each seedling were strongly correlated with the maximum relative embolized area (REA) in stem cross-sections. Embolism development and nematode reproduction were not restricted to the inoculation site, as any portion of the stem could be the initial point of a population burst. The nematode population in the stem xylem was strongly correlated with the REA and with the circumferential proportion of cambial death in cross-sections monitored by MRI. The proportion of second-stage juveniles was also correlated with the REA in the xylem. In contrast, the nematode population in bark tissue was not correlated with either the REA or cambial death. These results suggested that nematode reproduction in the cambial zone is the key step in pine wilt disease, and second-stage juveniles were suggested to induce massive embolisms in the advanced stage of the disease.

Discovery of novel nematicidal active ingredient 2,4,7-trichloroquinazoline as a potential succinic dehydrogenase inhibitor

Plant-parasitic nematodes seriously affect the growth and yield of crops, causing huge economic losses to world agriculture. One of the effective ways to manage nematode diseases is the use of nematicides. Compound B1 was found to have good nematicidal activity in the biological test. Therefore, some compounds with similar structures to B1 were tested for better nematicidal activities. Among them, the LC50 of compound C3 against pine wood nematode (Bursaphelenchus xylophilus), rice stem nematode (Aphelenchoides besseyi), and sweet potato stem nematode (Ditylenchus destructor) were 4.6, 22.4, and 16.9 mg/L, respectively, which are markedly better than tioxazafen (103.9, 56.2, and 61.0 mg/L). Compound C3 was capable of not only significantly promoting oxidative stress in Bursaphelenchus xylophilus (B. xylophilus), but also curtailing population growth by influencing the frequency of head swing frequency, feeding behaviors, reproductive capabilities, and egg hatching rates. Furthermore, Compound C3 has demonstrated a favorable binding affinity to the succinic dehydrogenase (SDH) protein and has significantly dampened the activity of the SDH in B. xylophilus. Therefore, compound C3 can be used as a potential lead compound to discover new nematicides.

Pinewood nematode induced changes in the assembly process of gallery microbiomes benefit its vector beetle's development.

Microbiomes play crucial roles in insect adaptation, especially under stress such as pathogen invasion. Yet, how beneficial microbiomes assemble remains unclear. The wood-boring beetle Monochamus alternatus, a major pest and vector of the pine wilt disease (PWD) nematode, offers a unique model. We conducted controlled experiments using amplicon sequencing (16S rRNA and ITS) within galleries where beetles and microbes interact. PWD significantly altered bacterial and fungal communities, suggesting distinct assembly processes. Deterministic factors like priority effects, host selection, and microbial interactions shaped microbiome composition, distinguishing healthy from PWN-infected galleries. Actinobacteria, Firmicutes, and Ophiostomataceae emerged as potentially beneficial, aiding beetle's development and pathogen resistance. This study unveils how nematode-induced changes in gallery microbiomes influence beetle's development, shedding light on microbiome assembly amid insect-pathogen interactions. Insights gleaned enhance understanding of PWD spread and suggest novel management strategies via microbiome manipulation.IMPORTANCEThis study explores the assembly process of gallery microbiomes associated with a wood-boring beetles, Monochamus alternatus, a vector of the pine wilt disease (PWD). By conducting controlled comparison experiments and employing amplicon approaches, the study reveals significant changes in taxonomic composition and functional adaptation of bacterial and fungal communities induced by PWD. It identifies deterministic processes, including priority effects, host selection, and microbial interactions, as major drivers in microbiome assembly. Additionally, the study highlights the presence of potentially beneficial microbes such as Actinobacteria, Firmicutes, and Ophiostomataceae, which could enhance beetle development and resistance to pathogens. These findings shed light on the intricate interplay among insects, microbiomes, and pathogens, contributing to a deeper understanding of PWD prevalence and suggesting innovative management strategies through microbiome manipulation.

Caffeic acid: A game changer in pine wood nematode overwintering survival.

Following the invasion by the pine wood nematode (PWN) into north-east China, a notable disparity in susceptibility was observed among Pinaceae species. Larix olgensis exhibited marked resilience and suffered minimal fatalities, while Pinus koraiensis experienced significant mortality due to PWN infection. Our research demonstrated that the PWNs in L. olgensis showed a 13.43% reduction in lipid content compared to P. koraiensis (p < 0.05), which was attributable to the accumulation of caffeic acid in L. olgensis. This reduction in lipid content was correlated with a decreased overwintering survival of PWNs. The diminished lipid reserves were associated with substantial stunting in PWNs, including reduced body length and maximum body width. The result suggests that lower lipid content is a major factor contributing to the lower overwintering survival rate of PWNs in L. olgensis induced by caffeic acid. Through verification tests, we concluded that the minimal fatalities observed in L. olgensis could be attributed to the reduced overwintering survival of PWNs, a consequence of caffeic acid-induced stunting. This study provides valuable insights into PWN-host interactions and suggests that targeting caffeic acid biosynthesis pathways could be a potential strategy for managing PWN in forest ecosystems.

