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Bursaphelenchus mucronatus is the sibling species of B. xylophilus, which causes pine wilt disease. Sex pheromone-mediated mating behavior underlies the development of B. xylophilus populations. The study of the molecular mechanism of sex pheromone receptor genes is vital for the control of B. xylophilus. The pivotal role of the 5-HT receptor ser-1 in Caenorhabditis elegans's mating has been demonstrated, but there is little known in B. mucronatus and B. xylophilus. In our study, the molecular features and biological functions of Bmu-ser-1 are explored. qPCR results showed that Bmu-ser-1 was expressed at all ages, especially at J2 and J4, with significantly high expression. Notably, the expression levels in males were significantly higher than that in females. The results of in situ hybridization suggest that the Bmu-ser-1 gene was expressed at the site of the digestive system during the embryonic stage, the whole body during the J2 stage, and mainly at the end of the germinal primordium during the J3 stage, as well as centrally in the vulva of females and in the gonads and tail of males during the J4 and adult stages. The RNAi results indicate a significant decrease in hatchability and stagnation at the J1 stage after interference. Treated J2 had reduced motility and stunted growth. Males after interference showed mismatch and females showed spawning difficulties. Sexual arousal experiments further validated Bmu-ser-1 as a receptor gene for males receiving female sex pheromones. Collectively, our findings strongly suggest that the Bmu-ser-1 gene has a classical role, the control of nematode growth; and a novel role involved in mating. The study on the molecular mechanisms of growth and reproduction in B. mucronatus could provide a reference for understanding the population expansion and disease epidemics of B. xylophilus. [ABSTRACT FROM AUTHOR]

Summary: Sperm development and structure in the wood-inhabiting fungal and plant-feeding nematode, Bursaphelenchus luxuriosae , were studied using transmission electron microscopy to evaluate interspecific similarities and differences of spermatozoa in nematodes. In general, spermatogenesis in B. luxuriosae fits the 'rhabditid' pattern supported by morphological and phylogenetic analysis of the order Rhabditida. Spermatocyte development includes formation of complexes of fibrous bodies (FBs) with membranous organelles (MOs), the complexes dissociate in the spermatids into separate components, and the immature sperm contain MOs but lack FBs, which transform into a dense matrix of sperm cytoplasm. The female spermatheca contains mature spermatozoa as bipolar cells subdivided into a pseudopod devoid of organelles and a main cell body containing a nucleus without a nuclear envelope, numerous mitochondria, and peripheral MOs as pouches opening to the exterior via pores. Data on B. luxuriosae are used for analysis of variable quantitative and morphological characteristics of spermatozoa in Aphelenchoidea. General size of spermatozoa and their MOs have little value for comparative analysis. The MO knobbles look uniform in immature spermatozoa of each aphelenchoidid species studied and may be considered as a taxonomically specific ultrastructural feature. The presence or absence of FBs in immature spermatozoa demonstrates the diagnostic value of the aphelenchoidid sperm structure at the species level. Analysis also shows close similarity of spermatozoa in Bursaphelenchus spp. and Caenorhabditis elegans , the model species with comprehensive data on sperm biology. This may be used to identify new ways for the control and suppression of harmful nematode species such as B. xylophilus. [ABSTRACT FROM AUTHOR]

Bursaphelenchus mucronatus is the sibling species of B. xylophilus, which causes pine wilt disease. Sex pheromone-mediated mating behavior underlies the development of B. xylophilus populations. The study of the molecular mechanism of sex pheromone receptor genes is vital for the control of B. xylophilus. The pivotal role of the 5-HT receptor ser-1 in Caenorhabditis elegans’s mating has been demonstrated, but there is little known in B. mucronatus and B. xylophilus. In our study, the molecular features and biological functions of Bmu-ser-1 are explored. qPCR results showed that Bmu-ser-1 was expressed at all ages, especially at J2 and J4, with significantly high expression. Notably, the expression levels in males were significantly higher than that in females. The results of in situ hybridization suggest that the Bmu-ser-1 gene was expressed at the site of the digestive system during the embryonic stage, the whole body during the J2 stage, and mainly at the end of the germinal primordium during the J3 stage, as well as centrally in the vulva of females and in the gonads and tail of males during the J4 and adult stages. The RNAi results indicate a significant decrease in hatchability and stagnation at the J1 stage after interference. Treated J2 had reduced motility and stunted growth. Males after interference showed mismatch and females showed spawning difficulties. Sexual arousal experiments further validated Bmu-ser-1 as a receptor gene for males receiving female sex pheromones. Collectively, our findings strongly suggest that the Bmu-ser-1 gene has a classical role, the control of nematode growth; and a novel role involved in mating. The study on the molecular mechanisms of growth and reproduction in B. mucronatus could provide a reference for understanding the population expansion and disease epidemics of B. xylophilus.

