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To investigate the effective technical measures for rapid surface cover and effective control of slope erosion in forest sites damaged by Bursaphelenchus xylophilus, this study used runoff plot location observations to compare runoff, sediment, and nutrient loss in pine wilt-damaged sites under different soil management conditions, including biodegradable films (SWM), weed-proof films (FCB), and polyacrylamide (PAM) . The results demonstrated that the SWM-95% treatment exhibited the most effective sediment loss control, with considerably lower nutrient loss in runoff and sediments than other treatments conversely, the SWM-67% treatment exhibited a significantly higher loss of sediment and nutrients than the other treatments. The control effects of runoff, sediment, and nutrient loss in the FCB and PAM treatments were superior to those of the other treatments at both 90 and 180 d. Furthermore, losses in both treatments were lower than those in the CK treatment. The PAM treatment exhibited the most favorable outcomes, with runoff and sediment loss in the PAM-95% treatment demonstrating 52. 16%, 75. 6% and 71. 7%, 73. 0% reductions, respectively, in comparison with the CK treatment at 90 and 180 d. Nevertheless, the controlling effects of the PAM treatment on different nutrients exhibited varying outcomes. Its impact on N and K losses was more pronounced, whereas the impact of P nutrient loss on sediment loss was less pronounced. Furthermore, the control effect of PAM on runoff, sediment, and nutrient loss decreased significantly after270 d. Contrarily, the control effect on water and soil loss and total and available nutrient loss was still superior in the FCB treatment compared to the other treatments, with a control effect of 95%＞67%. A comparison of the nutrient contents of the Chinese fir-afforested land with those of the SWM, FCB and PAM treatments revealed that the total N, P, and K contents were overall higher than those of the CK treatment. The total soil P and available P of the SWM-67% treatment in different soil layers were significantly higher than those of the other treatments. Conversely, the PAM and FCB treatments had a more pronounced effect on maintaining the total and available nutrients of N and K. The growth of newly planted trees also demonstrated that different treatments promoted the height growth of Chinese fir, with the FCB and PAM treatments being more effective. In conclusion, PAM-95% treatment can be employed to rapidly and effectively control soil and water loss in Bursaphelenchus xylophilus damaged land. Furthermore, the combination of the PAM-95% and FCB-67% treatment could be employed for afforested land with more favorable conditions, which would result in the most optimal outcome while minimizing expenditure. [ABSTRACT FROM AUTHOR]

The pine sawyer beetle (Monochamus galloprovincialis) is a European vector of the invasive pinewood nematode (Bursaphelenchus xylophilus), one of the most dangerous threats to pine forests worldwide. One recommended method for detecting the nematode is catching and inspecting the M. galloprovincialis beetles. Effective commercial lures are available for that insect but attract non‐target species, particularly beneficial Thanasimus species. Our study aimed to develop an equally effective M. galloprovincialis lure less attractive to beneficial predatory insects.We used electroantennography to test several compounds occurring in the pheromones, hindgut extracts or headspace emissions of non‐Ips bark beetles. The compounds that caused the strongest antennal responses were used along with α‐pinene (host‐tree volatile) and monochamol (2‐undecyloxy‐1‐ethanol, a male‐produced aggregation pheromone of M. galloprovincialis) to prepare the lures for olfactometric and field testing.In the field experiments, the lure containing (+)‐α‐pinene, monochamol, (1R)‐(−)‐myrtenal and (S)‐(−)‐trans‐verbenol was equally effective as the commercial lure Galloprotect Pack. It attracted significantly less Thanasimus formicarius and T. femoralis beetles.Thus, the new lure can serve as a basis for developing an operational lure for monitoring M. galloprovincialis with reduced bycatch of predatory beetles. [ABSTRACT FROM AUTHOR]

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 (BxVAPl') expression by RNA interference in vitro. Following BxVAPl silencing, the reproduction rate and migration rate of the PWN population in Pinus massoniana decreased, the expression of the a-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 BxVAPl in Nicotiana benthamiana revealed that BxVAPl 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 BxVAPl gene affects host defense responses, including teipenoid metabolism in P. massoniana, and that BxVAPl can be recognized by N. benthamiana as an effector to trigger its innate immunity, expanding our understanding of the parasitic mechanism of B. xylophilus. [ABSTRACT FROM AUTHOR]

Bursaphelenchus xylophilus is the pathogen of pine wilt disease, which can devastate the pine forest ecosystem. Usually, plant cells generate reactive oxygen species (ROS) as a defensive substance or signalling molecules to resist the infection of nematodes. However, little is known about how B. xylophilus effectors mediate the plant ROS metabolism. Here, we identified a pioneer B. xylophilus Prx3‐interacting effector 1 (BxPIE1) expressed in the dorsal gland cells and the intestine. Silencing of the BxPIE1 gene resulted in reduced nematode reproduction and a delay in disease progression during parasitic stages, with the upregulation of pathogenesis‐related (PR) genes PtPR‐3 (class Ⅳ chitinase) and PtPR‐9 (peroxidase). The protein–protein interaction assays further demonstrated that BxPIE1 interacts with a Pinus thunbergii class III peroxidase (PtPrx3), which produces H2O2 under biotic stress. The expression of BxPIE1 and PtPrx3 was upregulated during the infection stage. Furthermore, BxPIE1 effectively inhibited H2O2 generating from class III peroxidase and ascorbate can recover the virulence of siBxPIE1‐treated B. xylophilus by scavenging H2O2. Taken together, BxPIE1 is an important virulence factor, revealing a novel mechanism utilized by nematodes to suppress plant immunity. Summary statement: A pioneer B. xylophilus Prx3‐interacting effector 1 (BxPIE1) suppresses reactive oxygen species burst by interacting with the class III peroxidase (PtPrx3) in Pinus thunbergii. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Pine wilt disease (PWD) is a devastating disease of pine trees caused by the pine wood nematode (Bursapherenchus xylophilus, PWN). To study how Pinus tabulaeformis responds to PWD infection, we collected 3-year-old P. tabulaeformis seedlings at 2 days, 5 days, and 8 days after being infected with B. xylophilus. We identified genes and metabolites early responding to infection using transcriptome and metabolomic data obtained by high-throughput mRNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assays, respectively. The following results were obtained: (1) After inoculation with PWN, the average number of days taken for 3-year-old P. tabulaeformis seedlings to develop symptoms was 8 days. (2) Combined transcriptome and metabolome analysis revealed that phenylpropanoid biosynthesis and flavonoid biosynthesis are critically important pathways for P. tabulaeformis to respond to PWD. (3) The response of P. tabulaeformis to stress was mainly through positive regulation of gene expression, including some key genes related to plant hormones or transcription factors that have been widely studied. Genes related to pathways such as photosynthesis, plant-pathogen interactions, and DNA replication were downregulated. (4) Terpenoid biosynthesis genes involved during the development of pine wilt disease. This study demonstrated the defence and pathogenic mechanisms of P. tabulaeformis against PWD, providing a reference for the early diagnosis of PWD. [ABSTRACT FROM AUTHOR]

One of the most damaging pathogens of pinewood is the pinewood nematode, Bursaphelenchus xylophilus, which could cause an adverse effect on the ecosystems of forests and the commerce of timber. Therefore, it is crucial to realize rapid and accurate B. xylophilus detection. In this work, an accelerated strand exchange amplification method (ASEA) was established to detect B. xylophilus for the first time. By integrating with fast nucleic acid extraction, the whole detection procedure could be finished within 30 min, dramatically shortened the detection time. The ASEA method exhibited high specificity towards B. xylophilus and the detection limit for B. xylophilus plasmid DNA was as low as 1.0 × 100 copies/μL. Furthermore, the ASEA approach also exhibited accurate detection for B. xylophilus when applied to actual pinewood samples, meeting the demand of B. xylophilus detection in realistic scenario. We believe the ASEA method has significant potential for B. xylophilus detection, and it will be helpful for controlling forest pest and quarantine regulations. [ABSTRACT FROM AUTHOR]

