New Records of Wood- and Bark-Inhabiting Nematodes from Woody Plants with a Description of Bursaphelenchus zvyagintsevi sp. n. (Aphelenchoididae: Parasitaphelenchinae) from Russia

Wood- and bark-inhabiting parasitic nematodes are of great economic importance. Nematodes can cause wilt diseases in conifers and deciduous trees. In 2014–2022, during nematology surveys conducted in different regions of Russia and Belarus, adults and dauer juveniles of nematodes were collected from wood, bark and beetle vectors. Using traditional morphological taxonomic characters integrated with molecular criteria, we identified in the studied samples the following nematode species: Aphelenchoides heidelbergi, Bursaphelenchus eremus, B. fraudulentus, B. michalskii, B. mucronatus, B. willibaldi, Deladenus posteroporus, Diplogasteroides nix and Laimaphelenchus hyrcanus, several unidentified species: Aphelenchoides sp.1 and sp.2, Cryptaphelenchus sp.1, sp.2 and sp.3, Laimaphelenchus sp.1, Micoletzkya sp.1, Parasitaphelenchus sp.1, Parasitorhabditis sp.1, three unidentified tylenchid nematodes and a new species, Bursaphelenchus zvyagintsevi sp.n. Morphological descriptions and molecular characterization are provided for B. zvyagintsevi sp. n. belonging to the Abietinus group and B. michalskii belonging to the Eggersi group. Findings of Aphelenchoides heidelbergi, Bursaphelenchus eremus, B. michalskii, Deladenus posteroporus, Diplogasteroides nix and Laimaphelenchus hyrcanus are new records for Russia. Phylogenetic positions of studied species were reconstructed using D2–D3 expansion segments of 28S rRNA gene sequence analysis. The data obtained in this study may help to detect the refugia of opportunistic plant pests and find possible native biocontrol nematode agents of insect vectors causing diseases.


