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The development of xylem embolism in 1-year-old stems of Japanese black pine ( Pinus thunbergii ) seedlings was monitored by compact magnetic resonance imaging (MRI) after inoculation with the pinewood nematode ( Bursaphelenchus xylophilus ). In parallel, the nematode distribution and population structure in the stems were examined by isolating the nematodes using the Baermann funnel technique. The vertical length and volume of massive embolisms in each seedling were strongly correlated with the maximum relative embolized area (REA) in stem cross-sections. Embolism development and nematode reproduction were not restricted to the inoculation site, as any portion of the stem could be the initial point of a population burst. The nematode population in the stem xylem was strongly correlated with the REA and with the circumferential proportion of cambial death in cross-sections monitored by MRI. The proportion of second-stage juveniles was also correlated with the REA in the xylem. In contrast, the nematode population in bark tissue was not correlated with either the REA or cambial death. These results suggested that nematode reproduction in the cambial zone is the key step in pine wilt disease, and second-stage juveniles were suggested to induce massive embolisms in the advanced stage of the disease., Competing Interests: The author(s) declare no conflict of interest.

Background: Root-lesion nematodes (RLN) are the most economically important pathogenic nematodes attacking maize. Significant economic losses due to lesion nematodes have been reported in maize producing countries in the world., Methods and Results: This study was conducted to determine the distribution and identity of root-lesion nematodes (Pratylenchus spp.) (Tylenchida: Hoplolaimidae) in maize (Zea mays L.) (Poales: Poaceae) fields of the Black Sea region of Türkiye. For this purpose, 39 locations were surveyed and soil samples were taken from 17 regional provinces. Nematodes were extracted using the modified Baerman funnel technique. The species were identified based on sequences of the Internal Transcribed Spacer (ITS) region of ribosomal DNA, as well as morphological characters and morphometrics. In addition, species identifications were confirmed using species-specific primers in the D3 expansion region of 26 S rDNA. At the end of the study, 51.3% of the maize production areas sampled in the region were infected with root-lesion nematode species. Pratylenchus agilis, P. mediterraneus, P. neglectus, P. penetrans, P. thornei, and P. vulnus were identified, and were present in 25%, 5%, 25%, 10%, 15%, and 20% of samples, respectively. To our knowledge, this is the first report of P. agilis in Türkiye., Conclusion: The present study concluded that the molecular analysis of Pratylenchus sequences based on the ITS and D3 region of ribosomal RNA genes allowed the identification of six root lesion nematode species. This study is of great importance in terms of adding additional species to the root-lesion nematode fauna in Turkey and will provide data for future research on the management of these nematodes., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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

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

Rationale: The nematode Aphelenchoides besseyi is the causal agent of green stem and foliar retention, a soybean disease recently described in Brazil. This condition can reduce soybean yield by up to 100%. However, little is known about chemical interactions between the plant and pathogen. Therefore, this work aimed to investigate metabolites from healthy soybean roots and from soybean roots that were inoculated with A. besseyi., Methods: A. besseyi were multiplied in vitro with Fusarium sp. colonies in Petri dishes for 25 days, and were axenically inoculated into hydroponics healthy soybean plants. The metabolites were extracted from the roots of healthy and A. besseyi-infected plants 16 days post-inoculation. These extracts were analyzed using an untargeted metabolomic method with an ultra-high-performance liquid chromatography/electrospray ionization /tandem mass spectrometry (UHPLC/ESI-MS/MS) and molecular networking approach., Results: Roots from infected plants showed morphological alterations such as shrinkage, darkening, and arching. Similarly, they also showed an increased presence of flavonoids, compared with healthy roots. Compounds such as neobavaisoflavone, glycitin, genistin, and genistein were putatively identified and had greater intensity in inoculated roots. These compounds are linked to the defensive mechanisms in plants against nematodes. Moreover, coumaric acid, also exclusively putatively identified in inoculated roots, shows activity related to inhibition of root growth., Conclusions: Liquid chromatography, mass spectrometry, and molecular networking approaches proved to be a powerful tool for the metabolomic study of GSFR. This study showed metabolomics differences of protective substances in the roots, evidencing a quick response of the plant to the attack of A. besseyi., (© 2019 John Wiley & Sons, Ltd.)

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.

The Bursaphelenchus mucronatus , which was highly similar with Bursaphelenchus xylophilus in terms of morphological characteristics and biological properties-but had weaker pathogenicity to forests-was a native species often displaced by B. xylophilus when occupying the same niche. Since the draft genome of the invasive B. xylophilus has been published, the absence of a reference genome of B. mucronatus still prevents us from understanding the molecular evidences behind competitive displacement. In this study, we employed Single Molecule, Real-Time (SMRT) sequencing and a Hi-C scaffolding approach to yield a near chromosome-level assembly of B. mucronatus , including six pseudo-chromosomes. The assembly size is 73 Mb, with scaffold N50 of 11.50 Mb and contig N50 of 1.48 Mb. Comparative genomics results showed high similarity between B. xylophilus and B. mucronatus . However, the losing of orphan genes and species-specific orthologous genes in B. mucronatus may indicate weaker adaptability to the environment. The gene family contractions of GPCRs (G Protein-Coupled Receptors) and cellulases in B. mucronatus may jointly contribute to its displacement by B. xylophilus . Overall, we introduced a valuable genomic resource for molecular and evolutionary studies of B. mucronatus , especially for studying the competitive displacement by the pinewood nematode, which could help us control the pathogenicity of pine wilt diseases.