Pinus, a conifer, dominates the world's forest ecosystems. But it is seriously infected with pine wood nematode (PWN). Transcription factors (TFs) are key regulators in regulating plant resistance. However, the molecular mechanism of TFs remains thus far unresolved in P. thunbergii inoculated with Bursaphelenchus xylophilus. Here, we used RNA-seq technology to identify differentially expressed TFs in resistant and susceptible pines. The results show that a total of 186 differentially expressed transcription factors (DETFs), including 99 upregulated and 87 downregulated genes were identified. Gene ontology (GO) enrichment showed that the highly enriched differentially expressed TFs were responsible for secondary biosynthetic processes. According to KEGG pathway analysis, the differentially expressed TFs were related to chaperones and folding catalysts, phenylpropanoid biosynthesis, and protein processing in the endoplasmic reticulum. Many TFs such as NAC, LBD, MYB, bHLH, and WRKY were determined to be quite abundant in the DETFs. Moreover, the NAC transcription factor PtNAC9 was upregulated in PWN-resistant and susceptible P. thunbergii and especially distinctly upregulated in resistant pines. By purifying recombinant PtNAC9 protein in vitro, we found that overexpression of PtNAC9 at the early stage of B. xylophilus infection could reduce the degree of disease. We also demonstrated the content of salicylic acid (SA) and the related genes were increased in the PtNAC9 protein-treated plants. These results could be helpful in enhancing our understanding of the resistance mechanism underlying different resistant pine. [ABSTRACT FROM AUTHOR]

Abstract Pine wilt disease (PWD) is a devastating disease of pine trees caused by the pine wood nematode (Bursapherenchus xylophilus, PWN). To study how Pinus tabulaeformis responds to PWD infection, we collected 3-year-old P. tabulaeformis seedlings at 2 days, 5 days, and 8 days after being infected with B. xylophilus. We identified genes and metabolites early responding to infection using transcriptome and metabolomic data obtained by high-throughput mRNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assays, respectively. The following results were obtained: (1) After inoculation with PWN, the average number of days taken for 3-year-old P. tabulaeformis seedlings to develop symptoms was 8 days. (2) Combined transcriptome and metabolome analysis revealed that phenylpropanoid biosynthesis and flavonoid biosynthesis are critically important pathways for P. tabulaeformis to respond to PWD. (3) The response of P. tabulaeformis to stress was mainly through positive regulation of gene expression, including some key genes related to plant hormones or transcription factors that have been widely studied. Genes related to pathways such as photosynthesis, plant-pathogen interactions, and DNA replication were downregulated. (4) Terpenoid biosynthesis genes involved during the development of pine wilt disease. This study demonstrated the defence and pathogenic mechanisms of P. tabulaeformis against PWD, providing a reference for the early diagnosis of PWD.

Traditional pesticides are based on toxic compounds that can reduce biodiversity, degrade the environment, and contribute to less healthy living. Plant allelochemicals can provide more environmentally friendly and sustainable alternatives. Essential oils (EOs) are complex mixtures of plant secondary metabolites that show strong biological activities. In the present study, the EOs of Cymbopogon citratus were screened for activity against the pinewood nematode (PWN), the causal agent of pine wilt disease. To understand their nematicidal properties, EOs were fractioned into hydrocarbon molecules and oxygen-containing compounds, and their main compounds were acquired and tested separately against the PWN. The EO oxygen-containing molecules fraction was highly active against the PWN (EC50 = 0.279 µL/mL), with citral and geraniol showing higher activities (EC50 = 0.266 and 0.341 µL/mL, respectively) than emamectin benzoate (EC50 = 0.364 µL/mL), a traditional nematicide used against the PWN. These compounds were additionally reported to be less toxic to non-target organisms (fish, invertebrates, and algae) and safer to human health (with higher reported toxicity thresholds) and predicted to exert fewer environmental impacts than traditional nematicides. Resorting to approved natural compounds can quickly leverage the development of sustainable alternatives to traditional nematicides. [ABSTRACT FROM AUTHOR]

Pinus is an important economic tree species, but pine wilt disease (PWD) seriously threatens the survival of pine trees. PWD caused by Bursaphelenchus xylophilus is a major quarantine disease worldwide that causes significant economic losses. However, more information about its molecular pathogenesis is needed, resulting in a lack of effective prevention and treatment measures. In recent years, effectors have become a hot topic in exploring the molecular pathogenic mechanism of pathogens. Here, we identified a specific effector, BxNMP1, from B. xylophilus. In situ hybridization experiments revealed that BxNMP1 was specifically expressed in dorsal gland cells and intestinal cells, and RT–qPCR experiments revealed that BxNMP1 was upregulated in the early stage of infection. The sequence of BxNMP1 was different in the avirulent strain, and when BxNMP1-silenced B. xylophilus was inoculated into P. thunbergii seedlings, the disease severity significantly decreased. We demonstrated that BxNMP1 interacted with the thaumatin-like protein PtTLP-L2 in P. thunbergii. Additionally, we found that the β-1,3-glucanase PtGLU interacted with PtTLP-L2. Therefore, we hypothesized that BxNMP1 might indirectly interact with PtGLU through PtTLP-L2 as an intermediate mediator. Both targets can respond to infection, and PtTLP-L2 can enhance the resistance of pine trees. Moreover, we detected increased salicylic acid contents in P. thunbergii seedlings inoculated with B. xylophilus when BxNMP1 was silenced or when the PtTLP-L2 recombinant protein was added. In summary, we identified a key virulence effector of PWNs, BxNMP1. It positively regulates the pathogenicity of B. xylophilus and interacts directly with PtTLP-L2 and indirectly with PtGLU. It also inhibits the expression of two targets and the host salicylic acid pathway. This study provides theoretical guidance and a practical basis for controlling PWD and breeding for disease resistance. [ABSTRACT FROM AUTHOR]

Bursaphelenchus xylophilus is a pest that interferes with the health of forests and hinders the development of the forestry industry, and its spread is influenced by changes in abiotic factors and human activities. The potential distribution areas of B. xylophilus in China under four shared-economic pathways were predicted using the optimized MaxEnt model (version 3.4.3), combining data from a variety of environmental variables: (1) prediction of natural environmental variables predicted under current climate models; (2) prediction of natural environmental variables + human activities under current climate models; and (3) prediction of natural environmental variables under the future climate models (2050s and 2070s). Meanwhile, whether the niche of B. xylophilus has changed over time is analyzed. The results showed that human activities, precipitation in the driest month, annual precipitation, and elevation had significant effects on the distribution of B. xylophilus. In the current conditions, human activities greatly reduced the survival area of B. xylophilus, and its suitable distribution area was mainly concentrated in the southwestern and central regions of China. Under the influence of climate change in the future, the habitat of B. xylophilus will gradually spread to the northeast. In addition, the ecological niche overlap analysis showed that B. xylophilus in future climate was greater than 0.74. This study provides important information for understanding the ecological adaptation and potential risk of B. xylophilus, which can help guide the decision making of pest control and forest protection. [ABSTRACT FROM AUTHOR]

Bursaphelenchus mucronatus Mamiya & Enda, 1979 is a wood nematode widely distributed over Palearctic coniferous forests. It has two subspecies, that is, B. mucronatus mucronatus and B. mucronatus kolymensis, which present molecularly different East Asian and European genotypes, respectively. The European subspecies is found mainly in Europe and Siberia, while the East Asian subspecies occupies mostly Eastern regions of Asia. However, local isolates of both subspecies have been occasionally reported from various localities in Europe and Asia. Our field isolation and molecular (DNA ITS‐RFLP) identification of 21 recently collected populations of B. mucronatus revealed only 4 isolates representing clearly East Asian or European genotypes, while the remaining 17 isolates showed intermediate genotypes with electrophoretic band characters of both the above types. Further individual crossbreeding, in vitro, of nematodes representing European, East Asian, and intermediate genotypes provided experimental evidence for the process of intraspecific hybridization between both subspecies, spontaneously taking place in the forest. Such a widening of the B. mucronatus genetic variation may have its effect on a range of reported in the literature direct interactions between this native, nonpathogenic nematode species and the genetically similar, causative agent of the pine wilt disease B. xylophilus which can colonize the same host trees, use the same insect vectors, compete, and crossbreed inter‐specifically. [ABSTRACT FROM AUTHOR]