Introduction
Wood-and bark-inhabiting parasitic nematodes cause wilt and dieback diseases in conifers and deciduous trees.Their destructive impact on forestry leads to significant economic and social consequences worldwide.Protective measures against nematodes are obligatory in all countries.Due to the prohibition or restriction of many nematicides, alternative control strategies are needed to control these diseases.Accurate nematode diagnostics, early detection of the neglected nematode pest refugia, identification of insect vectors of nematodes and suppressive bacterial and fungal agents are important for the development of effective control measures [1][2][3][4][5].
The aim of the present study was to analyze the data on the systematics and distribution of wood-and bark-inhabiting nematodes collected in Russia and Belarus from 2014 to 2022 using an integrative approach based on analyses of the molecular and morphological characters of parasitic nematodes extracted from wood and bark samples.The main goal of the study was to identify the wood-and bark-inhabiting parasitic nematodes collected in natural forest refugia in Russia, especially in the Siberian and Pacific regions, where numerous opportunistic pathogens associated with insect vectors, fungi and bacteria are found in weak and dying old trees in forest ecosystems.In this study, the tree wood and bark samples collected from ecosystems under anthropogenic transformations in urban areas, parks and botanical gardens were also included in the analysis.Under quickly changing climatic and biotic conditions and due to the anthropogenic disturbances of forest ecosystems, these nematodes may change their hosts and vectors and transform into the true pathogens [6][7][8][9].
In Russia, wood-and bark-inhabiting nematodes have been intensively studied and lists of associated species have been reported in several publications .However, with the exception of Bursaphelenchus, the identification of species was based on morphology only and, therefore, now requires molecular confirmation.
The data presented in this study may aid in detections of refuges of opportunistic plant pests and possible native biocontrol nematode agents of insect vectors of diseases.After consideration of the biology of nematodes associated with bark beetles, Polyanina et al. [31] revealed several types of relations in the nematode-beetle-tree associations.Following this classification, several ecological groups of nematodes associated with bark beetles could be distinguished among the studied samples: obligatory ectophoronts, fungal and plant feeders (Bursaphelenchus, Cryptaphelenchus, Aphelenchoides); facultative phoronts and fungivores (Laimaphelenchus); facultative phoronts, bacterial and fungal spore feeders (Diplogasteroides); facultative phoronts and carnivores (Micoletzkya); gut bark beetles endoparasites (Parasitorhabditis); hemocoel parasites of larval insect stage (Parasitaphelenchus); hemocoel parasites of adult insect stage (Deladenus and sphaerulariid nematodes).
Male: Similar to female in structure of anterior end.Testis situated on right subventral side of mid-intestine, long, anteriorly reflexed, zone of spermatids distinct, consisting of one or two quartets of large, separated cells, and a zone of granulated immature sperm cells located posterior to spermatids.Sperm gradually decreasing in size to spherical mature sperm cells filling posterior 20-25% part of testis.Tail strongly hooked, J-shaped, terminating with very small bursa 6-8 µm at borders, and 3-5 µm along mid-line.Bursa posterior edge truncate to conical.There are seven male tail papillae: mid-ventral unpaired P1 just anterior to cloacal opening, paired P2 almost at same level as P1 laterally (0-13% of tail length), paired P3 shifted to 50-59% of tail length from cloacal opening, and paired, small, pore-like P5 close to ventral mid-line at level of lateral edges of bursa 81 (74-86%).P5 pair may be considered 'gland papillae' because of their pore-like form, whereas other papillae are nipple-like.P4 pair absent.Spicule strong, narrow, J-shaped, rostrum and condylus well developed and separated.Angle between lines along capitulum (condylusrostrum) and extending spicule end = 13-23       Female: Ovary well developed, outstretched, extending to pharyngeal gland lobe, its distal end mostly reflexed, situated on right subventral side of mid-intestine.Oviduct straight and wide, with wrinkled surface.Spermatheca oval, situated ventrally and to left side of proximal part of oviduct, filled with ovoid cytoplasmic sperm 3 × 4 µm diam.Vagina 10-13 µm, sloping anteriorly to ventral body surface.Spermatheca opening from left side to pre-crustaformerial chamber via a spermathecal duct.Oviduct opening in pre-crustaformerial chamber from right side.Pre-crustaformerial chamber with small inner cavity, this chamber continuing proximally into crustaformeria.Pre-crustaformerial chamber and crustaformeria separated by sphincter.Crustaformeria formed by large cells containing cytoplasmic granules, joining with anterior uterus, walls consisting of large, flattened cells.Vagina 10-13 µm, sloping anteriorly to ventral body surface.Vulval anterior lip flap small, with apex directed anteriorly and long sides (lateral ridges) directed laterally.No vulval papillae or post-vulval surface transversal fold visible.Pair of three-celled structures situated laterally on both sides of vagina at uterus/post-uterine sac junction.Posterior vulval lip massive, expanded.Post-uterine sac (PUS) very wide, with sperm, posterior end hemispherical, not differentiated or with rudimentary ovary.Tail straight, hooked ventrally.Tail tip straight or slightly curved, conically rounded to digitate.
Type habitat and locality: Cultures on Botryotinia fuckeliana-potato dextrose agar medium (PDA) were started from individuals isolated from the phloem (0.5 cm deep) obtained from a dying ash, Fraxinus mandshurica (Oleaceae), showing symptoms of dieback, dark-colored ring in cross-section of wilted branches and trunk with galleries of larvae and pupae of Hylesinus laticollis Blandford (Curculionidae: Scolytinae).These were collected by Prof. Vy-acheslav.B. Zvyagintsev in the Arboretum of the Far Eastern Research Institute of Forestry, on the bank of the Amur River (GIS 48.463611, 135.084444),Khabarovsk city, Khabarovsk Krai, Russia, 25 August 2018.Several cultures were started from dauer juveniles extracted from the dissected female adults of H. laticollis collected simultaneously from galleries in phloem.
Etymology: The specific epithet was given in honor of Prof. Vyacheslav B. Zvyagintsev (Belarus), who collected these valuable samples with dieback symptoms during his research survey of ash Fraxinus spp.dieback infections in plantations in Belarus and Russia.
Type materials: Type nematode material obtained from 2-week-old Botryotinia fuckeliana cultures.Holotype male (slide P-4522), 20 paratype females, 20 paratype males (slides P-4524-4528) deposited in the Nematode Collection of the Zoological Institute RAS, Saint Petersburg, Russia.Four paratype males and four paratype females also deposited in the Nematode Collection of Wageningen Agricultural University, The Netherlands, and four paratype males and four paratype females in the Nematode Collection of the University of California, Riverside, CA, USA.
Diagnosis and relationships: Bursaphelenchus zvyagintsevi sp.n. belongs to the Abietinus group [32] according to molecular phylogenetic analyses and morphological characters.The new species is characterized by body length of 470-676 µm, stylet = 12-15 µm, with flexible shaft, stylet base slightly and smoothly expanded into three ridges, but without knobs, lateral field with two incisures.Median bulb oval, pharyngeal dorsal lobe 4-6 body diam.long.Spermatheca oval filled with ovoid sperm 3-4 µm diam.Female PUS 53-83% of the vulva-anus distance and 3.6-5.8vulval body diam.(VBD) long, filled with sperm, female tail straight, its tip conically rounded.Male with seven caudal papillae: 1 + 2 + 2 + 2, the mid-ventral unpaired P1 just anterior to the cloacal opening, the paired P2 level with the cloacal aperture, the paired P3 and paired pore-like 'gland papilla' P5 at the lateral edges of the very small conically rounded bursal flap.P4 pair absent.
The new species differs from B. abietinus, Braasch and Schmutzenhofer, 2000, in spicule devoid of cucullus vs. cucullus present in B. abietinus; PUS (post-vulval uterus sac) more than half of the vulva-anus distance vs. PUS less than 50% of the vulva-anus length; secretory-excretory pore at nerve ring or anterior vs. secretory-excretory pore posterior to nerve ring [36].
Bursaphelenchus zvyagintsevi sp.n. differs from B. rainulfi, Braasch and Burgermeister, 2002, in PUS more than half of the vulva-anus distance vs. PUS less than 50% of the vulva-anus length; spicule narrow with short condylus and one ridge along lamina vs. wide spicule with long prominent condylus and 2-3 ridges along lamina; female tail almost straight vs. female tail recurved [39].
Bursaphelenchus zvyagintsevi sp.n. differs from B. varicauda, Thong and Webster, 1983, in spicule narrow with short condylus vs. stout spicule with long prominent condylus; female tail tip without appendages vs. female tail tip with three finger-like processes in B. varicauda [40].
Molecular characterization: One D2-D3 of 28S and one ITS rRNA gene sequences were generated for this species.Phylogenetic position of B. zvyagintsevi sp.n. is given within some representatives of Aphelenchoidea (Figure 3) and the Abietinus group members (Figure 4) based on the analysis of D2-D3 of 28S and ITS rRNA gene sequences.Sequences of B. zvyagintsevi sp.n. clustered with that of B. sinensis in the D2-D3 28 rRNA gene tree (Figures 3 and 4A

