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Plant-parasitic nematodes are highly damaging pests responsible for heavy losses in a considerable number of plant crops. Common pest management strategies rely on the use of synthetic chemical nematicides, which have led to serious concerns regarding their impact on human health and the environment. The essential oils (EOs) obtained from aromatic plant species can provide a good source of agents for the sustainable control of nematodes, due to higher biodegradability, generally low toxicity for mammals, fish, and birds, and lower bioaccumulation in the environment. This study aimed to evaluate the nematicidal and nematostatic properties of EOs extracted from plant species relevant to Macaronesia flora or with widespread use as culinary herbs in Mediterranean cuisine. Eighteen EOs were chemically characterized and evaluated by direct contact and hatching bioassays on the root-knot nematode Meloidogyne javanica. The EOs that showed a significant effect on M. javanica second-stage juveniles' (J2) mortality (≥40%) were also used in chemotaxis assays. From the eighteen EOs, seven showed strong nematicidal activity (>80%) and hatching inhibition. The chemotaxis assays revealed that only Mentha pulegium exhibited repellent behavior for M. javanica J2, and the rest of EOs had attractive behavior. Furthermore, EOs were assessed against the root-lesion nematode Pratylenchus penetrans and the pinewood nematode Bursaphelenchus xylophilus. Cinnamomum burmanni was the EO with the highest nematicidal activity for the three nematode species. Among the terpene-rich EOs, high mortality values and hatching inhibition for M. javanica were observed for the carvacrol chemotype Origanum vulgare, albeit with low activity for P. penetrans and B. xylophilus. Mentha pulegium, mainly composed of monoterpene ketones and monoterpenoids, demonstrated moderate-to-high mortality activity (from 30% for P. penetrans to 99% for M. javanica) for the three nematode species. [ABSTRACT FROM AUTHOR]

The European Commission requested the EFSA Panel on Plant Health to deliver a risk assessment on the likelihood of pest freedom from regulated EU quarantine pests, with emphasis on Bursaphelenchus xylophilus and its vectors Monochamus spp. of debarked conifer wood chips fumigated with sulfuryl fluoride as proposed by the United States (US) and as outlined in ISPM 28 - PT23 of sulfuryl fluoride (SF) fumiga- tion treatment for nematodes and insects in debarked wood. The assessment con- sidered the different phases in the wood chips' production, with special emphasis on the SF treatment. In addition to B. xylophilus and its vectors Monochamus spp., 22 EU quarantine pests and protected zone quarantine pests, some of which are regulated as groups of pests by the Commission Implementing Regulation (EU) 2019/2072, are present in the US and are potentially associated with the commodity. For these pests an expert judgement is given on the likelihood of pest freedom taking into con- sideration the available scientific information and technical information provided by the US, including uncertainties associated with the assessment. The likelihood of pest freedom varies among the pests evaluated, with B. xylophilus being the pest most frequently expected on the commodity. The Expert Knowledge Elicitation (EKE) indicated with 95% certainty that between 9491 and 10,000 m³ of debarked conifer wood chips treated with SF per 10,000 m³ will be free from B. xylophilus, and that between 9987 and 10,000 m³ of wood chips per 10,000 m³ will be free from Monochamus spp. Technical elements which are critical for a successful treatment and for minimising the presence of Union quarantine pests on the commodity are identified and described in the opinion. In particular, it is important to note that SF treatments are generally less effective in eliminating fungi than insects, the required parameters of the fumigation should be met at all points of the pile of wood chips and the time of storage of wood chips before treatment should be kept as short as possible because B. xylophilus can easily reproduce and spread throughout the pile under conducive conditions. [ABSTRACT FROM AUTHOR]

To study the trapping effectiveness of various attractants on Monochamus alternatus carrying pathogenic levels of Bursaphelenchus xylophilus, five attractants:APF-Ⅰ(A), Shuiguang(B), F2 (C), Zhongjie Sifang(D), and F8 (E) were selected for comparison. The results showed that a high number of dead trees caused by pine wilt disease in pine forests led to an increase in M. alternatus population density. The severity of pinewood nematode disease was positively correlated with the population density of M. alternatus carrying B. xylophilus. For trapping M. alternatus with pathogenic levels of B. xylophilus, attractant A was most effective in forests with a high incidence of pine wilt disease, while attractants B and D showed better performance in forests with a low incidence. In controlling pine wilt disease, attractant A is recommended for areas with severe outbreaks, whereas attractants B and D are suited for areas with early outbreaks. Selecting attractants based on the disease incidence in different forest areas can effectively and accurately reduce the density of M. alternatus carrying pathogenic levels of B. xylophilus, which is crucial for controlling the development and spread of pinewood nematode disease. [ABSTRACT FROM AUTHOR]