Interspecific competition occurs fiercely between invasive and native species which occupy similar niche. Here, we report the competitive mechanism underlying the displacement of Bursaphelenchus mucronatus by Bursaphelenchus xylophilus. In the early stage of the competition, population of both species increase significantly, but their abundance rates were differentiated, B. xylophilus increased, whereas B. mucronatus decreased. Unexpectedly, sex ratio of female to male of both species increased sharply and peaked at 4.87 and 3.88, respectively, which were outclassed that of noncompetitive conditions (2.77 and 2.21). Thereafter, the sex ratio of B. xylophilus declined and stabilized at 2.81, but the sex ratio of B. mucronatus declined till no B. mucronatus was left, indicating the interspecific competition could be dominated by female. The mating behaviors showed that for both species, the mating success rate of one couple dropped slightly when one male or female of other species was added, but dropped significantly when two males or females were added. Surprisingly, almost no successful mating was observed when three males or females of other species were added. We therefore hypothesize that the mating opportunity of one couple could be deprived by the other species once three or more male or female were added. Further, threshold value of competitive displacement experiments and model analysis were completely consistent with the above experiments. Our results elucidate the crucial role of sex ratio in interspecific competition for the first time, as well as their mating disruption, directing a wonderful story of female-dominated war between two xylophilus group nematode species. [ABSTRACT FROM AUTHOR]

Summary: The pine wood nematode (PWN), Bursaphelenchus xylophilus , is the causative agent of pine wilt disease and is transmitted by cerambycid adults of the genus Monochamus. It is believed to have been introduced into Japan from North America in the 1900s and since then it has been devastating the native pine forests. By contrast, the epidemics of the disease have never occurred in pine forests native to North America in the original habitats, indicating no expression of virulence. To determine the evolutionary change in the transmission traits of PWN, the temporal pattern of PWN transmission to fresh branch sections of Pinus sylvestris was studied using 38 Monochamus carolinensis adults in Illinois, USA, and compared with previous studies on the PWN- M. alternatus and B. mucronatus - M. saltuarius systems in Japan. A great difference was observed in the initial nematode load among vectors irrespective of the three systems. The maximal numbers of PWN transmitted (5 days)−1 by individual vectors were greater and the efficiencies of nematode transmission and invasion were higher for the Japanese PWN- M. alternatus system than for the North American PWN- M. carolinensis system in two heavy classes of initial load of over 1000 nematodes. The proportion of PWN transmitted to healthy pine trees by reproductively immature female vectors was small (2.5-11.5%) in the three nematode-vector systems in most cases. The evolutionary changes in the transmission traits and the control of the range expansion of the PWN were discussed in relation to the expression of virulence. [ABSTRACT FROM AUTHOR]

The pinewood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine-wilt disease (PWD), is a threat to Pinus forests in Asia and Europe. Bursaphelenchus mucronatus and B. fraudulentus are closely related non-pathogenic pine-wood nematodes. In the present work, four medium-chain aliphatic alcohols (C10 to C13) were evaluated in direct-contact bioassays against B. xylophilus, B. mucronatus and B. fraudulentus. The compounds showed high nematicidal activity against the species tested. The lowest values for half-maximal effective concentrations (EC50) were determined in B. xylophilus and B. fraudulentus, suggesting a higher sensibility to these compounds. Further bioassays will include compounds with different chain lengths and functional groups to explore the diversity in the activity of oxygen-containing aliphatic compounds for a more targeted sustainable-control strategy for the PWN.

