Temporal Transcriptome Profiling of Pinus densiflora Infected with Pine Wood Nematode Reveals Genetically Programmed Changes upon Pine Wilt Disease.

Pine, an evergreen conifer, is widely distributed worldwide. It is economically, scientifically, and ecologically important. However, pine wilt disease (PWD) induced by the pine wood nematode (PWN) adversely affects pine trees. Many studies have been conducted on the PWN and its beetle vectors to prevent the spread of PWD. However, studies providing a comprehensive understanding of the pine tree transcriptome in response to PWN infection are lacking. Here, we performed temporal profiling of the pine tree transcriptome using PWD-infected red pine trees, Pinus densiflora, inoculated with the PWN by RNA sequencing. Our analysis revealed that defense-responsive genes involved in cell wall modification, jasmonic acid signaling, and phenylpropanoid-related processes were significantly enriched 2 weeks after PWD infection. Furthermore, some WRKY-type and MYB-type transcription factors were upregulated 2 weeks after PWD infection, suggesting that these transcription factors might be responsible for the genome-wide reprogramming of defense-responsive genes in the early PWD stage. Our comprehensive transcriptome analysis will assist in developing PWD-resistant pine trees and identifying genes to diagnose PWD at the early stage of infection, during which large-scale phenotypic changes are absent in PWD-infected pine trees.

Pinus thunbergii Parl. Somatic Plants' Resistance to Bursaphelenchus xylophilus Depends on Pathogen-Induced Differential Transcriptomic Responses.

Pinus thunbergii Parl. is an economically and medicinally important plant, as well as a world-renowned horticultural species of the Pinus genus. Pine wilt disease is a dangerous condition that affects P. thunbergii. However, understanding of the genetics underlying resistance to this disease is poor. Our findings reveal that P. thunbergii's resistance mechanism is based on differential transcriptome responses generated by the early presence of the pathogen Bursaphelenchus xylophilus, also known as the pine wood nematode. A transcriptome analysis (RNA-seq) was performed to examine gene expression in shoot tissues from resistant and susceptible P. thunbergii trees. RNA samples were collected from the shoots of inoculated pines throughout the infection phases by the virulent Bursaphelenchus xylophilus AMA3 strain. The photosynthesis and plant-pathogen interaction pathways were significantly enriched in the first and third days after infection. Flavonoid biosynthesis was induced in response to late infestation (7 and 14 days post-infestation). Calmodulin, RBOH, HLC protein, RPS, PR1, and genes implicated in phytohormone crosstalk (e.g., SGT1, MYC2, PP2C, and ERF1) showed significant alterations between resistant and susceptible trees. Furthermore, salicylic acid was found to aid pine wood nematodes tolerate adverse conditions and boost reproduction, which may be significant for pine wood nematode colonization within pines. These findings provide new insights into how host defenses overcame pine wood nematode infection in the early stage, which could potentially contribute to the development of novel strategies for the control of pine wilt disease.

The Neurotranscriptome of Monochamus alternatus.

The Japanese pine sawyer Monochamus alternatus serves as the primary vector for pine wilt disease, a devastating pine disease that poses a significant threat to the sustainable development of forestry in the Eurasian region. Currently, trap devices based on informational compounds have played a crucial role in monitoring and controlling the M. alternatus population. However, the specific proteins within M. alternatus involved in recognizing the aforementioned informational compounds remain largely unclear. To elucidate the spatiotemporal distribution of M. alternatus chemosensory-related genes, this study conducted neural transcriptome analyses to investigate gene expression patterns in different body parts during the feeding and mating stages of both male and female beetles. The results revealed that 15 genes in the gustatory receptor (GR) gene family exhibited high expression in the mouthparts, most genes in the odorant binding protein (OBP) gene family exhibited high expression across all body parts, 22 genes in the odorant receptor (OR) gene family exhibited high expression in the antennae, a significant number of genes in the chemosensory protein (CSP) and sensory neuron membrane protein (SNMP) gene families exhibited high expression in both the mouthparts and antennae, and 30 genes in the ionotropic receptors (IR) gene family were expressed in the antennae. Through co-expression analyses, it was observed that 34 genes in the IR gene family were co-expressed across the four developmental stages. The Antenna IR subfamily and IR8a/Ir25a subfamily exhibited relatively high expression levels in the antennae, while the Kainate subfamily, NMDA subfamily, and Divergent subfamily exhibited predominantly high expression in the facial region. MalIR33 is expressed only during the feeding stage of M. alternatus, the MalIR37 gene exhibits specific expression in male beetles, the MalIR34 gene exhibits specific expression during the feeding stage in male beetles, the MalIR8 and MalIR39 genes exhibit specific expression during the feeding stage in female beetles, and MalIR8 is expressed only during two developmental stages in male beetles and during the mating stage in female beetles. The IR gene family exhibits gene-specific expression in different spatiotemporal contexts, laying the foundation for the subsequent selection of functional genes and facilitating the full utilization of host plant volatiles and insect sex pheromones, thereby enabling the development of more efficient attractants.