Pines are the most economically important trees in the world and, together with eucalyptus, they dominate commercial forests. But the success of a relatively small number of widely planted species, such as Pinus pinaster, the maritime pine, comes at a price. Pines are attractive to damaging pathogens and insect pests, including the pinewood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD). Originally described in Japan, PWD has caused widespread destruction to forests in countries such as China, Taiwan, Portugal, Spain and the United States. PWN causes irreparable damage to the vascular system of its pine hosts, leading to mortality within 3 months. Pine sawyer beetles (Monochamus spp.) are key vectors of PWD, introducing the PWN to healthy trees during feeding. Other organisms contribute to PWD spread and development, including bacteria, fungi and bark beetles. Control measures include tree felling to prevent vector transmission of PWN, insecticide treatments, trapping of Monochamus spp. and tree breeding for plant resistance. The PWN is a quarantine pathogen and subject to regular legislation and phytosanitary measures aimed at restricting movement and preventing introduction to new areas. Current research is investigating the use of biopesticides against PWN and Monochamus spp. This review examines the biology, epidemiology, impact and management of PWD through published research, grey literature and interviews with people directly involved in the management of the disease in Portugal. [ABSTRACT FROM AUTHOR]

Bursaphelenchus xylophilus is a pathogenic nematode responsible for pine wilt disease, which can cause the demise of pine trees and discoloration of trunks. As rotenone is an important botanical pesticide, its impact on B. xylophilus was investigated through RNA-seq to understand the response mechanism of nematode. The bioassay results yielded the 12-h LC30 (1.35 mg L−1) and LC50 (2.60 mg L−1) values for rotenone. Differential gene expression analysis identified 172 and 614 differentially expressed genes (DEGs) in B. xylophilus exposed to two different concentrations of rotenone (1.35 and 2.60 mg L−1). To validate these findings, the expression patterns of 10 DEGs were confirmed through RT-qPCR. Additionally, all DEGs were categorized into eight gene expression profiles using STEM. Notably, the gene ontology (GO) processes of ''single-organism process,'' ''metabolic process,'' and ''catalytic activity'' were prominently enriched in rotenone-treated samples, suggesting a role for metabolic and catalytic pathways in the nematode's response to rotenone stress. KEGG pathways related to ''carbon metabolism,'' ''drug metabolism-cytochrome P450,'' and ''metabolism of xenobiotics by cytochrome P450'' were similarly enriched, indicating potential mechanisms for detoxification resistance and oxidative stress resistance. The analysis pointed to the pivotal roles of detoxification- and oxidoreduction-related genes, as well as signal transduction-related genes, in enabling B. xylophilus to adapt to rotenone exposure. These insights could markedly enhance our understanding of nematode resistance mechanisms and potentially inform the development of more effective rotenone-based strategies for controlling B. xylophilus.

Pine wilt disease (PWD) is a globally significant quarantine forest disease caused by Bursaphelenchus xylophilus (PWN), resulting in substantial ecological and economic losses. Traditional nematode management practices are neither cost-effective nor environmentally friendly, prompting the exploration of biocontrol as a promising alternative for managing this devastating forest disease. Obtaining novel and specific biocontrol agents is extremely crucial for the effective and precise control of PWD. In the present study, a total of 136 endophytic isolates were obtained from the roots, stems and needles of Pinus tabuliformis in the Qinling Mountains of China, which were then subjected to nematocidal activity assay against PWN in vitro. Nine endophytic bacterial isolates exhibited exceptionally strong nematocidal capacity, with a corrected mortality rate exceeding 90 %, which were then identified as the genus of Bacillus through morphological features, endospore staining, and 16S rDNA sequencing, with one strain as B. mycoides, two as B. cereus, and six as B. velezensis. Additionally, the inhibition effects of the three Bacillus species on the reproduction of PWN in vitro was assessed using an original detection model, with B. velezensis Pt-RP9 identified as the most promising strain. Subsequently, the biocontrol efficacy of B. velezensis Pt-RP9 against PWD was evaluated in greenhouse experiments. Pt-RP9 demonstrated significant biocontrol effectiveness against PWD, with control efficiencies ranging from 31.25 % to 68.89 % across all treatments, particularly showing improved efficacy when pine seedlings were pre-treated with Pt-RP9 before PWN inoculation. Furthermore, pine seedlings treated with Pt-RP9 exhibited significantly reduced PWN density and lipid peroxidation levels in cell membranes compared to the control groups, along with increased activities of peroxidase, catalase, and polyphenol oxidase. To our knowledge, this study is the first to showcase the nematocidal activity of endophytes from P. tabuliformis against PWN and their biocontrol efficacy against PWD, marking a significant advancement in the field. The findings highlight the potential of B. velezensis Pt-RP9 as a crucial biological control agent against PWD, presenting a novel and sustainable disease management approach for pine forests.

Abstract Key message The invasive pine wood nematode is a major threat to pine forests worldwide, causing extensive tree mortality. Although scientific knowledge and control measures are continuously improving, important gaps remain. We argue that some key questions, notably related to early detection and pest management, need to be urgently tackled in countries at risk of invasion such as France.

Pine wilt disease caused by Bursaphelenchus xylophilus poses a serious threat to the economic and ecological value of forestry. Nematode trapping fungi trap and kill nematodes using specialized trapping devices, which are highly efficient and non-toxic to the environment, and are very promising for use as biological control agents. In this study, we isolated several nematode-trapping fungi from various regions and screened three for their high nematocidal efficiency. However, the effectiveness of these fungi as nematicides is notably influenced by temperature and exhibits different morphologies in response to temperature fluctuations, which are categorized as "NA," "thin," "dense," and "sparse." The trend of trap formation with temperature was consistent with the trend of nematocidal efficiency with temperature. Both of which initially increased and then decreased with increasing temperature. Among them, Arthrobotrys cladodes exhibited the highest level of nematocidal activity and trap formation among the tested species. Transcriptome data were collected from A. cladodes with various trap morphologies. Hydrolase activity was significantly enriched according to GO and KEGG enrichment analyses. Eight genes related to hydrolases were found to be consistent with the trend of trap morphology with temperature. Weighted gene co-expression analysis and the Cytoscape network revealed that these 8 genes are associated with either mitosis or autophagy. This suggests that they contribute to the formation of "dense" structures in nematode-trapping fungi. One of these genes is the serine protein hydrolase gene involved in autophagy. This study reveals a potentially critical role for hydrolases in trap formation and nematocidal efficiency. And presents a model where temperature affects trap formation and nematocidal efficiency by influencing the serine protease prb1 involved in the autophagy process. [ABSTRACT FROM AUTHOR]

Pinus sibirica is primarily distributed in Siberia. Owing to its excellent cold resistance and development potential, it has become an important introduced tree species in the Greater Xing'an area of China. Pine wilt disease, triggered by the pine wood nematode (PWN, Bursaphelenchus xylophilus), constitutes a profoundly critical affliction within forest ecosystems. Its incidence has extended to the northeastern region of China in recent years. To explore the potential host status of P. sibirica in the Greater Xing'an area for PWN and to elucidate the responses following inoculation, artificial inoculation, transcriptomics, and metabolomics methods were used. In the artificial inoculation experiments, quantitative analysis of nematode populations within the trees demonstrated that PWN exhibited normal growth and reproductive capabilities within P. sibirica. Subsequently, transcriptome and metabolome sequencing were conducted at four time points before disease onset (3-, 5-, 7-, and 9-days post inoculation). Gene trend analysis and differentially expressed gene screening were employed and the results indicated that genes associated with the flavonoid biosynthesis pathway exhibited predominant enrichment among the upregulated genes. Metabolome analysis showed that the abundance of flavonoidrelated metabolites in P. sibirica increased after inoculation with PWN. Integrated analysis of transcriptome and metabolome revealed that after PWN inoculation in P. sibirica, two chalcone synthase (chs) genes and a chalcone isomerase (chi) gene were significantly upregulated, and the upregulation should accumulate naringenin, pinocembrin, and apigenin to help P. sibirica resist infection of PWN. The results suggested that flavonoid biosynthesis pathway continued to respond after P. sibirica was infected with PWN and played an important role in the interaction between P. sibirica and PWN. [ABSTRACT FROM AUTHOR]

The pine wood nematode (PWN) uses several Monochamus species as vehicles, through a temporary hitchhiking process known as phoresy, enabling it to access new host plant resources. Monochamus saltuarius acts as a new and major vector of the PWN in Northeastern China, showing lower PWN carrying capacity and a shorter transmission cycle compared to established vectors. The apparently altered symbiotic relationship offers an interesting area for researching the costs and adaptions involved in nematode–beetle, a specialized phoresy. We analyzed the response and fitness costs of M. saltuarius through physiological measurements and transcriptomics. The PWN exerted adverse repercussions on the growth and development of M. saltuarius. The PWN accelerated larval development into pupae, while beetle adults carrying the PWN exhibited an elevated abnormality rate and mortality, and reduced starvation resistance. During the pupal stage, the expression of growth-related genes, including ecdysone-inducible genes (E74EA), cuticle proteins, and chitin genes (CHTs), markedly increased. Meanwhile, the induced immune response, mainly by the IMD and Toll signaling pathways, could be a contributing factor to adult abnormality and mortality. Adult gonads and trachea exhibited enrichment in pathways related to fatty acid elongation, biosynthesis, and metabolism. FASN, ELOVL, and SCD possibly contributed to resistance against PWN. Our research indicated that phoretic interactions between vector beetles and PWN vary throughout the vector's lifespan, particularly before and after entry into the trachea. This study highlighted the fitness costs of immunity and metabolism on the vector beetle, indicating the adaptation mechanisms and evolutionary trade-offs to PWN. [ABSTRACT FROM AUTHOR]