Summary: To clarify the species composition and residency of Bursaphelenchus nematodes in the bodies of Monochamus saltuarius adults, we dissected 70 adults that had emerged from cut pine bolts set at two locations at the frontline of pine wilt disease spread in Nagano Prefecture, Japan. Fourth-stage dispersal juveniles of Bursaphelenchus nematodes were detected in 39 insects; PCR-based identification showed that the insects mainly harboured B. mucronatus , but could sometimes harbour B. xylophilus or both. Detailed dissection of 17 insects revealed that hundreds of Bursaphelenchus nematodes were often found in the reproductive organs of both sexes, including the inner parts such as the internal sac and the ejaculatory ducts in males. Thus, the residence of Bursaphelenchus nematodes in the reproductive organs of their vectors is not an accidental invasion but indicative of the phoretic nature of the nematodes. [ABSTRACT FROM AUTHOR]

Summary: Spermatogenesis in Paraphelenchus myceliophthorus is similar to that of the 'rhabditid' nematodes. The sperm development includes formation of complexes of fibrous bodies (FB) and membranous organelles (MO), which appear in spermatocytes; the complexes dissociate in the spermatids and the immature sperm contains separate FB and MO. Mature spermatozoa from the uterus are bipolar cells subdivided into anterior pseudopod and posterior main cell body containing a nucleus without a nuclear envelope, numerous mitochondria and peripheral MO opening to the exterior via pores. The mature spermatozoa in the uterus form chains of more than ten flattened cells. Spermatogenesis characters, such as nuclear satellites, presence of FB in immature spermatozoa, small knobbles of MO, specific dense inclusions, absence of filopodia and sperm conjugation, make spermatozoa of P. myceliophthorus morphologically specific for comparative analysis with representatives of Aphelenchoidea and other rhabditids. [ABSTRACT FROM AUTHOR]

Competitive displacement is not only the most extreme outcome of interspecific competition, but also an important strategy for invasive species to be successful invaders. Pinewood nematode, Bursaphelenchus xylophilus, the causal agent for pine wilt disease and global quarantine pest, usually displaces Bursaphelenchus mucronatus, a native sympatric sibling species, during its invasion process. Despite this prevalent outcome, the driving forces behind this displacement remain elusive. Ascarosides, an evolutionarily conserved family of nematode pheromones, are versatile in structure and function. We hypothesize these nematode pheromones play a role in species displacement. To investigate this hypothesis, we compared the ascarosides composition of B. xylophilus and B. mucronatus by LC–MS/MS followed by bioassays to test the responses of two nematodes to both crude and synthetic ascarosides. We found that asc-C5 (ascr#9) was the most abundant component and that there were no differences in pheromone composition between the two nematode species. B. xylophilus had faster growth rates under competition conditions. Furthermore, low concentrations of both crude and synthetic ascarosides [asc-C5, asc-C6 (ascr#12) and their mixture] enhanced female fecundity and body length growth in B. xylophilus but not in B. mucronatus. In contrast, body length of B. mucronatus was suppressed by a crude extract of its own ascarosides as well as by synthetic ascarosides (asc-C5, asc-C6 and their mixture). Our results strongly suggest that ascarosides play a role in the competitive displacement between two nematode species, which could explain the phenomena observed in B. xylophilus-invaded forests where B. mucronatus widely existed prior to B. xylophilus invasion. [ABSTRACT FROM AUTHOR]

We determined the mitochondrial gene sequence of Monochamus alternatus and three other mitogenomes of Lamiinae (Insect: Coleoptera: Cerambycidae) belonging to three genera (Aulaconotus, Apriona and Paraglenea) to enrich the mitochondrial genome database of Lamiinae and further explore the phylogenetic relationships within the subfamily. Phylogenetic trees of the Lamiinae were built using the Bayesian inference (BI) and maximum likelihood (ML) methods and the monophyly of Monochamus, Anoplophora, and Batocera genera was supported. Anoplophora chinensis, An. glabripennis and Aristobia reticulator were closely related, suggesting they may also be potential vectors for the transmission of the pine wood pathogenic nematode (Bursaphelenchus xylophilus) in addition to M. alternatus, a well-known vector of pine wilt disease. There is a special symbiotic relationship between M. alternatus and Bursaphelenchus xylophilus. As the native sympatric sibling species of B. xylophilus, B. mucronatus also has a specific relationship that is often overlooked. The analysis of mitochondrial gene expression aimed to explore the effect of B. mucronatus on the energy metabolism of the respiratory chain of M. alternatus adults. Using RT-qPCR, we determined and analyzed the expression of eight mitochondrial protein-coding genes (COI, COII, COIII, ND1, ND4, ND5, ATP6, and Cty b) between M. alternatus infected by B. mucronatus and M. alternatus without the nematode. Expression of all the eight mitochondrial genes were up-regulated, particularly the ND4 and ND5 gene, which were up-regulated by 4–5-fold (p < 0.01). Since longicorn beetles have immune responses to nematodes, we believe that their relationship should not be viewed as symbiotic, but classed as parasitic.