Bursaphelenchus michalskii Tomalak and Filipiak, 2018
Adults (Figure 5, Table 1): Body very narrow and long, curved ventrally.Lateral field with three equidistant lateral incisures (two bands).Stylet base slightly expanded, but without distinct knobs.Cephalic annuli weakly visible under light microscopy, at least five annuli distinct.Lip region lobed, lateral lobes narrower than subventral and subdorsal ones.Median bulb pyriform, median valve located in posterior third of bulb, anterior third narrow, glandular, posterior two thirds spherical.Secretory-excretory pore located at nerve ring to one-half length of median bulb anterior to bulb.
Male: Similar to female in structure of anterior end.Testis situated on right subventral side of mid-intestine, long, anteriorly reflexed, zone of spermatids distinct, consisting of one or two quartets of large, separated cells, and a zone of granulated immature sperm cells located posterior to spermatids.Sperm gradually decreasing in size to ellipsoid mature sperm cells filling posterior 20% part of testis.Vas deferens with a wall of flat large polygonal cells, four cloacal lengths long.Tail strongly hooked, J-shaped, terminating with very small bursa 9-15 μm at borders, and 3-6.5 μm along midline.Bursal flap truncate with roundish concavity at center of distal end with two pointed tips on each lateral side.There are seven male tail papillae: mid-ventral unpaired P1 just anterior to cloacal opening, paired P2 almost at same level as P1 laterally (0-13% of tail length),