Assessing global pine wilt disease risk based on ensemble species distribution models

Pine wilt disease (PWD), caused by the invasive pine wood nematode, is a major threat to global pine forests. This study utilized global PWD occurrence data alongside climatic, soil, and topographic variables to develop an ensemble of species distribution models. Using this ensemble model, we identified key factors influencing PWD and assessed the risk for current conditions and future periods (2041–2060 and 2071–2090) under three climate scenarios (SSP126, SSP370, and SSP585). The results indicate that key factors include the average temperature during the hottest quarter, clay content in soil, and precipitation during the hottest quarter, total annual precipitation, and precipitation during the coldest quarter. Currently, southern and northeastern China, central-southern Europe, and Southeast Asia are at high risks. With future climate changes, potential risk areas are expected to expand to higher latitudes, affecting regions like Hokkaido, Canada, and Northern Europe, especially under the SSP585 scenario. This study offers essential insights for global PWD prevention and forests resource conservation.

Genetic Structure of Monochamus alternatus (Hope) in Qinling-Daba Mountains and Expansion Trend: Implications for Pest Prevention and Management.

Pine wilt disease (PWD), caused by Bursaphelenchus xylophilus, severely threatens global pine forests. Monochamus alternatus is the primary vector of B. xylophilus in East Asia. Understanding the population structure and evolutionary forces of vector insects is critical for establishing effective PWD management strategies. The present work explores the genetic structure and phylogenetic relationships of 20 populations of M. alternatus from the Qinling-Daba Mountains (QDM) in China using the mitochondria DNA dataset, supplemented by ecological niche modeling (ENM). All M. alternatus populations were categorized into three phylogeographic clusters (Clade A, Clade B, and Clade C), with Clade A and Clade B corresponding to the western and eastern QDM, respectively. The results of divergence time estimation concur with environmental changes induced by Quaternary glacial climate oscillations in QDM of China. M. alternatus populations exhibited significant genetic differentiation, with expansion in their population size. Ecological niche modeling (ENM) demonstrated that precipitation and temperature significantly influence the distribution of M. alternatus and the species is anticipated to grow into higher latitude and higher altitude regions in the future. In a nutshell, exploring the genetic structure and evolutionary dynamics of M. alternatus can provide valuable insights into the prevention and occurrence of B. xylophilus. These findings also serve as a reference for research on population differentiation and phylogeography of other species in QDM and adjacent areas.

Bursaphelenchus xylophilus Venom Allergen-Like Protein BxVAP1, Triggering Plant Defense-Related Programmed Cell Death, Plays an Important Role in Regulating Pinus massoniana Terpene Defense Responses.

Bursaphelenchus xylophilus (pine wood nematode, PWN), a migratory plant-parasitic nematode, acts as an etiological agent, inflicting considerable damage to pine forests worldwide. Plant immunity constitutes a crucial factor in resisting various pathogenic invasions. The primary defensive responses of host pines against PWN infection encompass terpene accumulation, defense response-related gene expression, and programmed cell death. Venom allergen-like proteins (VAPs), as potential effectors, are instrumental in facilitating the successful colonization of PWNs. In this study, we investigated the inhibition of B. xylophilus VAP (BxVAP1) expression by RNA interference in vitro. Following BxVAP1 silencing, the reproduction rate and migration rate of the PWN population in Pinus massoniana decreased, the expression of the α-pinene synthase gene was induced, other terpene synthase and pathogenesis-related genes were inhibited and delayed, the peak times and levels of terpene-related substances were changed, and the degree of cavitation in P. massoniana was diminished. Transient expression of BxVAP1 in Nicotiana benthamiana revealed that BxVAP1 was expressed in both the cell membrane and nucleus, inducing programmed cell death and the expression of pathogen-associated molecular pattern-triggered immunity marker genes (NbAcre31 and NbPTI5). This study is the first to demonstrate that silencing the BxVAP1 gene affects host defense responses, including terpenoid metabolism in P. massoniana, and that BxVAP1 can be recognized by N. benthamiana as an effector to trigger its innate immunity, expanding our understanding of the parasitic mechanism of B. xylophilus.

Whole-Genome Sequences of 13 Chinese Indigenous Pinewood Nematodes, Bursaphelenchus xylophilus.