Pinewood nematode [Bursaphelenchus xylophilus (Steiner & Buhrer, 1934)] is considered a significant threat to European forests. Functional monitoring of its vector, the longhorn beetle Monochamus galloprovincialis (Olivier, 1795), represents an efficient method of pinewood nematode detection and needs to be adopted across European countries. Hence, the effectiveness of M. galloprovincialis trapping under different attractants and trap heights above the ground was investigated at four forest study sites in the Czech Republic in 2015–2016. Additionally, the effects of the calendar week and meteorological variables were assessed. In total, 24 Teflon-coated cross-vane traps (Crosstrap® traps) captured 1524 M. galloprovincialis individuals and 36,896 nontarget insects. Traps placed in the canopy (18–24 m above the ground) captured sevenfold more M. galloprovincialis individuals than those installed 2 m above the ground, and the Spanish attractant Galloprotect Pack® resulted in three times more catches than the Austrian attractant Gallopro Pinowit®. The highest maximum daily temperature during trap exposure was also an important factor, with most catches occurring above 27 °C. The results showed that Teflon-coated cross-vane traps with Galloprotect Pack® attractant are efficient tools for M. galloprovincialis monitoring in Central European forests when installed in the tree canopy. Additionally, we suggest using traps with larger sieve mesh sizes at the bottom to enable the escape of small nontarget taxa. During the 2-year study, the pinewood nematode was not found. However, 38 specimens of M. galloprovincialis were shown to be vectors of Bursaphelenchus mucronatus (Mamiya & Enda, 1979) dauers. [ABSTRACT FROM AUTHOR]

Bursaphelenchus xylophilus is the pathogen that causes pine wilt disease, which has greatly damaged forests and ecosystems in countries of East Asia and Europe. Bursaphelenchus mucronatus is closely related to B. xylophilus in morphology and host plant specificity. A long-running debate has existed regarding whether these two species can successfully produce hybrid offspring. In the present study, we performed in the laboratory, hybridization of two B. xylophilus nematode isolates from China and Japan and three B. mucronatus isolates from China, Japan and France. Nematode isolates of B. xylophilus were successfully crossed with B. mucronatus isolates, and the rate of hybridization was relatively high; however, some hybrid offspring died. Successful hybridization occurred between B. xylophilus and B. mucronatus isolates from China, and 22 generations of hybrids were produced. All F1 hybrids could be backcrossed with their parents and produce offspring. Variation in mucro length among the hybrid offspring and their parents was observed. The hybrid offspring and their parents were inoculated into 3-months-old black pine (Pinus tunbergii) seedlings. Weaker pathogenicity of hybrid offspring was observed compared with that of their parents, and significantly fewer offspring nematodes than parents were reisolated from pines. Therefore, the offspring of B. xylophilus and B. mucronatus may exist in the forest and could influence disease epidemics. [ABSTRACT FROM AUTHOR]

Quantitative reverse transcription polymerase chain reaction (RT-qPCR), a sensitive technique for gene expression analysis, depends on the stability of the reference genes used for data normalization under different experimental conditions. Bursaphelenchus mucronatus, a pine-parasitic nematode varying in virulence, is widely distributed in natural pine forests throughout the northern hemisphere, but has not been investigated with respect to the identification of reference genes suitable for the normalization of RT-qPCR data. In the present study, eight candidate reference genes were analyzed in B. mucronatus under different habitat conditions and at different developmental stages. The expression stability of these genes was assessed by geNorm, NormFinder, BestKeeper, delta Cq, and RefFinder algorithms. In general, our results identified encoding beta-tubulin as the most stable gene. Moreover, pairwise analysis showed that three reference genes were sufficient to normalize the gene expression data under each set of conditions, with genes encoding beta-tubulin, 18S ribosomal RNA and ubiquitin-conjugating enzyme being the most suitable reference genes for different habitat conditions, whereas genes encoding beta-tubulin, histone, and 18S ribosomal RNA exhibited the most stable expression at different developmental stages. Validation of the selected reference genes was performed by profiling the expression of the fatty acid- and retinol-binding protein gene in different habitats, and by profiling the expression of the arginine kinase gene at different developmental stages. This first systematic analysis for the selection of suitable reference genes for RT-qPCR in B. mucronatus will facilitate future functional analyses and deep mining of genetic resources in this nematode.

