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Anna Filipiak and Marek Tomalak

Abstract

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) μm (as measured along arc) and shape, moderate length, i.e., L = 702 (603-946) μm in females and L = 679 (543-828) μm 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.

Marek Tomalak and Anna Filipiak

Abstract

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) μm (as measured along arc) and shape, and a relatively long (i.e., L,= 1020 (909-1111) μm in females), and L = 850 (756-1055) μm 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.

Marek Tomalak and Tadeusz Malewski

Bursaphelenchus tiliae sp. n. is described from the bark of dead branches of the small-leaved lime, Tilia cordata, infested with the lime bark beetle, Ernoporus tiliae. The nematode adults and propagative juveniles were present in larval galleries, while dispersal dauer juveniles colonised the Malpighian tubules of adult beetles. Bursaphelenchus tiliae sp. n. is characterised by the body length of 664 (568-817) μm in female and 622 (515-754) μm in male, moderately slender body (a = 41.0 (34.7-47.9) and 43.1 (37.8-51.9) in female and male, respectively), and spicules 20.0 (18.1-21.8) μm long. The extended anterior vulval lip in female, lateral fields with four incisures, long post-uterine sac, and number (7) and arrangement of male caudal papillae, may indicate that B. tiliae sp. n. is closely related to the xylophilus group. It differs from this group by smaller, arcuate spicules with relatively short rostrum, narrow capitulum (5.2-6.9 μm), and indistinct condylus in an almost continuous line with dorsal lamina. Distal third of spicule dorsal contour is usually almost straight. The spicules have a distinct, disk-like cucullus at the terminus. The status of the new species is confirmed by the unique molecular profile of the ITS region (ITS-RFLP) and its close relation to the xylophilus group by DNA sequencing and phylogenetic analysis of the 28S rDNA region. The presence of B. tiliae sp. n. dauer juveniles in Malpighian tubules of their vector bark beetle, E. tiliae, caused limited pathogenicity expressed as a local expansion of the tubule basement membrane and partial degradation of the cellular epithelium.

Marek Tomalak and Anna Filipiak

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.

Marek Tomalak and Anna Filipiak

Summary

Bursaphelenchus michalskii sp. n. is described from the bark of the European white elm, Ulmus laevis. All propagative stages of the nematode were found in larval galleries of the large elm bark beetle, Scolytus scolytus, and in overlapping gallery systems of this species and the small European elm bark beetle, S. multistriatus. Dauer juveniles of the new nematode are transmitted to new breeding trees under elytra of adult S. scolytus. Bursaphelenchus michalskii sp. n. is characterised by the female body length of 953 (838-1108) μm and male body length of 893 (811-971) μm, very slender body (a = 53.9 (46.1-58.5) and 60.9 (52.2-72.0) in female and male, respectively), lateral fields with three incisures (two bands), excretory pore usually located anterior to the median bulb, lack of vulval flap, long post-uterine sac, relatively small spicules 12.3 (10.8-13.3) μm long with no cucullus and with distinct, somewhat thorn-like, dorsally bent or reflexed condylus and a conical or digitate rostrum, and the arrangement of the seven male caudal papillae (i.e., a single precloacal ventromedian papilla (P1), one pair of adcloacal ventrosublateral papillae (P2) at or just anterior to cloacal slit, one ventrosublateral, postcloacal pair (P3) located at ca 60% of the tail length, posterior to cloacal slit, and one pair (P4) of ventrosublateral papillae located near the base of the bursa). The newly described species shares most of the key morphological characters with members of the eremus-group (sensu Braasch et al., 2009). However, B. michalskii sp. n. is unique amongst Bursaphelenchus species by a combination of female tail and spicule shape, excretory pore position, and other morphometric characters. These findings were confirmed by DNA sequencing and phylogenetic analysis of the 18S and 28S rDNA regions and by the unique molecular profile of the ITS region (ITS-RFLP).