Bursaphelenchus xylophilus (pine wood nematode, PWN) has been present in China for over 40 years and has spread to northeast China, where native pine species are key components of the local top community. Pinus thunbergii is known to be susceptible to PWN among local conifer species, whereas research on PWN's pathogenicity in Larix remains limited. Furthermore, there are no research reports on PWN infestation in Picea and Abies species within China. This study conducted a detailed analysis of phenotypic changes and temporal spectral reflectance variations in four conifer species in northeast China—P. thunbergii, Larix kaempferi, Picea koraiensis, and Abies holophylla—following artificial inoculation with PWN. The aim of this study is to establish a theoretical basis for identifying the potential hosts and threats of PWN. The study incorporated a 60-day post-inoculation observation period to systematically monitor and compare temporal changes in external morphology, disease susceptibility (incidence and mortality rates), spectral reflectance, and the normalized wilt index (NWI) in 2–3-year-old seedlings of P. thunbergii, L. kaempferi, P. koraiensis, and A. holophylla after inoculation with PWN. The results showed that P. thunbergii displayed the earliest infection symptoms, followed by L. kaempferi, A. holophylla, and finally P. koraiensis. After inoculation, P. thunbergii was the first to experience mortality, followed by L. kaempferi, P. koraiensis, and A. holophylla. Following inoculation, P. thunbergii exhibited the earliest significant increase in NWI (p < 0.001), followed by L. kaempferi and A. holophylla; P. koraiensis showed the latest increase (p < 0.001). In conclusion, the experiment identified P. koraiensis as having the strongest resistance to PWN among the four species, followed by A. holophylla. P. thunbergii showed the weakest resistance, while L. kaempferi exhibited moderate resistance. The ranking of PWN susceptibility for the four conifer species, from highest to lowest, is as follows: P. thunbergii, L. kaempferi, A. holophylla, and P. koraiensis. [ABSTRACT FROM AUTHOR]

Bursaphelenchus xylophilus is one of the most destructive quarantine pests, causing irreversible damage to pine trees. However, the unexpected identification of pine wilt disease in Northern China indicates that Bursaphelenchus xylophilus can survive under low temperatures. In this study, we analyzed the reproductivity variations among 18 different isolates, and SC13 was identified to have excellent low temperature resistance. Subsequent molecular analysis of SC13 indicated its distinct gene expression under low temperatures. The epidermal growth factor, nematode cuticle collagen and G-protein-coupled receptor genes with environmental adaptation functions were demonstrated to be differentially expressed under low temperatures. Meanwhile, morphological observations also indicated that SC13 contained significantly more lipid drops in low-temperature treatments. Generally, the identification of representative Bursaphelenchus xylophilus isolates will facilitate relevant studies in the future, and the discovery of the gene expression and morphological changes of Bursaphelenchus xylophilus under low temperatures could expand the current understanding of the environmental adaption abilities of such invasive nematodes. [ABSTRACT FROM AUTHOR]

Invasive pests and plant pathogens pose a significant threat to ecosystems and economies worldwide, prompting the need of anticipatory strategies. Preventing their introduction by detection at the ports of entry has been proven extremely difficult. This review explores the potential of biogenic volatile detection as a reliable preventive solution. It underscores the importance of early detection and rapid response as integral components of effective invasive pest management, and it discusses the limitations of current control measures and the increasing globalization that facilitates the spread of pests and pathogens. Through a synthesis of existing literature, this review analyzes the Volatile Organic Compound (VOC) emissions in five invasive model species: three insects, Halyomorpha halys , Spodoptera frugiperda , Helicoverpa armigera , a nematode, Bursaphelenchus xylophilus , and an oomycete, Phytophthora ramorum. The review focuses on the specific volatiles, released by both the invasive organisms and the infested host plants. If available, the volatiles emitted from similar species were considered for comparison. Ultimately, this review highlights specific pest volatile and shared Herbivore Induced Plant Volatiles (HIPVs) as a reliable and innovative solution in pest detection. If possible, candidate compounds are provided, whilst the lack of some emphasizes the urge of expanding the information available. [ABSTRACT FROM AUTHOR]

