Anna Filipiak

Description of Bursaphelenchus populi sp. n. (Nematoda: Parasitaphelenchidae), a new member of the xylophilus group from aspen, Populus tremula L., in Europe.

Bursaphelenchus populi sp. n. is described from dying and dead aspen trees, Populus tremula , in Poland. The nematode was found in trees infested with a long-horn beetle, Saperda perforata , and is vectored in the insect haemocoel. The characteristic morphology of male spicules, extended anterior vulval lip, lateral fields with four incisures, and number and arrangement of male caudal papillae, place B. populi sp. n. in the xylophilus group. Bursaphelenchus populi sp. n. can be separated from all other species in that group by the distinctive vulval flap, which is always bent with its distal half sunken in a conspicuous, sharp depression posterior to the vulva, and other morphological and morphometric characters, i.e. , female tail shape, excretory pore position, spicule length (32.1 (25.7-37.0) μ m (as measured along arc) and shape, and a relatively long ( i.e. , L,= 1020 (909-1111) μ m in females), and L = 850 (756-1055) μ m in males), and slender body (a = 45.4 (40.2-52.4) and 42.0 (36.1-49.3) in female and male, respectively). The status of the new species is confirmed by the unique molecular profile of the ITS region (ITS-RFLP). DNA sequencing and phylogenetic analysis of 28S rDNA region placed the new species close to B. fraudulentus in the xylophilus group. In vitro cross-breeding of the new nematode with B. xylophilus , B. mucronatus , B. fraudulentus and B. doui revealed full reproductive incompatibility between these species. In laboratory experiments on 2-year-old seedlings B. populi sp. n. was specific to aspen. It did not develop on pine or oak and reproduced only in weakened or dead aspen seedlings. No pathogenicity to the tree host or vector insect was observed. In Botrytis cinerea PDA cultures, body dimensions were significantly altered in both sexes and female tail morphology differed when compared to individuals extracted from aspen wood.

Bursaphelenchus trypophloei sp. n. (Nematoda: Parasitaphelenchinae) - an associate of the bark beetle, Trypophloeus asperatus (Gyll.) (Coleoptera: Curculionidae, Scolytinae), in aspen, Populus tremula L.

Bursaphelenchus trypophloei sp. n. is described from weakened and dead twigs of living or recently fallen aspen trees, Populus tremula , in Poland. All propagative stages of the nematode were present in larval galleries of a bark beetle, Trypophloeus asperatus , and its dauer juveniles could be found in the haemocoel of older larvae, pupae and adult beetles. Characteristic morphology of the male spicules with distinct cucullus, extended anterior vulval lip, lateral fields with four incisures, and number and arrangement of male caudal papillae indicate that B. trypophloei sp. n. is closely related to the xylophilus group. This relation has been further confirmed by DNA sequencing and phylogenetic analysis of the 28S and ITS-1 rDNA region. Bursaphelenchus trypophloei sp. n. can be separated from other species in the xylophilus group by the morphology of spicules which have a shorter capitulum and unique rostrum which is pointed somewhat anteriorly, relatively thick vulval flap, which is straight, parallel to the body long axis or bent towards the body wall at its distal end, and other morphological and morphometric characters, i.e. , shape of female tail, position of excretory pore at or posterior to nerve ring, spicule length of 25.7 (23.1-28.0) μ m (as measured along arc) and shape, moderate length, i.e. , L = 702 (603-946) μ m in females and L = 679 (543-828) μ m in males, and slender body (a = 39.2 (33.0-45.2) and 40.9 (32.0-46.4) in female and male, respectively). The taxonomic separation of the new species is also confirmed by the unique molecular profile of the ITS region (ITS-RFLP). Full reproductive incompatibility of B. trypophloei sp. n. with B. xylophilus , B. mucronatus , B. fraudulentus , B. populi and B. doui has been demonstrated in in vitro diallelic cross-breeding. The newly described nematode revealed limited ability to develop and reproduce on Botrytis cinerea cultures although it produced large populations on laboratory cultures of Cytospora chrysosperma – a fungus naturally associated with galleries of the nematode vector, T. asperatus .

