Received: 8 August 2018 | Revised: 22 April 2019 | Accepted: 10 June 2019 DOI: 10.1111/efp.12534

ORIGINAL ARTICLE

Report of Bursaphelenchus crenati Rühm, 1956 (Nematoda: Parasitaphelenchinae) from Belarus and Russia with a diagnostic key and phylogeny of the Sexdentati group

Alexander Yu. Ryss1 | Kristina S. Polyanina1 | Alexander V. Petrov2 | Alexander A. Sazonov3 | Michail Yu. Mandelshtam4 | Sergei A. Subbotin5,6

1Zoological Institute of the Russian Academy of Sciences, St. Petersburg, Russia Abstract 2Institute of Forest Science of the Russian The endoparasitic nematode, Bursaphelenchus crenati, in beetle tunnels of Hylesinus Academy of Sciences (ILAN), Uspenskoe, crenatus from the ash Fraxinus excelsior L. with signs of ash dieback, is recorded for the Russia 3RUE "Belgosles", Minsk, Belarus first time in Central Russia and Belarus. Third‐stage dauer juveniles were extracted 4Saint Petersburg State Forest Technical from the bark beetle, H. crenatus, and cultured on Botrytis cinerea. Morphological, University, St. Petersburg, Russia morphometric and molecular analyses of these populations are presented here. Some 5Plant Pest Diagnostics Center, California morphological differences of dauers and adults were found between Belarusian and Department of Food and Agriculture, Sacramento, California Russian populations, which may be attributed to seasonal variations. For description 6 Center of Parasitology of A.N. Severtsov of new Bursaphelenchus species, it is recommended to indicate not only the num‐ Institute of Ecology and Evolution of the Russian Academy of Sciences, Moscow, ber of lateral incisures but also the number of bands. Recommendations are made Russia to classify the male caudal papillae of Bursaphelenchus into two groups: papilliform

Correspondence and gland papillae with separate numerals. Emended diagnosis, tabular diagnostic Alexander Yu. Ryss, Zoological Institute polytomous key for the species of Sexdentati group with lists of their vectors and of the Russian Academy of Sciences, Universitetskaya Naberezhnaya 1, St plants, is given. Phylogenetic relationships between some species of the Sexdentati Petersburg 199034, Russia. group are also presented based on the analysis of partial 18S, ITS and 28S rRNA gene Email: [email protected] sequences. Funding information The study was funded by State KEYWORDS Academic Programs FSR: AAAA‐ A17‐117030310322‐3, AAAA‐ ash dieback, Bursaphelenchus, dauer juveniles, Fraxinus, Hylesinus, molecular phylogeny A17‐117080110040‐3; grant RFBR 17‐04‐00360a.

Editor: S. Woodward

1 | INTRODUCTION record of this species in Russia and Belarus. The species was de‐ scribed in Bavaria, Germany, by Rühm in 1956 on the basis of adult In 2017, during a deciduous forest survey in Belarus and Voronezh nematode specimens which were collected from larval galleries of Regions of Russia, dauer juveniles of an Aphelenchoidid nematode the Greater ash bark beetle, H. crenatus, developing in the phloem were found under elytra of the Greater ash bark beetle, Hylesinus and bark of trunks in older European ash, F. excelsior L. Dauer ju‐ crenatus Fabricius, constructing galleries in ash, Fraxinus excelsior veniles were collected from the beetle larvae, pupae and imagoes L. infected with the ash dieback fungus Hymenoscyphus fraxineus (Rühm, 1956). Later, B. crenati was re‐described on the similar ma‐ (T. Kowalski) Baral et al. The nematodes were cultured in the lab‐ terial from Poland by Gu, Tomalak, He, and Fang (2017). Detailed oratory on the fungus Botrytis cinerea. The nematode adults were descriptions of nematode populations from Belarus and Russia with identified as Bursaphelenchus crenati Rühm, 1956. This is the first a phylogenetic analysis of the Sexdentati group are provided here.