Key Message: Resistance QTL to root lesion nematode (Pratylenchus thornei) in wheat (Triticum aestivum), QRlnt.sk-6D and QRlnt.sk-2B, were mapped to intervals of 3.5 cM/1.77 Mbp on chromosome 6D and 1.4 cM/2.19 Mbp on chromosome 2B, respectively. Candidate resistance genes were identified in the QTL regions and molecular markers developed for marker-assisted breeding. Two previously known resistance QTL for root lesion nematode (Pratylenchus thornei) in bread wheat (Triticum aestivum), QRlnt.sk-6D and QRlnt.sk-2B, were fine-mapped using a Sokoll (moderately resistant) by Krichauff (susceptible) doubled haploid (DH) population and six newly developed recombinant inbred line populations. Bulked segregation analysis with the 90K wheat SNP array identified linked SNPs which were subsequently converted to KASP assays for mapping in the DH and RIL populations. On chromosome 6D, 60 KASP and five SSR markers spanned a total genetic distance of 23.7 cM. QRlnt.sk-6D was delimited to a 3.5 cM interval, representing 1.77 Mbp in the bread wheat cv. Chinese Spring reference genome sequence and 2.29 Mbp in the Aegilops tauschii genome sequence. These intervals contained 42 and 43 gene models in the respective annotated genome sequences. On chromosome 2B, 41 KASP and 5 SSR markers produced a map spanning 19.9 cM. QRlnt.sk-2B was delimited to 1.4 cM, corresponding 3.14 Mbp in the durum wheat cv. Svevo reference sequence and 2.19 Mbp in Chinese Spring. The interval in Chinese Spring contained 56 high-confidence gene models. Intervals for both QTL contained genes with similarity to those previously reported to be involved in disease resistance, namely genes for phenylpropanoid biosynthetic pathway-related enzymes, NBS-LRR proteins and protein kinases. The potential roles of these candidate genes in P. thornei resistance are discussed. The KASP markers reported in this study could potentially be used for marker-assisted breeding of P. thornei-resistant wheat cultivars.

The pine wood nematode Bursaphelenchus xylophilus is a destructive species affecting pine trees worldwide; however, the underlying mechanism leading to pathogenesis remains unclear. In this study, a B. xylophilus gene encoding thaumatin-like protein-1 ( Bx-tlp-1 ) was silenced by RNA interference to clarify the relationship between the Bx-tlp-1 gene and pathogenicity. The in vitro knockdown of Bx-tlp-1 with double-stranded RNA (dsRNA) decreased B. xylophilus reproduction and pathogenicity. Treatments with dsRNA targeting Bx-tlp-1 decreased expression by 90%, with the silencing effect maintained even in the F3 offspring. Pine trees inoculated with B. xylophilus treated with Bx-tlp-1 dsRNA decreased the symptom of wilting, and the disease severity index was 56.7 at 30 days after inoculation. Additionally, analyses of the cavitation of intact pine stem samples by X-ray microtomography revealed that the xylem cavitation area of pine trees inoculated with B. xylophilus treated with Bx-tlp-1 dsRNA was 0.46 mm 2 at 30 days after inoculation. Results from this study indicated that the silencing of Bx-tlp-1 has effects on B. xylophilus fitness. The data presented here provide the foundation for future analyses of Bx-tlp-1 functions related to B. xylophilus pathogenicity.

Cytochrome P450 genes are very important for plant-parasitic nematodes to reproduce and to metabolize xenobiotic compounds generated by their host plants. The pine wood nematode (PWN), Bursaphelenchus xylophilus , causes very high annual economic losses by killing large numbers of pine trees across Asia and into Europe. In this study, we used RNA interference (RNAi) to analyze the function of the cyp-33C9 gene of PWN. Our results showed that expression of the cyp-33C9 gene was suppressed successfully after soaking nematodes for 24 h in cyp-33C9 double-stranded RNA (dsRNA). The silencing of the cyp-33C9 gene significantly decreased the feeding, reproduction, oviposition and egg hatch of B. xylophilus . Meanwhile, the migration speed of B. xylophilus in Pinus thunbergii was reduced in the early stages when the cyp-33C9 gene was silenced in the nematodes. Moreover, knockdown of the cyp-33C9 gene in B. xylophilus caused a decrease in pathogenicity to pine trees. These results suggest that the cyp-33C9 gene plays an important role in the reproduction and pathogenicity of B. xylophilus . This discovery identified several functions of the cyp-33C9 gene in B. xylophilus and provided useful information for understanding the molecular mechanism behind pine wilt disease caused by PWN.

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 Author(s) 2019. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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's parasitic adaptability.

The pine wood nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease, one of the most devastating forest diseases in East Asian and West European countries. The lifecycle of B. xylophilus includes four propagative larval stages and gonochoristic adults which are involved in the pathogenicity, and two stages of dispersal larvae involved in the spread of the disease. To elucidate the ecological roles of each developmental stage in the pathogenic life cycle, we performed a comprehensive transcriptome analysis using RNA-seq generated from all developmental stages of B. xylophilus and compared transcriptomes between stages. We found more than 9000 genes are differentially expressed in at least one stage of the life cycle including genes involved in general nematode biology such as reproduction and moulting but also effector genes likely to be involved in parasitism. The dispersal-stage transcriptome revealed its analogy to C. elegans dauer and the distinct roles of the two larval stages from each other regarding survival and transmission. This study provides important insights and resources to understand B. xylophilus parasitic biology.