Generation of a novel antibody against BxPrx, a diagnostic marker of pine wilt disease.

BACKGROUND: Bursaphelenchus xylophilus is a pathogenic nematode that causes pine wilt disease (PWD). To prevent the rapid spread of this pathogen, developing a method for rapid and accurate detection of B. xylophilus is required. METHODS AND RESULTS: In this study, we produced a B. xylophilus peroxiredoxin (BxPrx), which is a protein that is overexpressed in B. xylophilus. Using recombinant BxPrx as an antigen, we generated and selected a novel antibody that binds to BxPrx via phage display and biopanning. We subcloned the anti-BxPrx single-chain variable fragment-encoding phagemid DNA to mammalian expression vector. We transfected the plasmid into mammalian cells and produced a highly sensitive recombinant antibody that enabled nanogram order detection of BxPrx. CONCLUSION: The sequence of anti-BxPrx antibody as well as the rapid immunoassay system described here can be applied for rapid and accurate diagnosis of PWD.

Functional importance of groups I and II chitinases in cuticle chitin turnover during molting in a wood-boring beetle, Monochamus alternatus.

Insects must periodically replace their old cuticle/exoskeleton with a new one in a process called molting or ecdysis to allow for continuous growth through sequential developmental stages. Many RNA interference (RNAi) studies have demonstrated that certain chitinases (CHTs) play roles in this vital physiological event because knockdown of these CHT genes resulted in developmental arrest during the ensuing molting period in several insect species. In this research we analyzed the functions of group I (MaCHT5) and group II (MaCHT10) CHT genes in molting of the Japanese pine sawyer, Monochamus alternatus, an important forest pest known as a major vector of the pinewood nematode. Real-time qPCR revealed that these two CHT genes differ in their expression patterns during late stages of development. Depletion of either MaCHT5 or MaCHT10 transcripts by RNAi resulted in lethal larval-pupal and pupal-adult molting defects depending on the double-stranded RNA (dsRNA) injection timing during development. The insects were unable to shed their old cuticle and died. Furthermore, transmission electron microscopic analysis revealed that, unlike dsEGFP-treated controls, dsMaCHT5- and dsMaCHT10-treated pharate adults exhibited a failure of degradation of the endocuticular layer of their old pupal cuticle, retaining nearly intact horizontal chitinous laminae and vertical pore canal fibers. Both enzymes were indispensable for complete turnover of the chitinous old endocuticle, which is critical for insect molting. The possible functions of two spliced variants of MaCHT10, namely, MaCHT10a and MaCHT10b, are also discussed. Our results add to the knowledge base for further functional studies of insect chitin catabolism by revealing the relative importance of both MaCHT5 and MaCHT10 in chitin turnover with subtle differences in their action. These essential genes and their encoded proteins are potential targets to manipulate for controlling populations of M. alternatus and other pest insects.

CRISPR-Cas Detection Coupled with Isothermal Amplification of Bursaphelenchus xylophilus.

