Pine Wood Nematode Bursaphelenchus Xylophilus Research Articles

Stability analysis of a pine wood nematode prevention and control model with delay

Pine wilt disease is a destructive forest disease with strong infectivity, a wide spread range and high difficulty in prevention and control. Since controlling Monochamus alternatus, the vector of pine wood nematode (Bursaphelenchus xylophilus) can reduce the occurrence of pine wilt disease efficiently, the parasitic natural enemy of M. alternatus, Dastarcus helophoroides, is introduced in this paper. Considering the influence of parasitic time of D. helophoroides on the control effect, based on the mutualistic symbiosis and parasitic relationship among pine wood nematode, M. alternatus and D. heloporoides, this paper establishes a pine wood nematode prevention and control model with delay. Then, the stability of positive equilibrium and the existence of Hopf bifurcation are discussed. Besides, we obtain the normal form of Hopf bifurcation by applying the multiple time scales method. Finally, numerical simulations with two sets of meaningful parameters selected by means of statistical analysis are carried out to support the theoretical findings. Through the comparative analysis of numerical simulations, the factors affecting the control effect of pine wilt disease are obtained, and some suggestions are put forward for practical control in the forest.

Identification of pine wood nematode (Bursaphelenchus xylophilus) loading response genes in Japanese pine sawyer (Monochamus alternatus) through comparative genomics and transcriptomics.

Pine wood nematode (PWN; Bursaphelenchus xylophilus) is the causative agent of pine wilt disease (PWD), which is considered the most dangerous biohazard to conifer trees globally. The transmission of PWN relies on insect vectors, particularly the Japanese pine sawyer (JPS; Monochamus alternatus). However, the molecular mechanism underlying PWN-JPS assembly remains largely unknown. Here, we found that both geographical and gender could significantly affect the PCA (PWN carrying amount) of JPS; thus, JPS transcriptomes from diverse locations and genders were explored regard to PWN loading. Due to the shortage of genomes, we developed a full-length reference transcriptome for analyzing next-generation sequencing data. A comparative genomic study was performed, and 11 248 potential PWN-carrying associate genes (β) were nominated in JPS by using the reported genomes of PWN and non-PWN carrier insect species. Then, 151 differentially expressed transcripts (DETs), 28 of them overlapped with β, correlated with the PCA of JPS were nominated by RNA-Seq, and found that fatty acid β-oxidation might be the key factor that affected the PCA of JPS. Furthermore, JPS fatty acid β-oxidation rates were experimentally decreased using the inhibitor Etomoxir, leading to an increased PCA of JPS. Meanwhile, silencing MaCPT1 in JPS by RNA interference led to a decreased fatty acid β-oxidation rate and increased PCA of JPS. In conclusion, MaCPT1 was able to decrease the PWN-JPS assembly formation through the fatty acid β-oxidation of JPS. These results provide new insights for exploring the impact of PWN invasion on JPS. © 2024 Society of Chemical Industry.

Phytochemical profile of secondary metabolites in the phloem of mature Pinus pinaster trees attacked by the pine wood nematode

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.

Chromosome-level genome assembly of the Japanese sawyer beetle Monochamus alternatus

The Japanese sawyer beetle Monochamus alternatus (Coleoptera: Cerambycidae) is a pest in pine forests and acts as a vector for the pine wood nematode Bursaphelenchus xylophilus, which causes the pine wilt disease. We assembled a high-quality genome of M. alternatus at the chromosomal level using Illumina, Nanopore, and Hi-C sequencing technologies. The assembled genome is 767.12 Mb, with a scaffold N50 of 82.0 Mb. All contigs were assembled into ten pseudo-chromosomes. The genome contains 63.95% repeat sequences. We identify 16, 284 protein-coding genes in the genome, of which 11,244 were functionally annotated. The high-quality genome of M. alternatus provides an invaluable resource for the biological, ecological, and genetic study of this beetle and opens new avenues for understanding the transmission of pine wood nematode by insect vectors.

Parasitism of Hirsutella rhossiliensis on Different Nematodes and Its Endophytism Promoting Plant Growth and Resistance against Root-Knot Nematodes.

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.

Application of dsRNA in the Pine Wood Nematode, Bursaphelenchus xylophilus.

