To clarify the movement of Bursaphelenchus xylophilus in the tracheal system of adult Monochamus alternatus, 49 adult beetles were dissected 0, 15, 30, 45 or 60 days after emergence and the nematodes heading inwards to the distal end and outwards to spiracles in tracheae were counted. In adult beetles just after emergence, the majority of nematodes headed inwards. The proportion of tracheae with nematodes heading outwards was significantly higher for beetles aged 15, 30 or 45 days than for newly-emerged beetles. The proportion of nematodes pointing outwards tended to increase with beetle age and was significantly greater in 45-day old beetles than in newly emerged ones. A comparison of prediction with observation suggests that the nematodes move forwards in the tracheal system from spiracles and exit from spiracles after turning round in the tracheae. Le déplacement de Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) dans le système trachéen des adultes de Monochamus alternatus (Coleoptera: Cerambycidae) - Pour mieux comprendre le déplacement de Bursaphelenchus xylophilus dans le système trachéen de Monochamus alternatus 49 insectes ont été disséqués 0, 15, 30, 45 ou 60 jours après l'émergence et les nématodes se dirigeant dans les trachées vers l'intérieur - partie distale - et vers l'extérieur - stigmates - ont été comptés. Chez les insectes adultes, immédiatement après l'émergence la majorité des nématodes se dirigent vers l'intérieur. La proportion de trachées contenant des nématodes se dirigeant vers l'extérieur est significativement plus élevée chez les insectes âgés de 15, 30 ou 45 jours que chez ceux ayant émergé depuis peu. La proportion de nématodes se dirigeant vers l'avant a tendance à croître avec l'âge de l'insecte: elle est en effet significativement plus élevée chez les insectes âgés de 45 jours que chez ceux dont l'émergence est récente. La comparaison entre projection et observations suggère que les nématodes progressent dans le système trachéen à partir des stigmates et sortent par ces derniers après s'être retournés dans la trachée.
Bursaphelenchus tokyoensis n. sp. is described and figured from a dead Japanese red pine, Pinus densiflora. Despite several attempts, the new species was not successfully cultured and so individuals isolated from the original dead log were used as type specimens. The new species has a body length of ca 710 μm (male) and 770 μm (female), medium values of ratio a (ca 32-38 for males and ca 29-41 for females), b ratio of ca 10-12 (male) and 11-14 (female), c ratio of ca 24-29 (male) and 30-41 (female), c′ ratio of ca 2.3 (male) and ca 2.0 (female), ca 11-14 μm long stylet, four lines in the lateral field, relatively small (ca 14-16 μm), smoothly arcuate spicule lacking clear condylus, rostrum and cucullus, seven male caudal papillae and short and broad female tail with variable-shaped terminus. The new species is typologically close to B. idius but can be distinguished by male caudal papillae arrangement, female tail length and number of lateral lines. Inferred trees based upon molecular phylogenetic analysis of near full length (ca 1.6 kb) small subunit and ca 0.7 kb of the D2/D3 expansion segment of the large subunit of ribosomal DNA placed B. tokyoensis n. sp. closest to the xylophilus- and africanus-groups. However, the new species is distinguished from members of these groups by its characteristic spicule morphology and relative molecular phylogenetic placement.
As a means for estimating the virulence level of Bursaphelenchus xylophilus, we focused on its reproductive ability. Using six nematode isolates, which had high, intermediate or low levels of virulence, the number of nematodes that reproduced on Botrytis cinerea and in branch sections of Pinus thunbergii during 5 days was investigated. A significant positive correlation was found between nematode virulence and the number of nematodes reproducing either on the fungal culture or in the branch section. However, the rate of population increase with rise of virulence was clearly faster on the fungal culture than in the branch section. Our results suggest that the virulence level of B. xylophilus can be estimated approximately by evaluating the reproductive ability on B. cinerea or in branch sections of P. thunbergii tree. The effectiveness of branch section as a material for estimating the nematode virulence is discussed, taking into account the various factors associated with nematode virulence.
Bursaphelenchus kesiyae n. sp. is described. The new species was isolated from dead wood of Pinus kesiya during a field survey of nematodes associated with dead pine trees (Pinus spp.). The new species is medium- to large-sized for the genus, with males 690-1059 μm and females 837-1122 μm in body length, and has four lateral lines, six male genital papillae (P1 ventral single papilla is missing or vestigial), a mitten-shaped spicule with clear dorsal and ventral limbs, an indistinctive small and narrow bursal flap, vulva lacking any flap apparatus, and female tail long, tapering and straight or slightly ventrally curved. Based upon its diagnostic morphological characters, the new species belongs to the B. fungivorus group and is closely related to B. thailandae and B. parathailandae, with which it forms a cryptic species complex. However, the new species is distinguished from these two species by the morphology of the male bursal flap and several morphometric values, i.e., the bursal flap of the new species is inconspicuous, or almost lost in many individuals. Molecular phylogenetic analysis inferred from near-full-length SSU and D2-D3 LSU supported the morphological observations, i.e., the new species is molecularly similar to B. thailandae and B. parathailandae, but could be distinguished phylogenetically. Further, differences in molecular sequences in SSU and D2-D3 LSU between the new species and its close relatives are slightly higher than those between B. xylophilus and B. mucronatus, thus, identification of the species status for B. kesiyae n. sp. is considered warranted.
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.
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.
Detection of pine wood nematode, Bursaphelenchus xylophilus, is fundamental for effective control of pine wilt disease. Recent molecular techniques, such as DNA detection, have enhanced detectability of the nematodes whereas appropriate field sampling has received less attention. In order to elucidate a sampling design that most efficiently detects B. xylophilus using a commercially-distributed DNA detection kit, we compared detection levels of B. xylophilus using wood chips taken from various positions on dead trees. Results showed that the DNA kit had a higher detection level than the conventional method, and that trunk samples had higher levels than branch samples. Statistical model revealed that among-tree variation influenced the detectability more strongly than within-tree variation. Our results suggest that, in practice, with limited resources for control, it is more efficient to take samples from many trees with a minimum number from each tree, rather than taking many samples from a small number of trees.
To clarify the ability of Monochamus grandis to carry Bursaphelenchus xylophilus, we transferred B. xylophilus to M. grandis and M. alternatus (the vector beetle of B. xylophilus) using a simple nematode-loading method. We examined: i) the effects of M. grandis on the formation of the fourth-stage dispersal juvenile (JIV) of B. xylophilus, the phoretic stage carried by vector beetles; and ii) whether JIV of B. xylophilus transferred to M. grandis, and compared the ability of M. grandis to carry B. xylophilus with that of M. alternatus. Monochamus grandis induced JIV formation and carried similar numbers of JIV as M. alternatus. Moreover, the percentage of JIV transferred to M. grandis to total JIV was higher than for M. alternatus. We concluded that M. grandis had potentially an equal ability to M. alternatus to carry B. xylophilus.
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.