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Pine wilt disease (PWD) is one of the most devastating diseases of Pinus spp. and is caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle. To study adaptation of PWN to survive in hosts that differ in resistance, we examined the self-regulatory characteristics of PWN at the biological and molecular levels early in the interaction. Two-year-old susceptible Pinus thunbergii and resistant Pinus taeda were selected for this experiment, and changes in PWNs after inoculation were assessed. qRT-PCR was used to detect changes in genes related to PWN pathogenicity and detoxification. The results showed that the migration and reproductive abilities of PWNs in P. thunbergii were stronger than those of PWNs in P. taeda. After 7 d, the number of nematodes in P. thunbergii was approximately 3.2-fold higher than in P. taeda. After 15 d, the number of nematodes in P. thunbergii was approximately twofold higher than that in P. taeda. Because PWN can adjust its sex ratio after infection, we compared the sex ratio of uninoculated PWNs, to that in the two pine species. In P. thunbergii, the female to male ratio first decreased and then stabilized over time; in P. taeda first decreased and then increased. Relative fat accumulation in PWNs increased significantly after the PWNs entered the tree body; the accumulation rate in P. thunbergii was higher than in P. taeda at 7 d, but lower after 15 d. Scanning electron microscopy (SEM) showed significantly more bacteria on the surface of PWNs in P. taeda compared with PWNs in susceptible P. thunbergii. At 12 h after inoculation, the expression of genes related to cell-wall degradation (BxBeta1-4 and Bxpel1), effectors (BxCDP1, BxSapB1), and active oxygen metabolism (Bxy-ctl-1 and BxGST3) was 2–6 × higher in the resistant pine than in the susceptible one. In contrast, in PWNs, the expression of autophagy-related genes BxATG1 and BxATG16 was 1.5–2 times higher in P. thunbergii than in P. taeda. These results indicate that the interaction between PWNs and pine trees with different resistance levels elicits a series of physiological and molecular adaptations that affect nematode reproduction and virulence. This study will help elucidate the adaptive mechanisms of PWN in different pine trees.

The plant‐parasitic nematode Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), causes enormous economic loss every year. Currently, little is known about the pathogenic mechanisms of PWD. Several effectors have been identified in B. xylophilus, but their functions and host targets have yet to be elucidated. Here, we demonstrated that BxSCD1 suppresses cell death and inhibits B. xylophilus PAMP BxCDP1‐triggered immunity in Nicotiana benthamiana and Pinus thunbergii. BxSCD1 was transcriptionally upregulated in the early stage of B. xylophilus infection. In situ hybridization experiments showed that BxSCD1 was specifically expressed in the dorsal glands and intestine. Cysteine residues are essential for the function of BxSCD1. Transient expression of BxSCD1 in N. benthamiana revealed that it was primarily targeted to the cytoplasm and nucleus. The morbidity was significantly reduced in P. thunbergii infected with B. xylophilus when BxSCD1 was silenced. We identified 1‐aminocyclopropane‐1‐carboxylate oxidase 1, the actual ethylene‐forming enzyme, as a host target of BxSCD1 by yeast two‐hybrid and coimmunoprecipitation. Overall, this study illustrated that BxSCD1 played a critical role in the B. xylophilus–plant interaction., BxSCD1 suppressed plant immunity in Nicotiana benthamiana and Pinus thunbergii. We report for the first time that BxSCD1 interacts with an ethylene‐forming enzyme in pine.

The pine wood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, is the pathogen of pine wilt disease (PWD) which can devastate forests. PWN can be of high or low severity and the mechanisms underlying the differences in virulence are unclear. Therefore, it is necessary to study the relationship between differentiation of PWN severity and its resistance to the main defensive substances of pine species (i.e., α-pinene and H2O2). The feeding rate and fecundity of PWN was examined at different levels of virulence under conditions of α-pinene and H2O2 stress. Moreover, the expression patterns of the main resistance genes of PWN with different virulence were determined under conditions of α-pinene and H2O2 stress. The feeding rate and fecundity of the high virulence strain AMA3 were higher than those of the low virulence strain YW4. The expression levels of the autophagy gene BxATG5, cytochrome P450 gene BxCYP33D3, and glutathione S-transferase genes BxGST1 and BxGST3 in AMA3 increased significantly upon exposure to α-pinene for 2 h, while these genes showed smaller degrees of upregulation in YW4. Under conditions of H2O2 stress, the expression levels of BxATG5, catalase genes Bxy-ctl-1and Bxy-ctl-2, and the 2-cysteine peroxiredoxin gene BxPrx in AMA3 were higher than those in YW4. These findings suggest that high virulence PWN has greater resistance to pine defensive substances α-pinene and H2O2 than low virulence PWN, and resistance genes mediate the differential resistance of PWN strains. This study will contribute to the clarification of the mechanism underlying virulence differentiation of PWN and will advance understanding of the pathogenic mechanism of PWD.