Bursaphelenchus michalskii Tomalak and Filipiak, 2018
Adults (Figure 5, Table 1): Body very narrow and long, curved ventrally.Lateral field with three equidistant lateral incisures (two bands).Stylet base slightly expanded, but without distinct knobs.Cephalic annuli weakly visible under light microscopy, at least five annuli distinct.Lip region lobed, lateral lobes narrower than subventral and subdorsal ones.Median bulb pyriform, median valve located in posterior third of bulb, anterior third narrow, glandular, posterior two thirds spherical.Secretory-excretory pore located at nerve ring to one-half length of median bulb anterior to bulb.Remarks: There are some differences between the Dagestan population and Poland population.The spermatheca is not axial, but a side branching sac of the genital tube, sperm is ellipsoid and not spherical, and the median bulb is distinctly pyriform with posteriorly shifted inner valve vs. median bulb ovoid with central valve.Such new Male: Similar to female in structure of anterior end.Testis situated on right subventral side of mid-intestine, long, anteriorly reflexed, zone of spermatids distinct, consisting of one or two quartets of large, separated cells, and a zone of granulated immature sperm cells located posterior to spermatids.Sperm gradually decreasing in size to ellipsoid mature sperm cells filling posterior 20% part of testis.Vas deferens with a wall of flat large polygonal cells, four cloacal lengths long.Tail strongly hooked, J-shaped, terminating with very small bursa 9-15 µm at borders, and 3-6.5 µm along midline.Bursal flap truncate with roundish concavity at center of distal end with two pointed tips on each lateral side.There are seven male tail papillae: mid-ventral unpaired P1 just anterior to cloacal opening, paired P2 almost at same level as P1 laterally (0-13% of tail length), paired P3 shifted to 41-58% of tail length from cloacal opening, and paired, small, pore-like P5 close to ventral mid-line at level of lateral edges of bursa 69 (66-75%).P5 pair may be considered as 'gland papillae' because of pore-like form, whereas other papillae are nipple-like.P4 pair absent.Spicule strong, wide, J-shaped, rostrum and condylus well developed and separated.Angle between lines along capitulum (condylus-rostrum) and extending spicule end = 13-23 • , point of intersection ventral.Rostrum acute, sharp, offset from the spicule contour.Junction of rostrum and calomus rectangular.Condylus rounded, dorsally bent from contour of lamina.Capitulum of spicule with a distinct depression its depth one fifth of the capitulum length.Spicular tip with small flat cucullus.Spicular lamina mid-point widened with two or three central ridges along lamina.Dorsal spicular lamina smoothly and symmetrically curved.
Female: Ovary well developed, outstretched, extending to pharyngeal gland lobe, its distal end mostly reflexed, situated on right subventral side of mid-intestine.Oviduct straight and wide, with wrinkled surface.Spermatheca round to oval, situated ventrally and to left side of proximal part of oviduct, with oval granulated cytoplasmic sperm 7 × 5 µm.Spermatheca opening from left side to crustaformerial chamber via a spermathecal duct.Oviduct opening in pre-crustaformerial chamber from right side.Pre-crustaformerial chamber with small inner cavity, this chamber continuing proximally into crustaformeria.Pre-crustaformerial chamber and crustaformeria separated by sphincter.Crustaformeria formed by large cells containing cytoplasmic granules, joining with anterior uterus, walls consisting of large, flattened cells.Vagina 10-13 µm, cuticular, sloping anteriorly to ventral body surface, vulval flap absent.No vulval papillae or post-vulval fold visible.Pair of three-celled structures situated laterally on both sides of vagina at uterus/post-uterine sac junction.Posterior vulval lip massive, expanded.Post-vulval uterine sac (PUS) very wide, filled with sperm, its end hemispherical, not differentiated and devoid of rudimentary ovary.Tail straight.Tail tip straight, rounded to conically rounded, sometimes with a 2-3 µm mucron.
Habitat and locality: Cultures on B. fuckeliana-PDA medium were started from individuals isolated from the phloem (0.5 cm deep) obtained from a dying elm, Ulmus minor Miller, 1768 (Ulmaceae), showing symptoms of dieback, dark-colored ring in cross-section of wilted branches, trunk with galleries of larvae and pupae of Scolytus jaroschewskii Schevyrew, 1893 (Curculionidae: Scolytinae).These were collected by Dr. Alexander V. Petrov in the Samursky Forest Reserve, near the Caspian Sea (41.844639, 48.544833), Republic of Dagestan, Russia, 27 August 2021.Several cultures were started from dauer juveniles extracted from the dissected female adults of S. jaroschewskii collected simultaneously from galleries in phloem.
Materials: Material was obtained from 2-week-old fungus cultures.20 females and 20 males (slides P-4529-4534) were deposited in the Nematode Collection of the Zoological Institute RAS, Saint Petersburg, Russia.
Remarks: There are some differences between the Dagestan population and Poland population.The spermatheca is not axial, but a side branching sac of the genital tube, sperm is ellipsoid and not spherical, and the median bulb is distinctly pyriform with posteriorly shifted inner valve vs. median bulb ovoid with central valve.Such new morphological features expand the characteristics of B. michalskii.The host plant and vector in the Dagestan population are also different: the vector Scolytus jaroschewskii and the host Ulmus minor for the new record vs. S. scolytus and U. laevis in the original description [47].

Characterization of Other Nematodes from Woody Plants
Molecular characterization of nematodes was carried out based on analysis of the D2-D3 of 28S rRNA gene.The phylogenetic positions of studied samples within closely related species and genera are given in Figure 3 and Figures S1-S3.