The pine wood nematode (PWN), Bursaphelenchus xylophilus, causes significant damage to pine trees and, thus, poses a serious threat to pine forests worldwide, particularly in China, Korea, and Japan. A fast, affordable, and ultrasensitive detection of B. xylophilus is urgently needed for disease diagnosis. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have reshaped molecular diagnosis, with high speed, precision, specificity, strength, efficiency, and versatility. Herein, we established two isothermal diagnostics methods based on CRISPR-based platforms (CRISPR/Cas12a and CRISPR/Cas13a) for B. xylophilus-specific detection via fluorescence or lateral-flow strip readout. The guide RNA and CRISPR RNA were designed to target the 5S ribosomal DNA intergenic spacer sequences region of B. xylophilus. Recombinase-aided amplification was used for preamplification whose reaction condition was 37°C for 15 min. The sensitivity of CRISPR/Cas12a could reach 94 copies/µl of plasmid DNA, or 2.37 copies/µl of purified genomic DNA (gDNA) within 45 min at 37°C, while the sensitivity of CRISPR/Cas13a was 1,000 times higher than that of CRISPR/Cas12a of plasmid DNA in 15 min or 100 times higher of purified gDNA at the minimum reaction time of 4 min via fluorescence measurement. The CRISPR/Cas12a assay enabled the detection of 0.01 PWNs per 100 mg of pine wood, 10 times higher than that of the CRISPR/Cas13a assay. This work enriches molecular detection approaches for B. xylophilus and provides huge potential for ultrasensitive and rapid methods to detect B. xylophilus in pine wood, facilitating point-of-sample diagnostic processing for pine wilt disease management.

Resistant and Susceptible Pinus thunbergii ParL. Show Highly Divergent Patterns of Differentially Expressed Genes during the Process of Infection by Bursaphelenchus xylophilus.

Pine wilt disease (PWD) is a devastating disease that threatens pine forests worldwide, and breeding resistant pines is an important management strategy used to reduce its impact. A batch of resistant seeds of P. thunbergii was introduced from Japan. Based on the resistant materials, we obtained somatic plants through somatic embryogenesis. In this study, we performed transcriptome analysis to further understand the defense response of resistant somatic plants of P. thunbergii to PWD. The results showed that, after pine wood nematode (PWN) infection, resistant P. thunbergii stimulated more differential expression genes (DEGs) and involved more regulatory pathways than did susceptible P. thunbergii. For the first time, the alpha-linolenic acid metabolism and linoleic acid metabolism were intensively observed in pines resisting PWN infection. The related genes disease resistance protein RPS2 (SUMM2) and pathogenesis-related genes (PR1), as well as reactive oxygen species (ROS)-related genes were significantly up-expressed in order to contribute to protection against PWN inoculation in P. thunbergii. In addition, the diterpenoid biosynthesis pathway was significantly enriched only in resistant P. thunbergii. These findings provided valuable genetic information for future breeding of resistant conifers, and could contribute to the development of new diagnostic tools for early screening of resistant pine seedlings based on specific PWN-tolerance-related markers.

Expression of PmACRE1 in Arabidopsis thaliana enables host defence against Bursaphelenchus xylophilus infection.

BACKGROUND: Pine wilt disease (PWD) is a destructive disease that endangers pine trees, resulting in the wilting, with yellowing and browning of the needles, and eventually the death of the trees. Previous studies showed that the Avr9/Cf-9 rapidly elicited (PmACRE1) gene was downregulated by Bursaphelenchus xylophilus infection, suggesting a correlation between PmACRE1 expression and pine tolerance. Here, we used the expression of PmACRE1 in Arabidopsis thaliana to evaluate the role of PmACRE1 in the regulation of host defence against B. xylophilus infection. RESULTS: Our results showed that the transformation of PmACRE1 into A. thaliana enhanced plant resistance to the pine wood nematode (PWN); that is, the leaves of the transgenic line remained healthy for a longer period than those of the blank vector group. Ascorbate peroxidase (APX) activity and total phenolic acid and total flavonoid contents were higher in the transgenic line than in the control line. Widely targeted metabolomics analysis of the global secondary metabolites in the transgenic line and the vector control line showed that the contents of 30 compounds were significantly different between these two lines; specifically, the levels of crotaline, neohesperidin, nobiletin, vestitol, and 11 other compounds were significantly increased in the transgenic line. The studies also showed that the ACRE1 protein interacted with serine hydroxymethyltransferase, catalase domain-containing protein, myrosinase, dihydrolipoyl dehydrogenase, ketol-acid reductoisomerase, geranylgeranyl diphosphate reductase, S-adenosylmethionine synthase, glutamine synthetase, and others to comprehensively regulate plant resistance. CONCLUSIONS: Taken together, these results indicate that PmACRE1 has a potential role in the regulation of plant defence against PWNs.

Faunimonas pinastri gen. nov., sp. nov., an endophyte from a pine tree of the family Pleomorphomonadaceae, class Alphaproteobacteria.