Forest Pathology. 2019;00:e12534. wileyonlinelibrary.com/journal/efp © 2019 Blackwell Verlag GmbH | 1 of 16 https://doi.org/10.1111/efp.12534 2 of 16 | RYSS et al.

2 | MATERIALS AND METHODS living but stressed with infections of the ash dieback fungus, Hymenoscyphus fraxineus. 2.1 | Nematode isolation and morphological Dauer juveniles of nematodes were found under elytra of the observation beetles after the latter were submersed in 0.9% NaCl solution. Alive dauers were transferred to 1 week‐old Botrytis cinerea cultures Bark beetles, Hylesinus crenatus, infested with nematodes were (sporeless white strain) seeded in 2% potato dextrose agar which collected from tunnels in sapwood and phloem tissues of liv‐ prepared from fresh potato tubers, agar and sugar (Ryss, 2015). ing, diseased ash trees, Fraxinus excelsior. Samples were taken in Nematodes increased in numbers in 2 weeks after transfer, and the two locations: (a) 52°47'32.2"N 27°59'36.0"E. Forestry planta‐ culture isolates were deposited in the Nematode Collection of the tion “Lyubanskoye Lesnichestvo,” Lyuban, Minsk Region, Belarus. Zoological Institute of the Russian Academy of Sciences (ZIN RAS). November 2017; (b) 51°21'46.8"N 42°02'40.3"E. Forestry plan‐ Nematodes were fixed in hot TAF (4% formaldehyde with addition tation “Tellermanovskoye Opytnoye Lesnichestvo ILAN RAS,” of 2 ml of triethanolamine for 100 ml of solution) according to the Tellermanovsky settlement, Gribanovsky District, Voronezh technique developed by Ryss (2015, 2017b). Fixed nematodes were Region, Russia, June 2017. At both locations, the ash trees were processed into glycerine according to Ryss (2017a) and embedded

TABLE 1 Morphometrics of Bursaphelenchus crenati. All measurements are in µm and in the form: mean ± s.d. (range)