Pine wilt disease, a devastating infectious disease of pine trees, is caused by the pinewood nematode, Bursaphelenchus xylophilus. It is important to understand the spatial dispersal processes of B. xylophilus within host-tree tissues to assess its pathogenic mechanism. However, this is not feasible with conventional microscopy-imaging techniques. In this study, we showed that X-ray micro-computed tomography (micro-CT) imaging can be a powerful tool for visualizing infected nematodes within host-tree tissues. We visualized 161 nematodes and 11 eggs within a Pinus thunbergii stem section, 47.3 mm3 in volume, using an appropriate segmentation of the micro-CT images. Quantitative measurements of the segmented region corresponding to the nematodes allowed for the calculation of the longitudinal length and a-value, which were similar to previous morphological descriptions of B. xylophilus. The technique adopted in this study can aid in understanding the behavior of and obtaining quantitative information on B. xylophilus within tree tissues. [ABSTRACT FROM AUTHOR]

Pine wilt disease, caused by Bursaphelenchus xylophilus, is a major quarantine forest disease that has resulted in massive economic losses as well as ecological disaster. Therefore, it is imperative to study the defence response mechanism of host pine trees to infection by B. xylophilus to understand further the internal causes of pine tree death and to find control strategies. For many years, systematic research has been carried out on the pathophysiological response of pine trees to pine wilt disease. However, due to the complexity of the occurrence and development of pine wilt disease, the specific response mechanisms of different tree species have remained unclear. This paper attempts to provide an overview of the resistance mechanism of host pine trees against B. xylophilus infection through the perspectives of histopathological characteristics, physiological and biochemical responses, and molecular response mechanisms, which provide a theoretical reference for further investigating the disease resistance mechanism of pine trees to pine wilt disease and lay the foundation for the prevention and quarantine of pine wilt disease. [ABSTRACT FROM AUTHOR]

Key message: The invasive pine wood nematode is a major threat to pine forests worldwide, causing extensive tree mortality. Although scientific knowledge and control measures are continuously improving, important gaps remain. We argue that some key questions, notably related to early detection and pest management, need to be urgently tackled in countries at risk of invasion such as France. [ABSTRACT FROM AUTHOR]

The migratory endoparasitic phytonematodes Bursaphelenchus xylophilus is the causal agent of pine wilt disease and causes significant economic damage to pine forests in China. Effectors play a key role in the successful parasitism of plants by phytonematodes. In this study, 210 genes obtained by transcriptomics analyses were found to be upregulated in B. xylophilus infecting Pinus massoniana that were not functionally annotated nor reported previously in B. xylophilus infecting P. thunbergii. Among these differentially expressed genes, a novel effector, BxICD1, that could induce cell death in the extracellular space of Nicotiana benthamiana was identified. BxICD1 was upregulated in the early stages of infection, as shown by RT-qPCR analyses. In situ hybridization analysis showed that BxICD1 was expressed in the esophageal gland of nematodes. The yeast signal sequence trap system indicated that BxICD1 possessed an N-terminal signal peptide with secretion functionality. Using an Agrobacterium-mediated transient expression system, it was demonstrated that the cell death-inducing activity of BxICD1 was dependent on N. benthamiana brassinosteroid-insensitive 1-associated kinase 1 (NbBAK1). Finally, BxICD1 contributed to B. xylophilus virulence and migration in host pine trees, as demonstrated by RNAi silencing assays. These findings indicate that BxICD1 both induces plant cell death and also contributes to nematode virulence and migration in P. massonian. [ABSTRACT FROM AUTHOR]

The present study highlights, for the first time, the chemical composition of secondary metabolites (lipophilic and hydrophilic compounds) present in the phloem tissues of infected mature Pinus pinaster Ait. trees (on average 74 years old), which may be potentially determinant in the development of the pine wood nematode (PWN) Bursaphelenchus xylophilus. The levels of secondary metabolites in the phloem of infected trees were fairly constant along the height of the trees (an average of 30% dry phloem), but slightly lower compared with total extractives content in the phloem of mature healthy P. pinaster trees (30.0 vs. 39.9%). The hydrophilic fraction represented 91% of the total soluble compounds (27.3% of phloem mass) and it was mainly composed of phenolic compounds and reducing sugars (44.8% and 58.8% of the ethanol-water extracts). The lipophilic fraction (2.7% of phloem mass), was dominated by tricyclic diterpenic compounds (54.2–63.2% of the total lipophilic extracts) and fatty acids. Differences in the relative concentrations of individual compounds were observed compared to healthy pines, suggesting a response to the biotic stress imposed by nematode damage by an increased content of diterpenic resin acids, specifically dehydroabietic and abietic acids, and of total unsaturated fatty acids with a predominance of 9-octadecenoic acid. [ABSTRACT FROM AUTHOR]

Bursaphelenchus xylophilus, the pine wood nematode (PWN), is the causal agent of pine wilt disease (PWD), which causes enormous economic loss annually. According to our previous research, fomepizole, as a selective inhibitor of PWN alcohol dehydrogenase (ADH), has the potential to be a preferable lead compound for developing novel nematicides. However, the underlying molecular mechanism is still unclear. The result of molecular docking showed that the stronger interactions between fomepizole and PWN ADH at the active site of ADH were attributed to hydrogen bonds. Low-dose fomepizole had a substantial negative impact on the egg hatchability, development, oviposition, and lifespan of PWN. Transcriptome analysis indicated that 2,124 upregulated genes and 490 downregulated genes in fomepizole-treated PWN were obtained. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed genes indicated that fomepizole could be involved in controlling PWN vitality mainly by regulating key signaling pathways, such as the ribosome, hippo signaling pathway, and lysosome. Remarkably, the results of RNA interference indicated that the downregulated serine/threonine-protein phosphatase gene (stpp) could reduce the egg hatchability, development, oviposition, and lifespan of PWN, which was closely similar to the consequences of nematodes with low-dose fomepizole treatment. In addition, the silencing of stpp resulted in weakness of PWN pathogenicity, which indicated that stpp could be a potential drug target to control PWN. [ABSTRACT FROM AUTHOR]

Pine wilt disease is a major biological disaster caused by Bursaphelenchus xylophilus. This study establishes a high-quality and well-annotated genome sequence of B. xylophilus strain TS-1 from Mountain Tai in Shandong province, China. The 75-Mbp assembly containing six chromosomes was established. These genome data represent a valuable new resource for future studies on B. xylophilus and the management of pine wilt disease. [Figure: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Pinewood nematodes threaten forest health and continue to interfere with international trade because they can be spread around the globe via nematode-infested wood. International Standards for Phytosanitary Measure (ISPM-15) requires that all pine wood be treated at 56°C for 30 min to ensure that all pests and pathogens are killed within sawn wood, whereas fumigation with methyl bromide is the currently approved practice and widely used in treating whole logs. A method of treatment that uses less energy and time or does not rely on environmentally damaging gases is urgently needed. Because vacuum with steam has shown promise in treating several different commodities, the purpose of this study was to use it to eradicate pinewood nematodes in whole logs. Three protocols were applied: 1) 48°C for 15 min., 2) 56°C for 30 min., and 3) 60°C for 60 min. The third protocol reduced the population to statistically zero; however, some samples contained at least 1 survivor. Unfortunately, these surviving nematodes increased in number one month after treatment, and one year later, they continued to reproduce in the wood. Therefore, this protocol needs to be further refined to remove pinewood nematodes completely. Explanation of the survival of individual nematodes within whole logs remains a matter for conjecture: 1) certain portions of the wood were somehow insulated from the heat and did not achieve the lethal temperature, and 2) survival stages may be able to survive temperatures that are deadly to the normal life stages.

