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Kazuyoshi Futai and Natsumi Kanzaki

Abstract

The pine wood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease. It can invade healthy pine trees through the feeding wound of its vector beetle, Monochamus alternatus, and can enter a dead or dying tree through the vector's oviposition mark. By contrast, B. mucronatus, which is closely related to B. xylophilus but has been considered to have no pathogenicity to pine species, also invades living host trees through the same process as B. xylophilus, although the transmission efficiency is lower than that of B. xylophilus. In order to clarify the reason why Bursaphelenchus mucronatus invades a living host, we inoculated shaded and unshaded host seedlings of Pinus densiflora with B. mucronatus. Bursaphelenchus mucronatus displayed pathogenicity in the host seedlings but only with those that were shaded. In addition, B. mucronatus was recovered from the seedlings that had survived until the end of the experiment. Bursaphelenchus mucronatus seems to survive in the living healthy host tree without causing disease symptoms. These results suggest a mutualistic relationship between B. mucronatus and its vector beetle.

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Kazuyoshi Futai and Yuko Takeuchi

Abstract

A rapid and accurate diagnostic method is needed to detect asymptomatic carrier trees to assist in exterminating pine wilt disease caused by the pathogenic pine wood nematode (PWN), Bursaphelenchus xylophilus. We extracted DNA by the CTAB method from pine trees located in two natural stands, a Japanese black pine stand and a Japanese red pine stand, known to be infected with PWN, and detected the PWN by the nested-PCR method. Many trees of both pine species harboured PWN, although some of them had no external and/or internal symptoms such as yellowing of needles and disrupted resin exudation. Some Japanese black pine trees remained asymptomatic during the study and survived for 1 or more years after PWN infection without displaying any wilting symptoms. The diagnostic method adopted in the present study is useful and sensitive enough for practical use to detect the asymptomatic carrier trees and may prove to be an effective method to assist in control of the pest in the field.

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Noritoshi Maehara and Kazuyoshi Futai

Abstract

Although fourth-stage dispersal juveniles (JIV) of Bursaphelenchus xylophilus, the pinewood nematode (PWN), developed in the presence of both Monochamus alternatus and Psacothea hilaris, the numbers and the percentage of JIV were far higher in the presence of the former than of the latter. JIV first appeared 7 days after pupation of M. alternatus and the number increased from the day of beetle eclosion to the third day thereafter, then remained stable. We conclude that the presence of specific vectors affects both the life history of the PWN and the numbers of nematodes carried by vectors emerging from killed pine trees.

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Natsumi Kanzaki and Kazuyoshi Futai

Abstract

The life history of the nematode Bursaphelenchus conicaudatus and its phoretic relationship to the yellow-spotted longicorn beetle, Psacothea hilaris, were studied, and are discussed by comparison with those of the pinewood nematode B. xylophilus, the pathogen of the pine wilt disease, and its vectors Monochamus species. The life history of B. conicaudatus was found to be very similar to that of B. xylophilus in dispersal pattern, vector selection and feeding preference, although the average number of B. conicaudatus carried by the yellow-spotted longicorn beetle, the proportion of beetles infested with the nematodes, and the nematode release pattern differed from those of B. xylophilus. These findings indicate a close relationship between the two species of Bursaphelenchus and the cerambycid beetles, as well as the adaptability of these two nematode species to the life cycles of their vectors.

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Natsumi Kanzaki and Kazuyoshi Futai

Abstract

The phylogenetic relationships of Bursaphelenchus conicaudatus with B. abruptus, B. fraudulentus, B. mucronatus and B. xylophilus species were analysed based on the DNA base sequences of 18S, 5.8S, ITS1 and ITS2 of rDNA and the partial code of mitochondrial cytochrome oxidase subunit I (COI) gene. The monophyly of B. abruptus and the other species in the xylophilus group was not supported, and B. abruptus could be excluded from the xylophilus group. The other species, i.e ., B. conicaudatus, B. fraudulentus, B. mucronatus and B. xylophilus were assumed to be monophyletic. The ancestor of these four species is assumed to have originated in the eastern part of the Eurasian continent as a free-living nematode inhabiting broad-leaved trees. First, B. conicaudatus branched from the ancestor in eastern Asia. Then B. fraudulentus and B. mucronatus separated from the ancestor. B. mucronatus changed its host from broad-leaved trees to conifers and spread throughout the coniferous forest over the Eurasian continent and North America. B. xylophilus might originate from a population of B. mucronatus remaining in North America after B. mucronatus had diversified.

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Noritoshi Maehara and Kazuyoshi Futai

Abstract

Numerous pinewood nematodes (Bursaphelenchus xylophilus) aggregated around pupal chambers of Monochamus alternatus in pine wilt-killed trees and the beetle carried many nematodes. However, few nematodes aggregated around pupal chambers of the other beetles, i.e., Acanthocinus griseus, Pissodes obscurus and Trogossita japonica, and these beetles carried few or no nematodes. The higher percentage of M. alternatus pupal chambers with intense blue-stain of wood would cause more nematodes to aggregate around such chambers, as blue-stain fungi are suitable for B. xylophilus propagation.