Male Female

Character Central Russia Belarus Central Russia Belarus

n 20 20 25 20 L 698 ± 69 (607—804) 689 ± 69 (590—755) 806 ± 90 (659—1,008) 710 ± 88 (529—827) a 40 ± 5 (34—50) 38 ± 3 (34—43) 48 ± 7 (39—60) 40 ± 3 (36—45) b 9.7 ± 1.2 (7.7—11.3) 11.6 ± 0.5 (10.9—12.4) 10.9 ± 1.6 (8.8—14.1) 11.9 ± 1.1 (9.6—12.9) b' 5.8 ± 0.6 (4.9—6.6) 6.4 ± 0.6 (5.6—7.0) 6.0 ± 1.1 (4.5—7.6) 6.9 ± 0.8 (5.4—8.4) c 24.3 ± 2.9 (20.4—30.5) 23.0 ± 2.0 (20.2—25.2) 22.4 ± 4.8 (17.9—37.2) 23.4 ± 2.8 (17.6—29.1) c' 1.8 ± 0.2 (1.4—2.2) 2.4 ± 0.1 (2.3—2.5) 3.1 ± 0.6 (2.4—4.3) 3.9 ± 0.5 (3.3—5.2) V – – 74 ± 1 (72—77) 74 ± 1 (73—76) Stylet 10 ± 0.1 (8—12) 11 ± 1 (9—12) 12 ± 1 (10–13) 10 ± 1 (8–12) Cephalic region diam. 7.0 ± 0.6 (7—8) 5.9 ± 0.3 (5.4—6.2) 6.6 ± 0.4 (6.1—7.5) 6.1 ± 0.5 (5.0—7.0) Cephalic region height 4 ± 0.5 (3—5) 2.8 ± 0.3 (2.5—3.3) 3.2 ± 0.3 (3—4) 2.7 ± 0.4 (2.1—3.3) Median bulb length (L) 15 ± 2 (13—18) 13 ± 1 (11—15) 16 ± 3 (12—23) 14 ± 1 (12—15) Median bulb diam. (D) 12 ± 1 (11—14) 9 ± 1 (8—9) 13 ± 1 (10—14) 10 ± 1 (8—11) Median bulb L: D 1.3 ± 0.1 (1.1 ± 1.5) 1.5 ± 0.1 (1.3—1.6) 1.2 ± 0.2 (0.9—1.8) 1.4 ± 0.1 (1.2—1.6) Excretory pore from anterior 63 ± 2 (60—77) 57 ± 8 (44—63) 71 ± 8 (59—90) 53 ± 4 (43—58) Nerve ring from anterior 76 ± 5 (66—82) 66 ± 7 (53—70) 79 ± 8 (70—99) 63 ± 5 (55—71) Pharynx 72 ± 8 (58—85) 60 ± 8 (47—66) 75 ± 7 (63—92) 60 ± 4 (52—66) Anterior to gland lobe end 122 ± 12 (105—144) 108 ± 2 (105—111) 137 ± 21 (108—178) 103 ± 9 (89—122) Gland lobe 50 ± 6 (41—64) 48 ± 8 (42—58) 62 ± 16 (40—91) 44 ± 7 (29—60) Gland lobe/body diam. 2.9 ± 0.6 (2.3—4.5) 2.7 ± 0.5 (2.2—3.3) 3.6 ± 0.6 (2.9—4.6) 2.5±0.5 (1.6—3.5) Max. body diam. 17 ± 3 (14—21) 18 ± 1 (17—19) 17 ± 2 (13—20) 18 ± 2 (13—20) Posterior genital branch — – 113 ± 17 (88—145) 101 ± 15 (69—118) Posterior genital branch/vulval diam. – – 6.7 ± 1.1 (5.0—9.2) 5.7 ± 0.6 (4.4—6.6) Posterior genital branch/vulva–anus – – 66 ± 4 (61—79) 66 ± 6 (56—75) distance (%) Tail 29 ± 3 (26—35) 30 ± 0.6 (29—31) 37 ± 7 (20—48) 30 ± 2.4 (25—35) Tail diam. 17 ± 2 (12—19) 13 ± 0.6 (12—13) 12 ± 2 (8—14) 8 ± 0.9 (6—9) Annuli (width of 10 at midbody) 11 ± 2 (9—15) 11 ± 2 (10—13) 11 ± 2 (9—15) 11 ± 2 (9—13) Spicule length (arc) 17 ± 1 (16—19) 17 ± 0.7 (16—18) – – Capitulum width 8.4 ± 0.9 (7.3—10.0) 7.6 ± 0.3 (7.0—8.5) – – RYSS et al. | 3 of 16 in permanent slides. Nematodes impregnated with glycerine were photographed under an automated Leica DM5000 B microscope stained with methylene blue for the enhanced distinction of geni‐ with differential interference contrast (DIC) and a Leica DFC320 tal, glandular and excretory structures in adults and dauers. Before (R2) digital camera with Leica DFC Twain Software for PC and Leica staining, nematodes were transferred from glycerine suspension IM50 Image Manager for PC. Illustrations were made using a camera to a 1 μl drop of glycerine on a glass slide, to which a 5 μl drop of lucida and series of photographs. All measurements were analysed saturated methylene blue water solution was added. The drop was using the software ImageJ 1.48v (National Institute of Health, USA, placed on a laboratory table at room temperature for 12 hr for water http://imagej.nih.gov/ij). Permanent slides were deposited in the evaporation and staining. Then, nematodes were transferred to an‐ Nematode Collection of ZIN RAS, St. Petersburg. other small glycerine drop on a different glass slide, heated gently for 1 min for stain contrasting and differentiation, covered with a 2.2 | DNA extraction, PCR, sequencing and cover slip and sealed with nail polish. Caudal papillae of males were phylogenetic analysis studied by the microscopic examination of living males placed ven‐ trally in a drop of water on a glass slide and covered with a cover DNA from nematode samples was extracted from several individuals slip according to the technique used by Braasch & Braasch‐Bidasak, using proteinase K. PCR and sequencing protocols were described 2002; Braasch & Burgermeister, 2002; and personal communication by Tanha Maafi, Subbotin, and Moens (2003). The primer set: D2A of Dr. H. Braasch. All slide‐mounted nematodes were measured and (5'‐ACA AGT ACC GTG AGG GAA AGT TG–3') and D3B (5'‐TCG