Wood-decay fungi, including white- and brown-decay fungi, are well known for their ability to degrade lignin and cellulose, respectively. The combined use of these fungi can increase the decomposition of woody substrates. Research has indicated that these fungi also exhibit inhibitory effects against Bursaphelenchus xylophilus, the causative agent of pine wilt disease (PWD). In this study, we investigated a composite microbial formulation that efficiently decomposes pine wood while inhibiting B. xylophilus. We initially established a correlation between the degradation rate of wood blocks and fungal biomass, underscoring the necessity of optimizing biomass for effective treatment. A systematic approach involving a one-way test, a Plackett–Burman design, a steepest ascent experiment, and a Box–Behnken design, was employed to optimize the fermentation process. Validation trials were conducted in a 10-L fermenter. The bioagent's efficacy and safety were assessed through field applications in a forest, with a focus on wood degradation capacity and B. xylophilus mortality rate. Additionally, the environmental impact of the microbial products was evaluated by analysing soil quality around treated areas to ensure that the formulation did not adversely affect soil health. [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.

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]

This study focused on the persistence, distribution, and efficacy of trunk-injected pesticides in Pinus koraiensis (Korean pine) with regard to controlling pinewood nematodes (PWNs, Bursaphelenchus xylophilus), the causative agent of pine wilt disease (PWD). In this study, we compared pesticide residues in the needles and branches of Korean pine, revealing significant declines in pesticide residues over time after treatments were applied. Notably, abamectin residues decreased from 0.2325 mg/kg to 0.0901 mg/kg in branches over a period of 18 months. In contrast, emamectin benzoate showed a variation in residue decline depending on the formulation, with the residue level in branches decreasing from 0.1220 mg/kg to 0.0328 mg/kg over the same period. From a spatial perspective, the results revealed minimal differences in pesticide residue at varying tree heights, although a decrease in upper canopy residue was observed in some cases. The nematicidal efficacy test demonstrated that none of the treated trees developed PWN symptoms. Overall, the findings suggest that the trunk-injected pesticides abamectin and emamectin benzoate can persist for two years, with the residue levels being sufficient to prevent PWN propagation, even when the levels are below critical inhibition concentrations. [ABSTRACT FROM AUTHOR]

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

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]

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 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 (PWD), caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus), is one of the most serious threats to pine forests worldwide. The fungus Esteya vermicola, with its lunate conidia capable of parasitizing the PWN, has shown promise as an efficient biological control agent against PWD. Solid-state fermentation (SSF) is preferred for large-scale applications in the field, as it facilitates microbial agent transport and ensures a long shelf life. However, research on enhancing the yield of lunate conidia from E. vermicola through SSF is limited. In this study, we initially achieved a yield of 3.04 × 108 conidia/g using a basic SSF medium composed of wheat bran, corn flour, and soybean flour. To improve this yield, we employed an orthogonal experimental design (OED) to identify the optimal medium composition, which required a wheat bran-to-corn flour-to soybean flour ratio of 7:2:1 (w/w/w), a substrate-to-water ratio of 1:0.7 (w/v), and the addition of 1.33% (w/w) glucose, 1.33% (w/w) yeast extract fermentation, and 1.33% (w/w) MgSO4. Using the response surface methodology (RSM), we calculated the optimal fermentation conditions, which were 24.9 °C, 78.0% relative humidity (RH), an inoculation volume of 16.3% (v/w), and a fermentation time of 7.1 days. Under these conditions, the yield of lunate conidia reached a maximum of 16.58 × 108 conidia/g, a 4.45-fold increase after optimization. This study improved the yield of E. vermicola lunate conidia and provides insights for developing biopesticides based on this strain.