Bursaphelenchus xylophilus is an economically important pathogen that has caused serious damage to pine forests. Fatty acid and retinol-binding proteins (FARs) are nematode-specific proteins. One FAR gene, Bx-FAR-1, was identified from the transcriptome of B. xylophilus as a putative effector. It was upregulated in both highly and weakly virulent isolates in early infection stages. Transient expression assays showed that Bx-FAR-1 inhibited BAX- and INF1-induced cell death in Nicotiana benthamiana when secreted into the intracellular site. Bx-FAR-1 was expressed in the glandular tissues, intestines, and seminal vesicles of nematodes by in situ hybridization. Silencing Bx-FAR-1 via in vitro RNA interference attenuated the reproductive ability and pathogenicity of B. xylophilus as well as increased the expression of the pathogenesis-related gene 6 and JA biosynthesis gene LOX-5 (lipoxygenase-5) in pine trees. These results suggest that Bx-FAR-1 suppresses the plant immune response in the N. benthamiana intracellular site and may contribute to the pathogenicity of B. xylophilus in the early infection stages.

The migratory plant‐parasitic nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease, which causes serious damage to pine forests in China. Plant immunity plays an important role in plant resistance to multiple pathogens. Activation of the plant immune system is generally determined by immune receptors, including plant pattern recognition receptors, which mediate pattern recognition. However, little is known about molecular pattern recognition in the interaction between pines and B. xylophilus. Based on the B. xylophilus transcriptome at the early stages of infection and Agrobacterium tumefaciens‐mediated transient expression and infiltration of recombinant proteins produced by Pichia pastoris in many plant species, a novel molecular pattern (BxCDP1) was characterized in B. xylophilus. We found that BxCDP1 was highly up‐regulated at the early infection stages of B. xylophilus, and was similar to a protein in Pararhizobium haloflavum. BxCDP1 triggered cell death in Nicotiana benthamiana when secreted into the apoplast, and this effect was dependent on brassinosteroid‐insensitive 1‐associated kinase 1, but independent of suppressor of BIR1‐1. BxCDP1 also exhibited cell death‐inducing activity in pine, Arabidopsis, tomato, pepper, and lettuce. BxCDP1 triggered reactive oxygen species production and the expression of PAMP‐triggered immunity marker genes (NbAcre31, NbPTI5, and NbCyp71D20) in N. benthamiana. It also induced the expression of pathogenesis‐related genes (PtPR‐3, PtPR‐4, and PtPR‐5) in Pinus thunbergii. These results suggest that as a new B. xylophilus molecular pattern, BxCDP1 can not only be recognized by many plant species, but also triggers innate immunity in N. benthamiana and defence responses of P. thunbergii., BxCDP1 can not only trigger innate immunity of the nonhost plant Nicotiana benthamiana, which depends on co‐receptors NbBAK1, but also induces defence responses of the host Pinus thunbergii.

Background Bursaphelenchus xylophilus, the pinewood nematode, kills millions of pine trees worldwide every year, and causes enormous economic and ecological losses. Despite extensive research on population variation, there is little understanding of the population-wide variation spectrum in China. Results We sequenced an inbred B. xylophilus strain using Pacbio+Illumina+Bionano+Hi-C and generated a chromosome-level assembly (AH1) with 6 chromosomes of 77.1 Mb (chromosome N50: 12 Mb). The AH1 assembly shows very high continuity, completeness and contains novel genes with potentially important functions compared with previous assemblies. Subsequently, we sequenced 181 strains from China and USA and found ~7.8 million SNPs. Analysis shows that the B. xylophilus population in China can be divided into geographically bounded subpopulations with severe cross infection and potential migrations. Besides, distribution of B. xylophilus is dominated by temperature zones while geographically associated SNPs are mainly located on adaptation related GPCR gene families, suggesting the nematode has been evolving to adapt to different temperatures. A machine learning based epidemic tracking method has been established to predict their geographical origins which can be applied to any other species. Conclusion Our study provides the community with the first high-quality chromosome-level assembly which includes a comprehensive catalogue of genetic variations. It provides insights into population structure and effective tracking method for this invasive species which facilitates future studies to address a variety of applied, genomic and evolutionary questions in the B. xylophilus as well as related species. This article is protected by copyright. All rights reserved.

Background The pine wood nematode (PWN), Bursaphelenchus xylophilus, is a devastating pathogen of many Pinus species in China. The aim of this study was to understand the interactive molecular mechanism of PWN and its host by comparing differentially expressed genes and candidate effectors from three transcriptomes of B. xylophilus at different infection stages. Results In total, 62, 69 and 46 candidate effectors were identified in three transcriptomes (2.5 h postinfection, 6, 12 and 24 h postinoculation and 6 and 15 d postinfection, respectively). In addition to uncharacterized pioneers, other candidate effectors were involved in the degradation of host tissues, suppression of host defenses, targeting plant signaling pathways, feeding and detoxification, which helped B. xylophilus survive successfully in the host. Seven candidate effectors were identified in both our study and the B. xylophilus transcriptome at 2.5 h postinfection, and one candidate effector was identified in all three transcriptomes. These common candidate effectors were upregulated at infection stages, and one of them suppressed pathogen-associated molecular pattern (PAMP) PsXEG1-triggered cell death in Nicotiana benthamiana. Conclusions The results indicated that B. xylophilus secreted various candidate effectors, and some of them continued to function throughout all infection stages. These various candidate effectors were important to B. xylophilus infection and survival, and they functioned in different ways (such as breaking down host cell walls, suppressing host defenses, promoting feeding efficiency, promoting detoxification and playing virulence functions). The present results provide valuable resources for in-depth research on the pathogenesis of B. xylophilus from the perspective of effectors.