Quantitative reverse transcription polymerase chain reaction (RT-qPCR), a sensitive technique for gene expression analysis, depends on the stability of the reference genes used for data normalization under different experimental conditions. Bursaphelenchus mucronatus, a pine-parasitic nematode varying in virulence, is widely distributed in natural pine forests throughout the northern hemisphere, but has not been investigated with respect to the identification of reference genes suitable for the normalization of RT-qPCR data. In the present study, eight candidate reference genes were analyzed in B. mucronatus under different habitat conditions and at different developmental stages. The expression stability of these genes was assessed by geNorm, NormFinder, BestKeeper, delta Cq, and RefFinder algorithms. In general, our results identified encoding beta-tubulin as the most stable gene. Moreover, pairwise analysis showed that three reference genes were sufficient to normalize the gene expression data under each set of conditions, with genes encoding beta-tubulin, 18S ribosomal RNA and ubiquitin-conjugating enzyme being the most suitable reference genes for different habitat conditions, whereas genes encoding beta-tubulin, histone, and 18S ribosomal RNA exhibited the most stable expression at different developmental stages. Validation of the selected reference genes was performed by profiling the expression of the fatty acid- and retinol-binding protein gene in different habitats, and by profiling the expression of the arginine kinase gene at different developmental stages. This first systematic analysis for the selection of suitable reference genes for RT-qPCR in B. mucronatus will facilitate future functional analyses and deep mining of genetic resources in this nematode. [ABSTRACT FROM AUTHOR]

The internal transcribed spacer ( ITS) regions of rDNA have been routinely employed for identification and phylogenetic analysis of many nematode species. In this study, the intra- and interspecies ITS genetic diversity of Bursaphelenchus xylophilus and Bursaphelenchus mucronatus was evaluated. Ninety-one isolates of the two nematode species collected from 14 Chinese provinces, Japan and Korea were used for ITS- PCR and sequencing. An unweighted pair group cluster analysis dendrogram clustered them as two B. mucronatus and one B. xylophilus independent clades. Principal component analysis showed the phylogenetic relationship of the two nematode species more clearly; B. mucronatus isolates were separated into more than four groups, whereas B. xylophilus isolates still clustered into a group. The results of the Mantel test indicated the correlation of genetic distance matrices and geographic distance matrices was significant for both nematode species. The genetic differentiation coefficient ( Gst) and gene flow ( Nm) of B. mucronatus were 0.341 and 1.091, respectively, suggesting the importance of landscape heterogeneity and considerable obstacles for genetic exchange among B. mucronatus isolates in China. However, Gst and Nm of B. xylophilus were 0.188 and 2.151, respectively, very different compared to B. mucronatus. This could be owing to the short-term introduction of B. xylophilus into China and a rapid spread through anthropogenic pathways. Our work adds to the understanding of the genetic diversity and genetic relationship of the two pine-parasitic nematode species, and will aid in controlling them in the future. [ABSTRACT FROM AUTHOR]

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 acidand 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. [ABSTRACT FROM AUTHOR]

The real-time PCR-HRM analysis was developed for the detection and discrimination of the quarantine nematode Bursaphelenchus xylophilus and Bursaphelenchus mucronatus. A set of primers was designed to target the ITS region of rDNA. The results have demonstrated that this analysis is a valuable tool for differentiation of these both species. [ABSTRACT FROM AUTHOR]

A total of 33 pine trees with symptoms of decline were collected in Jeonnam Province, South Korea, and were examined for the presence of nematodes. About 20% of the trees sampled were positive with Bursaphelenchus species. All Bursaphelenchus species were found in recently dead or dying trees. Based on morphological observations, the nematode extracted from the declining pine trees was identified as B. mucronatus. The highly pathogenic pine wood nematode B. xylophilus was not found in any pine trees sampled. B. mucronatus was easily reared on fungus Botrytis cinerea. Twenty one fungal isolates were isolated from dead trees, fallen twigs, and healthy pine trees. The fungal isolates belonged to Trichoderma genus and were dominant in the wood of partially declining pines. The blue-stain fungi transmitted by the Monochamus beetle were not detected. The B. mucronatus population decreased markedly on Auxarthron reticulatum DY-2 isolated from soils. The number of nematodes also reduced on Verticillium saksenae A-1, a nematophagous fungus, and Beauveria bassiana, an entomopathogenic fungus. This observation suggested the fungal production of nematicidal activity against B. mucronatus. When the fungal culture filtrates were also used for nematicidal activity on B. mucronatus, the culture filtrates of A-1, DY-2 and B. bassiana showed over 50% mortality within 48 h exposure. The fungi BC4, BC5 and BC6 isolated from declining pine trees inhibited the reproduction of B. mucronatus, and their culture filtrates also expressed nematicidal activity, indicating a possible interaction between the fungi in pine trees and nematodes at microhabitat level. [ABSTRACT FROM AUTHOR]