Bursaphelenchus masseyi sp. n. (Nematoda: Parasitaphelenchinae) – a nematode associate of the bark beetle, Trypophloeus populi Hopkins (Coleoptera: Curculionidae: Scolytinae), in aspen, Populus tremuloides Michx. affected by sudden aspen decline in Colorado

Bursaphelenchus masseyi sp. n. is described from trunks of unhealthy trembling aspen, Populus tremuloides, affected by sudden aspen decline (SAD) in Colorado, USA. All propagative stages of the nematode were present in larval galleries of a bark beetle, Trypophloeus populi. The dauer juveniles occupy the haemocoel of older larvae, pupae and adult beetles. The characteristic morphology of the male spicules with small but distinct cucullus, extended anterior vulval lip in female, lateral fields with four incisures, and number and arrangement of male caudal papillae, indicate that B. masseyi sp. n. is closely related to the xylophilus group. This relation has been confirmed by DNA sequencing and phylogenetic analysis of the 28S rDNA region. The new species is characterised by the body length of 958 (765-1203) μm in female and 874 (691-1122) μm in male, moderately slender body (a = 38.2 (33.5-41.1) and 38.0 (33.1-44.1) in female and male, respectively), and spicules 29.2 (26.2-34.7) μm long with a small cucullus (1.3-1.5 μm in diam.) at their tips. Bursaphelenchus masseyi sp. n. can be separated from other species in the xylophilus group by the morphology of spicules which have a short capitulum and unique rostrum that is pointed somewhat anteriorly, relatively thick vulval flap, which is straight, parallel to the body long axis and bent towards the body wall at its distal end, and other morphological and morphometric characters. The new species most closely resembles B. trypophloei, but differs by the morphology of spicules (short but distinct condylus vs condylus in a continuous line with dorsal lamina, and smaller cucullus). The taxonomic separation of the new species is also confirmed by the unique molecular profile of the ITS region (ITS-RFLP). Diallelic cross-breeding in vitro revealed also that B. masseyi sp. n. and B. trypophloei are reproductively incompatible. The new species showed poor ability to develop and reproduce on Botrytis cinerea cultures, although it grew vigorously on laboratory cultures of Polish and Colorado isolates of Cytospora chrysosperma, the fungus naturally associated with galleries of the nematode vector, T. populi.

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.

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.

Species-specific polymerase chain reaction primers for simple detection of Bursaphelenchus fraudulentus (Nematoda: Parasitaphelenchidae)

Bursaphelenchus fraudulentus Rühm, 1956 (Nematoda: Parasitaphelenchidae) is a tree-inhabiting species frequently found in Europe (Rühm, 1956; Schauer-Blume & Sturhan, 1989; Tomalak, 2004; Carletti et al., 2005) and Asia (Braasch et al., 2001). It is primarily found in dying or dead deciduous trees (Rühm, 1956; Schauer-Blume & Sturhan, 1989), although occasionally reported from conifers (Braasch et al., 2001). Within the genus the specific morphological characters (i.e., vulval flap, shape of spicules, position of caudal papillae and presence of four incisures in the lateral fields) place B. fraudulentus into a distinct xylophilus-group which comprises eight other, morphologically similar, species, including the quarantine pest B. xylophilus (Gu et al., 2008). Reliable methods of taxonomic identification of these species are therefore of particular interest to plant quarantine services. Several molecular techniques have been developed and used for identification of Bursaphelenchus spp. (Wang et al., 1999; Matsunaga & Togashi, 2004; Burgermeister et al., 2005; Castagnone et al., 2005; Leal et al., 2005, 2007) with ITS-RLFP analysis the most widely used in research and quarantine practice. An alternative method, based on a simple PCR amplification with primers specific for B. xylophilus and B. mucronatus (Matsunaga & Togashi, 2004), enables rapid and precise species identification, even from a single nematode (Filipiak et al., 2007). Both native European species from the xylophilusgroup, i.e., B. mucronatus and B. fraudulentus, are relatively common and harmless to trees. They can be morphologically difficult to distinguish from each other and from the quarantine pest B. xylophilus. The provision of PCR primers specific for the DNA of B. fraudulentus, in addition to those already existing for B. xylophilus and B.