Simple Summary: Pine wilt disease is a devastating disease, and its causal pathogen, pine wood nematode (PWN), is extremely challenging to manage. This study identified that PWN enters cryptobiosis under high-osmotic-pressure stress. The phenotypic changes and survival of the nematodes during each developmental stage were significantly different. Among them, the third-stage dispersal juvenile had the highest survival and body length change rates. By comparing the gene expression differences between the third-stage dispersal juvenile and other stages, it was found that they exhibited expression differences in genes in energy compound synthesis and anti-reversal signal pathway. The results of this study provide a theoretical basis for the prevention and treatment of PWN by exploiting the differences in high osmotic pressure tolerance at different developmental stages. Under ion imbalance, water deficiency, and salt stress, the osmotic pressure of the tree sap increases, and pine wood nematodes (Bursaphelenchus xylophilus, PWN) parasitizing in the trees may be subjected to high-osmotic-pressure stress. KCl, L-malic acid, sucrose, and glycerol solutions were used as osmolytes to explore the highest osmotic concentration that PWN can tolerate. Survival analysis showed that when the treatment concentration exceeded 90%, only a few nematodes in the glycerol group survived under 6 h treatment, and most of the survivors were third-stage dispersal juveniles (DJ3). Further examination revealed that under different concentrations of glycerol-induced high osmotic pressure, the survival rate and body length change rate were the highest in the DJ3 and the lowest in the second-stage propagative juveniles. In order to explore the molecular mechanism of resistance of DJ3 to high osmotic stress, transcriptome sequencing was performed at each developmental stage of PWN and differentially expressed genes that were up-regulated or down-regulated only in DJ3 were screened. The expression of genes related to CoA in DJ3, a key enzyme in metabolism, was significantly higher than the other developmental stages. In addition, the expression of the anti-reversal signal pathway-related gene AKT-1 in DJ3 was significantly lower than in the other development stages. Therefore, the specific expression of genes in DJ3 under high osmotic pressure may help them rapidly produce and accumulate energy-related compounds and activate the adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) pathway to respond to damage caused by high-osmotic-pressure stress in time, thus promoting survival. [ABSTRACT FROM AUTHOR]

In our previous screening of the transcriptome of the causal agent of the devastating pine wilt disease, pine wood nematode (PWN, Bursaphelenchus xylophilus), after treatment with the nematicide fomepizole, Surfeit locus gene sft-4, which encodes a regulatory factor, was found to be downregulated. In situ hybridization results showed that the sft-4 was continuously expressed from egg to adult and was especially high in the reproductive system. Here in a study of the effect of RNA interference (RNAi) of sft-4 and recombinant SFT-4 on PWN activity, treatment with sft-4 dsRNA inhibited feeding, reproduction, oviposition and egg hatching of PWN with the greatest inhibition on reproduction and oviposition, whereas recombinant SFT-4 had the opposite effect. In addition, RNAi of sft-4 changed the female–male ratio and lifespan of PWN. In bioassays of PWNs, with RNAi of sft-4 on seedlings and 2-year-old Pinus thunbergii trees, none of the treated plants developed symptoms during the monitoring period, indicating that virulence of PWNs was either significantly weakened. These results indicate that the influence of sft-4 on PWN pathogenicity may be mainly through regulating reproductive function of PWN and its lifespan. [ABSTRACT FROM AUTHOR]

BACKGROUND: The pinewood nematode (Bursaphelenchus xylophilus) causes severe damage to pine trees. The nematophagous fungus, Esteya vermicola, exhibits considerable promise in the biological control of Bursaphelenchus xylophilus due to its infectivity. Notably, the lunate conidia produced by E. vermicola can infect Bursaphelenchus xylophilus. In the study, we aim to investigate the genes involved in the formation of the lunate conidia of E. vermicola CBS115803. RESULTS: Esteya vermicola CBS115803 yielded 95% lunate conidia on the complete medium (CM) and 86% bacilloid conidia on the minimal medium (MM). Transcriptomic analysis of conidia from both media revealed a significant enrichment of differentially expressed genes in the pathway related to 'cellular amino acid biosynthesis and metabolism'. Functional assessment showed that the knockout of two arginine biosynthesis genes (EV232 and EV289) resulted in defects in conidia germination, mycelial growth, lunate conidia formation, and virulence of E. vermicola CBS115803 in Bursaphelenchus xylophilus. Remarkably, the addition of arginine to the MM improved mycelial growth, conidiation and lunate conidia formation in the mutants and notably increased conidia yield and the lunate conidia ratio in the wild‐type E. vermicola CBS115803. CONCLUSION: This investigation confirms the essential role of two arginine biosynthesis genes in lunate conidia formation in E. vermicola CBS115803. The findings also suggest that the supplementation of arginine to the culture medium can enhance the lunate conidia yield. These insights contribute significantly to the application of E. vermicola CBS115803 in managing Bursaphelenchus xylophilus infections. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The endoparasitic fungus Hirsutella rhossiliensis is an important biocontrol agent of cyst nematodes in nature. To determine the potential parasitism of the fungus on a non-natural host, the pinewood nematode (Bursaphelenchus xylophilus) living in pine trees and the endophytic ability of the fungus on plants, in this paper, we first constructed and utilized a green fluorescent protein (GFP)-tagged H. rhossiliensis HR02 transformant to observe the fungal infection process on B. xylophilus and its colonization on Arabidopsis roots. Then, we compared the fungal parasitism on three species of nematodes with different lifestyles, and we found that the fungal parasitism is correlated with nematode species and stages. The parasitic effect of H. rhossiliensis on adults of B. xylophilus is similar to that on second-stage juveniles (J2) of the root-knot nematode Meloidogyne incognita after 24 h of inoculation, although the virulence of the fungus to second-stage juveniles of M. incognita is stronger than that to those of B. xylophilus and Caenorhabditis elegans. Moreover, the endophytism of H. rhossiliensis was confirmed. By applying an appropriate concentration of H. rhossiliensis conidial suspension (5 × 106 spores/mL) in rhizosphere soil, it was found that the endophytic fungus can promote A. thaliana growth and reproduction, as well as improve host resistance against M. incognita. Our results provide a deeper understanding of the fungus H. rhossiliensis as a promising biocontrol agent against plant-parasitic nematodes. [ABSTRACT FROM AUTHOR]

Knottin-type antimicrobial peptides possess exceptional attributes, such as high efficacy, low vulnerability to drug resistance, minimal toxicity, and precise targeting of drug sites. These peptides play a crucial role in the innate immunity of insects, offering protection against bacteria, fungi, and parasites. Knottins have garnered considerable interest as promising contenders for drug development due to their ability to bridge the gap between small molecules and protein-based biopharmaceuticals, effectively addressing the therapeutic limitations of both modalities. This work presents the isolation and identification of a novel antimicrobial peptide derived from Monochamus alternatus. The cDNA encodes a 56-amino acid knottin propeptide, while the mature peptide comprises only 34 amino acids. We have labeled this knottin peptide as MaK. Using chemically synthesized MaK, we evaluated its hemolytic activity, thermal stability, antibacterial properties, and efficacy against nematodes. The results of this study indicate that MaK is an exceptionally effective knottin-type peptide. It demonstrates low toxicity, superior stability, potent antibacterial activity, and the ability to suppress pine wood nematodes. Consequently, these findings suggest that MaK has potential use in developing innovative therapeutic agents to prevent and manage pine wilt disease. [ABSTRACT FROM AUTHOR]

DNA methylation is a pivotal process that regulates gene expression and facilitates rapid adaptation to challenging environments. The pinewood nematode (PWN; Bursaphelenchus xylophilus), the causative agent of pine wilt disease, survives at low temperatures through third-stage dispersal juvenile, making it a major pathogen for pines in Asia. To comprehend the impact of DNA methylation on the formation and environmental adaptation of third-stage dispersal juvenile, we conducted whole-genome bisulfite sequencing and transcriptional sequencing on both the third-stage dispersal juvenile and three other propagative juvenile stages of PWN. Our findings revealed that the average methylation rate of cytosine in the samples ranged from 0.89% to 0.99%. Moreover, we observed significant DNA methylation changes in the third-stage dispersal juvenile and the second-stage propagative juvenile of PWN, including differentially methylated cytosine (DMCs, n = 435) and regions (DMRs, n = 72). In the joint analysis of methylation-associated transcription, we observed that 23 genes exhibited overlap between differentially methylated regions and differential gene expression during the formation of the third-stage dispersal juvenile of PWN. Further functional analysis of these genes revealed enrichment in processes related to lipid metabolism and fatty acid synthesis. These findings emphasize the significance of DNA methylation in the development of third-stage dispersal juvenile of PWN, as it regulates transcription to enhance the probability of rapid expansion in PWN.