Pine wilt disease, caused by the pine wood nematode Bursaphelenchus xylophilus, leads to severe damage to pine forests in China. In our previous study, effectors secreted by this pathogen were shown to play roles in the different infection stages of pine wilt disease, and a series of candidate effectors were predicted by transcriptome sequencing. This study identified and characterized a novel effector, BxSapB3, which was among these candidate effectors. Agrobacterium-mediated transient expression was used to identify BxSapB3. BxSapB3 was secreted by B. xylophilus and found to be capable of inducing cell death in Nicotiana benthamiana. Quantitative real-time PCR (qRT-PCR) analysis revealed that BxSapB3 was upregulated in a highly virulent strain of B. xylophilus and expressed at lower levels in a weakly virulent strain at the early stages of infection. When BxSapB3 was silenced in B. xylophilus, the process of infection was delayed. These results indicate that BxSapB3 acts as an effector and contributes to virulence at the early stages of B. xylophilus infection.

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is the pathogen causing pine wilt disease (PWD), which is a devastating forest disease. At present, little is known about the defense mechanisms of the PWN, which limits PWD control. Although autophagy plays an important role in the physiological and pathological processes of eukaryotes, its significance in the PWN remains unknown. In this study, we prepared an anti-BxATG8 polyclonal antibody and identified two PWN autophagy marker proteins: BxATG8-I and BxATG8-II. By western blot analysis, we found that the ratio of BxATG8-II to BxATG8-I, which represents autophagic activity, was decreased significantly when samples were treated with the autophagy inhibitor 3-methyladenine. As such, we were able to successfully detect and quantify autophagic activity in the PWN. Thereafter, we investigated the effects of low and high temperatures on PWN growth and reproduction. The results revealed that feeding rate, reproduction rate, and mobility decreased at 15°C and increased at 35°C. By contrast, autophagic activity was high at 15°C and low at 35°C, suggesting that the PWN regulates autophagic activity in response to changes in temperature to maintain physiological homeostasis. When autophagy was inhibited at 15°C, feeding rate, reproductive rate, and mobility declined further, indicating that autophagy is crucial for PWN growth and reproduction at low temperature. These results indicate that autophagy in the PWN is an important response mechanism to temperature changes.

The pine wood nematode (PWN) Bursaphelenchus xylophilus has caused serious damage to pine forests in China. Effectors secreted by phytonematodes play a role in host infection. We identified and characterized an effector, BxSapB1, based on the B. xylophilus transcriptome at the early stages of infection and the transient expression of proteins in Nicotiana benthamiana. BxSapB1 triggered cell death in N. benthamiana when secreted into the apoplast, and this effect was independent of N. benthamiana brassinosteroid-insensitive 1–associated kinase 1 (NbBAK1) and suppressor of BIR1-1 (NbSOBIR1). The signal peptide of BxSapB1 was proven to be functional in yeast using the yeast signal sequence trap system and BxSapB1 was strongly expressed in the subventral gland cells of B. xylophilus, as revealed by in-situ hybridization. In addition, based on local BLAST analysis, the BxSapB1 showed 100% identity to BUX.s00139.62, which was identified from the B. xylophilus secretome during Pinus thunbergii infection. BxSapB1 was upregulated in a highly virulent strain and downregulated in a weakly virulent strain of PWN at the early stages of infection. RNA interference assays showed that silencing BxSapB1 resulted in decreased expression of pathogenesis-related genes (PtPR-1b, PtPR-3, and PtPR-5) as well as delayed onset of symptoms in P. thunbergii infected by B. xylophilus. The combined data suggest that BxSapB1 can trigger cell death in N. benthamiana and that it contributes to the virulence in B. xylophilus during parasitic interaction.