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.

Reactive oxygen species (ROS) play important roles in defence response of host plants versus pathogens. While generation and detoxification of ROS is well understood, how varied ability of different isolates of pathogens to overcome host ROS, or ROS contribution to a particular isolate's pathogenicity, remains largely unexplored. Here, we report that transcriptional regulation of the ROS pathway, in combination with the insulin pathway, increases the pathogenicity of invasive species Bursaphelenchus xylophilus. The results showed a positive correlation between fecundity and pathogenicity of different nematode isolates. The virulent isolates from introduced populations in Japan, China and Europe had significantly higher fecundity than native avirulent isolates from the USA. Increased expression of Mn-SOD and reduced expression of catalase/ GPX-5 and H 2 O 2 accumulation during invasion are associated with virulent strains. Additional H 2 O 2 could improve fecundity of Bu. xylophilus. Furthermore, depletion of Mn-SOD decreased fecundity and virulence of Bu. xylophilus, while the insulin pathway is significantly affected. Thus, we propose that destructive pathogenicity of Bu. xylophilus to pines is partly owing to upregulated fecundity modulated by the insulin pathway in association with the ROS pathway and further enhanced by H 2 O 2 oxidative stress. These findings provide a better understanding of pathogenic mechanisms in plant-pathogen interactions and adaptive evolution of invasive species. This article is part of the theme issue 'Biotic signalling sheds light on smart pest management'.

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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-cpl s 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-cpl s could accumulate specifically in the egg, intestine, and genital system of PWN. During different developmental stages of PWN, the expression of Bx-cpl s in the egg stage was highest. After infection, the expression levels of Bx-cpl s 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-cpl s play multiple roles in the development and pathogenic processes of PWN.

During a survey in agricultural fields of the sub-humid region of Meerut district, India, two strains of entomopathogenic nematodes, labelled CS31 and CS32, were isolated using the Galleria baiting technique. Based on morphological and morphometric studies, and molecular data, the nematodes were identified as Steinernema pakistanense, making this finding the first report of this species from India. For the first time, we performed a molecular and biochemical characterization of the bacterial symbiont of S. pakistanense. Furthermore, a co-phylogenetic analysis of the bacteria from the monophyletic clade containing a symbiont of S. pakistanense, together with their nematode hosts, was conducted, to test the degree of nematode-bacteria co-speciation. Both isolates were also tested in a laboratory assay for pathogenicity against two major pests, Helicoverpa armigera and Spodoptera litura. The morphology of the Indian isolates corresponds mainly to the original description, with the only difference being the absence of a mucron in first-generation females and missing epiptygmata in the second generation. The sequences of bacterial recA and gyrB genes have shown that the symbiont of S. pakistanense is closely related to Xenorhabdus indica, which is associated with some other nematodes from the 'bicornutum' group. Co-phylogenetic analysis has shown a remarkable congruence between the nematode and bacterial phylogenies, suggesting that, in some lineages within the Steinernema / Xenorhabdus complex, the nematodes and bacteria have undergone co-speciation. In the virulence assay, both strains caused a 100% mortality of both tested insects after 48 h, even at the lowest doses of 25 infective juveniles per insect, suggesting that S. pakistanense could be considered for use in the biocontrol of these organisms in India.

Pine wilt disease caused by pine wood nematode (Bursaphelenchus xylophilus, PWN) is a severe forest disease of the genus Pinus. Masson pine as an important timber and oleoresin resource in South China, is the major species infected by pine wilt disease. However, the underlying mechanism of pine resistance is still unclear. Here, we performed a transcriptomics analysis to identify differentially expressed genes associated with resistance to PWN infection. By comparing the expression profiles of resistant and susceptible trees inoculated with PWN at 1, 15, or 30 days post-inoculation (dpi), 260, 371 and 152 differentially expressed genes (DEGs) in resistant trees and 756, 2179 and 398 DEGs in susceptible trees were obtained. Gene Ontology enrichment analysis of DEGs revealed that the most significant biological processes were "syncytium formation" in the resistant phenotype and "response to stress" and "terpenoid biosynthesis" in the susceptible phenotype at 1 and 15 dpi, respectively. Furthermore, some key DEGs with potential regulatory roles to PWN infection, including expansins, pinene synthases and reactive oxidation species (ROS)-related genes were evaluated in detail. Finally, we propose that the biosynthesis of oleoresin and capability of ROS scavenging are pivotal to the high resistance of PWN.

During sampling of several Coffea arabica plantations in Tanzania severe root galling, caused by a root-knot nematode was observed. From pure cultures, morphology and morphometrics of juveniles and females matched perfectly with Meloidogyne africana, whereas morphology of the males matched identically with those of Meloidogyne decalineata. Based on their Cox1 sequence, however, the recovered juveniles, females and males were confirmed to belong to the same species, creating a taxonomic conundrum. Adding further to this puzzle, re-examination of M. oteifae type material showed insufficient morphological evidence to maintain its status as a separate species. Consequently, M. decalineata and M. oteifae are synonymized with M. africana, which is herewith redescribed based on results of light and scanning electron microscopy, ribosomal and mitochondrial DNA sequences, isozyme electrophoresis, along with bionomic and cytogenetic features. Multi-gene phylogenetic analysis placed M. africana outside of the three major clades, together with M. coffeicola, M. ichinohei and M. camelliae. This phylogenetic position was confirmed by several morphological features, including cellular structure of the spermatheca, egg mass position, perineal pattern and head shape. Moreover, M. africana was found to be a polyphagous species, demonstrating that "early-branching" Meloidogyne spp. are not as oligophagous as had previously been assumed. Cytogenetic information indicates M. africana (2n = 21) and M. ardenensis (2n = 51-54) to be a triploid mitotic parthenogenetic species, revealing at least four independent origins of mitotic parthenogenesis within the genus Meloidogyne. Furthermore, M. mali (n = 12) was found to reproduce by amphimixis, indicating that amphimictic species with a limited number of chromosomes are widespread in the genus, potentially reflecting the ancestral state of the genus. The wide variation in chromosome numbers and associated changes in reproduction modes indicate that cytogenetic evolution played a crucial role in the speciation of root-knot nematodes and plant-parasitic nematodes in general.

