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

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

The use of real-time polymerase chain reaction with high resolution melting (real-time PCR-HRM) analysis for the detection and discrimination of nematodes Bursaphelenchus xylophilus and Bursaphelenchus mucronatus.

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.

Identifying Virulence-Associated Genes Using Transcriptomic and Proteomic Association Analyses of the Plant Parasitic Nematode Bursaphelenchus mucronatus.

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 ß-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.

Mitogenome Analysis of Four Lamiinae Species (Coleoptera: Cerambycidae) and Gene Expression Responses by Monochamus alternatus When Infected with the Parasitic Nematode, Bursaphelenchus mucronatus.

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.

A Reference Genome of Bursaphelenchus mucronatus Provides New Resources for Revealing Its Displacement by Pinewood Nematode.

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.

Selection of Reliable Reference Genes for RT-qPCR Analysis of Bursaphelenchus mucronatus Gene Expression From Different Habitats and Developmental Stages.

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.

Temperature-mediated Behavioral Relationships in Bursaphelenchus xylophilus, B. mucronatus, and Their Hybrids.

The influence of temperature on reproduction and movement was examined for seven geographic isolates of Bursaphelenchus xylophilus, three of B. mucronatus, and two of their interspecific hybrids. All nematode isolates tended to be more active and fecund the higher the temperature, with the isolates of B. xylophilus reaching a reproductive peak at higher temperatures than isolates of B. mucronatus. Most isolates of B. xylophilus and B. mucronatus did not produce significantly more progeny at higher male-to-female ratios. The interspecific hybrids appear to possess temperature-related characteristics of either B. xylophilus or both of the parents.

Cryopreservation of the Pinewood Nematode, Bursaphelenchus spp.

Populations of three isolates of Bursaphelenchus xylophilus, the pinewood nematode, and one of B. mucronatus were treated with three cryoprotectants at -70 C for 24 hours followed by deep freezing at -180 C in liquid nitrogen for different periods of time. A solution of 15% glycerol, 35% buffer S, and 50% M9, or 1% aqueous solution of dimethylsulfoxide (DMSO), or a mixture of 60% M9 and 40% S buffer were used as cryoprotectants. A significantly larger number of juveniles than adults survived deep freezing. Significantly more nematodes were motile after cryopreservation in the 15% glycerol-S-M9 soludon than in the M9-S buffer solution or the DMSO aqueous solution. When cryopreserved nematodes that had been treated with glycerol solution were plated onto Botrytis cinerea, they reproduced rapidly over several generations. Cryopreserved nematodes were as pathogenic as untreated nematodes to Scots pines.

Inhibitory Effect of Bursaphelenchus mucronatus (Nematoda: Aphelenchoididae) on B. xylophilus Boarding Adult Monochamus alternatus (Coleoptera: Cerambycidae).

Inhibitory effects of Bursaphelenchus mucronatus on the number of B. xylophilus carried by an adult Monochamus alternatus were investigated using artificial pupal chambers. When pupal chambers were infested with either B. xylophilus or B. mucronatus, the load of B. xylophilus onto the beetle was greater (P < 0.001) than that of B. mucronatus. However, within the pupal chamber there was no difference in the abundance of the third-stage dispersal juveniles, which would molt to the fourth-stage dispersal juveniles to board beetles. The nematode load on beetles that emerged from pupal chambers infested with both Bursaphelenchus species was smaller (P = 0.015) than that of beetles with B. xylophilus alone but greater (P < 0.001) than that of beetles with B. mucronatus alone, suggesting an inhibitory effect of B. mucronatus. As a result of this study, the rate of inhibition of B. mucronatus on molting of third-stage dispersal juveniles of B. xylophilus to fourth-stage dispersal juveniles was 0.65, which resulted in great inhibition on boarding beetles at a rate of 0.7.

Genomic Differences among Pathotypes of Bursaphelenchus xylophilus.

Total genomic DNA from Bursaphelenchus xylophilus pathotypes MPSy-1 and VPSt-1 and from B. mucronatus was digested with restriction endonucleases. DNA fragments were electrophoretically separated, Southern blotted to nitrocellulose, and hybridized to genomic DNA from one of the isolates. The resulting hybridization patterns indicate genomic differences in repetitive DNA sequences among these populations. Greatest differences were seen between B. xylophilus and B. mucronatus, but genomic differences were also apparent between B. xylophilus pathotypes MPSy-1 and VPSt-1 and between a population from P. nigra in New Jersey and a population of a mucronate form from Abies balsamea in Quebec, Canada.

Change in Water Status of Pinus thunbergii Parl. Inoculated with Species of Bursaphelenchus.

Maximum and minimum xylem pressure potentials of needles were measured to evaluate water status of Pinus thunbergii Parl. after inoculation with the virulent or avirulent populations of Bursaphelenchus xylophilus or B. mucronatus. In virulent B. xylophilus-inoculated pines, the water status changed abruptly and needle chlorosis occurred by day 29 after inoculation. Similar changes were not seen in B. mucronatus-inoculated and uninoculated control pines. Oleoresin flow ceased in virulent B. xylophilus-inoculated pines. Avirulent B. xylophilus-inoculated pines responded very little to nematode invasion by a slight decrease in oleoresin flow. Oleoresin flow did not vary in B. mucronatus-inoculated and uninoculated control pines. A decrease in soil water potential below field capacity seemed to accelerate the development of pine wilt disease.

Characterization of a Nonparasitic Isolate of Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus isolate MPSy-1av was subcultured from pathotype MPSy-1. MPSy-1av is nonparasitic and does not establish in Pinus sylvestris, P. strobus, P. nigra, or P. taeda. This isolate produces ethanol as an end product of carbohydrate metabolism, whereas its parent pathotype, MPSy-1, does not. Alcohol dehydrogenase activity was easily detectable in homogenates of MPSy-1av but barely detectable in some homogenates of MPSy-1. Genomic differences were seen between MPSy-1 and M PSy-1av by restriction endonuclease analysis of total nematode DNA, and hybridization of DNA fragments to the alcohol dehydrogenase gene from Drosophila.

Nematicidal metabolites from roots of Stellera chamaejasme against Bursaphelenchus xylophilus and Bursaphelenchus mucronatus.

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.