The pine wood nematode Bursaphelenchus xylophilus is one of the most destructive invasive species worldwide, causing the wilting and eventual death of pine trees. Despite recognition of their economic and environmental significance, it has thus far been impossible to study the detailed gene functions of plant parasitic nematodes through conventional forward genetics and transgenic methods. RNA interference (RNAi), as a reverse genetics technology, offers great convenience for studying the functional genes of nematodes, including B. xylophilus. We here outline a protocol for RNAi of the ppm-1 gene in B. xylophilus, which has been reported to play crucial roles in the development and reproduction of other pathogenic nematodes. For RNAi, the T7 promoter was linked to the 5'-terminal of the target fragment by polymerase chain reaction (PCR), and then double-stranded RNA (dsRNA) was synthesized by in vitro transcription. Subsequently, dsRNA delivery was accomplished by soaking nematodes with the dsRNA solution mixed with synthetic neurostimulants. Synchronized eggs, juveniles, and adults of B. xylophilus (approximately 20,000 individuals of each stage) were washed and soaked in dsRNA (0.8μg/mL) with the soaking buffer for 24h in the dark at 25°C. The same quantity of nematodes was placed in the soaking buffer without dsRNA or with green fluorescent protein dsRNA as a control. After soaking, the expression level of the target transcripts was determined by real-time quantitative PCR. The effects of RNAi were then confirmed by microscopic observation of the phenotypes and comparison of the body size of adults among groups. The current protocol can help to progress research to understand the functions of the genes of B. xylophilus and other parasitic nematodes toward developing control strategies through genetic engineering.

Design, Synthesis, Nematicidal Activity, and Mechanism of Novel Amide Derivatives Containing an 1,2,4-Oxadiazole Moiety.

To discover new nematicides, a series of novel amide derivatives containing 1,2,4-oxadiazole were designed and synthesized. Several compounds showed excellent nematicidal activity. The LC50 values of compounds A7, A18, and A20-A22 against pine wood nematode (Bursaphelenchus xylophilus), rice stem nematode (Aphelenchoides besseyi), and sweet potato stem nematode (Ditylenchus destructor) were 1.39-3.09 mg/L, which were significantly better than the control nematicide tioxazafen (106, 49.0, and 75.0 mg/L, respectively). Compound A7 had an outstanding inhibitory effect on nematode feeding, reproductive ability, and egg hatching. Compound A7 effectively promoted the oxidative stress of nematodes and caused intestinal damage to nematodes. Compound A7 significantly inhibited the activity of succinate dehydrogenase (SDH) in nematodes, leading to blockage of electron transfer in the respiratory chain and thereby hindering the synthesis of adenosine triphosphate (ATP), which consequently affects the entire oxidative phosphorylation process to finally cause nematode death. Therefore, compound A7 can be used as a potential SDH inhibitor in nematicide applications.

Calcium stress reduces the reproductive capacity and pathogenicity of the pine wood nematode (Bursaphelenchus xylophilus) by inhibiting oxidative phosphorylation reaction

The continuous use of chemical pesticides to control nematodes could result in the developing of pesticide-resistant nematodes. Novel nucleic acid pesticides are becoming the focus of pesticide research due to their strong specificity, high efficiency, and environmental friendliness. However, the limited known biochemical targets restrict the development of target pesticides for nematodes. The calcium stress experiments on pine wood nematodes (PWN) showed that 100 mmol/L Ca2+ resulted in longitudinal depression on the PWN body wall, reduced oviposition, and increased corrected mortality. To enrich the biological targets of nematode pesticides, we further investigated the response mechanism of PWN to calcium stress at the molecular level. Differentially expressed gene analysis showed that genes involved in the oxidative phosphorylation (OXPHOS) pathway were significantly enriched. RNA interference results of 6 key genes belonging to four mitochondrial complex I (BXNDUFA2), III (BXQCR8), IV (BXCOX17), V (BXV-ATPaseB, BXV-ATPaseE, BXV-ATPaseε) in non-stressed nematodes showed reduction in PWN oviposition, population size, feeding ability, and pathogenicity. The BXNDUFA2 gene interference had the highest inhibitory impact by decreasing the oviposition from 31.00 eggs to 6.75 eggs and PWN population size from 8.27 × 103 nematodes to 1.64 × 103 nematodes, respectively. Interestingly, RNA interference of these 6 key genes in calcium-stressed nematodes also led to increased mortality and decreased oviposition of PWN. In summary, calcium stress inhibited the reproductive capacity of PWN by down-regulating key genes BXNDUFA2, BXQCR8, BXV-ATPaseB, BXV-ATPaseE, BXV-ATPaseε, and BXCOX17, thereby reducing the pathogenicity. The current results enrich the RNAi targets in PWN and provide a scientific basis for developing novel nucleic nematicides.