Pine wilt disease (PWD) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, is a devastating disease for Pinus spp. The virulence and resilience of PWN are closely linked to the spread and development of PWD. Numerous studies have shown that autophagy has important physiological and pathological functions in eukaryotes. But little is known about the relationships between autophagy and PWNs' virulence and resistance. In this study, through observation under the microscope and recording, we found the induction of autophagy by rapamycin could dramatically improve movement ability of PWNs with different virulence, and the highly virulent AMA3 isolate moved more than the low virulent YW4 isolate when autophagy was over-induced. High concentrations of rapamycin substantially improved the feeding and reproduction of AMA3 but not YW4. Conserved domains of autophagy genes BxATG3, BxATG4, and BxATG7 were first cloned from PWNs by reverse transcription-polymerase chain reaction (RT-PCR). Expression profiling of these three autophagy genes under biotic and abiotic stresses in PWNs with different virulence was determined by quantitative RT-PCR. The results revealed the expression levels of these three autophagy genes in PWNs with different virulence were increased significantly when nematodes were subject to high and low temperatures, oxidative stress, and defensive responses of pine trees. The expression levels of autophagy genes under biotic and abiotic stresses in AMA3 were higher than those in YW4, and different genes showed different performance. Our study clarified that autophagy was closely related to virulence and resistance of PWN, and the ability of a highly virulent isolate to regulate autophagy activity under stresses was stronger than that of a low virulent isolate.

Stenotrophomonas maltophilia influences the reproduction, pathogenicity, and gene expression of aseptic Bursaphelenchus xylophilus after inoculation of aseptic Pinus massoniana. Pine wilt disease is a destructive pine forest disease caused by B. xylophilus, and its pathogenesis is unclear. The role of bacteria associated with B. xylophilus in pine wilt disease has attracted widespread attention. S. maltophilia is one of the most dominant bacteria in B. xylophilus, and its effect is ambiguous. This study aims to explore the role of S. maltophilia in pine wilt disease. The reproduction and virulence of aseptic B. xylophilus and B. xylophilus containing S. maltophilia were examined by inoculating aseptic P. massoniana seedlings. The gene expressions of two nematode treatments were identified by transcriptome sequencing. The reproduction and virulence of B. xylophilus containing S. maltophilia were stronger than that of aseptic nematodes. There were 4240 differentially expressed genes between aseptic B. xylophilus and B. xylophilus containing S. maltophilia after inoculation of aseptic P. massoniana, including 1147 upregulated genes and 2763 downregulated genes. These differentially expressed genes were significantly enriched in some immune-related gene ontology (GO) categories, such as membrane, transporter activity, metabolic processes, and many immune-related pathways, such as the wnt, rap1, PI3K-Akt, cAMP, cGMP-PKG, MAPK, ECM-receptor interaction, and calcium signaling pathways. The polyubiquitin-rich gene, leucine-rich repeat serine/threonine-protein kinase gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, acetyl-CoA carboxylase gene, and heat shock protein genes were the key genes associated with immune resistance. Moreover, there were four cell wall hydrolase genes, thirty-six detoxification- and pathogenesis-related protein genes, one effector gene and ten cathepsin L-like cysteine proteinase genes that were differentially expressed. After inoculation of the host pine, S. maltophilia could affect the virulence and reproduction of B. xylophilus by regulating the expression of parasitic, immune, and pathogenicity genes of B. xylophilus.

Bursaphelenchus xylophilus is the pathogen that causes pine wilt disease (PWD). The disease has caused significant economic losses and damage to forests. However, the pathogenic mechanism of B. xylophilus remains unclear. Cystatins are involved in various biological processes where they regulate normal proteolysis and also play a role in pathogenicity, but their functions in B. xylophilus are unknown. Therefore, we cloned the full-length cDNA of a cystatin gene of B. xylophilus (Bx-cpi-1) by rapid-amplification of cDNA ends and analyzed its characteristics with bioinformatic methods. In situ mRNA hybridization analyses showed that transcripts of Bx-cpi-1 were abundantly expressed in the reproductive organs of B. xylophilus. The expression of Bx-cpi-1 was investigated using qPCR. Bx-cpi-1 was expressed during each of the different developmental stages of B. xylophilus. The highest gene expression was at the egg stage. After infection of Pinus massoniana, the expression of Bx-cpi-1 increased. The functions of Bx-cpi-1 were verified by RNA interference. The feeding rate, reproduction and pathogenicity of B. xylophilus all decreased as a result of silencing of the Bx-cpi-1 gene. These results revealed that Bx-cpi-1 may be a variant of a type II cystatin gene which is involved in the development and pathogenic process of B. xylophilus., Summary: Bx-cpi-1, a variant of a type II cystatin gene, was abundantly expressed in the reproductive organs of Bursaphelenchus xylophilus, and involved in the development and pathogenicity of B. xylophilus.

Bursaphelenchus xylophilus, the causal agent of pine wilt disease, is a destructive threat to pine forests. The role of bacteria associated with B. xylophilus in pine wilt disease has attracted widespread attention. This study investigated variation in bacterial communities and the virulence of surface-sterilized B. xylophilus from different Pinus spp. The predominant culturable bacteria of nematodes from different pines were Stenotrophomonas and Pseudomonas. Biolog EcoPlate analysis showed that metabolic diversity of bacteria in B. xylophilus from P. massoniana was the highest, followed by P. thunbergii and P. densiflora. High-throughput sequencing analysis indicated that bacterial diversity and community structure in nematodes from the different pine species varied, and the dominant bacteria were Stenotrophomonas and Elizabethkingia. The virulence determination of B. xylophilus showed that the nematodes from P. massoniana had the greatest virulence, followed by the nematodes from P. thunbergii and P. densiflora. After the nematodes were inoculated onto P. thunbergii, the relative abundance of the predominant bacteria changed greatly, and some new bacterial species emerged. Meanwhile, the virulence of all the nematode isolates increased after passage through P. thunbergii. These inferred that some bacteria associated with B. xylophilus isolated from different pine species might be helpful to adjust the PWN&rsquo, s parasitic adaptability.