Abstract The pinewood nematode (PWN) Bursaphelenchus xylophilus is a forestry quarantine pest and causes an extremely dangerous forest disease that is spreading worldwide. Due to the complex pathogenic factors of pine wood nematode disease, the pathogenesis is still unknown. B. xylophilus ultimately invades a host and causes death. However, little is known about the defence-regulating process of host pine after infection by B. xylophilus at the molecular level. Therefore, we wanted to understand how Pinus massoniana regulates its response to invasion by B. xylophilus. P. massoniana were artificially inoculated with B. xylophilus solution, while those without B. xylophilus solution were used as controls. P. massoniana inoculated with B. xylophilus solution for 0 h, 6 h, 24 h, and 120 h was subjected to high-throughput sequencing to obtain transcriptome data. At various time points (0 h, 6 h, 24 h, 120 h), gene transcription was measured in P. massoniana inoculated with PWN. At different time points, P. massoniana gene transcription differed significantly, with a response to early invasion by PWN. According to Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, P. massoniana response to PWN invasion involves a wide range of genes, including plant hormone signal transformation, flavonoid biosynthesis, amino sugar and nucleoside sugar metabolism, and MAPK signalling pathways. Among them, inoculation for 120 hours had the greatest impact on differential genes. Subsequently, weighted gene coexpression network analysis (WGCNA) was used to analyse transcriptional regulation of P. massoniana after PWN infection. The results showed that the core gene module of P. massoniana responding to PWN was “MEmagenta”, enriched in oxidative phosphorylation, amino sugar and nucleotide sugar metabolism, and the MAPK signalling pathway. MYB family transcription factors with the highest number of changes between infected and healthy pine trees accounted for 20.4% of the total differentially expressed transcription factors. To conclude, this study contributes to our understanding of the molecular mechanism of initial PWN infection of P. massoniana. Moreover, it provides some important background information on PWN pathogenic mechanisms.

Traditional pesticides are based on toxic compounds that can reduce biodiversity, degrade the environment, and contribute to less healthy living. Plant allelochemicals can provide more environmentally friendly and sustainable alternatives. Essential oils (EOs) are complex mixtures of plant secondary metabolites that show strong biological activities. In the present study, the EOs of Cymbopogon citratus were screened for activity against the pinewood nematode (PWN), the causal agent of pine wilt disease. To understand their nematicidal properties, EOs were fractioned into hydrocarbon molecules and oxygen-containing compounds, and their main compounds were acquired and tested separately against the PWN. The EO oxygen-containing molecules fraction was highly active against the PWN (EC50 = 0.279 µL/mL), with citral and geraniol showing higher activities (EC50 = 0.266 and 0.341 µL/mL, respectively) than emamectin benzoate (EC50 = 0.364 µL/mL), a traditional nematicide used against the PWN. These compounds were additionally reported to be less toxic to non-target organisms (fish, invertebrates, and algae) and safer to human health (with higher reported toxicity thresholds) and predicted to exert fewer environmental impacts than traditional nematicides. Resorting to approved natural compounds can quickly leverage the development of sustainable alternatives to traditional nematicides.

The pinewood nematode (PWN) Bursaphelenchus xylophilus is a forestry quarantine pest and causes an extremely dangerous forest disease that is spreading worldwide. Due to the complex pathogenic factors of pine wood nematode disease, the pathogenesis is still unknown. B. xylophilus ultimately invades a host and causes death. However, little is known about the defence-regulating process of host pine after infection by B. xylophilus at the molecular level. Therefore, we wanted to understand how Pinus massoniana regulates its response to invasion by B. xylophilus. P. massoniana were artificially inoculated with B. xylophilus solution, while those without B. xylophilus solution were used as controls. P. massoniana inoculated with B. xylophilus solution for 0 h, 6 h, 24 h, and 120 h was subjected to high-throughput sequencing to obtain transcriptome data. At various time points (0 h, 6 h, 24 h, 120 h), gene transcription was measured in P. massoniana inoculated with PWN. At different time points, P. massoniana gene transcription differed significantly, with a response to early invasion by PWN. According to Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, P. massoniana response to PWN invasion involves a wide range of genes, including plant hormone signal transformation, flavonoid biosynthesis, amino sugar and nucleoside sugar metabolism, and MAPK signalling pathways. Among them, inoculation for 120 hours had the greatest impact on differential genes. Subsequently, weighted gene coexpression network analysis (WGCNA) was used to analyse transcriptional regulation of P. massoniana after PWN infection. The results showed that the core gene module of P. massoniana responding to PWN was "MEmagenta", enriched in oxidative phosphorylation, amino sugar and nucleotide sugar metabolism, and the MAPK signalling pathway. MYB family transcription factors with the highest number of changes between infected and healthy pine trees accounted for 20.4% of the total differentially expressed transcription factors. To conclude, this study contributes to our understanding of the molecular mechanism of initial PWN infection of P. massoniana. Moreover, it provides some important background information on PWN pathogenic mechanisms. [ABSTRACT FROM AUTHOR]

In order to explore the changes in the fatty acid metabolism of Bursaphelenchus xylophilus under the treatment of Bacillus cereus NJSZ-13, the surface changes in lipid droplets were observed under a Zeiss microscope after the B. xylophilus was stained with modified Oil Red O. The triglyceride (TG) content in B. xylophilus was determined according to the TG kit instructions. The type and content of fatty acids in B. xylophilus were detected by gas chromatography–mass spectrometry (GC-MS); the stearyl coenzyme A of B. xylophilus was analyzed by real-time fluorescence quantitative PCR. The change in Bx-SCD (gene regulating stearyl CoA desaturase in B. xylophilus) expression was observed. The results showed that the lipid droplets of B. xylophilus treated with NJSZ-13 were broken to varying degrees, and the TGs in B. xylophilus decreased continuously. The total fatty acid content in the bodies of treated B. xylophilus decreased: the difference between the fermentation broth treatment and the control was extremely significant (p < 0.01); that between the fermentation filtrate and the control was significant (p < 0.05); and that between the bacterial suspension and the control was not significant (p > 0.05). Saturated fatty acids decreased in all treatments, but not significantly. Compared with the control group, the unsaturated fatty acid content in fermentation broth and fermentation filtrate treatments was extremely significantly reduced, and the unsaturated fatty acid content of the bacterial suspension group was significantly decreased, which indicated that NJSZ-13 mainly caused a decrease in the unsaturated fatty acids in B. xylophilus. The trend in changes in monounsaturated fatty acids and unsaturated fatty acids was the same, but for polyunsaturated fatty acids, the fermentation broth and fermentation filtrate treatments caused a significant decrease in content, but the bacterial suspension resulted in no significant change. The results showed that NJSZ-13 mainly caused a decrease in monounsaturated fatty acid content in B. xylophilus. In addition, the contents of C16:1, C18:1, and C18:2 fatty acids were significantly decreased after treatment with strain NJSZ-13 for 48 h, and the contents of C16:1, C18:1, C18:2, and C20:4 were extremely significantly decreased after the fermentation broth and filtrate treatments. The expression of Bx-SCD in B. xylophilus was significantly lower than that of the control (p < 0.0001). This study analyzed the changes in the content of related substances and relative gene expression in fatty acid metabolism of B. xylophilus treated with strain NJSZ-13, and preliminarily reveals the nematicidal mechanism of strain NJSZ-13 against B. xylophilus. This provides a theoretical basis for further exploration of the key cause of death induced by this strain in B. xylophilus. [ABSTRACT FROM AUTHOR]

