Competitive displacement of the native species <Emphasis Type="Italic">Bursaphelenchus mucronatus</Emphasis> by an alien species <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis> (Nematoda: Aphelenchida: Aphelenchoididae): a case of successful invasion

The presence of alien invasive species has serious negative impact on endemic biodiversity, especially on native species that occupy the same niche in the ecosystem. To study the influence of the alien invasive species Bursaphelenchus xylophilus on its native sister species B. mucronatus, the two nematode species were mix-cultured in a fungal mat and mix-inoculated into a susceptible host. By comparing the propagation parameters of both species under competitive and noncompetitive conditions it was shown that the propagation level of B. xylophilus was clearly higher than that of B. mucronatus under laboratory culture. Furthermore, the propagation capacity of B. xylophilus under competitive conditions was much higher than that under noncompetitive conditions, both in laboratory culture and with host inoculation. Bursaphelenchus xylophilus also excluded B. mucronatus when the two species were cultured as a mixture for a longer time. The relative abundance ratios of the two species in natural pinewoods were also determined by random sampling of dying pine trees from regions with different invasion histories. It was noted that with an increase in invasion years the distribution frequency of B. xylophilus increased while that of B. mucronatus decreased. Experimental tests verified our hypothesis that because of its high fecundity and strong competitive ability, the invasive species B. xylophilus out-competed the native species B. mucronatus and displaced it in natural ecosystems. The successful invasion of B. xylophilus is attributed to competitive displacement, which may be one of the ecological invasive mechanisms.     

Comparison of complete mitochondrial genomes of pine wilt nematode Bursaphelenchus xylophilus and Bursaphelenchus mucronatus (Nematoda: Aphelenchoidea) and development of a molecular tool for species identification

We determined the complete mitochondrial genome sequences for Bursaphelenchus mucronatus, one species of pinewood nematode. The genome is a circular-DNA molecule of 14,583 bp (195 bp 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.     

First Insights into the Genetic Diversity of the Pinewood Nematode in Its Native Area Using New Polymorphic Microsatellite Loci

The pinewood nematode, Bursaphelenchus xylophilus, native to North America, is the causative agent of pine wilt disease and among the most important invasive forest pests in the East-Asian countries, such as Japan and China. Since 1999, it has been found in Europe in the Iberian Peninsula, where it also causes significant damage. In a previous study, 94 pairs of microsatellite primers have been identified in silico in the pinewood nematode genome. In the present study, specific PCR amplifications and polymorphism tests to validate these loci were performed and 17 microsatellite loci that were suitable for routine analysis of B. xylophilus genetic diversity were selected. The polymorphism of these markers was evaluated on nematodes from four field origins and one laboratory collection strain, all originate from the native area. The number of alleles and the expected heterozygosity varied between 2 and 11 and between 0.039 and 0.777, respectively. First insights into the population genetic structure of B. xylophilus were obtained using clustering and multivariate methods on the genotypes obtained from the field samples. The results showed that the pinewood nematode genetic diversity is spatially structured at the scale of the pine tree and probably at larger scales. The role of dispersal by the insect vector versus human activities in shaping this structure is discussed.     

Occurrence of Bursaphelenchus mucronatus (Nematoda: Aphelenchoididae) and the microhabitat distribution of fungi in declining pine trees in a locale in Korea

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.     

New Insights into the Phylogeny and Worldwide Dispersion of Two Closely Related Nematode Species,Bursaphelenchus xylophilus and Bursaphelenchus mucronatus

The pinewood nematode, Bursaphelenchus xylophilus, is one of the greatest threats to coniferous forests worldwide, causing severe ecological damage and economic loss. The biology of B. xylophilus is similar to that of its closest relative, B. mucronatus, as both species share food resources and insect vectors, and have very similar morphological characteristics, although little pathogenicity to conifers has been associated with B. mucronatus. Using both nuclear and mitochondrial DNA markers, we show that B. xylophilus and B. mucronatus form distinct phylogenetic groups with contrasting phylogeographic patterns. B. xylophilus presents lower levels of intraspecific diversity than B. mucronatus, as expected for a species that evolved relatively recently through geographical or reproductive isolation. Genetic diversity was particularly low in recently colonised areas, such as in southwestern Europe. By contrast, B. mucronatus displays high levels of genetic diversity and two well-differentiated clades in both mitochondrial and nuclear DNA phylogenies. The lack of correlation between genetic and geographic distances in B. mucronatus suggests intense gene flow among distant regions, a phenomenon that may have remained unnoticed due to the reduced pathogenicity of the species. Overall, our findings suggest that B. xylophilus and B. mucronatus have different demographic histories despite their morphological resemblance and ecological overlap. These results suggest that Bursaphelenchus species are a valuable model for understanding the dispersion of invasive species and the risks posed to native biodiversity and ecosystems.     