Impact of Climate Change on the Habitat Suitability of Monochamus saltuarius Gebler (Coleoptera; Cerambycidae) and Its Natural Enemies in China

The longicorn beetle Monochamus saltuarius (Coleoptera; Cerambycidae) is an insect vector that transmits the pine wood nematode Bursaphelenchus xylophilus, a serious issue in pine forests in China, Japan, and South Korea. Dastarcus helophoroides and Scleroderma guani are effective in controlling M. saltuarius. However, the application of these parasites for the direct control of vector insects requires detailed analyses of the suitability of the selected natural enemies in potential pest areas, particularly under climate change. Based on species distribution records as well as the current and projected climate change trends up to 2050, we utilized an optimized Maxent algorithm to predict the potential distribution patterns of M. saltuarius and its natural enemies in China. Furthermore, we estimated Schoener’s D to quantify the overlap in adaptability between M. saltuarius and its natural enemies. The potentially suitable areas predicted under the current climate conditions were concentrated mainly in northeast and north China. Additional suitable areas were detected in central, eastern, northwestern, and southwestern China. Under future climate conditions, the suitable range of M. saltuarius gradually shifted southward, with a tendency to expand into high-altitude and colder regions, such as Sichuan, Qinghai, and Tibet. The potential distribution of the natural enemies D. helophoroides and S. guani continued to expand under climate change. A comparison of the appropriate habitats of M. saltuarius and natural enemy insects indicated that D. helophoroides is more suitable for initial biological control in north, northeast, and central China as well as certain northwest and coastal regions. S. guani could be prioritized for nationwide release, while joint control using both species may be effective in Liaoning, Hebei, and Tianjin provinces. These analyses demonstrate that future climate change is likely to exert adverse effects on the potential distribution of M. saltuarius but is favorable for the potential distributions of natural enemies. This study offers important insights into the effective control of M. saltuarius and provides an experimental basis for the utilization of natural enemies according to the characteristics of different regions.

Transcriptomic response of Pinus massoniana to infection stress from the pine wood nematode Bursaphelenchus xylophilus

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.

NTR-1′s essential contribution to asymmetric mating between two sibling nematode Species: Bursaphelenchus xylophilus and B. Mucronatus

The pine-wood invasive species nematode Bursaphelenchus xylophilus causes great forestry damage globally, particularly in Eurasia. B. xylophilus can hybridize with its native sibling, Bursaphelenchus mucronatus, with whom it shares an interestingly asymmetric mating behavior. However, the molecular mechanism underlying interspecific asymmetric mating has yet to be clarified. ntr-1, a nematocin receptor gene, is involved in an oxytocin/vasopressin-like signaling system that can regulate reproduction. Structural analysis using bioinformatics revealed that both Bxy- and Bmu-ntr-1 encode 7TM-GPCR, a conserved sequence. In situ hybridization and qPCR showed that both Bxy- and Bmu-ntr-1 were highly expressed in adult nematodes. Specifically, Bxy-ntr-1 was expressed in the vulva of females and caudal gonad of males, whereas Bmu-ntr-1 was expressed in the postal vulva and uterus of females and the whole gonads of males. Furthermore, RNAi of ntr-1 further demonstrated the biological function of interspecific mating: ntr-1 can regulate mating behavior, lead to male–female specificity, and ultimately result in interspecific differences. In B. mucronatus, ntr-1 influenced male mating more than female mating success, while downregulation of ntr-1 in B. xylophilus resulted in a significant decline in the female mating rate. Competitive tests revealed that the mating rate of the cross significantly declined after downregulation of Bxy♀- and Bmu♂-ntr-1, but no obvious change occurred in the reciprocal cross. Thus, we speculate that ntr-1 may be the key factor behind interspecific asymmetric mating. The current study (1) demonstrated the regulatory function of ntr-1 on mating behavior and (2) theoretically revealed the molecular basis of interspecific asymmetric mating.

The essential role of arginine biosynthetic genes in lunate conidia formation, conidiation, mycelial growth, and virulence of nematophagous fungus, Esteya vermicola CBS115803.

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. 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. 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.

