Takuya Aikawa

A family of glycosyl hydrolase family 45 cellulases from the pine wood nematode Bursaphelenchus xylophilus

We have characterized a family of GHF45 cellulases from the pine wood nematode Bursaphelenchus xylophilus. The absence of such genes from other nematodes and their similarity to fungal genes suggests that they may have been acquired by horizontal gene transfer (HGT) from fungi. The cell wall degrading enzymes of other plant parasitic nematodes may have been acquired by HGT from bacteria. B. xylophilus is not directly related to other plant parasites and our data therefore suggest that horizontal transfer of cell wall degrading enzymes has played a key role in evolution of plant parasitism by nematodes on more than one occasion.

A Rapid and Precise Diagnostic Method for Detecting the Pinewood Nematode Bursaphelenchus xylophilus by Loop-Mediated Isothermal Amplification

ABSTRACT Bursaphelenchus xylophilus is the causal agent of pine wilt disease, which is a major forest disease in Japan, Korea, China, Taiwan, and Portugal. A diagnostic method which is rapid, precise, and simple could greatly help the proper management of this disease. Here, we present a novel detection method using loop-mediated isothermal amplification (LAMP) targeting the internal transcribed spacer region of ribosomal DNA of the nematode. Specificity of the primers and LAMP was confirmed using DNA from various nematode species related to B. xylophilus. Our experimental results suggest that LAMP can detect B. xylophilus faster and with higher sensitivity than the traditional diagnostic method. Moreover, because it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions.

Bursaphelenchus firmae n. sp. (Nematoda: Aphelenchoididae), isolated from Monochamus grandis Waterhouse that emerged from dead firs, Abies firma Sieb. et Zucc.

Bursaphelenchus firmae n. sp. is described. This new species was isolated during a field survey of longhorn beetle-associated nematodes. The fourth-stage dispersal (dauer) juveniles of the new species were recovered from dissected bodies (tracheal system) of Monochamus grandis, which emerged from dead logs of Japanese fir, Abies firma, collected from Hachioji, Tokyo, Japan. The new species is mid-sized for the genus, with females 603-828 μm and males 530-698 μm long. Four lateral lines occur on the body surface and seven genital papillae are found in males (P1 ventral single papilla and P2-P4 pairs). A long and arcuate spicule forms a trapezium in lateral view and a rather large, sub-squared, bursal flap and a vulva with conspicuous vulval flap are present. The female tail is smoothly tapered and possesses a conspicuous and blunt mucro. Based upon its diagnostic morphological characters, the new species belongs to the B. xylophilus group of the genus, and is closely related to B. fraudulentus, B. mucronatus, B. doui, B. macromucronatus and B. populi. It is distinguished from these five species by the morphology of the male bursal flap and the female mucro and several morphometric values. Molecular phylogenetic analyses inferred from D2/D3 LSU suggest that the new species is close to B. mucronatus and B. xylophilus, i.e., these three species form a well supported monophyletic clade within the genus. Although the new species has a weak pathogenicity to pine trees, it does not seem to be a severe risk to native pine forests.

Description of Bursaphelenchus kiyoharai n. sp. (Tylenchina: Aphelenchoididae) with remarks on the taxonomic framework of the Parasitaphelenchinae Rühm, 1956 and Aphelenchoidinae Fuchs, 1937

A Bursaphelenchus species was isolated from an ambrosia beetle, Xyleborus seriatus, during a biodiversity survey of entomophilic nematodes in a conserved forest in the Shirakami mountain range, Aomori, Japan. The nematode has several interesting morphological characters that do not fit the morphological definition of Bursaphelenchus, e.g., males have a tail spike instead of a bursal flap and lack the P1 ventral single papilla, and females have a very long and tapering tail (filiform). The molecular phylogenetic status of the new species inferred by the near-full-length small subunit (SSU: 18S) and the D2/D3 expansion segments of the large subunit (LSU: 28S) suggests, however, that the nematode is included in the B. fungivorus group, and is most closely related to B. thailandae and B. willibaldi. Despite these unique morphological characters, its spicule morphology, which is almost identical to that of B. gonzalezi, B. thailandae and B. willibaldi, and vulval structure, i.e., lacking a vulval flap, are in accordance with the molecular phylogenetic status of the nematode. The new species, which shows evidence of secondary loss of the bursal flap and P1 papilla, is described herein as B. kiyoharai n. sp. The typological descriptions of the genus and Parasitaphelenchinae were reconsidered and the taxonomic framework of the Aphelenchoidinae and Parasitaphelenchinae revised. Bursaphelenchus asiaticus n. comb. is proposed for Ruehmaphelenchus asiaticus.