The rice white tip nematode (RWTN), Aphelenchoides besseyi and the chrysanthemum foliar nematode (CFN), Aphelenchoides ritzemabosi are migratory plant parasitic nematodes that infect the aboveground parts of plants. In this research, Arabidopsis thaliana was infected by RWTN and CFN under indoor aseptic cultivation, and the nematodes caused recognizable symptoms in the leaves. Furthermore, RWTN and CFN completed their life cycles and proliferated. Therefore, A. thaliana was identified as a new host of RWTN and CFN. The optimum inoculum concentration for RWTN and CFN was 100 nematodes/plantlet, and the optimum inoculum times were 21 and 24 days, respectively. For different RWTN populations, the pathogenicity and reproduction rates were different in the A. thaliana Col-0 ecotype and were positively correlated. The optimum A. thaliana ecotypes were Col-0 and WS, which were the most susceptible to RWTN and CFN, respectively. Additionally, RWTN was ectoparasitic and CFN was ecto- and endoparasitic in A. thaliana. The RWTN and CFN migrated from inoculated leaves to the entire plantlet, and the number of nematodes in different parts of A. thaliana was not correlated with distance from the inoculum point. This is a detailed study of the behavior and infection process of foliar nematodes on A. thaliana.

Pine wilt disease (PWD) results from the interaction of three elements: the pathogenic nematode, Bursaphelenchus xylophilus; the insect-vector, Monochamus sp.; and the host tree, mostly Pinus species. Bacteria isolated from B. xylophilus may be a fourth element in this complex disease. However, the precise role of bacteria in this interaction is unclear as both plant-beneficial and as plant-pathogenic bacteria may be associated with PWD. Using whole genome sequencing and phenotypic characterization, we were able to investigate in more detail the genetic repertoire of Serratia marcescens PWN146, a bacterium associated with B. xylophilus. We show clear evidence that S. marcescens PWN146 is able to withstand and colonize the plant environment, without having any deleterious effects towards a susceptible host (Pinus thunbergii), B. xylophilus nor to the nematode model C. elegans. This bacterium is able to tolerate growth in presence of xenobiotic/organic compounds, and use phenylacetic acid as carbon source. Furthermore, we present a detailed list of S. marcescens PWN146 potentials to interfere with plant metabolism via hormonal pathways and/or nutritional acquisition, and to be competitive against other bacteria and/or fungi in terms of resource acquisition or production of antimicrobial compounds. Further investigation is required to understand the role of bacteria in PWD. We have now reinforced the theory that B. xylophilus-associated bacteria may have a plant origin.

During a screening program for new agrochemicals from Chinese medicinal herbs, the ethanol extract of Notopterygium incisum rhizomes was found to possess strong nematicidal activity against the two species of nematodes, Bursaphelenchus xylophilus and Meloidogyne incognita. Based on bioactivity-guided fractionation, the four constituents were isolated from the ethanol extract and identified as columbianetin, falcarindiol, falcarinol, and isoimperatorin. Among the four isolated constituents, two acetylenic compounds, falcarindiol and falcarinol (2.20-12.60 μg/mL and 1.06-4.96 μg/mL, respectively) exhibited stronger nematicidal activity than two furanocoumarins, columbianetin, and isoimperatorin (21.83-103.44 μg/mL and 17.21-30.91 μg/mL, respectively) against the two species of nematodes, B. xylophilus and M. incognita. The four isolated constituents also displayed phototoxic activity against the nematodes. The results indicate that the ethanol extract of N. incisum and its four isolated constituents have potential for development into natural nematicides for control of plant-parasitic nematodes.

Bursaphelenchus mucronatus (B. mucronatus) isolates that originate from different regions may vary in their virulence, but their virulence-associated genes and proteins are poorly understood. Thus, we conducted an integrated study coupling RNA-Seq and isobaric tags for relative and absolute quantitation (iTRAQ) to analyse transcriptomic and proteomic data of highly and weakly virulent B. mucronatus isolates during the pathogenic processes. Approximately 40,000 annotated unigenes and 5000 proteins were gained from the isolates. When we matched all of the proteins with their detected transcripts, a low correlation coefficient of r = 0.138 was found, indicating probable post-transcriptional gene regulation involved in the pathogenic processes. A functional analysis showed that five differentially expressed proteins which were all highly expressed in the highly virulent isolate were involved in the pathogenic processes of nematodes. Peroxiredoxin, fatty acid- and retinol-binding protein, and glutathione peroxidase relate to resistance against plant defence responses, while β-1,4-endoglucanase and expansin are associated with the breakdown of plant cell walls. Thus, the pathogenesis of B. mucronatus depends on its successful survival in host plants. Our work adds to the understanding of B. mucronatus' pathogenesis, and will aid in controlling B. mucronatus and other pinewood nematode species complexes in the future., Competing Interests: The authors declare no conflict of interest.