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is the pathogen of pine wilt disease (PWD), resulting in huge losses in pine forests. However, its pathogenic mechanism remains unclear. The cathepsin L-like cysteine proteinase (CPL) genes are multifunctional genes related to the parasitic abilities of plant-parasitic nematodes, but their functions in PWN remain unclear. We cloned three cpl genes of PWN (Bx-cpls) by rapid amplification of cDNA ends (RACE) and analyzed their characteristics using bioinformatic methods. The tissue specificity of cpl gene of PWN (Bx-cpl) was studied using in situ mRNA hybridization (ISH). The functions of Bx-cpls in development and pathogenicity were investigated using real-time quantitative PCR (qPCR) and RNA interference (RNAi). The results showed that the full-length cDNAs of Bx-cpl-1, Bx-cpl-2, and Bx-cpl-3 were 1163 bp, 1305 bp, and 1302 bp, respectively. Bx-cpls could accumulate specifically in the egg, intestine, and genital system of PWN. During different developmental stages of PWN, the expression of Bx-cpls in the egg stage was highest. After infection, the expression levels of Bx-cpls increased and reached their highest at the initial stage of PWD, then declined gradually. The silencing of Bx-cpl could reduce the feeding, reproduction, and pathogenicity of PWN. These results revealed that Bx-cpls play multiple roles in the development and pathogenic processes of PWN.

The pine wood nematode (PWN),Bursaphelenchus xylophilus, the pathogen of pine wilt disease, has propagative and dispersal forms in its life cycle, which is associated with the insect vectorMonochamus alternatus. Pine wood nematode-associated bacteria could play important roles in pine wilt disease. In this study, the diversity and population dynamics of bacteria in propagative and dispersal stages of PWN were analysed using cultural and Biolog Eco™ methods coupled with high-throughput sequencing. The results revealed that culturableStenotrophomonasin PWN from diseased pine was not found fromM. alternatus. Bacteria in dispersal PWN carried byM. alternatusshowed the highest total carbon utilisation. The high-throughput analysis showed thatStenotrophomonas,AchromobacterandSphingobacteriumwere more abundant in propagative PWN than in dispersal PWN, whereasCitrobacter,Vibrioand Enterobacteriaceae were more abundant in dispersal PWN. The results showed that bacteria of PWN in different developmental forms differed. This change of bacteria might help PWN to adapt to the changing environment.

The pine wood nematode (PWN) Bursaphelenchus xylophilus is the pathogen that causes pine wilt disease (PWD), a devastating forest disease. PWN-associated bacteria may play a role in PWD. However, little is known about the endo-bacteria in PWN. We analyzed the diversity of endo-bacteria in nine isolates of PWNs from Pinus massoniana Lamb. in nine epidemic areas from three Chinese provinces by high-throughput sequencing of 16S rDNA and isolated and identified culturable endo-bacteria through construction of a 16S rDNA phylogenetic tree and Biolog microbial identification. We also examined the effects of endo-bacteria on PWN fecundity, antioxidant capacity, and virulence using sterile nematodes as a control. While the dominant endo-bacteria in PWNs from different regions exhibited no significant difference in the classification levels of class and genus, their proportions differed. Pseudomonas and Stenotrophomonas were highly abundant in all PWN isolates. A total of 15 endo-bacterial strains were successfully isolated and identified as six species: Stenotrophomonas maltophilia, Pseudomonas fluorescens, Kocuria palustris, Microbacterium testaceum, Rhizobium radiobacter, and Leifsonia aquatica. We also found that P. fluorescens significantly increased the egg production of PWN, and that both P. fluorescens and S. maltophilia enhanced the mobility of PWN under oxidative stress and reduced the content of reactive oxygen species by increasing antioxidant enzyme activity in PWN. These strains also accelerated the development of PWD, and P. fluorescens had a more beneficial effect on PWN than S. maltophilia. Diversity exists among the endo-bacteria in PWNs from different regions, and some endo-bacteria can promote PWN infestation by enhancing the fecundity and antioxidant capacity of the nematode. Our study contributes to clarifying the interaction between endo-bacteria and PWN.