Aphelenchoides
Aphelenchoides heidelbergi (Figure S4D-G)-Nematodes were extracted from galleries of bark beetles and maintained on Botryotinia fuckeliana culture.Sequences obtained from two nematode samples (Saint Petersburg and Dagestan) of this species were identical to each other and to that from Portugal.The percentage of its identity shared with other published sequences of this species from Australia, Turkey, the USA and China varied from 99.01 to 99.86%.The finding of A. heidelbergi is a new report of this species in Russia.This species was originally described from wood of the exotic pine, Pinus radiata, in Victoria, Australia, and then in the USA and Portugal [48][49][50].
Two unidentified Aphelenchoides sp.1 (Figure S4A-C) and sp.2 were reported from Quercus and Betula, respectively.Sequences of Aphelenchoides sp.1 differed in 1.4% (10 bp) from each other and had the highest percentage of identity (86.6%) with Aphelenchoides eldaricus.The sequence of Aphelenchoides sp.2 was identical to that of unidentified Aphelenchoides sp. from Belgium (KX356787) (Figure 3).
Aphelenchoides is one of the largest genera under the order Aphelenchida and has the large number of hosts as well as a wide distribution [51].According to the literature data, the following Aphelenchoides species were reported from living and freshly fallen wood in Russia: A. clarus, A. hamatus, A. macromucrons, A. paramonovi. A. parasexlineatus, A. rhytium, A. saprophilus and several other unidentified species [12,15,20,21,52,53].

Bursaphelenchus
Bursaphelenchus eremus (Figure S5D-F)-Nematodes were collected both from beetle galleries and beetles in oak groves, where most trees were dying.Its detection is a new report of this species in Russia.The sequence of this sample from Nizhny Novgorod clustered with that of B. eremus (AM396568) from Germany and differed from it by absence of an insertion (~19 bp) (Figure 3).The insertion was not observed in other Bursaphelenchus and aphelenchoid species and, perhaps, it is the result of a sequence reading mistake.
Bursaphelenchus fraudulentus (Figure S5A-C)-Nematodes were extracted from galleries of the Cerambycidae beetle.Two sequences obtained from samples collected in Belarus and Moscow, Russia, were different in 1 bp (0.1%) and showed higher identity with sequences of this species deposited in the GenBank (Figure 3).
Bursaphelenchus mucronatus (Figure S5G-I)-Samples were taken from galleries and living beetles Polygraphus proximus.The sequence of this sample collected in Buryatia showed the highest identity of 99.72% with sequences of this species from Germany (AM396572) and Mexico (EU295494) (Figure 3).
Bursaphelenchus willibaldi (Figure S6A-C)-Specimens were extracted from wilted and dying Quercus robur trees, and bark with galleries of Scolytus intricatus.The sequence of this sample collected in Nizhny Novgorod was identical to that from Romania (MN879886) (Figure 3).

Deladenus
Deladenus posteroporus-Nematodes were obtained from galleries of Scolytinae beetles.Two sequences of this species collected in the Magadan region are identical to each other and that of D. posteroporus described from packaging wood originating from Canada (KX094978) [64] (Figure S1).It is the first report of this species in Russia.The genus Deladenus contains more than 30 valid species [65,66].A few species, including D. laricis [67], D. obesus [19], and several unidentified species [21] were reported in Russia.

Diplogasteroides
Diplogasteroides nix-Nematodes were extracted from galleries of Scolytinae beetles.This sequence is identical to that of this species (LC145090) from Japan and very similar to those of D. andrassyi and D. asiaticus [68] (Figure S2).This is the first report in Russia and the second recorded outside the type locality in Japan.Another species, Diplogasteroides sexdentati, was reported in the former USSR and found in Russia [69].

Laimaphelenchus
Laimaphelenchus hyrcanus-Specimens were extracted from galleries of the bark beetle Scotylus sp.The sequence of this sample showed the highest similarity (99.45 and 99.86%) with that of L. hyrcanus from Iran (KJ567061) and Serbia (KF881746), respectively (Figure 3).It is the first report of this species in Russia.This species was described from bark samples of cypress (Cupressus sp.) and jujube (Ziziphus jujube) from Iran [70] and bark of a wilting black pine (Pinus nigra) from Serbia [71,72].

Micoletzkya
The genus Micoletzkya includes more than 20 nominal species and most of these species were isolated directly from the wood-boring Curculionidae including Scolytinae or from their breeding galleries.The association seems to be very specific and obligatory for the nematodes [76].In this study, one unidentified Micoletzkya sp. was found as adult individuals and dauer juveniles collected on beetle surface in Buryatia, Baikal Nature Reserve.The sequence showed the highest similarity (98.87%) with that of Micoletzkya sp.(JX163966) from the bark beetle Dryocoetes autographus from Germany (Figure S2).

Parasitaphelenchus
Parasitaphelenchus sp.1-Nematodes were extracted from galleries of Ips amitinus and from live beetles.The sequence showed the highest similarity (93.70%) with that of Parasitaphelenchus costati (LC269967) from Japan (Figure 3).Currently, the genus Parasitaphelenchus contains 16 species.One species, P. macrohami, was described in Russia by Lazarevskaya [10].The majority of the members were described in the early-middle 20th century.Therefore, many important typological characters are missing and no molecular information is available for these nominal species [77].