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.

Pinus massoniaia Lamb has gained more and more attention as the most important tree species for timber and forestation in South China. Gene expression studies are of great importance to identify new and elite cultivars. Real-time quantitative PCR, a highly sensitive and specific method, is commonly used in the analysis of gene expression. The appropriate reference genes must be employed to normalize the calculation program for ascertaining repeatable and significant results. Herein, eleven housekeeping genes were evaluated during different stages of P. massoniana post nematode inoculation in this study. Three statistical approaches such as geNorm, NormFinder and BestKeeper were selected to analyze the stability of candidate genes. The results indicated that U2af and β-TUB were the most stable reference genes. These two genes could be used for the normalization in most of the experiments of P. massoniana, while Histone and AK were the least stable ones. In addition, EF expressed at the lowest average Ct value was the most abundant candidate gene. As an important gene associated with defense mechanisms, ABC transporter was analyzed by qRT-PCR, and the results were used to confirm the reliability of two genes. The selected reference genes in the present study will be conducive to future gene expression normalized by qRT-PCR in P. massoniana.

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.

It is known that several bacteria are adherent to the surface coat of pine wood nematode (Bursaphelenchus xylophilus), but their function and role in the pathogenesis of pine wilt disease remains debatable. The Pseudomonas fluorescens GcM5-1A is a bacterium isolated from the surface coat of pine wood nematodes. In previous studies, GcM5-1A was evident in connection with the pathogenicity of pine wilt disease. In this study, we report the de novo sequencing of the GcM5-1A genome. A 600-Mb collection of high-quality reads was obtained and assembled into sequence contigs spanning a 6.01-Mb length. Sequence annotation predicted 5,413 open reading frames, of which 2,988 were homologous to genes in the other four sequenced P. fluorescens isolates (SBW25, WH6, Pf0-1 and Pf-5) and 1,137 were unique to GcM5-1A. Phylogenetic studies and genome comparison revealed that GcM5-1A is more closely related to SBW25 and WH6 isolates than to Pf0-1 and Pf-5 isolates. Towards study of pathogenesis, we identified 79 candidate virulence factors in the genome of GcM5-1A, including the Alg, Fl, Waa gene families, and genes coding the major pathogenic protein fliC. In addition, genes for a complete T3SS system were identified in the genome of GcM5-1A. Such systems have proved to play a critical role in subverting and colonizing the host organisms of many gram-negative pathogenic bacteria. Although the functions of the candidate virulence factors need yet to be deciphered experimentally, the availability of this genome provides a basic platform to obtain informative clues to be addressed in future studies by the pine wilt disease research community.

Background: Bursaphelenchus xylophilus is an emerging pathogenic nematode that is responsible for a devastating epidemic of pine wilt disease across Asia and Europe. In this study, we report the first genome-wide variation analysis of the nematode with an aim to obtain a full picture of its diversity., Methods: We sequenced six key B. xylophilus strains using Illumina HiSeq sequencer. All the strains were isolated in Japan and have been widely used in previous studies. Detection of genomic variations were done by mapping the reads to the reference genome., Results: Over 3 Mb of genetic variations, accounting for 4.1 % of the total genome, were detected as single nucleotide polymorphisms or small indels, suggesting multiple introductions of this invaded species from its native area into the country. The high level of genetic diversity of the pine wood nematode was related to its pathogenicity and ecological trait differences. Moreover, we identified a gene set affected by genomic variation, and functional annotation of those genes indicated that some of them had potential roles in pathogenesis., Conclusions: This study provides an important resource for understanding the population structure, pathogenicity and evolutionary ecology of the nematode, and further analysis based on this study with geographically diverse B. xylophilus populations will greatly accelerate our understanding of the complex evolutionary/epidemic history of this emerging pathogen.

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&#95;Unclassified or Rhizobiaceae&#95;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.

Mountain ranges may delimit the distribution of native species as well as constitute potential barriers to the spread of invasive species. The invasive pinewood nematode, Bursaphelenchus xylophilus, is a severe forest pest inducing pine wilt disease. It is vectored in Europe by a native long-horned beetle, Monochamus galloprovincialis. This study explored the potential of the Pyrenean chain to slow or prevent the natural spread of nematode-infested beetles from the Iberian Peninsula, where the nematode is established and is expanding its range, towards France and the rest of Europe. An analysis of the genetic structure and migration patterns of the beetle populations throughout the Pyrenean mountain range was combined with a spread model simulating the potential movements of nematode-infested beetles across it. The central part of the Pyrenees, which corresponds to the highest elevation zone, was shown to prevent gene flow between the French and Spanish populations of M. galloprovincialis on each side of the mountains. Conversely, strong admixture was detected between populations located on both sides of low elevation hills, and especially at the east and west extremities of the mountain range. Simulations of the spread of nematode-infested beetles under various thresholds of beetle survival and pine wilt disease expression gave results consistent with the variation in genetic make-up, suggesting that western and eastern hillsides may represent corridors favoring natural spread of the nematode from the Iberian Peninsula to France. Simulations also showed that temperature rise due to climate change may significantly reduce the extent of the barrier formed by highest elevations. Our results support the hypothesis that the Pyrenean chain represents a partial barrier to the natural spread of nematode-infested beetles. These results, which have to be considered together with potential human-assisted long-distance spread of the nematode, highlight priority zones for future pest monitoring and management programs. More generally, such an integrated approach could be used to assess the role of mountain chains in the potential spread of other invasive pests.