(a) (b) (c)

(d)

(e)

(f) (g) (h)

FIGURE 1 Bursaphelenchus crenati. Belarusian isolate. Male. (a)—general body outline. (b)—anterior body part. (c, d, e)— tail tip with caudal papillae and spicules, at different focal points. (f, g, h)—anterior part of testis (reversed laterally) at different focal points. Scale 50 µm for (a) and 20 µm for the rest 4 of 16 | RYSS et al.

FIGURE 2 Bursaphelenchus crenati. Belarusian isolate. Male, living specimen flattened under coverslip (a‐f), specimen in glycerine (g). A— spicules moved forward from cloaca. (b,c)—head and stylet. (c, d, e, f, g)—tail, caudal papillae and spicules. bu—bursal flap, cap—capitulum, cd—condylus, dn—denticles at bursa border, GP—gland papillae, la—lamina, lic—lamina central core incisures, lig—spicule rostrum ligament, P1,P2,P3—papilliform papillae, pl—spicule plate, prj—spicule projection, ro—rostrum, spt—spicule tip, versus—vesicle. Scale 10 µm

GAA GGA ACC AGC TAC TA–3') (De Ley et al., 1999), was used for Ronquist, 2001) under the GTR model. BI analysis for each gene was amplification of the D2‐D3 expansion segments of 28S rRNA gene, initiated with a random starting tree and was run with four chains the primer set: G18SU (5'–GCT TGT CTC AAA GAT TAA GCC‐3') for 1.0 × 106 generations. The Markov chains were sampled at inter‐ and R18Tyl1 (5'‐GGT CCA AGA ATT TCA CCT CTC‐3') (Chizhov, vals of 100 generations. Two runs were performed for each analysis. Chumakova, Subbotin, & Baldwin, 2006), was used for amplification After discarding burning samples and evaluating convergence, the of the partial 18S rRNA gene, and the primer set: F194 (5'‐CGT AAC remaining samples were retained for further analysis. The topologies AAG GTA GCT GTA G–3') (Ferris, Ferris, & Faghihi, 1993) and 5,368 were used to generate a 50% majority rule consensus tree. Posterior (5'‐TTT CAC TCG CCG TTA CTA AGG–3') (Vrain, 1993), was used probabilities (PP) are given on appropriate clades. for amplification of the ITS rRNA gene. The new sequences were submitted to the GenBank under accession numbers: MH666125, MH666163, MH666164, MH668884 and MH668885. 3 | RESULTS The D2‐D3 expansion segments of 28S rRNA, 18S rRNA and ITS rRNA gene sequences of Bursaphelenchus spp. from the GenBank Bursaphelenchus crenati 1956.

(Lange, Burgermeister, Metge, & Braasch, 2007; Ye, Giblin‐Davis, Description (emended). Braasch, Morris, & Thomas, 2007; Dayi et al., 2014; Marek, Zouhar, Adults (Measurements: Table 1). Douda, Gaar, & Rysanek, 2014, Gu et al., 2017, Tomalak, Malewski, Gu, & Zhan, 2017 and others) were also used for phylogenetic recon‐ 3.1 | General morphology struction. Outgroup taxa for each data set were chosen according to previously published data (Ryss, Polyanina, Popovichev, & Subbotin, Body C‐shaped when heat‐killed. Cephalic region set off, ellipsoid, 2015). The newly obtained and published sequences for each width twice that of height, bearing five to six weakly visible annuli gene were aligned using Clustal X. The alignments were analysed and anterior slit‐like sub‐dorsal amphids; cephalic and labial discs ab‐ with Bayesian inference (BI) using MrBayes 3.1.2 (Huelsenbeck & sent. Stylet conus 50% of entire stylet, posterior shaft with three RYSS et al. | 5 of 16