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Kazuyoshi Futai and Natsumi Kanzaki

Abstract

The phoretic relationships between the nematode Bursaphelenchus conicaudatus and the yellow-spotted longicorn beetle, Psacothea hilaris, collected at various districts in Japan were investigated. A high proportion of all ten subspecies of the beetle examined were infected with the nematodes, suggesting the universality of this phoretic association. Molecular analysis based on the partial base sequence of cytochrome oxidase subunit I in the mitochondrial DNA revealed that the phylogenetic relationships among ten subspecies of the beetle was similar to that among associated nematode isolates obtained from each beetle subspecies. Intraspecific vector replacements were supposed to occur in the isolates of B. conicaudatus on two islands. The phoretic association between the nematode and the beetle is assumed to have been established before the divergence of P.hilaris into subspecies.

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Natsumi Kanzaki and Kazuyoshi Futai

Abstract

Bursaphelenchus luxuriosae n. sp. is described and figured. Specimens were collected from a 2-week-old culture on Botrytis cinerea. The new species is characterised by a body length of 897 (710-1159) μm in the female and 745 (621-887) μm in the male, relatively robust body (a = 33-39 in the female and 27-30 in the male), stylet ca 14 (11-16) μm long, four lines in the lateral field, the large (27-30 μm) arcuate spicule with a terminal cucullus, seven (2 + 1 + 2 + 2) male caudal papillae, the long, well developed vulval flap and the shape of the female tail which is long, tapered, and ventrally bent when killed by heat with an irregular or roughened dorsal contour near the tip and an irregular terminus. The new species is considered to belong to the Bursaphelenchus xylophilus group of the genus Bursaphelenchus and is most closely related to B. conicaudatus and B. fraudulentus in spicule shape, vulval flap and 'a' values of males and females. It is easily distinguished from these two species by the morphology of female tail. The RFLP profile confirms the distinctness of the new species within the B. xylophilus group. The phylogenetic status of B. luxuriosae n. sp. within the B. xylophilus group is indicated by molecular phylogenetic analysis. Bursaphelenchus luxuriosae n. sp. is assumed to be close to B. conicaudatus and to have diverged from the ancestor of the B. xylophilus group early in the speciation of the group.

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Natsumi Kanzaki and Kazuyoshi Futai

Abstract

Bursaphelenchus parvispicularis n. sp. is described and figured. The new species was isolated from the bark of a dead oak tree, Quercus mongolica var. grosseserrata, and reared on Botrytis cinerea. Specimens were collected from a 2-week-old culture on Botrytis cinerea. The new species has a body length of 894 (711-1012) μm in the female and 764 (600-870) μm in the male, a ratio of 33.4 (29.7-37.1) in the female and 35.6 (31.4-45.3) in the male, and c and c′ ratios of 16.7 (14.8-19.0) and 4.4 (3.9-5.1), respectively, in the female and 24.3 (20.0-28.4) and 2.5 (2.2-2.8), respectively, in the male. The stylet is 14-16 μm long in the female and 13-15 μm in the male; there are three lines in the lateral field; the spicules are small (13 (13-15) μm long) and broad; there are seven (2 + 1 + 2 + 2) male caudal papillae; the 'bursa' varies from roundish trapezoid to rectangular with a concave terminus, and the female tail is tapered with clearly annulated dorsal surface near the tail tip and a rounded terminus. Based upon morphology the new species belongs to the B. hofmanni-group sensu Braasch. However, the new species is distinguished from the other B. hofmanni-group species by the size and form of the spicule, female tail shape, and morphometric values. Based on the molecular phylogenetic analysis of the DNA base sequences of ribosomal DNA and mtCOI gene, the new species was close to two B. hofmanni-group species (B. paracorneolus and B. hofmanni) and to B. hylobianum. This result is in accordance with their morphological similarity.

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Kazuyoshi Futai, Natsumi Kanzaki and Kaku Tsuda

Abstract

Bursaphelenchus conicaudatus n. sp. is described and figured. Specimens were collected from a 3-week-old culture on Botrytis cinerea. The new species is characterised by the roundish trapezoid bursa of males and tapered and mucronate tail of females. The new species is closely related to B. xylophilus, B. mucronatus and B. fraudulentus in the shapes of spicule and vulval flap, but was easily distinguished from these three species by the shapes of the bursa and female tail. Bursaphelenchus conicaudatus n. sp. est décrit et illustré. Des spécimens ont été collectés à partir d’une culture de 3 semaines sur Botrytis cinerea. La nouvelle espèce est caractérisée par une bourse trapézoïdate et arrondie chez le mâle et par l’extrémité mucronée de la queue chez la femelle. La nouvelle espèce est très proche de B. xylophilus, B. mucronatus et B. fraudulentus par la forme des spicules et du volet vulvaire mais elle a été facilement différenciée de ces trois espèces par la forme de la bourse du mâle et de la queue de la femelle.