Bursaphelenchus niphades n. sp. (Tylenchina: Aphelenchoididae) amensally associated with Niphades variegatus (Roelofs) (Coleoptera: Curculionidae)

A Bursaphelenchus species was isolated from a Japanese native wood-boring weevil, Niphades variegatus, and dead Pinaceae trees. The nematode is associated with the weevils as dauer (dispersal third stage) juveniles and the dauers enter the weevil tracheal system forming an abnormal expansion on the weevil trachea (atrium). Thus, the nematode is hypothesised to be an amensal/phoretic associate of the weevil because the abnormal expansion appeared to inhibit weevil respiration. The propagative stages of the nematode are associated with dead trees (wood and bark materials) and are thought to feed there on naturally propagated fungi. Morphologically, the new species is considered an undescribed species close to B. antoniae, B. chengi and B. hylobianum. Within these four species, the new species, which is described herein as B. niphades n. sp., is closest to B. chengi, i.e., the typological character of these two species are almost identical to each other and is distinguished by some minor characters (structure of the male P4 genital papillae and spicule length). The molecular phylogenetic analysis supported the morphological observations. Bursaphelenchus niphades n. sp. formed a well supported subclade with the four species and is intermediate between B. hylobianum and B. chengi; however, it is distinguished by the molecular sequences of some ribosomal RNA genes. Because three of these four species are associated with weevil species, the subclade is considered a ‘weevil-associated’ species group.

Different Developmental Responses of Virulent and Avirulent Isolates of the Pinewood Nematode, Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae), to the Insect Vector, Monochamus alternatus (Coleoptera: Cerambycidae)

Abstract The pinewood nematode (Bursaphelenchus xylophilus) is known to be the causative agent of pine wilt disease, which is transmitted from wilt-killed to healthy pine trees by the insect vector Monochamus alternatus. The second-stage propagative juvenile develops into third-stage dispersal (JIII) or propagative juveniles. The JIII develops into the fourth-stage dispersal juvenile (JIV), a special stage for transportation, then the JIV enters the tracheal system of adult beetles in pupal chambers in the xylem. To determine the differences in some life history parameters related to transmission between a virulent and an avirulent isolate of B. xylophilus, progression to the different life stages was investigated using two nematode isolates of different virulence, M. alternatus larvae, and pine bolts with an artificial pupal chamber. The numbers of JIIIs and JIVs produced around the pupal chamber by the time of beetle emergence were much smaller in the avirulent isolate than in the virulent one. The proportion of JIIIs produced in a population around the pupal chamber (the JIII and JIV number/the total population) and the probability of JIIIs developing into JIVs (the JIV number/the JIII and JIV number) were also smaller in the avirulent isolate. Although the probability of JIVs boarding beetles (the initial nematode load/the JIV number) was equal between the two isolates, the mean initial nematode load was much smaller in the avirulent isolate. The smaller initial nematode load of the avirulent isolate was ascribed to its smaller rate of reproduction and to lower production rates of JIII and JIV. A trade-off between the virulence and transmission rate of B. xylophilus is discussed herein, taking into account the effect of the initial nematode load on the vector’s longevity and flight performance.

Bursaphelenchus doui Braasch, Gu, Burgermeister & Zhang, 2005 (Aphelenchida: Parasitaphelenchidae), an associate of Monochamus subfasciatus Bates (Coleoptera: Cerambycidae) and Pinus densiflora Sieb. & Zucc.

Summary – Bursaphelenchus doui was isolated from a dead Japanese red pine, Pinus densiflora, in Shizuoka, and from the tracheal system of a species of longhorn beetle, Monochamus subfasciatus, collected at Tama Forest Science Garden of Forestry and Forest Products Research Institute, Tokyo, Japan. The Japanese populations of B. doui were compared with the original description of material obtained from coniferous packaging materials imported from Taiwan and Korea to continental China. Additional characters from the Japanese population include a constricted female mucron with a step-like appearance and several morphometric values. The molecular profiles of the Japanese B. doui populations were determined by DNA sequencing and ITS-RFLP profiles and were compared with those of the Taiwanese and Chinese populations of B. doui and other species in the genus. The phylogenetic analysis of the small subunit and large subunit ribosomal DNA indicated that B. doui is clearly included in the xylophilus-group of the genus Bursaphelenchus and may be close to B. conicaudatus and B. luxuriosae. The potential risk of B. doui for pine species is considered to be relatively low because B. doui did not display any pathogenicity to Japanese black pine in an inoculation test.