Bacterial strain A52C2T was isolated from the endophytic microbial community of a Pinus pinaster tree trunk and characterized. Strain A52C2T stained Gram-negative and formed rod-shaped cells that grew optimally at 30 °C and at pH 6.0-7.0. The G+C content of the DNA was 65.1 mol %. The respiratory quinone was ubiquinone 10, and the major fatty acids were cyclo-C19:0 ω8c and C18:0, representing 70.1 % of the total fatty acids. Phylogenetic analyses based on the 16S rRNA gene sequences placed strain A52C2T in a distinct lineage within the order Hyphomicrobiales, family Pleomorphomonadaceae. The 16S rRNA gene sequence similarities of A52C2T to that of Mongoliimonas terrestris and Oharaeibacter diazotrophicus were 93.15 and 93.2 %, respectively. The draft genome sequence of strain A52C2T comprises 4 196 045 bases with a 195-fold mapped coverage of the genome. The assembled genome consists of 43 contigs of more than 1 000 bp (N50 contig size was 209 720 bp). The genome encodes 4033 putative coding sequences. The phylogenetic, phenotypic and chemotaxonomic data showed that strain A52C2T (=UCCCB 130T=CECT 8949T=LMG 29042T) represents the type of a novel species and genus, for which we propose the name Faunimonas pinastri gen. nov., sp. nov.

Optimization of a solid culture medium based on Monochamus alternatus for Cordyceps militaris fruiting body formation.

Monochamus alternatus (Coleoptera: Cerambycidae; M. alternatus), popularly known as the Japanese pine sawyer, is a vector of pinewood nematode (Bursaphelenchus xylophilus) that causes pine wilt disease. A solid medium culture with M. alternatus produced Cordyceps militaris fruiting bodies with the longest strips and the highest biological efficiency. Supplementing the original form of M. alternatus with oats resulted in slightly enhanced fruiting body production. The original form of M. alternatus showed higher production than its powder form. The solid culture medium was optimized using a response surface methodology, and the optimal medium contained the following: 8·5 g per bottle of M. alternatus and 11·5 g per bottle of oats mixed with 22·4 ml of water in a 300-ml cylindrical plastic bottle. The optimal culturing period for the fruiting body formation was 37·1 days. Under these conditions, a fruiting body dry weight of 38·0 g per bottle (actual value) was attained. The fruiting body produced using a solid culture medium based on M. alternatus had a cordycepin content of about 25 µg g-1 . The solid culture medium containing M. alternatus is highly efficient and eco-friendly, and its effectiveness in large-scale fruiting body production from C. militaris has been demonstrated.

The Detection of Pine Wilt Disease: A Literature Review.

Pine wilt disease (PWD) is a global quarantine disease of forests that mainly affects Pinaceae species. The disease spreads rapidly. Once infected, pine trees have an extremely high mortality rate. This paper provides a summary of the common techniques used to detect PWD, including morphological-, molecular-, chemical- and physical-based methods. By comprehending the complex relationship among pinewood nematodes, vectors and host pine trees and employing the available approaches for nematode detection, we can improve the implementation of intervention and control measures to effectively reduce the damage caused by PWD. Although conventional techniques allow a reliable diagnosis of the symptomatic phase, the volatile compound detection and remote sensing technology facilitate a rapid diagnosis during asymptomatic stages. Moreover, the remote sensing technology is capable of monitoring PWD over large areas. Therefore, multiple perspective evaluations based on these technologies are crucial for the rapid and effective detection of PWD.

Nematicidal Activities of Saccharin and Erythritol Against Pinewood Nematode.

The pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Bührer), causes pine wilt disease (PWD) resulting in severe environmental damage to pine forest ecosystems worldwide. To develop alternative strategies for managing PWD, the nematicidal activities of two sweeteners, erythritol and saccharin, were investigated. Among these two sweeteners, saccharin induced higher mortality in a dose-dependent manner. The LC50 and LC90 values of saccharin were estimated to be 0.321 M and 0.615 M, respectively. However, erythritol did not exhibit nematicidal activities. The results of our study demonstrated that saccharin is lethal to PWN and shows nematicidal effects in a dose-dependent manner. Although the mechanisms of saccharin toxicity are not yet investigated, saccharin could be used as an effective alternative for the management of PWN.

Trehalose in pine wood nematode participates in DJ3 formation and confers resistance to low-temperature stress.