The pinewood nematode (Bursaphelenchus xylophilus, PWN) induces pine wilt disease in Pinaceae plants, causing severe destruction to pine forests. Previous studies report that by 2023, 663 cities across 18 provinces in China had been infested by the PWN, necessitating immediate control measures. To identify the genetic variations associated with the PWN's adaptation to new environments, we conducted whole-genome sequencing (WGS) on 13 indigenous PWN samples from two distinct geographic zones within China, specifically Anhui, Liaoning, and Jiangxi provinces. We identified genetic variants and analyzed the genetic structure of these populations, followed by functional gene enrichment analyses. Our findings reveal genetic variants associated with secretion, immune system function, membrane processes, metabolism, catabolism, and cell wall regulation, supporting the hypothesis that the PWN genome has been shaped by local ecosystems.

Infection of In Vivo and In Vitro Pines with the Pinewood Nematode Bursaphelenchus xylophilus and Isolation of Induced Volatiles.

The pinewood nematode (PWN) is a phytoparasite that causes pine wilt disease (PWD) in conifer species. This plant parasitic nematode has heavily contributed to pine deforestation in Asian countries, e.g., Japan, China, and Korea. Over the last two decades, in Europe, Portugal and Spain have been greatly affected. Research on the mechanisms of PWN infection and/or PWD progression in susceptible host species relies on the controlled infection of pine seedlings under greenhouse conditions. This technique is laborious and mobilizes substantial economic and human resources. Additionally, it can be prone to variability that results from the genetic diversity associated with some pine species but also from the interference of external factors. As an alternative, in vitro co-cultures of pine with PWNs offer a more advantageous system for studying biochemical changes since they a) allow controlling single environmental or nutritional variables, b) occupy less space, c) require less time to obtain, and d) arefree from contamination or from host genetic variation. The following protocol details the standard in vivo PWN infection of Pinus pinaster, the maritime pine, and the establishment of the novel in vitro co-cultures of pine shoots with the PWN as an improved methodology to study this phytoparasite influence on pine volatiles. PWN-induced volatiles are extracted from in vivo and in vitro infected pines by hydrodistillation and distillation-extraction, and the emitted volatiles are captured by solid phase microextraction (SPME), using fiber or packed column techniques.

A novel BH3DNet method for identifying pine wilt disease in Masson pine fusing UAS hyperspectral imagery and LiDAR data

Pine Wilt Disease (PWD) is a forest infectious disease that inflicts substantial economic losses to China’s forestry. Its rapid spread and the significant challenges associated with its control make early detection of infected trees crucial for disaster prevention. Unmanned aerial systems (UASs) hyperspectral imaging (HSI) and light detection and ranging (LiDAR) technologies provide high-resolution spectral diagnostic information coupled with intricate three-dimensional structural data, which has potential for fine grained monitoring of PWD. However, how to fuse HSI and LiDAR data to identify the early infected individual trees is still a challenge. This study presents a novel instance segmentation network, BH3DNet, to identify individual trees at different PWD-infected stages by extracting high-level abstract features based on the fusion of drone HSI and LiDAR data. BH3DNet introduces the PointNet++ model as the base network, and incorporates a shared encoder and twin parallel decoders to align semantic category prediction and instance segmentation of individual trees in an end-to-end approach. By applying an enhanced point cloud dataset that fuses drone HSI and LiDAR point cloud data, this model facilitates the identification of PWD infection stages at the individual tree scale. We evaluated the proposed model in a Masson pine forest stand sparsely mixed with broadleaf trees in a variety of infection states ranging from healthy to severely infected by PWD, and compared the performance of the model using the RGB bands, full HSI bands and screened bands as inputs, respectively. BH3DNet achieves an overall accuracy of 89.65 % with a Kappa × 100 of 87.29 for identifying individual trees using screened HSI bands and LiDAR point cloud, significantly outperforming the Mask R-CNN using only HSI data (overall accuracy: 70.81 %, Kappa × 100: 64.16). Moreover, BH3DNet’s accuracy at the early infection stage reaches 83.75 %. It proves that fusing HSI and point cloud data reflects the information of individual trees distribution and infection status, and the BH3DNet is suitable for high-precision monitoring of PWD.

Establishment of a Sensitive and Reliable Droplet Digital PCR Assay for the Detection of Bursaphelenchus xylophilus.