Based on the forest woody species wilt areassurvey in Nizhniy Novgorod region in August 2014, the possible factors of the pest spread risk modelling were analysed on six species of the genus Bursaphelenchus and Devibursaphelenchus teratospicularis using six parameters: plant host species, beetle vector species, average temperatures in July and January, annual precipitation. It was concluded that these parameters in the evaluated wilt spots correspond to climatic and biological data of the already published woody plants wilt records in Europe and Asia caused by the same nematode pest species. It was speculated that the annual precipitation of 600 mm and average July temperature of 25 degrees C or higher, are the critical combination that may be used to develop the predicative risk modelling in the forests' and parks' wilt monitoring.

The pinewood nematode, Bursaphelenchus xylophilus, is native to North America but it only causes damaging pine wilt disease in those regions of the world where it has been introduced. The accurate detection of the species and its dispersal routes are thus essential to define effective control measures. The main goals of this study were to analyse the genetic diversity among B. xylophilus isolates from different geographic locations and identify single nucleotide polymorphism (SNPs) markers for geographic origin, through a comparative transcriptomic approach. The transcriptomes of seven B. xylophilus isolates, from Continental Portugal (4), China (1), Japan (1) and USA (1), were sequenced in the next generation platform Roche 454. Analysis of effector gene transcripts revealed inter-isolate nucleotide diversity that was validated by Sanger sequencing in the genomic DNA of the seven isolates and eight additional isolates from different geographic locations: Madeira Island (2), China (1), USA (1), Japan (2) and South Korea (2). The analysis identified 136 polymorphic positions in 10 effector transcripts. Pairwise comparison of the 136 SNPs through Neighbor-Joining and the Maximum Likelihood methods and 5-mer frequency analysis with the alignment-independent bilinear multivariate modelling approach correlated the SNPs with the isolates geographic origin. Furthermore, the SNP analysis indicated a closer proximity of the Portuguese isolates to the Korean and Chinese isolates than to the Japanese or American isolates. Each geographic cluster carried exclusive alleles that can be used as SNP markers for B. xylophilus isolate identification.

The advent of next generation sequencing has revolutionized research approaches to biology by making entire genome sequences available and marking a new age in biology that has the potential to open innovative research avenues in various fields. Genome sequencing is now being applied in the fields of forest ecology and forest pathology, which previously had limited access to molecular techniques. One of the most advanced areas of progress is the study of "pine wilt disease", which is caused by the parasitic nematode, Bursaphelenchus xylophilus. The entire genome sequence of B. xylophilus was determined in 2011, and since then, proteomic studies have been conducted to understand the molecular basis of the parasitism and pathogenicity of B. xylophilus. These postgenomic studies have provided numerous molecular insights and greatly changed our understanding of the pathogenesis of pine wilt disease. Here, we review the recent advances in genomic and proteomic approaches that address some of the longstanding questions behind the pathogenesis of pine wilt disease and have identified future questions and directions in this regard., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

After devastating vast areas of pine forests in Asian countries, the pine wilt disease spread into European forests in 1999 and is causing worldwide concern. This disease involves very complicated interactions between a pathogenic nematode, its vector beetle, host pine species, and fungi in dead hosts. Pathogenicity of the pine wood nematode is determined not only by its physical and chemical traits but also by its behavioral traits. Most life history traits of the pine wood nematode, such as its phoretic relationship with vector beetles, seem to be more effective in virulent than in avirulent isolates or species. As the pathogenicity determinants, secreted enzymes, and surface coat proteins are very important, they have therefore been studied intensively. The mechanism of quick death of a large pine tree as a result of infection by a tiny nematode could be ascribed to the dysfunction of the water-conducting system caused by the death of parenchyma cells, which must have originally evolved as an inherent resistant system.

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.

The pinewood nematode, Bursaphelenchus xylophilus, is a destructive pest in several countries including Japan, China and Korea. Of three genes encoding the venom allergen-like protein in B. xylophilus, Bxvap-1 showed the highest transcript levels at the pine-grown propagative stage. In addition, western blot and immunohistochemical analyses using anti-BxVap-1 polyclonal antibody verified a specific increase in BxVap-1 expression levels at the pine-grown propagative stage. Using immunohistochemistry, BxVap-1 was detected around the putative oesophageal glands and metacarpus, suggesting that BxVap-1 is secreted into the host pine tree and is involved in the parasitic mechanism. To explain the parasitic role of BxVap-1, we measured the migration rate inside pine seedlings of B. xylophilus either with or without Bxvap-1 knockdown by RNA interference. Bxvap-1 knockdown resulted in a significantly lower migration rate in the >6cm region compared with the control B. xylophilus. These results suggest that BxVap-1 is involved in B. xylophilus migration, perhaps by suppressing the pine tree defence mechanism., (Copyright © 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Pine wilt is a disease of pine (Pinus spp.) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus. However, the pathogenic mechanism of pine wilt disease (PWD) remains unclear. Although the PWN was thought to be the only pathogenic agent associated with this disease, a potential role for bacterial symbionts in the disease process was recently proposed. Studies have indicated that aseptic PWNs do not cause PWD in aseptic pine trees, while PWNs associated with bacteria cause wilting symptoms. To investigate the pathogenicity of the PWN and its associated bacteria, 3-month-old microcuttings derived from certain clones of Pinus densiflora Siebold & Zucc. produced in vitro were inoculated under aseptic conditions with aseptic PWNs, non-aseptic PWNs and bacteria isolated from the nematodes. Six-month-old aseptic P. densiflora microcuttings and 7-month-old P. massoniana seedlings were also inoculated under aseptic conditions with aseptic PWNs and non-aseptic PWNs. The results showed that the aseptic microcuttings and seedlings inoculated with aseptic PWNs or non-aseptic PWNs wilted, while those inoculated with bacterial isolates did not wilt. Nematodes were recovered from wilted microcuttings and seedlings inoculated with aseptic PWNs and non-aseptic PWNs, and the asepsis of nematodes recovered from aseptic PWN-inoculated microcuttings and seedlings was reconfirmed by culturing them in NB liquid medium at 30°C for more than 7 days. Taken together, the results indicate that the asepsis of PWN did not cause the loss of pathogenicity.