Lipase is involved in lipid hydrolysis, which is related to nematodes' energy reserves and stress resistance. However, the role of lipases in Bursaphelenchus xylophilus, a notorious plant‐parasitic nematode responsible for severe damage to pine forest ecosystems, remains largely obscure. Here, we characterized a class III lipase as a candidate effector and named it BxLip‐3. It was transcriptionally up‐regulated in the parasitic stages of B. xylophilus and specifically expressed in the oesophageal gland cells and the intestine. In addition, BxLip‐3 suppressed cell death triggered by the pathogen‐associated molecular patterns PsXEG1 and BxCDP1 in Nicotiana benthamiana, and its Lipase‐3 domain is essential for immunosuppression. Silencing of the BxLip‐3 gene resulted in a delay in disease onset and increased the activity of antioxidant enzymes and the expression of pathogenesis‐related (PR) genes. Plant chitinases are thought to be PR proteins involved in the defence system against pathogen attack. Using yeast two‐hybrid and co‐immunoprecipitation assays, we identified two class I chitinases in Pinus thunbergii, PtChia1‐3 and PtChia1‐4, as targets of BxLip‐3. The expression of these two chitinases was up‐regulated during B. xylophilus inoculation and inhibited by BxLip‐3. Overall, this study illustrated that BxLip‐3 is a crucial virulence factor that plays a critical role in the interaction between B. xylophilus and host pine. [ABSTRACT FROM AUTHOR]

The northward expansion of Bursaphelenchus xylophilus has caused devastating disasters in the northeastern region, posing a significant threat to Pinus koraiensis, which is the major component species of the top ecological community of the Northeast China forest area. This study aims to enrich the molecular mechanism of the interaction between P. koraiensis and B. xylophilus, thus laying a theoretical foundation for the breeding of disease-tolerant P. koraiensis. The disease tolerance of 20 families of P. koraiensis to B. xylophilus was identified by artificial inoculation and different families of P. koraiensis were divided into three categories: tolerant, susceptible and high-susceptible. The RNA of tolerant and high-susceptible P. koraiensis was extracted for transcriptome sequencing. The α-terpineol synthase gene (Pk-αts) related to disease tolerance was screened and cloned. The expression level of Pk-αts in 4 tolerant, 2 susceptible, and 4 high-susceptible P. koraiensis was randomly detected by RT-qPCR, and the relative tolerance index of these 10 P. koraiensis families was calculated. The correlation between Pk-αts expression level and the relative tolerance index of P. koraiensis was analyzed. The results showed that after inoculating B. xylophilus for 20 days, only a few needles of tolerant P. koraiensis turned yellow, with an average relative tolerance index of 0. 88, while high-susceptible P. koraiensis showed partial needle redness, with an average relative tolerance index of 0. 09. Transcriptome sequencing identified 1 770 genes that were up-regulated in tolerant P. koraiensis and had no change or down-regulated in high-susceptible P. koraiensis. The relative expression of Pk-αts in tolerant P. koraiensis was 2. 83, which was extremely significant, but the relative expression in high-susceptible P. koraiensis was -1. 27. The cloned Pk-αts gene was 1 230 bp in length, encoding a protein composed of 409 amino acids, with an isoelectric point of 5. 55 and a relative molecular weight of 47. 60 ku. After comparison, it had the highest homology with the α-terpineol synthase gene and contained conserved motifs such as DDxxD and RRx8W of plant monoterpene synthases and Terpene_synth domain, which were consistent with the structural characteristics of monoterpene synthases. RT-qPCR detection showed that the expression of the Pk-αts gene was upregulated (the average expression level is 1. 39) in tolerant P. koraiensis, while it was unchanged or downregulated in susceptible and high -susceptible P. koraiensis, indicating that the upregulation of the Pk-αts gene expression was synchronized with the temporal and spatial of P. koraiensis tolerance to in the early stage of B. xylophilus infection. The expression level of Pk-αts was positively correlated with the relative tolerance index of P. koraiensis (Pearson's coefficient was 0. 911, P< 0. 01), indicating that the up-regulated expression of the Pk-αts gene improved the disease tolerance of P. koraiensis. In summary, different families of P. koraiensis have different tolerance to B. xylophilus, and some P. koraiensis have certain disease tolerance. The up-regulated expression of Pk-αts gene alleviates disease symptoms and helps to improve the disease tolerance of P. koraiensis to B. xylophilus. The Pk-αts gene can be used as a detection target for identifying tolerance of P. koraiensis to B. xylophilus, which is helpful for the breeding of tolerant P. koraiensis. [ABSTRACT FROM AUTHOR]

Pine wilt disease (PWD), caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus, poses a severe threat to pine forests worldwide. However, the understanding of the impact of PWD on the host microbiome remains limited. This study aimed to investigate the structure and function of the fungal community associated with Pinus thunbergii fine roots and the rhizosphere fungi and bacteria of the tree naturally infected by PWN and the healthy tree. We employed high-throughput sequencing in conjunction with functional prediction tools (Functional Annotation of Prokaryotic Taxa and Fungi Functional Guild) and soil enzyme activity measurements between the two treatments (disease vs. health). The results showed that PWD significantly decreased the activity of β-cellobiosidase (CEL) and β-glucosidase (GLS) enzymes involved in carbon cycling in the rhizosphere (p < 0.05). However, PWD did not alter the diversity of rhizosphere bacteria and fine root fungi, but it did cause a significant decrease in the richness of rhizosphere fungi (p < 0.05). Moreover, PWD significantly reduced the abundance of Actinobacteria and genus Gaiella (p < 0.05). Functionally, bacterial intracellular parasites exhibited a higher abundance in the rhizosphere after PWN infection, whereas ureolysis showed a lower abundance (p < 0.05). Fungal saprotroph–symbiotroph exhibited a higher abundance in the rhizosphere after PWN infection, whereas symbiotroph showed a lower abundance (p < 0.05). Additionally, it led to a significant reduction in the infection rate of ectomycorrhizal fungi (p < 0.05). Infected host fine root exhibited higher abundance of pathotroph–symbiotroph, whereas symbiotroph had a lower abundance (p < 0.05). These findings provided valuable insights into the interactions between pine wilt disease, plant microbial communities, and soil enzyme activity. [ABSTRACT FROM AUTHOR]

Abstract Key message This study takes stock of the first 20 years (2000–2019) of monitoring the pine wood nematode (PWN) in metropolitan France. While PWN was never found in the wild during this period, it was reported in some wood-based commodities entering or circulating on French territory. This stresses the importance of remaining extremely vigilant, as the conditions found in France, especially weather conditions, could be particularly suitable for the pest’s establishment. Context The pine wood nematode (PWN) Bursaphelenchus xylophilus, responsible for pine wilt disease (PWD), is one of the most important forest tree pests worldwide. It is thus the focus of many monitoring programmes. In the European Union, for example, it is categorised as a priority quarantine pest, so each member state is obliged to monitor the PWN on its territory. Aims The first objective of this paper was to describe PWN monitoring in metropolitan France, namely how it is organised and whether it has led to the nematode’s detection. Secondly, we wished to investigate what the levels of PWD expression for host pines infected by B. xylophilus would be in France. Thirdly, we wanted to find out whether other Bursaphelenchus species had been found on French territory during these two decades of PWN monitoring. Methods We analysed data from samples collected in the framework of the monitoring programme between 2000 and 2019 to track the PWN in its host pines, its insect vector (Monochamus spp.) and in wood-based commodities imported into or circulating in metropolitan France. We also generated risk maps of PWD expression based on an evapo-transpiration model using climate data for the period 2000–2019. Results This monitoring, which was regularly reinforced from 2000 to 2019, consisted of sampling and analysing around 18,000 wood samples and 66,000 insects over this period. Although the PWN was not detected in pine stands or within its insect vector, some wood-based commodities were found to be contaminated. Risk maps of PWD expression show that in the most recent years (2015–2019), the weather conditions in a large fraction of metropolitan France were suited to PWD expression, mostly with a delay (i.e., latency) between infection and observable wilt symptoms. PWN monitoring has also revealed the presence of other Bursaphelenchus species, most of which were discovered for the first time in metropolitan France and are described herein. Conclusion While metropolitan France is still free of the PWN, this study emphasises the need to remain cautious as the French territory appears particularly suitable for this pest’s establishment. Furthermore, our research has led us to propose some ideas on how to improve PWN monitoring.