FIGURE 3 Bursaphelenchus crenati. (b) Female. Belarusian isolate. (a)—posterior body part. (b)—General body outline. (c)—lateral field. (d)—tail. (e, f, g, h)—vulval (a) region at different focal planes. I—anterior (c) (d) (i) part. Scale 50 µm for (a and b); 25 µm for I; 10 µm for the rest

(e) (f) (g) (h)

moderately developed basal thickenings, posteriorly sloped. Lateral There are five papilliform papillae P1 (unpaired) 2 µm above cloaca, fields with two bands, usually three incisures, sometimes four in‐ P2 pair ventrolateral just posterior to cloaca and P3 pair ventrolat‐ cisures visible when the two bands are distanced. Median bulb well eral, at one bursa length anterior to bursal flap. Additionally, there developed, muscular, length 1.2–1.5 times more than width, with is one (GP) pair of small submerged gland papillae (papillae designa‐ moderately developed cuticular valve in the centre. Pharyngeal–in‐ tion according to Rühm, 1956) situated posterior to the P3 papillae testinal junction ½ to one median bulb diameter posterior to median closely to medial line of ventral surface. Each papilliform papilla has bulb. Pharyngeal glands in a single lobe overlapping intestine dor‐ small spherical ending surrounded with broad disc‐like base protrud‐ sally, 2.5–3.5 body diameter. Excretory pore located between the ing a papilla above the cuticle surface. Gland papillae have similar level of median bulb valve and nerve ring, mostly at the posterior small endings but devoid of elevated disc‐like base; both gland papil‐ third of median bulb. Tail tip pointed but without mucron or append‐ lae located on a small median cuticle plate bordered by shallow lat‐ age. Vulval flap very small, inverted “U‐shape” in ventral view; its eral folds. From inner side, each gland papilla connected with dense relatively long lateral wings continuing to one body diameter pos‐ filament bearing a small pellet. In glycerine, this pellet may be erro‐ terior to vulva. Male spicule tip sharply conical, cucullus absent, a neously considered as an additional gland papilla. Paired papilliform lateral projection near spicule tip present in each spicule. papillae and submerged gland papillae symmetrical or asymmetrical The general morphology is typical for the Sexdentati group with depending on the tail curvature; such variability may be difficult to the exception of the number of lateral incisures caused by the close interpret (see Kanzaki, 2008). The male tail tip continues to terminal positions of bands (in the species of Sexdentati group, lateral field bursal flap in the form of a short dense filament. Vesicle‐like pel‐ with two bands is visible as a four‐incisure pattern caused by a wide let situated anterior to filament terminus. Bursal flap in ventral view groove between the two bands, but in B. crenati, mostly closely lo‐ blunt petal‐shaped, posterior border truncates to slightly concave, cated bands are visible as a three‐incisure pattern). bearing four denticles. Spicules are paired, separated and typical for the Sexdentati group, 16–19 µm along curved arc, condylus prominent and blunt, 3.2 | Male posteriorly set off from smoothly curved dorsal outline of lamina, Figures 1 and 2; Table 1. Body strongly curved in posterior part. rostrum sharp, linked by a ligament with midline of ventral body Male tail with seven papillae: unpaired P1; paired P2; P3; and GP. wall. Capitulum flattened, ½ spicule length long. Spicule tip sharply 6 of 16 | RYSS et al.