BACKGROUND Bursaphelenchus xylophilus, the causal agent of pine wilt disease, severely damages pine forests, and Bursaphelenchus mucronatus, a species related to B. xylophilus, is weakly pathogenic. Because the use of synthetic pesticides to control pine wilt disease has raised concerns for human and environmental health, a search for useful botanically based compounds is needed. RESULTS The ethyl acetate layer from an ethanol extract of Stellera chamaejasme L. roots showed significant nematicidal activity against B. xylophilus and B. mucronatus, with LC50 values of 169.7 and 37.7 µg mL−1, respectively, at 72 h. Further purification of the active layer yielded eight effective nematicidal compounds, which were identified by mass and nuclear magnetic resonance spectral data. Among the eight bioactive compounds, chamaejasmenin C induced the highest mortality against B. xylophilus, with an LC50 value of 2.7 µM at 72 h, and chamaechromone exhibited strong nematicidal activity against B. mucronatus, with an LC50 value of 0.003 µM at 72 h. CONCLUSION The nematicidal metabolites from S. chamaejasme roots could be used as lead compounds for developing botanically based nematicides for the management of pine wilt disease. © 2013 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Abstract: We determined the complete mitochondrial genome sequences for Bursaphelenchus mucronatus, one species of pinewood nematode. The genome is a circular-DNA molecule of 14,583bp (195bp smaller than its congener Bursaphelenchus xylophilus) and contains 12 protein-coding genes (lacking atp8), 22 tRNA genes, and 2 rRNA genes encoded in the same direction, consistent with most other nematodes. Based on sequence comparison of mtDNA genomes, we developed a PCR-based molecular assay to differentiate B. xylophilus (highly pathogenic) and B. mucronatus (relatively less virulent) using species-specific primers. The molecular identification system employs multiplex-PCR and is very effective and reliable for discriminating these Bursaphelenchus species, which are economically important, but difficult to distinguish based on morphology. The comparison of the mitochondrial genomes and molecular identification system of the two species of Bursaphelenchus spp. should provide a rich source of genetic information to support the effective control and management (quarantine) of the pine wilt disease caused by pinewood nematodes. [Copyright &y& Elsevier]

Abstract: Bursaphelenchus xylophilus and Bursaphelenchus mucronatus are migratory endoparasitic nematodes that live in pine trees. To gain insight into their molecular similarities and differences, transcriptomes of the two nematodes were analysed. A total of 23,765 and 21,782 contigs (>300bp) were obtained from B. xylophilus and B. mucronatus, respectively. More than 80% of the contigs could map to each other''s transcriptome reciprocally. A total of 23,467 and 21,370 Open Reading Frames were predicted, respectively. Besides those known parasitism-related proteins, six new venom allergen-like proteins (VAPs) were found, which were not homologous to known VAPs. Enzymes involved in xenobiotic biodegradation were abundant in the two transcriptomes based on KEGG functional annotation. Metabolism of xenobiotics by cytochrome P450 comprised the main detoxification pathways. The mRNA expression levels of detoxification genes in nematodes living in the host were higher than those in nematodes feeding on fungus. However, there were fewer enzymes involved in the α-pinene degradation. Our results indicate that the two pinewood nematodes have evolved similar molecular mechanisms to adapt to life on pine hosts. [Copyright &y& Elsevier]

The vulval pattern of six species of the genus Bursaphelenchus (B. abruptus, B. conicaudatus, B. fraudulentus, B. luxuriosae, B. mucronatus and B. xylophilus) was studied using scanning electron microscopy. A terminology for the vulval region structures observed is proposed herein and illustrated by micrographs and line drawings. It was shown that, of the studied species, only B. mucronatus and B. xylophilus share an identical morphology of the vulval region, all other species differing significantly from each other and from both B. mucronatus and B. xylophilus. This study indicates the diagnostic potential for variation in vulval morphology within Bursaphelenchus and it is recommended that such features are recorded in all future descriptions. [ABSTRACT FROM AUTHOR]