The cloning and characterization of a cDNA encoding a calreticulin from the pinewood nematode Bursaphelenchus xylophilus is described herein. The full-length cDNA (Bx-crt-1) contained a 1200 bp open reading frame that could be translated to a 399 amino acid polypeptide. The deduced protein contained highly conserved regions of a calreticulin gene and had 66.2-70.1% amino acid sequence identity to other calreticulin sequences from nematodes. RNAi, RT-PCR amplification, and southern blot suggest that Bx-crt-1 may be important for the development of B. xylophilus., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Pinewood nematode (PWN) is the causal agent of the pine wilt disease. Previous studies have suggested that secretions from the esophageal glands of PWN play an important role in pathogenicity. A cluster of three venom allergen-like protein genes and one pseudogene, Bx-vap-1, Bx-vap-2, Bx-vap-3 and Bx-vap-P, were identified within a 3.7-kb region. Additionally, three putative modification, transport and regulatory protein genes were also detected in the same flanking region of the Bx-vap gene cluster. Genes vap-1, -2 and -3 are functional and encode three major allelic variants of PWN venom allergen-like proteins. But Bx-vap-P is an untranscribed pseudogene. Genes vap-1, -2 and -3 produce predicted products of 204, 206 and 203 amino acid residues, respectively, including the putative signal peptide sequence at the amino termini. In situ mRNA hybridization analysis showed that the transcripts of genes vap-1, -2 and -3 accumulated exclusively within the esophageal gland cells of Bursaphelenchus xylophilus., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is the most destructive pest of soybean worldwide. Host plant resistance is an effective approach to control this pest. Plant introduction PI 567516C has been reported to be highly resistant to multiple-HG types of SCN. The objectives of this study were to identify and map novel quantitative trait loci (QTL) for SCN resistance to six HG types (also known as races 1, 2, 3, 5, 14, and LY1). Mapping was conducted using 250 F(2:3) progeny derived from a Magellan (susceptible) × PI 567516C (resistant) cross. F(6:7) recombinant inbred lines (RILs) developed from the F(2:3) progeny were employed to confirm the putative QTL identified. A total of 927 polymorphic simple sequence repeats (SSR) and single nucleotide polymorphism (SNP) markers were genotyped. Following the genetic linkage analysis, permutation tests and composite interval mapping were performed to identify and map QTL. Four QTL were associated with resistance to either multiple- or single-SCN HG types. Two QTL for resistance to multiple-SCN HG types were mapped to Chromosomes 10 and 18 and have not been reported in other SCN resistance sources. New QTL were confirmed by analysis of 250 F(6:7) RILs from the same population. SSR and SNP markers closely associated with these QTL can be useful for the development of near-isogenic lines for fine-mapping and positional cloning of candidate genes for SCN resistance.

The pinewood nematode (PWN) is one of the worst tree-killing exotic pests in East-Asian countries. The first European record of establishment in Portugal in 1999 triggered extensive surveys and contingency plans for eradication in European countries, including immediate removal of large areas of conifer host trees. Using Norway as an example, we applied a simulation model to evaluate the chance of successful eradication of a hypothetical introduction by the current contingency plan in a northern area where wilting symptoms are not expected to occur. Despite a highly variable spread of nematode infestations in space and time, the probability of successful eradication in 20 years was consistently low (mean 0.035, SE 0.02). The low success did not change significantly by varying the biological parameters in sensitivity analyses (SA), probably due to the late detection of infestations by the survey (mean 14.3 years). SA revealed a strong influence of management parameters. However, a high probability of eradication required unrealistic measures: achieving an eradication probability of 0.99 in 20 years required 10,000 survey samples per year and a host tree removal radius of 8,000 m around each detection point., (© 2010 Society for Risk Analysis.)

Sphaerularia vespae sp. nov., an endoparasite of a common Japanese hornet, Vespa simillima is described from Hokkaido, Japan, and its molecular sequence profiles are given. This newly discovered nematode appears to belong to the genus Sphaerularia, judging from its characteristic parasitic form, the uterium, which looks like a sausage with many verrucae on its surface. Sphaerularia vespae sp. nov. is distinguished from S. bombi, the only other nominal species of the genus, by the morphology of the male bursa, female tail, and anterior end of parasitic juveniles. SSU, ITS, and D2D3 (LSU) DNA sequences were determined for S. vespae sp. nov. and compared with those from related nematodes obtained from the GenBank database. The sequences from S. vespae sp. nov. were close to those of S. bombi and several another tylenchid entomoparasitic nematodes. Sphaerularia vespae sp. nov. parasitizes overwintering gynes of the hornet and practically sterilizes them, as S. bombi does for bumblebee gynes.