BACKGROUND: Recently, pine wood nematode (PWN, Bursaphelenchus xylophilus) has been found in the extreme cold area of northeast China. The third-stage dispersal juvenile (DJ3) of PWN, which is a long-lived stress-resistant stage, plays an important role in the process of PWN spreading to low-temperature areas, as this stage can survive under unfavorable conditions. RESULTS: Weighted correlation network analysis (WGCNA) was used to analyze the expression patterns of 15,889 genes included in 21 RNA-Seq results of PWN at DJ3 and the other 6 different stages, and a total of 12 coexpression modules were obtained. Among them, the magenta module has the highest correlation with DJ3, which included a total of 652 genes. KEGG enrichment analysis showed that most of the genes in the magenta module were involved in metabolic processes, which were related to autophagy and longevity regulation. These pathways included starch and sucrose metabolism, which contains trehalose metabolism. To explore the function of trehalose in DJ3 formation and survival under - 20 °C, a trehalose-6-phosphate synthase encoding gene (Bx-tps), a trehalose-6-phosphate phosphatase encoding gene (Bx-tpp) and 7 trehalase encoding genes (Bx-tres) were identified and investigated. The expression of these 9 genes was related to the formation of DJ3. A treatment under - 20 °C induced the accumulation of trehalose. The survival rate of DJ3 at -20 °C reduced after silencing of any of these trehalose metabolism genes. Further analysis suggested that two trehalose synthesis genes were highly correlated with DJ3 and might be involved in autophagy by regulating with energy conversion related genes. CONCLUSIONS: The above results indicated that trehalose metabolism promotes DJ3 formation and helps DJ3 survive at -20 °C. Although trehalose accumulation is favorable for DJ3 to cope with low-temperature stress, multiple trehalose metabolism genes need to work together. There may be a multi-path regulated physiological process involving trehalose synthesis genes under low-temperature stress resistance. This physiological process may regulate the formation and maintenance of DJ3 through autophagy and energy conversion.

Molecular variation among virulent and avirulent strains of the quarantine nematode Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is an emerging pathogenic nematode that is responsible for a devastating epidemic of pine wilt disease worldwide, causing severe ecological damage and economic losses to forestry. Two forms of this nematode have been reported, i.e., with strong and weak virulence, commonly referred as virulent and avirulent strains. However, the pathogenicity-related genes of B. xylophilus are not sufficiently characterized. In this study, to find pathogenesis related genes we re-sequenced and compared genomes of two virulent and two avirulent populations. We identified genes affected by genomic variation, and functional annotation of those genes indicated that some of them might play potential roles in pathogenesis. The performed analysis showed that both avirulent populations differed from the virulent ones by 1576 genes with high impact variants. Demonstration of genetic differences between virulent and avirulent strains will provide effective methods to distinguish these two nematode virulence forms at the molecular level. The reported results provide basic information that can facilitate development of a better diagnosis for B. xylophilus isolates/strains which present different levels of virulence and better understanding of the molecular mechanism involved in the development of the PWD.

Effect of culture conditions on conidia production and enhancement of environmental stress resistance of Esteya vermicola in solid-state fermentation.

AIMS: Esteya vermicola is an endoparasitic fungus producing lunate conidia, which kill pine wood nematode (PWN), and PWN could cause pine wilt disease (PWD). The aims of this study were to increase production and confirm the resistance (temperature and UV irradiation) of lunate conidia, and further determine the effective concentrations of conidia infecting PWN. METHODS AND RESULTS: In this study, rice was used as a carrier to absorb conidial suspension to propagate conidia. The optimal conditions for lunate conidia production were 25°C temperature, 9 days of culture time, 2 : 1 rice/distilled water ratio and 10% inoculum size. The germination rate of E. vermicola cultured on potato dextrose agar was influenced by UV irradiation, similar to growth on rice. Esteya vermicola cultured on rice under heat stress might be more suitable for application in the field. The concentration (1 × 108 conidia per ml) to kill PWN had the highest infectivity among the four conidia concentrations tested after 3 days of inoculation. CONCLUSIONS: This study showed a rice substrate-supported high-quality conidia production and the optimal infectivity concentration of E. vermicola. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide the necessary process of an economical and efficient biological control strategy against PWD.

Bursaphelenchus xylophilus: An Important Pathogenic Factor of Pine Wilt Disease and Its Relationship with Bursaphelenchus mucronatus.