Interactions between pinewood nematodes and the fungal community of pine trees

The pinewood nematode Bursaphelenchus xylophilus, is the pathogenic agent of pine wilt disease and a globally notable pine pest. Despite being a plant pathogen, B. xylophilus has a mycophagous phase during its life cycle. We assessed the capacity for polyphagy of mycetophagous pinewood nematodes, testing which of the common species of fungi in pine trees provide better food and higher population growth rates. B. xylophilus performed particularly well on airborne fungi, namely the endophytes Botrytis cinerea and Cladosporium herbarum, and the pathogens Sirococcus conigenus and Sphaeropsis sapinea. Surprisingly, growth performance was not as good on the blue stain species (Ophiostoma spp. and Leptographium spp.) which are considered natural associates of B. xylophilus in the wild. Most of the fungi nonetheless permitted positive population growth of B. xylophilus, which is polyphagous and capable of feeding on numerous fungal species with diverse ecological niches.     

Bacteria associated with the pinewood nematode <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis> collected in Portugal

In this study, we report on the bacterial community associated with the pinewood nematode Bursaphelenchus xylophilus from symptomatic pine wilted trees, as well as from long-term preserved B. xylophilus laboratory collection specimens, emphasizing the close bacteria–nematode associations that may contribute to pine wilt disease development.     

Phenotypic plasticity of reproductive traits in response to food availability in invasive and native species of nematode

Invasive species cause severe ecological and economic damage; however, the mechanisms underlying their successful invasion often remain elusive. In the case of Bursaphelenchus xylophilus, a global quarantine pest which invaded Asia and Europe, it has been suggested that this species possesses highly competitive abilities, which promotes its establishment and rapid spread. To explore biological traits that may explain its highly competitive abilities, we focused on expression of phenotypic plasticity in response to the food conditions experienced by the females during their development as juveniles in the invasive species B. xylophilus and native species Bursaphelenchus mucronatus. We report an unexpected significant difference of phenotypic trade-off between egg number and egg size in the invasive species B. xylophilus and native species B. mucronatus. This leads to superior propagation ability of invasive species, under high and low food conditions in culture. These effects reflect adaptive optimal resource allocation where more eggs are produced in favorable environments to enhance population viability. Furthermore, we show that B. xylophilus eggs hatched earlier than B. mucronatus when their parents experienced high food availability. Thus, this study revealed, for the first time, phenotypic plasticity of reproductive traits in B. xylophilus which empowers the species a competitive advantage relative to their native counterpart B. mucronatus when they are under different range of food availability. These results are a step towards answering the vital question of how an exotic invasive species exclude a native species from its original niche.     

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.     

Geographical variation in seasonality and life history of pine sawyer beetles Monochamus spp: its relationship with phoresy by the pinewood nematode Bursaphelenchus xylophilus

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Evolutionary change in a pine wilt system following the invasion of Japan by the pinewood nematode, <Emphasis Type="Italic">Bursaphelenchus</Emphasis><Emphasis Type="Italic">xylophilus</Emphasis>

Pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causative agent of pine wilt disease (PWD) of pine trees and is transmitted by cerambycid beetles belonging to the genus Monochamus. PWN is believed to have been introduced into Japan from North America at the beginning of the 20th century. In this article, we first provide an outline of the PWD system and the range expansion of PWN in Japan and then review the literature, focusing on the virulence of PWN. Virulence is a heritable trait in PWN, with high virulence being closely related to a high rate of reproduction and within-tree dispersal. When two PWN isolates with different virulence levels are inoculated into pine seedlings, the more virulent nematodes always dominate in dead seedlings. In a laboratory setting, many more virulent nematodes board the insect vectors than avirulent ones. The age at which vectors transmit the most abundant PWNs to pine twigs changes during the course of a PWD epidemic. However, the relation between virulence and transmission of PWN remains as yet relatively unknown. Such information would enable ecologists to predict the evolution of the PWD system. In this review we also compare ecological traits between the PWN and the avirulent congener, B. mucronatus.     

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.     