(a) (b) (a)

(b)

(c) (d)

(e)

FIGURE 4 Bursaphelenchus crenati. Belarusian isolate. Dauer juvenile. (a)—anterior part. (b)—general body outline. (c)—excretory pore (exp). (d)—central part of the body with four‐celled genital primordium (gp). (e)—tail tip. Scale 20 µm for (a, c and d); 50 µm for (b); and 5 µm for (e)

FIGURE 5 Bursaphelenchus crenati. Central Russian isolate. conical, devoid of cucullus. In the central core of spicule, two inner Dauer juvenile. (a)—photograph, methylene blue staining of TAF longitudinal incisures dividing spicule body into long median part fixed worm. (b)—drawing from (a). Arrows: genital primordium. with sharply conical spicule ending, and shorter lateral part forming Scale 50 µm a lateral projection near spicule tip. Such paired projections were described for protracted spicules of B. gerberae (Giblin‐Davies, 3.3 | Female Kanzaki, Ye, Center, & Kelley Thomas, 2006). Testis continues to ¾ body length, mostly reflexed ventrally in anterior end. Mature sperm Figure 3; Table 1. Vagina sloping ventrally, laterally bordered with three‐ in the posterior part of testis round, 2–3 µm diam. Spermatids as two celled plunged structures. Posterior uterine sac (PUS) located left to in‐ to three quartiles in the centre of the testis length. testine, mostly with round sperm, long, more than 60% of vulva–anus RYSS et al. | 7 of 16

TABLE 2 Measurements (in μm) and ratios of the dispersal diam. Ovary to the right side of intestine continues up to pharyngeal stages of Bursaphelenchus crenati gland lobe or slightly less. Oocytes in four to five rows anteriorly and one

Dauer J3d Dauer J3d row posteriorly in ovary. Tail short, three to four anal diameters, strongly Stage/Character Central Russia Belarus curved ventrally, its tip broadly conical with 2–3 µm hyaline part.