Parasitorhabditis
Parasitorhabditis sp.1-The sequence of this sample collected in Buryatia showed the highest identity in 93.51% with a sequence of Parasitorhabditis obtusa (EF990724) from Germany (Figure S3).Nearly 44 species are currently known in the genus Parasitorhabditis.Some of the species are inquirenda due to inadequate descriptions [78].The nematodes infect the digestive tract and occur in the lumen of the intestine of insects.Several species of Parasitorhabditis were reported in Russia, including P. palliati, P. acanthocini, P. fuchsia and P. sexdentati [19,52,62,[79][80][81][82].

Tylenchid nematodes
Sphaerulariid nematode sp.1, tylenchid nematodes sp.1 and sp.2-The sequences of these samples are clustered within Tylenchida and, based on phylogenetic analysis, they belong to unknown genera of the families Sphaerulariidae and Anguinidae, respectively (Figure S1).

General Discussion
Risk prediction at the early stages of biological invasions is the most cost-effective strategy to control invasive species [83].To assess such risks, it is necessary to find refugia of potentially expandable species and identify associations both within a refugium and at migration points outside the native habitat.
The case of transformation into a true pathogen is well known for Bursaphelenchus xylophilus, because in its native areas in North and Central America, this species does not kill healthy pines.This invasive species invaded Asia in 1905 and Europe in 1999, and then transformed from a weak opportunistic pathogen to a forest pest of world quarantine importance for conifers, causing the PWD [84][85][86].
It is difficult to predict with high probability which opportunistic pathogenic species will become a true pest.Therefore, the most economical method is to collect data on distant associations of opportunistic phytopathogenic and other entomochore species associated with xylobiont insects.These data will make it possible to evaluate the ability of a harmful nematode to change vector and host plants, as well as to identify refugia and pathways of pathogen migration along trade routes.
The identification of entomochore saprobiotic and predatory nematodes in the associations of bark and longhorn beetles in refugia allows us to understand which nematode species can be used for biological control of plant pathogens during accidental anthropogenic transmission and during timber trade or the use of wood packaging.Data on entomochore nematode associations are of particular value for Russia.The fauna of xylobiont nematodes in this vast area are poorly studied.It can be assumed that there are many refugia of potentially dangerous species, as well as agents of biocontrol of these species.Russia has an active timber trade, which increases the risks of potentially dangerous invasive species migrating to purchasing countries in Europe, North America and Asia.
Newly recorded associations of nematodes, vectors and hosts of the Bursaphelenchus species contribute to understanding the origin and evolution and life cycles within the lineages (species groups) of the pathogenic genus Bursaphelenchus and may be used for the mapping of possible refugia of the species.
Abietinus group: Bursaphelenchus zvyagintsevi sp.n. is most similar to B. sinensis and B. abberans, both in specular and general morphology and molecular characteristics.However, the data on the associations of the latter two species are not complete, because their vectors were not identified.Moreover, B. abberans is suspected to be conspecific with of B. sinesis [35,87].Both species were recorded only from wood of Pinus spp.trees.The species list of pine hosts for B. sinensis is as follows: Pinus densifora, P. thunbergii and Pinus sp.found in China, Japan and Korea [33,88,89] and all native records were from East Asia.The first detection of B. sinensis was on wood packaging material in Austria.It was intercepted by quarantine services, but other secondary dispersal cases to Europe are not excluded.Bursaphelenchus aberrans has been reported from Pinus massoniana in China [35,90] and Pinus merkusi in Thailand [91];.In Europe it was intercepted by quarantine services in Austria in load boards and pallets, tree not specified [92].The present record of B. zvyagintsevi sp.n. with exact vector data and the deciduous tree host Fraxinus gives new information on the B. sinensis clade within the Abietinus group.However, it cannot be interpreted phylogenetically because of a lack of data for vectors of B. aberrans and B. sinensis.The origin from native refugia in East Asia and the tree host shift from coniferous to deciduous hosts are evident for B. zvyagintsevi sp.n.
Eggersi group: The new Caucasian record of B. michalskii is the second-furthest distance record of this rare species that was originally found in the Malta urban forest, Poznan, Poland [47].The host plant and vector in our population are different but phylogenetically very close: the vector Scolytus jaroschewskii and the host Ulmus minor is a new record vs. S. scolytus and U. laevis in the original description [47].Based on the new Dagestan record, the conclusion of Kanzaki et al. [93,94] is confirmed: the subgroup 2 of the Eggersi group is associated with the Scolytus spp.vectors and the Ulmaceae woody plant hosts.The B. michalskii species in far-distanced natural refugia is associated with the Scolytus spp.vectors and elm hosts of the family Ulmaceae.It is highly likely that coevolution within nematode-vector-host association took place.
Another species within the Eggersi group is B. eremus, which was recorded in the Nizhni Novgorod region, in galleries of Scotylus intricatus in the wilted Quercus robur.All records of this nematode are known from the same vector species and Quercus spp.[95][96][97].
Fungivorus group: The B. willibaldi extracted from in Quercus robur bark and Scolytus intricatus vector is the first distinct identification of the vector species for this nematode.The original description of B. willibaldi from Germany [98] was based the Pinus sylvestris chips; other records are of its associations in Abies sp., Romania [99], and in Fagus sp., Iran [100].It may be concluded that B. willibaldi is presumably specific for the Pinaceae and Fagaceae host plants, and for Scolytus spp.vectors.Generally, the Fungivorus group is neither specific to hosts or vectors, nor distinctly pathogenic; these species are weak opportunistic pests participating in dead organic matter destruction [9].
Xylophilus group: The records of B. mucronatus in Pinaceae hosts and B. fraudulentus in deciduous hosts only confirmed the known host specificity and distribution areas of these species [101].
Among other taxa, the insect endoparasites Deladenus posteroporus, Parasitaphelenchus sp.1, and Parasitorhabditis sp.1 are possible biocontrol agents for insect vectors, especially Parasitorhabditis sp.1 for the invasive bark beetle Polygraphus proximus and Parasitaphelenchus sp.1 for Ips sp.The predator Micoletzkya sp.1 presumably may be used to control Bursaphelenchus spp.pending development of their cultivation.Species of the Aphelenchoides, Laimaphelenchus and Cryptaphelenchus are the fungal feeders associated with insect vectors and involved in the dead wood destruction; presumably, they restrict the multiplication of the pathogenic fungi in native habitats.