First Report of Parthenogenesis in the Genus Bursaphelenchus Fuchs, 1937: A Description of Bursaphelenchus okinawaensis sp. nov. Isolated from Monochamus maruokai (Coleoptera: Cerambycidae)

Abstract An evolutionarily interesting nematode, Bursaphelenchus okinawaensis sp. nov., is described and illustrated. The new species has several characteristic morphological traits, i.e., four lateral lines in both sexes, lack of a vulval flap in females, and a triangular spicule shape and stout P4 caudal papillae in males, and characteristic biological traits, including phoretic association with Monochamus maruokai, a species of longhorn beetle, parthenogenetic reproduction, and a high frequency of dauer production. Bursaphelenchus okinawaensis sp. nov. shares several important traits with various phylogenetic groups within the genus. The new species shares its spicule shape with B. hellenicus and B. hofmanni. It shares four lateral lines, P4 caudal papillar structure (size and position), and female vulval shape with the ‘hunti’ group, although it was molecularly inferred to be phylogenetically closer to the ‘xylophilus’ group and B. africanus. The autapomorphic traits of B. okinawaensis sp. nov. are parthenogenetic reproduction and high frequency of dauer production. All other nominal Bursaphelenchus nematodes have bisexual reproduction and tightly synchronized dauer production. The unique morphological and biological traits of B. okinawaensis sp. nov. suggest genetic flexibility within the genus. The importance of the morphology and arrangement of the caudal papillae is discussed relative to the phylogeny of the genus.

AN ENTOMOPARASITIC ADULT FORM IN BURSAPHELENCHUS DOUI (NEMATODA: TYLENCHOMORPHA) ASSOCIATED WITH ACALOLEPTA FRAUDATRIX

Abstract:  The nematode family Aphelenchoididae (Rhabditida: Tylenchomorpha) includes species with various feeding habitats. Bursaphelenchus, a member of the family, has for a long time been considered as a home for plant parasitic or mycophagous species (or both). However, recent intensive biological studies on the family revealed that the genus contains several insect parasitic species. Dauer juveniles of Bursaphelenchus doui were isolated from Acalolepta fraudatrix during a field study of longhorn beetle–Bursaphelenchus nematode associations. Two different insect-associated forms, an “entomoparasitic adult form” and a regular dauer juvenile, were isolated from a single individual beetle in a subsequent laboratory investigation of the B. doui–A. fraudatrix relationship. Thus these 2 distinct, insect-associated forms were confirmed to occur simultaneously. The entomoparasitic form is morphologically similar to that of Bursaphelenchus luxuriosae, with a dome-shaped head and vacuole-like spots assumed to be an internal structure of sensory organ, a stylet, a metacorpus (median bulb), and a moderately-developed and seemingly fully functional reproductive system. It is distinguishable from B. luxuriosae based on male spicule morphology and female tail morphology. A degenerate ingestive–digestive system distinguishes the entomoparasitic form from the propagative form and, unlike dauer juveniles, it has a moderately-developed reproductive system. The presence of this characteristic parasitic adult form is known only in these 2 Bursaphelenchus species. However, these 2 species did not form a clear monophyletic clade within the Bursaphelenchus xylophilus group and, thus, this characteristic parasitic form may occur independently in each species.

Worldwide invasion routes of the pinewood nematode: What can we infer from population genetics analyses?

Identifying the invasion routes and determining the origin of new outbreaks of invasive species are of crucial importance if we are to understand the invasion process, improve or establish regulatory measures and, potentially, limit the damage. We focused here on the invasion of Europe by the pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer, 1934; Nickle 1970; Nematoda: Aphelenchoididae), a major pest of forest ecosystems, native to North America and already invasive in Asia since the beginning of the twentieth century. We evaluated the genetic diversity and structure of worldwide field PWN samples by classical and Bayesian population genetics methods to determine the source of the European invasive populations and the number of introduction events in Europe. We found (1) a very strong spatial genetic structure in native PWN populations, (2) a very low level of polymorphism in each of the invaded areas and (3) contrasted results concerning the origin of European invasive populations. Our findings provide evidence for: (1) a large effect of genetic drift on the biological cycle of the PWN, due to intense demographic bottlenecks during tree infections, not compensated for by effective dispersal of its vector; (2) a single introduction event for each of the invaded areas in Japan and Europe and a small effective size for the introduced populations and (3) a mainland Portuguese origin for PWN populations from Madeira. However, more sophisticated methods of invasion route inference and broader sampling are required to conclusively determine the origin of the European outbreak.