n 20 20 L 413 ± 20 346 ± 38 3.4 | Dauer juvenile (392–463) (274–408) Body width 8 ± 1 (6–9) 11 ± 1.1 (9–12) Figures 4 and 5; Table 2. Dauer juveniles of the Belarusian popula‐ Pharynx 119 ± 9 (101–128) 101 ± 4 (90–109) tion differ from the Russian ones in smaller length (350 vs. 410 µm in average), shorter tail (21 vs. 37 µm in average), greater distance Pharyngeal gland 43 ± 7 (35–55) 45 ± 3 (40–51) of excretory pore from anterior end (52 vs. 40 µm average) and a Stylet 9.4 ± 0.4 (9–10) 7 ± 0.9 (6–9) curved, narrowly tapering tail versus straight, broadly conical tail. In Lip region width 4.8 ± 1.0 (4–7) 4.7 ± 0.5 (4–6) the Belarusian population, median bulb length‐to‐width ratio is 2.2 Lip region height 2.9 ± 0.3 (2.5–4) 2.7 ± 0.4 versus 1.4 (average) with weak valve versus moderately developed (2.5–3.5) valve in the Russian isolate. Differences in dauer juvenile morphology Median bulb from 49 ± 2 (45–51) 47 ± 3 (40–52) anterior may be due to seasonal effects on bark‐beetle development at the time of sampling (November in Belarus and June in Russia). Genital Median bulb length 11 ± 2 (9–15) 12.6 ± 0.9 (10–14) primordia of dauer juveniles of both populations showed good cor‐ Median bulb width 8 ± 2 (6–10) 5.8 ± 0.5 respondence in structure and sizes to J3D juveniles of the Sexdentati (4.4–6.5) group. Excretory pore from 40 ± 3 (37–44) 52 ± 3 (45–56) anterior 3.5 | The Sexdentati species group Tail 37 ± 3 (30–41) 21 ± 2 (17–26) Anal body width 8 ± 1 (5–9) 6.7 ± 0.9 (5–8) 3.5.1 | Emended diagnosis Genital primordium 14.8 ± 2.1 (11–18) 15.4 ± 1.8 length (GPL) (12–19) Lateral field with two bands (four or rarely three incisures, in Genital primordium width 3.9 ± 0.4 (3–5) 4.4 ± 1.2 (3–7) B. eroshenkii five incisures were detected): the bands commonly (GPW) distanced by a wide groove with a four‐incisure pattern of lateral Spicule length along arc – – field; sometimes (in B. crenati), the bands are situated close to each a 54 ± 8.3 32.0 ± 3.5 other appearing as a three‐incisure pattern. Male tail with seven (44.6–69.6) (26.6–39.0) caudal papillae of two types: papilliform papillae and gland papil‐ b 5.5 ± 0.4 (5.1–6.4) 6.2 ± 0.5 lae. Papilliform papillae: unpaired P1 just anterior to cloacal orifice; (5.3–7.1) paired P2 at the same level or slightly posteriorly to cloacal opening; b’ 3.5 ± 0.3 3.4 ± 0.2 and paired P3 on the posterior half of tail, usually near lateral wings (3.0–4.1) (3.0–3.7) of bursal flap. An additional pair of small round median submerged c 11.2 ± 0.9 16.7 ± 2.3 gland papillae (GP) situated just posterior to P3 at ventral tail midline, (9.9–13.2) (11.4–21.2) near the junction of tail tip and bursal flap (in earlier descriptions of c’ 4.7 ± 0.5 (4.3–5.5) 3.2 ± 0.3 B. eidmanni, B. piniperdae and B. poligraphi, two pairs of gland papillae (2.6–3.6) are indicated; for these species, the presence of a second pair of GP V % or (V) 62 ± 5 (56–72) 62 ± 8 (47–76) needs to be re‐examined). One pair (P4 papilliform), present in Kevini GPL/GPW 3.8 ± 0.5 3.7 ± 0.9 and Fungivorus groups, is absent. Caudal papilla formula: P1 + P2, P3, (3.0–4.5) (2.2–5.6) GP. Female: vulva with small vulval flap and rounded swollen pos‐ GPL/L, % 4 ± 1 (3–5) 5 ± 1 (3–6) terior vulval lip; short cuticular vagina sloping ventrally. Posterior Lip region length to its 1.6 ± 0.3 (1.4–2.4) 1.8 ± 0.3 uterine sack (PUS) occupies at least half of the vulva–anus distance width (1.4–2.6) or longer. Valve in median bulb well developed, central. Female tail Median bulb length to its 1.4 ± 0.2 (1.1–1.7) 2.2 ± 0.3 short, tail tip rounded, or bluntly sloped ventrally. width ratio (1.7–2.8) Life cycle trixenic with transmission (insect vectored) and prop‐ All values are given as mean ± SD (range). agative generations. Propagative generations of nematodes feed on fungal mycelium and in inner bark, cambium and sapwood of dying distance. Anterior uterus short, one body diameter long, attached to tree hosts with beetle tunnels under bark; mostly in conifers but spindle‐like crustaformeria appearing as a three‐section structure. sometimes (B. crenati) in deciduous tree hosts, the transmission gen‐ Spermatheca anterior to crustaformeria forms a blind two‐lobed sac to eration at dauer stage (J3d) is vectored under elytra of beetles of the the right side of genital tube and filled with round sperm of 2–3 µm in family Curculionidae. 8 of 16 | RYSS et al.

TABLE 3 Tabular diagnostic polytomous key to Bursaphelenchus species belonging to the Sexdentati group