Conclusions
The identification of natural refugia of potential plant pathogens, which include opportunistic pathogenic species of the genus Bursaphelenchus and other entomochore Aphelenchoididae, is necessary for risk assessment and the prevention of new biological invasions.In these natural habitats of the Bursaphelenchus spp., entomoparasitic nematodes may be potential biocontrol agents of their insect vectors, and mycophagous nematodes from the vector galleries presumably prevent the intense expansion of entomochore phytopathogenic fungi vectored by insects together with nematodes.All of these nematode species were identified, either as pathogens or as potential biocontrol agents.Simultaneously, the mapping of xylobiont nematode associations was carried out to understand directions of possible expansion pathways.An integrated approach was used involving both molecular and morphological identification.During the present study, the refugia of rare species of the genera Bursaphelenchus, Aphelenchoides, Laimaphelenchus, Parasitaphelenchus, Deladenus, Diplogasteroides, Parasitorhabditis and others were identified.Their genetic characterization and the detailed morphological description of two rare Bursaphelenchus species, B. zvyagintsevi sp.n. and B. michalskii, are given.It is revealed that the species of the Bursaphelenchus groups Abietinus, Eggersi and Xylophilus are presumably characterized by narrow vector and host plant specificity, restricted mostly at the genus level of the associates.

Sample Collection and Processing
Wood and bark samples were collected during the surveys conducted in different regions of Russia and Belarus in 2014-2022 (Table 1).The 150-500 cm 3 bark and wood samples with galleries of insects were obtained from trunks and 5-10 cm diam.branches using a folding garden saw, field axe, pruner, and slotted screwdriver with sharpened 3-5 mm tip.
Nematodes were extracted from wood and bark samples for 6-16 h at room temperature using a modified Baermann funnel technique [102].The bark galleries in the phloem and outer layer of wood were first cut with a scalpel and then put in an extraction chamber with a double cotton filter (6 cm diam.cotton pad and 10 × 10 cm cotton-cellulose tissue).This chamber was put into a plastic-bottomed glass into which tap water was poured, thus, the sample was covered with water rising from the bottom of the glass through a filter.Actively moving nematodes passed down through a filter, while the debris particles were trapped.It was determined that before sorting into species and processing, isolated nematode suspensions can be stored for up to 3-4 months at +8 • C in 0.5 mL tubes with a drop of 0.15-0.25 mL of tap water or Ringer's solution to ensure adequate oxygen access.
Dauer juveniles from bark and longhorn beetles collected from bark galleries and elytra of insects were submerged in Ringer's solution (0.9% NaCl) for 30 min.Additionally, insects and their senior larvae were cut in a drop of Ringer's solution on a plastic Petri dish with a scalpel.The suspensions extracted from bark and obtained from insects were studied under the stereomicroscope Mikromed MC-5 Zoom LED and the nematodes were picked out with a needle to select different taxa for propagation in vitro.Extracted nematodes were cultivated on laboratory cultures of the fungus Botryotinia fuckeliana, (de Bary) Whetzel, 1945 (=Botrytis cinerea Pers., 1794), growing on 2% potato dextrose agar medium at 22 • C [28,103].Aphelenchoides heidelbergi, Aphelenchoides sp.1, Bursaphelenchus eremus, B. fraudulentus, B. michalskii, B. mucronatus, B. willibaldi and B. zvyagintsevi sp.n. were maintained in the fungus culture and used for morphological and molecular studies.