Pinewood Nematode Species Complex: Interbreeding Potential and Chromosome Number

Interbreeding potential, chromosome number, and host range were compared among several isolates and species of Bursaphelenchus from diverse geographic areas. Some isolates from North America, Japan, and France had a wide-ranging interbreeding potential, whereas others were restricted in their potential to hybridize with other isolates. Although interbreeding occurred in the laboratory between some "M" and "R" forms of B. xylophilus, interbreeding of B. xylophilus and B. mucronatus was rare. The hybrids had the pathogenicity of the parent with the broader host range. This fact suggests that virulence may be inherited as a dominant character or that increased virulence may have resulted from differences in hybrid vigor. The haploid chromosome number of the different isolates separated the isolates into three groups and distinguished B. xylophilus from B. mucronatus. The findings suggest that the pinewood nematode species complex consists of sibling species that have evolved by reproductive isolation, that the French isolate is a new species, and that B. xylophilus and B. mucronatus have evolved from a common ancestor.     

Pathogenicity of Bursaphelenchus xylophilus on Pines in Minnesota and Wisconsin

The pinewood nematode, Bursaphelenchus xylophilus, was inoculated into established native jack and red pines (Pinus banksiana and P. resinosa) and exotic Austrian pine (P. nigra) in Minnesota and Wisconsin forests during summer 1981. The nematode isolates did not kill established nonstressed pine trees growing in the forest. However, the same nematode isolates killed pine seedlings under greenhouse conditions. Girdling the main stem of some trees to induce stress resulted in the death of the majority of inoculated and noninoculated branches of Austrian and jack pines, but no branch death was observed on red pine. Greater numbers of nematodes were extracted from branches of inoculated, girdled trees than from nongirdled trees. The mean number of nematodes extracted from branches of inoculated, nongirdled trees was 0.3 - 14 nematodes per gram of wood.     

Nematicidal Activity of Plant Essential Oils and Components From Ajowan (Trachyspermum ammi), Allspice (Pimenta dioica) and Litsea (Litsea cubeba) Essential Oils Against Pine Wood Nematode (Bursaphelenchus Xylophilus)

Commercial plant essential oils from 26 plant species were tested for their nematicidal activities against the pinewood nematode, Bursaphelenchus xylophilus. Good nematicidal activity against B. xylophilus was achieved with essential oils of ajowan (Trachyspermum ammi), allspice (Pimenta dioica) and litsea (Litsea cubeba). Analysis by gas chromatography-mass spectrometry led to identification of 12, 6 and 16 major compounds from ajowan, allspice and litsea oils, respectively. These compounds from three plant essential oils were tested individually for their nematicidal activities against the pinewood nematode. LC₅₀ values of geranial, isoeugenol, methyl isoeugenol, eugenol, methyl eugenol and neral against pine wood nematodes were 0.120, 0.200, 0.210, 0.480, 0.517 and 0.525 mg/ml, respectively. The essential oils described herein merit further study as potential nematicides against the pinewood nematode.     

Bacterial Diversity and Community Structure in the Pine Wood Nematode Bursaphelenchus xylophilus and B. mucronatus with Different Virulence by High-Throughput Sequencing of the 16S rDNA

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

Transmission of Bursaphelenchus xylophilus between Monochamus alternatus and Monochamus saltuarius through interspecific mating behaviour

Cerambycid adults of the genus Monochamus transmit the pine wood nematode (Bursaphelenchus xylophilus [Steiner et Buhrer] Nickle), the causative agent of pine wilt disease. To confirm the transmission of B. xylophilus between vector species, nematode‐infected Monochamus alternatus Hope and nematode‐free Monochamus saltuarius (Gebler) adults were paired and their behaviour was observed for 107–220 min. In three pairs that exhibited mounting without copulation, nematode transmission occurred, whereas it was not found in another pair without mountings. The effect of nematode transmission between different vector species on the invasion biology of B. xylophilus is discussed.     

Transmission of the Pinewood Nematode, Bursaphelenchus xylophilus,to Slash Pine Trees and Log Bolts by a Cerambycid Beetle,Monochamus titillator, in Florida

Field-collected adults of the southern pine sawyer, Monochamus titillator (F.) (Coleoptera: Cerambycidae), naturally infested with fourth-stage juveniles (dauerlarvae) of the pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer, 1934) Nickle, 1970, were maturation fed on excised shoots of typical slash pine, Pinus elliottii Engelm. var elliottii, for 21 days. During August 1981, a male and female adult beetle were held in a sleeve cage placed on the terminal of a side branch of each of seven replicate, healthy 10-year-old slash pine trees. All seven branch terminals showed evidence of beetle feeding on the bark after 1 week, and pinewood nematodes were present in wood samples taken near these feeding sites. Four of the seven trees showed wilt symptoms in 4-6 weeks and died about 9 weeks after beetle feeding. Pinewood nematodes were recovered from the roots and trunks of the dead trees. Each of seven replicate slash pine log bolts was enclosed in a jar with a pair of the same beetles used in the sleeve cages. After 1 week, wood underlying beetle oviposition sites in the bark of all replicate log bolts was infested with the pinewood nematode.