Bursaphelenchus xylophilus is the pathogen associated with pine wilt disease (PWD), an infectious disease of pine trees transmitted by cerambycid beetles of the genus Monochamus. Bursaphelenchus xylophilus is an invasive species, whilst B. mucronatus is a native congener and non-pathogenic to pine trees in Japan. To provide experiment evidence of the biotic and/or abiotic resistance to PWD expansion in a cool area of Japan, we inoculated B. xylophilus into healthy pine trees in a Japanese red pine (Pinus densiflora) stand with the indigenous pine tree-B. mucronatus-insect vector system before invasion of B. xylophilus in 1993 and 1994. Extremely cool air temperature with high precipitation and extremely high air temperature with low precipitation were observed in the summers of the 2 years of inoculation, respectively. The cool summer induced a low incidence of PWD and delayed disease development, resulting in the replacement of B. xylophilus by B. mucronatus within diseased trees and the emergence of Monochamus saltuarius beetles carrying B. mucronatus from the trees 2 years after the inoculation. The hot summer induced disease development in trees in the year of inoculation, but such diseased trees did not become infection sources because of the lack of M. alternatus, whose oviposition was synchronised with the period of disease development in pine trees. This study indicated that biotic factors were important in the inhibitory mechanism in a pine forest against the spread of PWD in the stand. Relevant biotic factors were the lack of M. alternatus and the presence of M. saltuarius carrying B. mucronatus. [ABSTRACT FROM AUTHOR]

Bursaphelenchus mucronatus Mamiya et Enda has been recovered for the first time from adults of the cerambycid beetle, Monochamus urussovi (Fischer), in Hokkaido, Japan. The nematode was also recovered from the inner bark of Picea jezoensis (Siebold et Zuccarini) Carrière and Abies sachalinensis (Fr Schmidt) Masters infested with M. urussovi larvae. PCR–RFLP analysis indicated that B. mucronatus in Hokkaido is the European type. [ABSTRACT FROM AUTHOR]

A real-time PCR polymerase chain reaction (real-time PCR) method was developed to detect and differentiate Bursaphelenchus xylophilus (pine wood nematode, PWN) from other wood-inhabiting nematode species. A primer set and a specific Taqman® fluorescent probe were designed to amplify target B. xylophilus heat shock protein 70 sequences. After optimization, this real-time PCR assay was shown to be highly specific and sensitive, detecting at least 0.005 ng of B. xylophilus genomic DNA, as well as DNA extracted from single nematodes. The practical application of this real-time PCR diagnostic method for the detection of B. xylophilus from actual wood samples of lodgepole pine (Pinus contorta, Dougl. var. latifolia) trees containing a heterogeneous population of nematodes, rather than pure cultures or individual nematodes, is demonstrated. This method works well in the presence of potential inhibitors associated with wood after Baermann extraction and thus eliminates the need to produce pure nematode samples through further culturing and/or isolation of nematodes with a high-power microscope. [ABSTRACT FROM AUTHOR]

A molecular diagnostic method has been designed for the detection and identification of Bursaphelenchus xylophilus. Heat shock protein 70 gene sequences from B. xylophilus and the closely related B. mucronatus were compared and used to design primers Bx701F and Bx701R which amplify a 171 base pair fragment from B. xylophilus by PCR. As a control, primers Bm701F and Bm701R were designed which specifically amplify a 168 base pair fragment from B. mucronatus. After optimisation, B. xylophilus primers were shown to be highly sensitive and could easily detect 23 target copies, or less than one nematode. Species-specific detection of B. xylophilus was carried out directly from concentrated Baermann funnel extracts using wood samples from lodgepole pine (Pinus contorta var. latifolia) trees from British Columbia, Canada, containing an unknown nematode population, thus bypassing the need for culturing or recovering the nematode before analysis. [ABSTRACT FROM AUTHOR]