Effects of Pine Wilt Disease on Rhizosphere Microbiota and Fine Root Fungi: Insights into Enzyme Activity, Ectomycorrhizal Infection and Microbial Composition

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.

Bursaphelenchus mucronatus (Nematoda: Parasitaphelenchidae) associated with Monochamus galloprovincialis from Bosnia and Herzegovina and Georgia.

Bursaphelenchus mucronatus was detected in association with the pine sawyer beetle (Monochamus galloprovincialis) during the implementation and testing of cross traps with insect attractants as an efficient tool for detection survey for pine wood nematode (Bursaphelenchus xylophilus) in Bosnia and Herzegovina and Georgia in 2017 and 2018, respectively. This nematode was characterized by morphological, morphometric and molecular features. This is the first report of B. mucronatus in association with a M. galloprovincialis in Bosnia and Herzegovina and in Georgia.

The effects of provenance, climate, and chemical defense on the resistance of Pinus pinaster Aiton to Bursaphelenchus xylophilus (Steiner and Buhrer)

Key messageThe resistance of Pinus pinaster Aiton to pinewood nematode Bursaphelenchus xylophilus (Steiner and Buhrer) varied among populations from the Iberian Peninsula, with survival rates for inoculated seedlings ranging from 5 to 100%. These differences in resistance were paralleled by differences in some constitutive chemical defenses. Populations from the southeastern Iberian Peninsula displayed higher resistance than northern populations.ContextThe presence of the pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner and Buhrer), in Portugal threatens Mediterranean pine species such as Pinus pinaster Aiton.AimsWe have focused on assessing the resistance of P. pinaster populations to B. xylophilus, looking for any relationship between the PWN resistance and some constitutive chemical defenses and geoclimatic parameters.MethodsTwo-year-old seedlings from 32 provenances and two seed orchards were evaluated in an experiment of artificial inoculation following a randomized complete block design under greenhouse conditions. We measured growth-related traits, response to B. xylophilus inoculations, and constitutive chemical compounds in needles of the evaluated seedlings and compiled geoclimatic data for each population. Mixed models, nonparametric tests, correlations, and PCA were used to analyze the data.ResultsSurvival, wilting symptoms, morphological traits, and nematode density varied significantly among populations. Lower concentrations of constitutive polyphenols, lipid-soluble substances, and tannins were related to higher PWN resistance. Populations from the southeast of the Iberian Peninsula showed higher survival rates than those from further north. Additionally, we observed that populations to warm, dry climates showed higher resistance to B. xylophilus than populations originating from humid, temperate climates.ConclusionHigher susceptibility to PWN is related to lower growth traits, to lower levels of certain constitutive chemical compounds, and to adaptations to harsher climate.

Antibacterial peptides from Monochamus alternatus induced oxidative stress and reproductive defects in pine wood nematode through the ERK/MAPK signaling pathway

Pine wilt disease is a devastating disease of pine caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus. Long-term use of chemical nematicides leads to the development of resistance in nematodes and harms the environment. Evaluations for green environmental protection agents, identified the antibacterial peptide, MaltDef1, from Monochamus alternatus which had nematicidal effect. We studied its nematicidal activity and action against PWN. In this study, the antibacterial peptide S-defensin was synthesized from M. alternatus. The results showed that S-defensin caused mortality to the PWN, causing shrinkage, pore, cell membrane dissolution and muscle atrophy. In addition, PWN reproduction was also affected by S-defensin; it decreased in a concentration dependent manner with increasing treatment concentration. By contrast, reactive oxygen species (ROS) in vivo increased in a concentration-dependent manner. We applied transcriptome to analyze the changes in gene expressions in S-defensin treated PWN, and found that the most significantly enriched pathway was the ERK/MAPK signaling pathway. RNAi was used to validate the functions of four differential genes (Let-23, Let-60, Mek-2 and Lin-1) in this pathway. The results showed that knockdown of these genes significantly decreased the survival rate and reproductive yield of, and also increased ROS in PWN. The antibacterial peptide S-defensin had a significant inhibitory effect on the survival and reproduction of PWN, shown by cell membrane damage and intracellular biological oxidative stress via regulating the ERK/MAPK signaling pathway. This indicates that S-defensin has a target in B. xylophilus, against which new green target pesticides can be developed.

First Report on the Synergistic Interaction between Essential Oils against the Pinewood Nematode Bursaphelenchus xylophilus.