Pine wilt disease (PWD), which poses a significant risk to pine plantations across the globe, is caused by the pathogenic agent Bursaphelenchus xylophilus, also referred to as the pine wood nematode (PWN). A droplet digital PCR (ddPCR) assay was developed for the quick identification of the PWN in order to improve detection sensitivity. The research findings indicate that the ddPCR assay demonstrated significantly higher analysis sensitivity and detection sensitivity in comparison to traditional quantitative PCR (qPCR). However, it had a more limited dynamic range. High specificity was shown by both the ddPCR and qPCR techniques in the diagnosis of the PWN. Assessments of reproducibility revealed that ddPCR had lower coefficients of variation at every template concentration. Inhibition tests showed that ddPCR was less susceptible to inhibitors. There was a strong linear association between standard template measurements obtained using ddPCR and qPCR (Pearson correlation = 0.9317; p < 0.001). Likewise, there was strong agreement (Pearson correlation = 0.9348; p < 0.001) between ddPCR and qPCR measurements in the evaluation of pine wood samples. Additionally, wood samples from symptomatic (100% versus 86.67%) and asymptomatic (31.43% versus 2.9%) pine trees were diagnosed with greater detection rates using ddPCR. This study's conclusions highlight the advantages of the ddPCR assay over qPCR for the quantitative detection of the PWN. This method has a lot of potential for ecological research on PWD and use in quarantines.

Transcription Factor and Protein Regulatory Network of PmACRE1 in Pinus massoniana Response to Pine Wilt Nematode Infection

Pine wilt disease, caused by Bursaphelenchus xylophilus, is a highly destructive and contagious forest affliction. Often termed the “cancer” of pine trees, it severely impacts the growth of Masson pine (Pinus massoniana). Previous studies have demonstrated that ectopic expression of the PmACRE1 gene from P. massoniana in Arabidopsis thaliana notably enhances resistance to pine wilt nematode infection. To further elucidate the transcriptional regulation and protein interactions of the PmACRE1 in P. massoniana in response to pine wilt nematode infection, we cloned a 1984 bp promoter fragment of the PmACRE1 gene, a transient expression vector was constructed by fusing this promoter with the reporter GFP gene, which successfully activated the GFP expression. DNA pull-down assays identified PmMYB8 as a trans-acting factor regulating PmACRE1 gene expression. Subsequently, we found that the PmACRE1 protein interacts with several proteins, including the ATP synthase CF1 α subunit, ATP synthase CF1 β subunit, extracellular calcium-sensing receptor (PmCAS), caffeoyl-CoA 3-O-methyltransferase (PmCCoAOMT), glutathione peroxidase, NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase 1, cinnamyl alcohol dehydrogenase, auxin response factor 16, and dehydrin 1 protein. Bimolecular fluorescence complementation (BiFC) assays confirmed the interactions between PmACRE1 and PmCCoAOMT, as well as PmCAS proteins in vitro. These findings provide preliminary insights into the regulatory role of PmACRE1 in P. massoniana’s defense against pine wilt nematode infection.

Molecular characterization and functional analysis of genes mediating emamectin benzoate action to the pinewood nematode (Bursaphelenchus xylophilus)

Emamectin benzoate (EB) is a highly effective and low-toxicity pesticide for the control of Bursaphelenchus xylophilus. However, its action mechanism in B. xylophilus has not yet been verified. Here, the genes (Bxy-glc-1, Bxy-glc-2, Bxy-glc-4, and Bxy-avr-14) encoding the glutamate-gated chloride channel (GluCl) of B. xylophilus were analysed and cloned. Functional validation of the target genes was conducted using RNAi and pathogenicity detection assays. The results of the bioinformatics analysis showed that the four GluCl genes contained the Cys-loop region and three transmembrane structural domains. Molecular docking and molecular dynamics simulation predictions revealed that BXY-GLC-2, BXY-GLC-4, and BXY-GLC-1 all had strong binding affinities to EB, and BXY-AVR-14 had no binding affinity to EB. The expression and in situ hybridisation of Bxy-glc-1, Bxy-glc-2, Bxy-glc-4, and Bxy-avr-14 was significantly higher in adult B. xylophilus than at other developmental stages. Interference of Bxy-glc-1, Bxy-glc-2, and Bxy-glc-4 significantly reduced adult mortality relative to the control group, and interference of Bxy-avr-14 did not have a significant on adult mortality. Adult mortality was lowest in the combined Bxy-glc-2 + Bxy-glc-4 treatment group, followed by the Bxy-glc-1 + Bxy-glc-2 and Bxy-glc-1 + Bxy-glc-4 groups. No significant changes were observed in the mortality rate of the Bxy-avr-14 group and the combination of the other three genes. The dsBxy-glc-1, dsBxy-glc-2, and dsBxy-glc-4 groups accelerated the progression of pine wilt disease induced by EB relative to the sole EB-treated group. Our results confirmed that Bxy-glc-1, Bxy-glc-2, and Bxy-glc-4 are target genes of GluCl in B. xylophilus.