Bursaphelenchus xylophilus, the causal agent of pine wilt disease, causes huge economic losses in pine forests. The high expression of cytochrome P450 genes in B. xylophilus during infection in P. thunbergii indicated that these genes had a certain relationship with the pathogenic process of B. xylophilus. Thus, we attempted to identify the molecular characterization and functions of cytochrome P450 genes in B. xylophilus. In this study, full-length cDNA of three cytochrome P450 genes, BxCYP33C9, BxCYP33C4 and BxCYP33D3 were first cloned from B. xylophilus using 3' and 5' RACE PCR amplification. Sequence analysis showed that all of them contained a highly-conserved cytochrome P450 domain. The characteristics of the three putative proteins were analyzed with bioinformatic methods. RNA interference (RNAi) was used to assess the functions of BxCYP33C9, BxCYP33C4 and BxCYP33D3. The results revealed that these cytochrome P450 genes were likely to be associated with the vitality, dispersal ability, reproduction, pathogenicity and pesticide metabolism of B. xylophilus. This discovery confirmed the molecular characterization and functions of three cytochrome P450 genes from B. xylophilus and provided fundamental information in elucidating the molecular interaction mechanism between B. xylophilus and its host plant.

Pine wilt disease (PWD) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, is a destructive forest disease. Recent studies showed that bacteria associated with PWN might play important roles in PWD. In this study, the effects of endobacteria isolated from PWN on the development and virulence of PWN were evaluated. The results revealed that endobacteria isolated from high virulence PWN could partially promote the development of the nematode, while endobacteria from PWN with low virulence could inhibit the development of nematode. Both bacteria-free and non-sterilised nematodes were able to infect microcuttings of Pinus densiflora. The endobacterial isolates, Stenotrophomonas maltophilia NSBx.14 and Achromobacter xylosoxidans ssp. xylosoxidans NSBx.22, enhanced the virulence of low virulence PWN but reduced the virulence of high virulence PWN. The results suggest the pathogenicity of PWN is not affected by loss of bacteria and endobacteria might play a role in the development and virulence of PWN.

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is the pathogen responsible for pine wilt disease (PWD), a devastating forest disease with a pathogenic mechanism that remains unclear. Autophagy plays a crucial role in physiological and pathological processes in eukaryotes, but its regulatory mechanism and significance in PWN are unknown. Therefore, we cloned and characterized three autophagy genes, BxATG5, BxATG9, and BxATG16, in PWN. BxATG9 and BxATG16 were efficiently silenced through RNA interference, and we found that BxATG16 positively regulated the expression of BxATG5. Silencing BxATG9 and BxATG16 severely inhibited feeding and reproduction in PWN, indicating that autophagy is essential for these processes. We then examined the expression patterns of these three autophagy genes in PWN under the stresses of &alpha, pinene and H2O2, the main defense substances of pine trees, and during the development of PWD using quantitative reverse transcription polymerase chain reaction. The expression levels of BxATG5, BxATG9, and BxATG16 all significantly increased after nematodes were stressed with &alpha, pinene and H2O2 and inoculated into pine trees, suggesting that autophagy plays an important role in the defense and pathogenesis of PWN. In this study, the molecular characteristics and functions of the autophagy genes BxATG5, BxATG9, and BxATG16 in PWN were elucidated.

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is the pathogen of pine wilt disease (PWD), resulting in huge losses in pine forests. However, its pathogenic mechanism remains unclear. The cathepsin L-like cysteine proteinase (CPL) genes are multifunctional genes related to the parasitic abilities of plant-parasitic nematodes, but their functions in PWN remain unclear. We cloned three cpl genes of PWN (Bx-cpls) by rapid amplification of cDNA ends (RACE) and analyzed their characteristics using bioinformatic methods. The tissue specificity of cpl gene of PWN (Bx-cpl) was studied using in situ mRNA hybridization (ISH). The functions of Bx-cpls in development and pathogenicity were investigated using real-time quantitative PCR (qPCR) and RNA interference (RNAi). The results showed that the full-length cDNAs of Bx-cpl-1, Bx-cpl-2, and Bx-cpl-3 were 1163 bp, 1305 bp, and 1302 bp, respectively. Bx-cpls could accumulate specifically in the egg, intestine, and genital system of PWN. During different developmental stages of PWN, the expression of Bx-cpls in the egg stage was highest. After infection, the expression levels of Bx-cpls increased and reached their highest at the initial stage of PWD, then declined gradually. The silencing of Bx-cpl could reduce the feeding, reproduction, and pathogenicity of PWN. These results revealed that Bx-cpls play multiple roles in the development and pathogenic processes of PWN.

