Bursaphelenchus fagi sp. n. (Nematoda: Parasitaphelenchidae), an insect-pathogenic nematode in the Malpighian tubules of the bark beetle, Taphrorychus bicolor (Herbst.) (Coleoptera: Curculionidae, Scolytinae), in European beech, Fagus silvatica L.

in Nematology
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Bursaphelenchus fagi sp. n. is described from the bark of European beech, Fagus silvatica. All propagative stages of the nematode are numerous in larval galleries of the beech bark beetle, Taphrorychus bicolor, while dauer juveniles aggregate in Malpighian tubules of adult beetles. The new species is characterised by the body length of 871 (763-1110) μm in female and 852 (718-992) μm in male, very slender body (a = 56.8 (50.4-67.1) and 64.5 (56.5-73.8) in female and male, respectively), and spicules 15.2 (14.0-17.0) μm long. The extended anterior vulval lip in female, lateral fields with four incisures, and number (7) and arrangement of male caudal papillae may indicate that B. fagi sp. n. is closely related to the xylophilus group. It differs from this group by the relatively small, claw-like spicules with narrow capitulum, indistinct condylus in almost continuous line with dorsal lamina, and lack of distinct cucullus at the spicule tip. The close relation of B. fagi sp. n. with the xylophilus group has been confirmed by DNA sequencing and phylogenetic analysis of the 28S rDNA region. Bursaphelenchus fagi sp. n. most closely resembles B. tokyoensis and B. idius, but can be separated by differences in the arrangement of the male caudal papillae, a unique feature for each species. The taxonomic separation of the new species is also confirmed by the unique molecular profile of the ITS region (ITS-RFLP). The mass presence of dauer juveniles of B. fagi sp. n. in Malpighian tubules of adult T. bicolor leads to degradation of the tubule cellular epithelium and local expansion of its basement membrane. These changes clearly indicate direct pathogenicity of the nematode to its vector insect. In laboratory rearing, B. fagi sp. n. can grow and reproduce on Botryotinia fuckeliana cultures.

Bursaphelenchus fagi sp. n. (Nematoda: Parasitaphelenchidae), an insect-pathogenic nematode in the Malpighian tubules of the bark beetle, Taphrorychus bicolor (Herbst.) (Coleoptera: Curculionidae, Scolytinae), in European beech, Fagus silvatica L.

in Nematology



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    Bursaphelenchus fagi sp. n. A: Female; B: Male; C: Anterior region (female); D: Vulval region (female); E: Male tail with spicules, bursa and positions of papillae (lateral view); F, G: Male tail showing shape of bursa and positions of two postcloacal pairs of papillae (partially ventral view, somewhat flattened); H-J: Female tail (range of forms); K: Dauer juvenile (dissected from a Malpighian tubule of the vector bark beetle, Taphrorychus bicolor).

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    Light (DIC) and SEM micrographs of Bursaphelenchus fagi sp. n. A-L: Adults. A, B: Anterior region: A: Head with stylet, median bulb and nerve ring; B: Median bulb and position of excretory pore; C: Vulva with extended anterior lip (flap); D: Vulva and postuterine sac with large sperm cells; E: Large sperm cells in male gonad; F: Lateral field with four incisures; G-I: Male tail; G: Spicules, lateral view; H: Shape of bursa (partially ventral view, somewhat flattened); I: Positions of caudal papillae; spicule terminus with somewhat widened and rounded tip (SEM); J-L: Female tail (range of forms); M-O: Dauer juvenile. M: Juvenile (entire view); N: Anterior region with median bulb; O: Tail.

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    ITS-RFLP profile of Bursaphelenchus fagi sp. n. Lanes 1, 8 = DNA marker (100 bp ladder, MassRuler™, Fermentas); Lane 2 = rDNA amplification product; Lanes 3-7 = restriction fragments: Lane 3 = RsaI; Lane 4 = HaeIII; Lane 5 = MspI; Lane 6 = HinfI; Lane 7 = AluI.

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    Maximum likelihood (ML) analysis of Bursaphelenchus fagi sp. n. and 38 other Bursaphelenchus species based on partial 28S rDNA. Aphelenchoides subtenuis served as outgroup species.

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    Neighbour-joining (NJ) analysis of Bursaphelenchus fagi sp. n. and 38 other Bursaphelenchus species based on partial 28S rDNA. Aphelenchoides subtenuis served as outgroup species.

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    Maximum parsimony (MP) analysis of Bursaphelenchus fagi sp. n. and 38 other Bursaphelenchus species based on partial 28S rDNA. Aphelenchoides subtenuis served as outgroup species.

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    Light micrographs (DIC) of Malpighian tubules of adult beech bark beetle, Taphrorychus bicolor. A-D: Uninfested tubules with cellular epithelium (micrographs taken at different levels to show cells and nuclei); E-J: Tubules colonised by dauer juveniles of Bursaphelenchus fagi sp. n. E: Overall view of tubule filled with nematodes – local expansion of the tubule diam.; F-J: Detailed view of tubule filled with nematodes showing a range of epithelial tissue degradation with clearly marked basement membrane and lack of cellular layer (J).

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