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Bursaphelenchus mucronatus Mamiya & Enda, 1979 is a wood nematode widely distributed over Palearctic coniferous forests. It has two subspecies, that is, B. mucronatus mucronatus and B. mucronatus kolymensis, which present molecularly different East Asian and European genotypes, respectively. The European subspecies is found mainly in Europe and Siberia, while the East Asian subspecies occupies mostly Eastern regions of Asia. However, local isolates of both subspecies have been occasionally reported from various localities in Europe and Asia. Our field isolation and molecular (DNA ITS‐RFLP) identification of 21 recently collected populations of B. mucronatus revealed only 4 isolates representing clearly East Asian or European genotypes, while the remaining 17 isolates showed intermediate genotypes with electrophoretic band characters of both the above types. Further individual crossbreeding, in vitro, of nematodes representing European, East Asian, and intermediate genotypes provided experimental evidence for the process of intraspecific hybridization between both subspecies, spontaneously taking place in the forest. Such a widening of the B. mucronatus genetic variation may have its effect on a range of reported in the literature direct interactions between this native, nonpathogenic nematode species and the genetically similar, causative agent of the pine wilt disease B. xylophilus which can colonize the same host trees, use the same insect vectors, compete, and crossbreed inter‐specifically. [ABSTRACT FROM AUTHOR]

The process of inter‐specific hybridization between the invasive, highly pathogenic pinewood nematode, Bursaphelenchus xylophilus and the Eurasian‐native, relatively harmless B. mucronatus has been examined with the aid of the B. xylophilus Roller (Bxy‐rol(tom3) mutation (Tomalak & Filipiak, 2019) as a morphological marker, in the breeding protocol. Validity of the morphologically based selection of the inter‐specific hybrids has been confirmed by examination of individual nematodes with the multiplex PCR technique with specific primers (Filipiak et al., 2017). The research showed that hybridization between both species was possible in F1 and to some extent in subsequent offspring generations. In the general body plan, the hybrids resembled the wild‐type parental species but differed in various morphological/anatomical details. The changes ranged from seemingly indistinct to heavily distorted or occasional missing of particular structures. The most affected were spicules, female tails, the development of gonads and the position of the excretory pore. The hybridization led to various distortions of mating, gametogenesis, embryogenesis and juvenile viability, and development in F1, F2 and subsequent generation offspring, which severely affected the nematode reproductive potential. Potential consequences of the spontaneous inter‐specific hybridization between both species are discussed. [ABSTRACT FROM AUTHOR]

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) μ m and male body length of 893 (811-971) μ m, 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) μ m 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). [ABSTRACT FROM AUTHOR]

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) µp\ in female and 852 (718-992) pm 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) p.m 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, clawlike spicules with narrow capituium, 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. [ABSTRACT FROM AUTHOR]

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) pm in female and 874 (691-1122) pm 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) ?? long with a small cucullus (1.3-1.5 ?? 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article examines the sensitivity of the species-specific polymerase chain reaction primers for Bursaphelenchus fraudulentus. Results of the DNA sequencing showed that the amplified fragment of Bursaphelenchus fraudulentus DNA has complete regions of ITS-1, 5.8S and ITS-2, along with parts of the 18S and 28S regions. Researchers found that Bursaphelenchus fraudulentus-specific primer was not redycd when using a combination of Bursaphelenchus fraudulentus, Bursaphelenchus xylophilus and Bursaphelenchus mucronatus DNA samples.