Plant-parasitic nematodes are highly damaging pests responsible for heavy losses in a considerable number of plant crops. Common pest management strategies rely on the use of synthetic chemical nematicides, which have led to serious concerns regarding their impact on human health and the environment. The essential oils (EOs) obtained from aromatic plant species can provide a good source of agents for the sustainable control of nematodes, due to higher biodegradability, generally low toxicity for mammals, fish, and birds, and lower bioaccumulation in the environment. This study aimed to evaluate the nematicidal and nematostatic properties of EOs extracted from plant species relevant to Macaronesia flora or with widespread use as culinary herbs in Mediterranean cuisine. Eighteen EOs were chemically characterized and evaluated by direct contact and hatching bioassays on the root-knot nematode Meloidogyne javanica. The EOs that showed a significant effect on M. javanica second-stage juveniles’ (J2) mortality (≥40%) were also used in chemotaxis assays. From the eighteen EOs, seven showed strong nematicidal activity (>80%) and hatching inhibition. The chemotaxis assays revealed that only Mentha pulegium exhibited repellent behavior for M. javanica J2, and the rest of EOs had attractive behavior. Furthermore, EOs were assessed against the root-lesion nematode Pratylenchus penetrans and the pinewood nematode Bursaphelenchus xylophilus. Cinnamomum burmanni was the EO with the highest nematicidal activity for the three nematode species. Among the terpene-rich EOs, high mortality values and hatching inhibition for M. javanica were observed for the carvacrol chemotype Origanum vulgare, albeit with low activity for P. penetrans and B. xylophilus. Mentha pulegium, mainly composed of monoterpene ketones and monoterpenoids, demonstrated moderate-to-high mortality activity (from 30% for P. penetrans to 99% for M. javanica) for the three nematode species.

Wood-decay fungi, including white- and brown-decay fungi, are well known for their ability to degrade lignin and cellulose, respectively. The combined use of these fungi can increase the decomposition of woody substrates. Research has indicated that these fungi also exhibit inhibitory effects against Bursaphelenchus xylophilus, the causative agent of pine wilt disease (PWD). In this study, we investigated a composite microbial formulation that efficiently decomposes pine wood while inhibiting B. xylophilus. We initially established a correlation between the degradation rate of wood blocks and fungal biomass, underscoring the necessity of optimizing biomass for effective treatment. A systematic approach involving a one-way test, a Plackett–Burman design, a steepest ascent experiment, and a Box–Behnken design, was employed to optimize the fermentation process. Validation trials were conducted in a 10-L fermenter. The bioagent’s efficacy and safety were assessed through field applications in a forest, with a focus on wood degradation capacity and B. xylophilus mortality rate. Additionally, the environmental impact of the microbial products was evaluated by analysing soil quality around treated areas to ensure that the formulation did not adversely affect soil health.

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]

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]

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]

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]

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.

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.

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.

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]

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]

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]

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.

Abstract Endophytic bacteria are an important biological control for nematodes. We isolated the nematicidal Bacillus cereus NJSZ-13 from healthy Pinus elliottii trunks. Bioassay experiments showed killing of all tested nematodes by proteins from the NJSZ-13 culture filtrate within 72 h. Degradation of the nematode cuticles was observed, suggesting the action of extracellular bacterial enzymes. The responsible protease was purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, ion-exchange chromatography, and SDS-PAGE. The protease had a molecular weight of 28 kDa and optimal activity at 55 °C and pH 9, indicating an alkaline protease. The study suggests the potential for using this B. cereus NJSZ-13 strain protease to prevent pinewood nematode infection.

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]

Pine wilt disease (caused by the nematode Bursaphelenchus xylophilus) is extremely harmful to pine forests in East Asia. As a low-resistance pine species, Pinus thunbergii is more vulnerable to pine wood nematode (PWN) than Pinus densiflora and Pinus massoniana. Field inoculation experiments were conducted on PWN-resistant and -susceptible P. thunbergii , and the difference in transcription profiles 24 h after inoculation was analyzed. We identified 2603 differentially expressed genes (DEGs) in PWN-susceptible P. thunbergii , while 2559 DEGs were identified in PWN-resistant P. thunbergii. Before inoculation, DEGs between PWN-resistant and PWN-susceptible P. thunbergii were enriched in the REDOX (Oxidation-Reduction) activity pathway (152 DEGs), followed by the oxidoreductase activity pathway (106 DEGs). After inoculation with PWN, however, the opposite was observed; DEGs were enriched in the oxidoreductase activity pathway (119 DEGs), followed by the REDOX activity pathway (84 DEGs). Before inoculation, according to the metabolic pathway analysis results, we found more genes upregulated in phenylpropanoid metabolic pathways and enriched in lignin synthesis pathways; cinnamoyl-CoA reductase-coding genes related to lignin synthesis were upregulated in PWN-resistant P. thunbergii and downregulated in PWN-susceptible P. thunbergii , and the lignin content was always higher in resistant than in susceptible P. thunbergii. These results reveal distinctive strategies of resistant and susceptible P. thunbergii in dealing with PWN infections. [ABSTRACT FROM AUTHOR]