Three bacterial strains Njh, Njt and Njw, have been isolated with a high frequency from the xylem of the wilted black pine ( Pinus thuntergii) and from the surface of the pine wood nematodes ( Bursaphelenchus xylophilus). These bacteria have been identified as Pseudomonas fluorescens biotype I, P. fluorescens biotype II and a species of the genus Pantoea respectively. To determine pathogenicity, callus and aseptic black pine seedlings were inoculated as follows: aseptic nematodes only (each of B. xylophilus and B. mucronatus); bacterium only (each of bacterial strain Njh, Njt, Njw) and aseptic nematodes plus bacterium (each of B. xylophilus and B. mucronatus in combination with each of Njh, Njt, Njw). The results showed that inoculation with either aseptic B. xylophilus or B. mucronatus did not lead to browning of the callus or wilt of aseptic black pine seedlings. But those inoculated with aseptic nematodes plus any one of Njh, Njt, Njw showed some wilting or browning symptoms. The combination of nematodes plus either Njh or Njt caused severe symptoms but the combination of nematodes plus Njw had fewer symptoms. The bacteria carried by pine wood nematodes played an important role in pathogenicity. In addition, bacteria were cultured in liquid media through shaking. The filtered liquid was directly applied to the callus and the ability of each kind of bacterium to induce browning was determined. The results showed that Njh and Njt caused severe browning but Njw did not. It is suspected that this is due to wilt related toxins in the bacterial culture fluid. Therefore, it's suggested that the disease was caused by co-infection of both pine wood nematodes and bacteria and possible toxic effect of bacteria. [ABSTRACT FROM AUTHOR]

Differences among four isolates of Bursaphelenchus xylophilus and one of B. mucronatus in vulnerability to the inhibitory effect of Pinus densiflora on nematode dispersal were investigated by inoculating boiled and living branch sections 5 cm long. The intrinsic dispersal ability and vulnerability to the inhibitory effect of living branches differed among isolates. Additionally, the degree of branch inhibition of nematode dispersal was examined by inoculating the five isolates on 2.5 and 5.0 cm long, P. densiflora living branch sections. An increase in section length induced an 80% decrease in the number of nematodes of all isolates passing through the branch. The extent to which passing nematode number decreased as branch section length increased can be used to express susceptibility of pine species to pine wilt disease. Virulence had no relation to intrinsic and realised dispersal rates in B. xylophilus. [ABSTRACT FROM AUTHOR]

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.

Simple Summary: The longicorn beetle, Monochamus alternatus, is a major vector for the transmission of pine wilt disease, which is caused by a nematode pathogen, Bursaphelenchus xylophilus (or also possibly by B. mucronatus) that is spread by the beetle as it feeds on pine trees. In this study, the mitochondrial genome sequences of four longicorn species (Coleoptera: Cerambycidae: Lamiinae) were determined to further elaborate the phylogenetic relationships of Lamiinae. RT-qPCR was also used to assess the expression of eight mitochondrial protein-coding genes in M. alternatus when carrying B. mucronatus or not, so as to explore the relationship between these two species. The results showed that expression of mitochondria-encoded genes was elevated in M. alternatus beetles that were infected with B. mucronatus, suggesting that B. mucronatus putatively activates an immune response, which significantly affects the metabolic processes of M. alternatus. These results are of significance for further understanding the phylogenetic relationships of longicorn beetles and controlling the spread of pine wilt disease. We determined the mitochondrial gene sequence of Monochamus alternatus and three other mitogenomes of Lamiinae (Insect: Coleoptera: Cerambycidae) belonging to three genera (Aulaconotus, Apriona and Paraglenea) to enrich the mitochondrial genome database of Lamiinae and further explore the phylogenetic relationships within the subfamily. Phylogenetic trees of the Lamiinae were built using the Bayesian inference (BI) and maximum likelihood (ML) methods and the monophyly of Monochamus, Anoplophora, and Batocera genera was supported. Anoplophora chinensis, An. glabripennis and Aristobia reticulator were closely related, suggesting they may also be potential vectors for the transmission of the pine wood pathogenic nematode (Bursaphelenchus xylophilus) in addition to M. alternatus, a well-known vector of pine wilt disease. There is a special symbiotic relationship between M. alternatus and Bursaphelenchus xylophilus. As the native sympatric sibling species of B. xylophilus, B. mucronatus also has a specific relationship that is often overlooked. The analysis of mitochondrial gene expression aimed to explore the effect of B. mucronatus on the energy metabolism of the respiratory chain of M. alternatus adults. Using RT-qPCR, we determined and analyzed the expression of eight mitochondrial protein-coding genes (COI, COII, COIII, ND1, ND4, ND5, ATP6, and Cty b) between M. alternatus infected by B. mucronatus and M. alternatus without the nematode. Expression of all the eight mitochondrial genes were up-regulated, particularly the ND4 and ND5 gene, which were up-regulated by 4–5-fold (p < 0.01). Since longicorn beetles have immune responses to nematodes, we believe that their relationship should not be viewed as symbiotic, but classed as parasitic. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]