Pine wilt disease (PWD) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, is one of the most devastating diseases of Pinus spp. The PWN was therefore listed as one of the most dangerous forest pests in China meriting quarantine. Virulence of the PWN is closely linked with the spread of PWD. However, main factors responsible for the virulence of PWNs are still unclear. Recently epiphytic bacteria carried by PWNs have drawn much attention. But little is known about the relationship between endophytic bacteria and virulence of B. xylophilus. In this research, virulence of ten strains of B. xylophilus from different geographical areas in six provinces of China and four pine species were tested with 2-year-old seedlings of Pinus thunbergii. Endophytic bacteria were isolated from PWNs with different virulence to investigate the relationship between the bacteria and PWN virulence. Meanwhile, the carbon metabolism of endophytic bacteria from highly and low virulent B. xylophilus was analyzed using Biolog plates (ECO). The results indicated that ten strains of PWNs showed a wide range of virulence. Simultaneously, endophytic bacteria were isolated from 90% of the B. xylophilus strains. The dominant endophytic bacteria in the nematodes were identified as species of Stenotrophomonas, Achromobacter, Ewingella, Leifsonia, Rhizobium, and Pseudomonas using molecular and biochemical methods. Moreover, S. maltophilia, and A. xylosoxidans subsp. xylosoxidans were the predominant strains. Most of the strains (80%) from P. massoniana contained either S. maltophilia, A. xylosoxidans, or both species. There was a difference between the abilities of the endophytic bacteria to utilize carbon sources. Endophytic bacteria from highly virulent B. xylophilus had a relatively high utilization rate of carbohydrate and carboxylic acids, while bacteria from low virulent B. xylophilus made better use of amino acids. In conclusion, endophytic bacteria widely exist in B. xylophilus from different pines and areas; and B. xylophilus strains with different virulence possessed various endophytic bacteria and diverse carbon metabolism which suggested that the endophytic bacteria species and carbon metabolism might be related with the B. xylophilus virulence.

Pine wilt disease (PWD) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, is responsible for devastating epidemics in pine trees in Asia and Europe. Recent studies showed that bacteria carried by the PWN might be involved in PWD. However, the molecular mechanism of the interaction between bacteria and the PWN remained unclear. Now that the whole genome of B. xylophilus (Bursaphelenchus xylophilus) is published, transcriptome analysis is a unique method to study the role played by bacteria in PWN. In this study, the transcriptome of aseptic B. xylophilus, B. xylophilus treated with endobacterium (Stenotrophomonas maltophilia NSPmBx03) and fungus B. xylophilus were sequenced. We found that 61 genes were up-regulated and 830 were down-regulated in B. xylophilus after treatment with the endobacterium; 178 genes were up-regulated and 1122 were down-regulated in fungus B. xylophilus compared with aseptic B. xylophilus. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were used to study the significantly changed biological functions and pathways for these differentially expressed genes. Many pathogenesis-related genes, including glutathinone S-transferase, pectate lyase, ATP-binding cassette transporter and cytochrome P450, were up-regulated after B. xylophilus were treated with the endobacterium. In addition, we found that bacteria enhanced the virulence of PWN. These findings indicate that endobacteria might play an important role in the development and virulence of PWN and will improve our understanding of the regulatory mechanisms involved in the interaction between bacteria and the PWN.

The pine wood nematode, Bursaphelenchus xylophilus, causes huge economic losses in pine forests, has a complex life cycle, and shows the remarkable ability to survive under unfavorable and changing environmental conditions. This ability may be related to autophagy, which is still poorly understood in B. xylophilus and no autophagy-related genes have been previously characterized. In this study, transmission electron microscopy was used to confirm that autophagy exists in B. xylophilus. The full-length cDNAs of BxATG1 and BxATG8 were first cloned from B. xylophilus, and BxATG1 and BxATG8 were characterized using bioinformatics methods. The expression pattern of the autophagy marker BxATG8 was investigated using in situ hybridization (ISH). BxATG8 was expressed in esophageal gland and hypodermal seam cells. We tested the effects of RNA interference (RNAi) on BxATG1 and BxATG8. The results revealed that BxATG1 and BxATG8 were likely associated with propagation of nematodes on fungal mats. This study confirmed the molecular characterization and functions of BxATG1 and BxATG8 in B. xylophilus and provided fundamental information between autophagy and B. xylophilus.

Bursaphelenchus xylophilus is the pathogen of pine wilt disease. Bursaphelenchus mucronatus is similar to B. xylophilus in morphology. Both species share a common niche, but they are quite different in pathogenicity. Presently, the role of bacteria in pine wilt disease development has been widely speculated. The diversity of bacteria associated with B. xylophilus and B. mucronatus with different virulence remains unclear. In this study, virulence of four B. xylophilus and four B. mucronatus strains were evaluated by inoculating Pinus thunbergii. High-throughput sequencing targeted 16S rDNA of different virulence nematode strains was carried out. The associated bacterial community structures of the eight strains were analyzed. The results showed that 634,051 high-quality sequences were obtained from the eight nematode strains. The number of OTUs of bacteria associated with B. mucronatus was generally greater than those of B. xylophilus. The richness of the community of bacteria associated with high virulent B. xylophilus ZL1 and AmA3 was higher than moderately virulent B. xylophilus AA3, HE2, and all B. mucronatus strains. While the diversity of bacteria associated with B. mucronatus was higher than B. xylophilus. Stenotrophomonas, Pseudomonadaceae_Unclassified or Rhizobiaceae_Unclassified were predominant in the nematode strains with different virulence. Oxalobacteraceae and Achromobacter were found more abundant in the low virulent B. xylophilus and non-virulent B. mucronatus strains.