Marek Tomalak and Anna Filipiak

Summary

The first spontaneous morphological/anatomical and behavioural mutation Bxy-rol(tom3) has been identified and characterised in the pine wood nematode, Bursaphelenchus xylophilus. The mutation is recessive to its wild-type counterpart. The mutant phenotype designated as Roller (Rol) is distinctive for the helical twist of the whole body around its longitudinal axis. The twist of the nematode body wall cuticle is followed by a similar twist of hypodermis, muscles and other organs and cuticular structures. These morphological and anatomical changes are responsible for the altered pattern of the nematode movement. Besides these changes, the Bxy-rol(tom3) mutation does not visibly affect any other essential character or function of B. xylophilus. Both wild-type and Rol nematodes present similar morphometrics, life cycle length, and reproductive potential. The Rol mutant easily cross-bred with individuals from any other tested strains/isolates of B. xylophilus and produced viable intra-specific hybrid offspring in vitro, on B. cinerea/potato dextrose agar plates. The mutants also seem to be well adapted to the nematode’s natural environment in pine wood. They dispersed inside pine seedlings, successfully mated, produced stable and fully viable offspring in this environment, and were as pathogenic to the host tree as the wild-type B. xylophilus. Potential of the characterised mutation as a marker for direct comparative study on strain/species reproductive compatibility, competition for space, food and vector insect, and for intra- and inter-specific hybridisation of B. xylophilus and B. mucronatus, is discussed.

Marek Tomalak and Jan J. Pomorski

Bursaphelenchus piceae sp. n. is described from the bark of Norway spruce, Picea abies. Adults and propagative juveniles of the nematode are found in larval galleries of the six-toothed spruce bark beetle, Pityogenes chalcographus, while its dauer juveniles aggregate in Malpighian tubules of older larvae, pupae and adult beetles. The new species is characterised by the body length of 719 (530-945) μm in female and 632 (474-808) μm in male, its moderately slender body (a = 41.4 (35.4-50.2) and 42.6 (35.1-47.2) in female and male, respectively), and small spicules (16.0 (14.0-18.3) μm along arc and 13.7 (12.2-15.5) μm along chord). The extended anterior vulval lip in female, lateral fields with four incisures, long post-uterine sac extending for 67-91% of vulva-anus distance, and number (7) and arrangement of male caudal papillae suggest that B. piceae sp. n. is closely related to the xylophilus-group yet it differs by the relatively small, claw-like spicules with no cucullus at the tip. The close relation of B. piceae sp. n. to the xylophilus-group has been confirmed by DNA sequencing and phylogenetic analysis. Morphologically B. piceae sp. n. most closely resembles B. tokyoensis and B. fagi but can be separated from both by the unique shape of the spicules. The taxonomic separation of the new species is also confirmed by the unique molecular profile of the ITS region (ITS-RFLP). The presence of dauer juveniles of B. piceae sp. n. in Malpighian tubules of adult P. chalcographus may lead to extensive damage to this organ as shown by local expansion of the tubule basement membrane and degradation of its cellular epithelium. Detailed phylogenetic analysis revealed that B. piceae sp. n. together with five other bark beetle-associated Bursaphelenchus species, namely B. trypophloei, B. masseyi, B. tiliae, B. tokyoensis and B. fagi, constitutes a small phylogenetic clade that is most closely located to, but separate from, the xylophilus-group.

Marek Tomalak, James Worrall and Anna Filipiak

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.

Anna Filipiak, Przemysław Wieczorek and Marek Tomalak

Differentiation between Bursaphelenchus xylophilus and other related, non-pathogenic species can be ambiguous when based exclusively on morphological characters. The morphology of B. mucronatus and B. fraudulentus most closely resembles that of B. xylophilus. Moreover, all of these nematodes are found in both Asia and Europe and can colonise various species of pine. Therefore, for phytosanitary purposes it is necessary to identify the three species precisely and rapidly. We report the results of a multiplex PCR that utilises four primers to identify and discriminate the three Bursaphelenchus species simultaneously. The multiplex PCR yielded DNA fragments of 767, 305 and 132 bp, for B. xylophilus, B. mucronatus and B. fraudulentus, respectively. This primer combination has produced reliable results in multiplex PCR assays with a number of different populations of the listed species, and no cross-reactions were observed with other Bursaphelenchus species. The described approach is simple, reliable and cheaper than other molecular methods presently used for simultaneous identification of the above three species within the xylophilus group.