Control of the pinewood nematode (PWN), the causal agent of pine wilt disease, can be achieved through the trunk injection of nematicides; however, many pesticides have been linked to environmental and human health concerns. Essential oils (EOs) are suitable alternatives due to their biodegradability and low toxicity to mammals. These complex mixtures of plant volatiles often display multiple biological activities and synergistic interactions between their compounds. The present work profiled the toxicity of eight EOs against the PWN in comparison to their 1:1 mixtures, to screen for successful synergistic interactions. Additionally, the main compounds of the most synergistic mixtures were characterized for their predicted environmental fate and toxicity to mammals in comparison to emamectin benzoate, a commercial nematicide used against PWN. The mixtures of Cymbopogon citratus with Mentha piperita and of Foeniculum vulgare with Satureja montana EOs showed the highest activities, with half-maximal effective concentrations (EC50) of 0.09 and 0.05 µL/mL, respectively. For these, complete PWN mortality was reached after only ca. 15 min or 2 h of direct contact, respectively. Their major compounds had a higher predicted affinity to air and water environmental compartments and are reported to have very low toxicity to mammals, with low acute oral and dermal toxicities. In comparison, emamectin benzoate showed lower nematicidal activity, a higher affinity to the soil and sediments environmental compartments and higher reported oral and dermal toxicity to mammals. Overall, uncovering synergistic activities in combinations of EOs from plants of different families may prove to be a source of biopesticides with optimized toxicity against PWNs.

Temperature-regulated metabolites of Serratia marcescens inhibited reproduction of pinewood nematode Bursaphelenchus xylophilus

The pinewood nematode Bursaphelenchus xylophilus is an invasive and destructive pathogen in forestry. Serratia marcescens AHPC29 was previously found to have nematicidal activity on B.xylophilus. The effect of AHPC29 growth temperature on B.xylophilus inhibition is unknown. Here we show that AHPC29 cultured at 15°C or 25°C, but not 37°C, inhibited B.xylophilus reproduction. Metabolomic analysis found 31 up-regulated metabolites as potential effective substances in this temperature-related difference, with five of them were tested to be effective in inhibiting B.xylophilus reproduction. Among the five metabolites, salsolinol was further verified in bacterial cultures with effective inhibition concentrations. This study found the inhibition of S.marcescens AHPC29 on B.xylophilus reproduction was temperature regulated and the differently expressed metabolites salsolinol played roles in this temperature-regulated effect, which implies the capability of S.marcescens and its metabolites as promising new agents for the management of B.xylophilus.

Two decades of epidemiological surveillance of the pine wood nematode in France reveal its absence despite suitable conditions for its establishment

Key messageThis 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.ContextThe 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.AimsThe 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.MethodsWe 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.ResultsThis 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.ConclusionWhile 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.

Resin Canal Traits Variation in Pinus spp. with Different Susceptibility to the Pine Wood Nematode

Different studies have emphasized the influence of resin canal traits in the susceptibility of pine trees to the pine wood nematode (PWN) Bursaphelenchus xylophilus. Resin canals can facilitate the PWN migration through the stem and are involved in the accumulation of volatile terpenes in the xylem in response to the pathogen, inducing tracheid embolisms. In this work, we conducted a PWN inoculation experiment under greenhouse conditions to investigate the anatomical traits of constitutive resin canals among seven Pinus species with different degrees of susceptibility to the PWN: P. canariensis, P. halepensis, P. pinea and P. taeda were grouped into a ‘low-susceptible group’, and P. pinaster, P. radiata, and P. sylvestris were grouped into a ‘high-susceptible group’. The high-susceptible group presented higher xylem radial growth, wider constitutive canals in the cortex, lower frequency of constitutive canals in the cortex, and smaller constitutive canals in the xylem than the low-susceptible group. The size of constitutive cortical canals was positively related to the number of seedlings colonized by the PWN, suggesting that wider canals facilitated migration. The inoculation of the PWN increased the frequency and diminished the mean area of canals in the xylem, and the high-susceptible group showed more frequency of induced xylem canals than the low-susceptible group. Additionally, the high-susceptible group presented larger radial growths in the xylem than the low-susceptible group. These results suggest a role of resin canal traits on Pinus spp. susceptibility to the PWN. Nevertheless, the high interspecific variability found in these traits within each susceptibility group evidences the importance of other factors in the susceptibility to the PWN.