The disease development and some physiobiochemical indices of 3-4-year-old Pinus thunbergii inoculated with Bursaphelenchus xylophilus were studied. The results showed that after nematode invasion, the disease expanded from the base to the upper part of the main stem, there was an interruption of resin exudation, drying and pith browning occurred sequentially in stem xylem, then the needles wilted and finally the plant died. The number of nematodes increased continuously and rapidly, and increased significantly just after symptoms appeared in pine seedlings. The stem water content decreased gradually, stem and leaf relative water content in a new season branch began to decrease not long before symptoms appeared, and the optical density of organic acid extract from the stem of pine seedlings increased. It is suggested that stem water content could be used as a disease indicator, and organic acid would be the most reliable physiobiochemical index for early diagnosis of the disease.

As the causal agent of pine wilt disease (PWD), the pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses by devastating pine forests worldwide. However, the pathogenesis-related genes of B. xylophilus are not well characterized. Thus, DNA microarrays were used to investigate differential gene expression in PWN where Pinus thunbergii was inoculated with nematodes, compared with those cultured on Botrytis cinerea. The microarrays comprised 31121 probes, 1310 (4.2%) of which were differentially regulated (changes of >2-fold, P < 0.01) in the two growth conditions. Of these 1310 genes, 633 genes were upregulated, whereas 677 genes were downregulated. Gene Ontology (GO) categories were assigned to the classes Cellular Component, Molecular Function, and Biological Process. The comparative gene expression analysis showed that a large number of the pathogenesis-related genes of B. xylophilus, such as pectate lyase genes, cytochrome P450s, UGTs, and ABC transporter genes, were highly expressed when B. xylophilus infected P. thunbergii. Annotation analysis indicated that these genes contributed to cell wall degradation, detoxification, and the reproduction process. The microarray results were validated using quantitative RT-PCR (qRT-PCR). The microarray data confirmed the specific expression of B. xylophilus genes during infection of P. thunbergii, which provides basic information that facilitates a better understanding of the molecular mechanism of PWD.

As the causal agent of pine wilt disease (PWD), the pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses by devastating pine forests worldwide. The pectate lyase gene is essential for successful invasion of their host plants by plant-parasitic nematodes. To demonstrate the role of pectate lyase gene in the PWD process, RNA interference (RNAi) is used to analyze the function of the pectate lyase 1 gene in B. xylophilus (Bxpel1). The efficiency of RNAi was detected by real-time PCR. The result demonstrated that the quantity of B. xylophilus propagated with control solution treatment was 62 times greater than that soaking in double-stranded RNA (dsRNA) after B. xylophilus inoculation in Botrytis cinerea for the first generation (F1). The number of B. xylophilus soaking in control solution was doubled compared to that soaking in Bxpel1 dsRNA four days after inoculation in Pinus thunbergii. The quantity of B. xylophilus was reduced significantly (p < 0.001) after treatment with dsRNAi compared with that using a control solution treatment. Bxpel1 dsRNAi reduced the migration speed and reproduction of B. xylophilus in pine trees. The pathogenicity to P. thunbergii seedling of B. xylophilus was weaker after soaking in dsRNA solution compared with that after soaking in the control solution. Our results suggest that Bxpel1 gene is a significant pathogenic factor in the PWD process and this basic information may facilitate a better understanding of the molecular mechanism of PWD.

The content of NO and H(2)O(2) as well as the activities of nitric oxide synthase (NOS)-like and nitrate reductase (NR) were monitored in the needles of Pinus thunbergii infected by Bursaphelenchus xylophilus. The results showed that the content of NO increased significantly only 8 h after the invasion of B. xylophilus, while H(2)O(2) increased 12 h after invasion. NO donor SNP could promote and NO scavenger cPTIO could prevent the production of NO and H(2)O(2). The content of NO changed earlier than that of H(2)O(2). In addition, the symptoms appeared 9, 5 and 12 days, respectively, after the inoculation with B. xylophilus, SNP pre-treatment and cPTIO pre-treatment followed by B. xylophilus infection. After B. xylophilus infection, the content of NO in P. thunbergii changed fiercely more earlier than the appearance of external symptoms, which indicated that the content of NO was related with the appearance and the development of the symptoms. The treatment with L-NNA (NOS inhibitor) inhibited the content of NO significantly, whereas, Na(2)WO(4) (NR inhibitor) had no effect. The further analysis of NOS revealed that NO changed in consistent with cNOS activity. To sum up, NO, as the upstream signal molecule of H(2)O(2), was involved in the pine early response to the invasion of B. xylophilus and influenced the accumulation of the content of H(2)O(2). Moreover, NOS-like rather than NR was responsible for the endogenous NO generation, which was modulated by cNOS during the interaction between P. thunbergii and B. xylophilus. Key message NO is involved in early response of P. thunbergii to the invasion of B. xylophilus and NOS is the key enzyme responsible for NO generation in P. thunbergii.