Morphological Study
For the morphological study, specimens were fixed in hot TAF (4% formaldehyde with addition of 2 mL of triethanolamine for 100 mL of solution) according to the technique developed by Ryss [102,104].Fixed nematodes were processed in glycerin according to Ryss [105] and embedded in permanent slides.Statistical morphometric parameters were determined in MS Excel.All slide-mounted nematodes were measured and photographed under an automated Leica DM5000 B microscope with differential interference contrast (DIC) and a Leica DFC320 (R2) digital camera with Leica DFC Twain Software for PC and Leica IM50 Image Manager for PC.Illustrations were made using a camera lucida and series of photographs.All measurements were analyzed using the software ImageJ 1.48v (http://imagej.nih.gov/ijaccessed on 10 January 2023).Permanent slides were deposited in the Nematode Collection of ZIN RAS, Saint Petersburg.

Molecular and Phylogenetic Study
DNA was extracted from several specimens using the proteinase K protocol.DNA extraction and PCR protocols were performed according to Subbotin [106].The following primer sets were used in this study: (i) D2A (5 -ACA AGT ACC GTG AGG GAA AGT TG-3 ) and D3B (5 -TCG GAA GGA ACC AGC TAC TA-3 ) amplifying the D2-D3 expansion segments of the 28S rRNA gene, and (ii) TW81 (5 -GTT TCC GTA GGT GAA CCT GC-3 ) and AB28 (5 -ATA TGC TTA AGT TCA GCG GGT-3 ) amplifying the ITS rRNA gene.The successfully amplified fragments were sequenced using the primer pairs used in PCR.The new sequences for each gene were edited, compared with other sequences available in GenBank database, and the relevant sequences were retrieved.New sequences were aligned using ClustalX 1.83 (parameters: gap opening 5.0 and gap extension-3.0)with corresponding selected and published gene sequences of nematodes [9,64,68,76,107].Sequence datasets were analyzed with Bayesian inference (BI) using MrBayes 3.1.2and PAUP 4.0 as described by Subbotin [108].The new sequences were submitted to the GenBank database under accession numbers indicated in Table 1 and phylogenetic trees.
Species delimitation was performed using an integrated approach that considered morphological and morphometric evaluation combined with molecular criteria based on phylogenetic inference and sequence analyses.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants12020382/s1, Figure S1: Phylogenetic positions of studied samples within some species from the order Tylenchida nematodes as inferred from Bayesian analysis of the D2-D3 of 28S rRNA (ntax = 46 nchar = 987) gene sequences.Posterior probability values more than 70% are given on appropriate clades.New sequences are indicated in bold; Figure S2
) or with those of B. sinensis and B. juglandis (Figure 4B) in the ITS rRNA gene tree.The D2-D3 28 rRNA gene sequence of B. zvyagintsevi sp.n. differed from that of B. sinensis in 9.3% (66 bp).The ITS rRNA gene sequence of B. zvyagintsevi sp.n. differed from those of B. sinensis in 26.7-28.0%(212-223 bp) and from that of B. juglandis in 33.3% (285 bp).

Figure 3 .
Figure 3. Phylogenetic position of the studied species relationships within some representat from the superfamily Aphelenchoidea as inferred from Bayesian analysis of the D2-D3 of rRNA gene sequences (ntax = 65 nchar = 913).Posterior probability values more than 70% are gi on appropriate clades.New sequences are indicated in bold.

Figure 3 .
Figure 3. Phylogenetic position of the studied species relationships within some representatives from the superfamily Aphelenchoidea as inferred from Bayesian analysis of the D2-D3 of 28S rRNA gene sequences (ntax = 65 nchar = 913).Posterior probability values more than 70% are given on appropriate clades.New sequences are indicated in bold.

Table 2 .
Measurements (in µm) and indices of Bursaphelenchus zvyagintsevi sp.n and B. michalskii.All measurements are in µm and in the form: mean ± s.d.(range).