Monochamus saltuarius Gebler was first identified as a new vector of pine wilt disease in Northeast China in 2018, and monitoring of M. saltuarius has become a key strategy to prevent and control the disease in this region. However, the potential distributions of M. saltuarius in China are unclear. In this study, we clarified bioclimatic environmental variables affecting the distribution of M. saltuarius, predicted the geographically suitable habitats of M. saltuarius in current and future climate conditions, and determined changes in the spatial pattern of a suitable distribution area of M. saltuarius under current and future climate conditions. This is the first study to use the optimized maximum entropy model and ArcGIS to accurately predict suitable geographical areas for M. saltuarius based on different climatic conditions in China. and the average area under the receiver operating characteristic curve reached 0.954 ± 0.0024. Of the 32 bioclimatic variables, temperature seasonality, precipitation of wettest month, precipitation seasonality, maximum temperature of the warmest month, and elevation played significant roles in determining the potential distribution of M. saltuarius, with contribution rates to the model of 32.1, 31.8, 11.5, 7.5, and 6.5%, respectively. Under the current climate scenario, the predicted suitable areas for M. saltuarius were mainly at latitudes north of 33° in China, and larger suitable areas were mainly distributed in Northeast China and North China, with areas of 87.04 × 104 and 73.15 × 104 km2, respectively. Using future climatic scenarios SSP126 and SSP585, the predicted suitable areas of M. saltuarius will continue to expand from that of 2040, 2070, and 2100, with highly and moderately suitable areas showing larger increasing trends but low suitable distribution areas will decrease to varying degrees. The potential suitable areas of M. saltuarius may increase greatly in Northwest, Central, and Eastern China. This study provides important scientific theoretical knowledge for effectively controlling and preventing M. saltuarius and pine wilt disease in northern China.

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Pine wilt disease (PWD) is a highly destructive disease in forest ecosystems, resulting in extensive forest decline and substantial economic losses. As soil pH plays a critical role in soil microbial activity and significantly impacts the prevalence and severity of diseases, we conducted an experiment to regulate soil pH for alleviating PWD in a black pine (Pinus thunbergii) forest. The result reveals that: (1) The pH of the soil under a P. thunbergii forest was 5.19 ± 0.40, which was significantly lower than that of soils under other vegetation types at 8.53 ± 0.44. (2) Finely ground shell powder (F-SP) was the optimal size for long-term and efficient regulation, but quicklime (QL) exhibited the strongest efficacy in raising soil pH, followed by F-SP and plant ash. The regulation effect strengthened with the dosage amount. (3) In the situ experiments, part of symptomatic black pine in F-SP or QL plots were apparently improved and converted to asymptomatic trees separately by 15.9% and 5.4%. Applying F-SP can alleviate PWD in a sustainable way. This paper presents the first investigation to assess the effects of regulating soil pH for controlling PWD. It holds significant practical value for the rational planning and the sustainable development of artificial forests in coastal regions. [ABSTRACT FROM AUTHOR]

Introduction: Pine wilt disease (Bursaphelenchus xylophilus) was recently detected in Liaoning Province, which was previously considered an unfavourable area for B. xylophilus due to its lowtemperatures. This studyaims tocomparethe reproductivity and genetic variations of B. xylophilus isolates from Liaoning Province and other parts of China to explore their phenotypic and genomic differences. Methods: The samples from Liaoning, Anhui, Hubei, Henan, Zhejiang and Jiangsu were isolated and purified to obtain the strains. The reproductivity of the strains was determined at 15 °C. The genetic structure was analyzed by using SNP molecular markers, and the whole genome association analysis was carried out by integrating SNP information and feculence traits. Results: A reproductivity experiment showed that Liaoning isolates have higher reproductive ability at 15 °C. Subsequent SNP profiling and population differentiation analysis revealed obvious genetic differentiation of Liaoning isolates from other isolates. A genome-wide association study showed that SNPs closely related to low-temperature tolerance were mainly located in GPCR, Acyl-CoA, and Cpn10, which are responsible for adaptation to environmental factors, such as temperature change. Discussion: Pine wood nematodes likely adapted to the climate in Liaoning and maintained a certain reproductive capacity at low temperature via variants of adaptation-related genes. This study provides a theoretical basis for elucidating the prevalence and diffusion status of B. xylophilus in China. [ABSTRACT FROM AUTHOR]

Hubei, Hunan and Henan Provinces are located in Central China, a region with extensive transport networks and trade. The pine wilt nematode (PWN), Bursaphelenchus xylophilus, the causative agent of pine wilt disease, is spread mainly through human activities. To further understand the genetic structure of PWN in Central China, we studied the genetic information of PWN populations in this region and compared the genetic relationship with strains from Guangdong and Jiangsu provinces. We found that the HB (Hubei) 15, HEN (Henan) 20, HN (Hunan) 07, HN08 and HN10 had significantly more SNPs and homozygotes than other strains from Central China, and their most frequent mutant genotypes also differed from other strains. The clustering results indicated that HB15, HEN 20, HN07, HN08 and HN10 were genetically distinct from other strains and closely related to Guangdong strains. We also observed significant genetic variation among strains in Henan province, suggesting that some of them might have different transmission sources than those from Hubei and Hunan provinces. Introgression analysis identified three possible pathways: (1) Guangdong to Henan; (2) Guangdong to Hunan; and (3) Jiangsu to Hubei. The results provide a basis for tracing the origin and spread of pine wood disease in China. [ABSTRACT FROM AUTHOR]

Pine wilt disease (PWD) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, is a globally distributed destructive disease of pine forest. To study the PWD pathogenic mechanism, the cinnamyl alcohol dehydrogenase gene (BxCAD-1) from B. xylophilus was selected. The BxCAD-1 gene was amplified by PCR from the cDNA library of B. xylophilus and cloned into the expression vector pET-15b to construct the recombinant vector pET-15b-BxCAD-1. The recombinant cinnamyl alcohol dehydrogenase protein was purified by Ni-NTA affinity chromatography from Escherichia coli BL21 (DE3) harboring pET-15b-BxCAD-1 induced by IPTG. The effects of pH, temperature, metal ions and substrates on the activity of BxCAD-1 were determined, showing the highest catalytic activity at pH 8.0 and 40 °C with cinnamyl alcohol as substrate and Zn2+ as an activator. To elucidate the functions of BxCAD-1 in B. xylophilus, the expression of the gene was down-regulated by RNA interference. Results showed that the movement, feeding, reproduction, spawning rate, hatching rate, lifespan, infectivity and sensitivity to ethanol decreased compared with negative controls. RNA interference also affected the development of B. xylophilus from the larval stage to the adult stage. In situ hybridization showed that the gene was expressed in the digestive tract of male and female adults. This study revealed a promising target for PWD control. [ABSTRACT FROM AUTHOR]

In previous research, the pine wood nematode Bxadh2 gene significantly increased its expression in pine seedlings inoculated with endophytic Bacillus cereus GD2 and pine wood nematode. To explore pine wood nematode Bxadh2 gene function, we cloned and analyzed its biological information, and we preliminarily studied its physiological function through RNA interference. We found that the Bxadh2 gene's full CDS length is 1269 bp, which encodes 422 amino acids, and presents a relatively stable hydrophobic protein. The protein encoded by the Bxadh2 gene has no signal peptide or transmembrane structure, and it is an intracellular protein that does not participate in transmembrane movement. The RNAi interference results showed that when the pine wood nematode's Bxadh2 gene was suppressed, its survival rate and fecundity significantly decreased, indicating that the expression of the Bxadh2 gene was related to the growth and development of pine wood nematodes. [ABSTRACT FROM AUTHOR]

Bursaphelenchus xylophilus, also known as the pine wood nematode (PWD), which causes pine wilt disease (PWD), is one of the most devastating diseases affecting pine forests globally. G protein-coupled receptors (GPCRs) are crucial in many biological processes and serve as privileged points of communication between cells and the surrounding environment. α-pinene has been found to play a crucial role in combating the infection, colonization, and early stages of pathogenesis caused by B. xylophilus. In this study, we investigated the molecular characteristics and biological functions of the GPCR gene Bx-srh-1 in B. xylophilus. Fluorescence in situ hybridization (FISH) was performed to determine the spatial expression patterns of Bx-srh-1 in B. xylophilus. The results indicated that Bx-srh-1 is expressed in the intestine and subcutaneous tissues of J2 and J3 juveniles and in the spicules of adult males and vulvae of adult females. RNA interference (RNAi) was used to analyze Bx-srh-1 gene function, and we examined the expression patterns of Bx-srh-1 in B. xylophilus under α-pinene stress. The RNA interference indicated that Bx-srh-1 was involved in the reproductive ability and pathogenicity of B. xylophilus; the expression levels of Bx-srh-1 significantly increased after the exposure to α-pinene for 12 h, and they peaked at 48 h. Silencing Bx-srh-1 may therefore lead to a reduction in B. xylophilus reproduction and pathogenicity. These results demonstrate that Bx-srh-1 is related to the feeding behavior, reproduction, pathogenicity, and resistance to α-pinene process of B. xylophilus. [ABSTRACT FROM AUTHOR]