Pine wilt disease is the most devastating pine disease caused by Bursaphelenchus xylophilus. Bursaphelenchus mucronatus is morphologically similar to B. xylophilus and geographically overlaps in its distribution. Although interspecific hybridization of the two nematodes has been performed in vitro, the dynamic regularity of hybrid formation and its risk in forests has not been well evaluated. In this study, a hybrid of B. xylophilus and Bursaphelenchus mucronatus mucronatus was identified in the laboratory and fields by molecular markers. The heterozygosity of ITS-5.8S loci for identification was unstable in the hybrid population, and the allele inherited from B. m. mucronatus was lost over several generations. We also provided evidence that hybrids existed in some new epidemic areas, while old epidemic areas were usually dominated by B. xylophilus. Hybrids could be generated when B. m. mucronatus was invaded by B. xylophilus, and the pathogenicity of the hybrids was similar to that of B. xylophilus. These findings may improve the understanding of the natural hybridization between B. xylophilus and B. m. mucronatus and pathogenic variation in pine wilt disease, providing new insights for future studies on disease detection, transmission, and quarantine.

Transcriptomic and Coexpression Network Analyses Revealed Pine Chalcone Synthase Genes Associated with Pine Wood Nematode Infection.

Pine wood nematode (PWN) causes serious diseases in conifers, especially pine species. To investigate the transcriptomic profiles of genes involved in pine-PWN interactions, two different pine species, namely, Pinus thunbergii and P. massoniana, were selected for this study. Weighted gene coexpression network analysis (WGCNA) was used to determine the relationship between changes in gene expression and the PWN population after PWN infection. PWN infection negatively affects the expression of most genes in pine trees, including plant defense-related genes such as genes related to plant hormone signal transduction, plant-pathogen interactions, and the MAPK signaling pathway in plants. However, the expression of chalcone synthase genes and their related genes were proportional to the changes in nematode populations, and chalcone synthase genes were dominant within the coexpression module enriched by genes highly correlated with the nematode population. Many genes that were closely related to chalcone synthase genes in the module were related to flavonoid biosynthesis, flavone and flavonol biosynthesis, and phenylpropanoid biosynthesis. Pine trees could actively adjust their defense strategies in response to changes in the number of invasive PWNs, but the sustained expression of chalcone synthase genes should play an important role in the inhibition of PWN infection.

Research Progress on the Early Monitoring of Pine Wilt Disease Using Hyperspectral Techniques.

Pine wilt disease (PWD) caused by pine wood nematode (PWN, Bursaphelenchus xylophilus) originated in North America and has since spread to Asia and Europe. PWN is currently a quarantine object in 52 countries. In recent years, pine wilt disease has caused considerable economic losses to the pine forest production industry in China, as it is difficult to control. Thus, one of the key strategies for controlling pine wilt disease is to identify epidemic points as early as possible. The use of hyperspectral cameras mounted on drones is expected to enable PWD monitoring over large areas of forest, and hyperspectral images can reflect different stages of PWD. The trend of applying hyperspectral techniques to the monitoring of pine wilt disease is analyzed, and the corresponding strategies to address the existing technical problems are proposed, such as data collection of early warning stages, needs of using unmanned aerial vehicles (UAVs), and establishment of models after preprocessing.

Major sperm protein BxMSP10 is required for reproduction and egg hatching in Bursaphelenchus xylophilus.

The pine wood nematode Bursaphelenchus xylophilus is a disastrous pathogen of pine forests in East Asia and Europe. Despite its decimating effect on pine forests, efficient and environmentally friendly methods available to control the pine wood nematode (PWN) are limited. The most abundant protein in nematode sperm, major sperm proteins (MSPs) have only been discovered in nematodes. In this study, phylogenetic analysis showed that BxMSP10 was highly conserved in the nematode and had a closer phylogenetic relationship with free-living nematodes than with plant-parasitic nematode species. BxMSP10 was specifically expressed in the seminal vesicle of male adults. dsRNA of BxMSP10 significantly decreased reproduction, egg hatching and population maintenance in B. xylophilus. These results indicated that BxMSP10 was a potential candidate for application in the control of B. xylophilus.

Identification of a novel effector BxSapB3 that enhances the virulence of pine wood nematode Bursaphelenchus xylophilus.

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.