The pine wilt disease caused by Bursaphelenchus xylophilus (BX), also known as the pine wood nematode (PWN), is the most devastating disease of pine trees. In this work, a high molecular weight B. xylophilus cellulase antigen (BXCa) was purified from total homogenates of nematodes. BXCa was found to be able to hydrolyze carboxymethyl cellulose (CMC) efficiently (155.65 U/mg) and to have an approximate molecular mass of 58.9 kDa. We harvested anti-BXCa antibodies and performed immunocytochemical assays, which revealed the localization of cellulase pools in the esophageal gland cells of the PWN. It was also discovered that cellulase was secreted from the stylet and was used to hydrolyze cellulose to facilitate the PWN entering host cells. These results are consistent with other plant parasitical nematodes. Interestingly, strong fluorescence signals from cellulase staining were observed in tracheid cells in naturally infected pine wood, in addition to ray cells and the resin canal zone. These results strongly suggest that the cellulase released by the PWN is one of the pathogenic substances of pine wilt disease and is responsible for the development of the early symptoms of the disease.

Serine proteinase inhibitors (IP's) are proteins found naturally in a wide range of plants with a significant role in the natural defense system of plants against herbivores. The question addressed in the present study involves assessing the ability of the serine proteinase inhibitor in combating nematode infestation. The present study involves engineering a plant serine proteinase inhibitor (pin2) gene into T. durum PDW215 by Agrobacterium-mediated transformation to combat cereal cyst nematode (Heterodera avenae) infestation. Putative T(0) transformants were screened and positive segregating lines analysed further for the study of the stable integration, expression and segregation of the genes. PCR, Southern analysis along with bar gene expression studies corroborate the stable integration pattern of the respective genes. The transformation efficiency is 3%, while the frequency of escapes was 35.71%. chi(2) analysis reveals the stable integration and segregation of the genes in both the T(1) and T(2) progeny lines. The PIN2 systemic expression confers satisfactory nematode resistance. The correlation analysis suggests that at p < 0.05 level of significance the relative proteinase inhibitor (PI) values show a direct positive correlation vis-à-vis plant height, plant seed weight and also the seed number.

The tomato Hero A gene is the only member of a multigene family that confers a high level (>80%) of resistance to all the economically important pathotypes of potato cyst nematode (PCN) species Globodera rostochiensis and G. pallida. Although the resistance levels of transgenic tomato lines were similar to those of the tomato line LA1792 containing the introgressed Hero multigene family, transgenic potato plants expressing the tomato Hero A gene are not resistant to PCNs. Comparative microscopy studies of in vitro infected roots of PCN-susceptible tomato cv. Money Maker, the resistant breeding line LA1792, and transgenic line L10 with Ro1 pathotype have revealed no statistically significant difference in the number of juveniles invading roots. However, syncytia (specialized feeding cells) induced in LA1792 and L10 roots mostly were found to have degenerated a few days after their induction, and a few surviving syncytia were able to support only the development of males rather than females. Thus, the ratio between males and females was biased towards males on LA1792 and L10 roots. A series of changes occur in resistant plants leading to formation of a layer of necrotic cells separating the syncytium from stellar conductive tissues and this is followed by degradation of the syncytium. Although the Hero A gene is expressed in all tissues, including roots, stems, leaves, and flower buds, its expression is upregulated in roots in response to PCN infection. Moreover, the expression profiles of the Hero A correlates with the timing of death of the syncytium.

Poria cocos, a famous traditional Chinese medicine, was found to have nematicidal activity in experiments searching for nematicidal fungi. The experiment showed it could kill 94.9% of the saprophytic nematode, Panagrellus redivivue, 92.6% of the root-knot nematode, Meloidogyne arenaria, and 93.5% of the pine nematode, Bursaphelenchus xylophilus, on PDA plate within 12 hours. According to the nematicidal activity, three new compounds, 2, 4, 6-triacetylenic octane diacid, 2, 4, 5, 6-tetrahydroxyhexanoic acid and 3, 4-dihydroxy-2-keto-n-butyl 2,4,5,6-tetrahydroxyhexanate, were isolated from submerged cultures of Poria cocos. Of these, 2, 4, 6-triacetylenic octane diacid could kill 83.9% Meloidogyne arenaria and 73.4% Panagrellus redivivus at 500 ppm within 12 hours. Here, it is reported for the first time that Poria cocos has nematicidal activity.

We isolated the nematode-trapping fungus Monacrosporium megalosporum from nature and examined its morphology, physiology and molecular characteristics. The nematode-trapping device of this fungus is a three-dimensional network. This fungus captures the pine wilt nematode (Bursaphelenchus xylophilus), but not a non-phytopathogenic nematode that is morphologically similar to B. xylophilus. The phylogenic relationship of the nucleotide sequence of the rDNA ITS region was close to those of M. thaumasium and Geniculifera eudermata, which also have nematode-trapping devices that are three-dimensional networks. Acidic pH inhibited both the liberation and regeneration of protoplasts. Moreover, cytoplasmic granulation of protoplasts was found below pH 6.0. Mycelial growth on agar media was also inhibited below pH 4, but not at pH 9. These results strongly suggest that the activity of this fungus is inhibited by acid rain in the field. Therefore, development of pine wilt disease might be a secondary effect of acid rain.