Species/Character C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14

B. andrassyi 1 3 4 3 1 3 2 1 3 3 12 2 1 2 B. borealis 2 1 2 2 2 1 3 2 2 3 1 2 1 2 B. crenati 12 3 1 2 123 3 2 2 23 3 12 23 1 1 B. eidmanni 2 2 2 2 3 3 4 2 2 1 1 2 2 2 B. eroshenkii 3 1 2 2 1 4 2 1 2 2 2 23 1 2 B. fuchsi 1 1 3 2 2 3 2 1 2 2 2 12 1 1 B. incurvus 1 1 4 3 1 3 2 2 2 1 2 12 ? 2 B. leoni 2 1 2 2 3 1 2 2 23 2 1 2 1 2 B. naujaci 1 2 4 3 1 3 2 2 23 2 12 2 1 2 B. nuesslini 1 2 1 2 2 4 2 2 3 2 ? 2 1 2 B. piniperdae 1 3 4 3 1 4 2 2 3 2 2 3 2 2 B. pinophilus 1 3 2 1 2 3 1 1 23 1 12 12 1 2 B. poligraphi 1 2 2 2 1 3 2 2 3 4 12 3 2 2 B. rufipennis 2 1 5 3 2 3 3 1 3 3 12 2 1 2 B. sexdentati 1 2 5 3 1 3 1 2 2 3 2 1 1 2 B. silvestris 1 1 2 2 3 1 3 2 1 3 1 2 1 2 B. sutoricus ? ? 2 1 2 4 3 2 2 2 1 2 1 2 B. talonus 2 2 4 3 1 4 3 2 3 3 2 3 ? 2 B. taphrorychi 1 3 2 2 12 4 2 2 3 3 1 2 1 1 B. uncispicularis 1 3 4 1 12 1 1 1 3 2 2 2 1 2 B. vallesianus 1 3 2 2 12 2 2 1 23 2 12 23 1 2

Note: Character 1. Number of lateral field incisures. 1:4; 2:3; 3:5. Character 2. Excretory pore position: (a): at nerve ring or posterior; (b): between nerve ring and median bulb; (c): at median bulb. Character 3. Tail shape in female. (a): conical, hook‐like; (b): conical, straight or very slightly curved; (c): subconical; (d): subcylindrical; (e): cylindrical. Character 4. Tail tip shape in female. (a): mucronate; (b): conical to digitate; (c): bluntly rounded. Character 5. c'‐value in female. 1:2–3; 2:3–4; 3:5 and more. Character 6. Spicule condylus. (a): digitate bent dorsally; (b): rectangular with backward processus; (c): rectangular, slightly bent dorsally; (d): thumb‐ like, straight continuation of the spicule dorsal outline. Character 7. Spicule rostrum. (a): thorn‐like; (b): pointed; (c): digitate; (d): small conical in the centre of elongated and almost straight spicule. Character 8. Spicule tip. (a): cucullus present. (b): cucullus absent. Character 9. Spicule length along arc. 1:26 µm and longer; 2:17–25 µm; 3:16 µm or less. Character 10. Number of incisures (lines) in the central core of spicule 1:1; 2:2; 3:3; 4:4. Character 11. Posterior uterine sack (PUS) length divided into vulva–anus distance, in %. 1:60% or less; 2: more than 60%. Character 12. Stylet length in female. 1:18 µm or longer; 2:13–16 µm; 3:12 µm or less. Character 13. Gland papillae at the tail tip of male just anterior to bursal flap. (a): one pair; (b): two pairs (in old descriptions of B. eidmanni, B. piniper‐ dae and B. poligraphi, two pairs of gland papillae are indicated; for these species, the presence of second pair of GP has to be re‐examined). Character 14. Female body shape after heat relaxation. (a): strongly coiled; (b): weakly curved.

Species list: 11. B. piniperdae Fuchs, 19373 12. B. pinophilus Brzeski & Baujard, 1997 1. B. andrassyi Dayi et al., 2014 13. B. poligraphi Fuchs, 1937 2. B. borealis Korentchenko, 1980 14. B. rufipennis Kanzaki et al., 2008 3. B. crenati Rühm, 1956 15. B. sexdentati Rühm, 1960 4. B. eidmanni Rühm, 1956 (Goodey, 1960)1 16. B. silvestris (Lieutier & Laumond, 1978) Baujard, 19801 5. B eroshenkii Kolossova, 19982 17. B. sutoricus Devdariani, 19744 6. B. fuchsi Kruglik &Eroshenko, 20043 18. B. talonus (Thorne, 1935) Massey, 19565 7. B. incurvus Rühm, 1956 (Goodey, 1960)3 19. B. taphrorychi Tomalak et al., 2017 8. B. leoni Baujard, 19801 20. B. uncispicularis Zhuo, Li X., Li S., Yu, & Liao, 20076 9. B. naujaci Baujard, 19803 21. B. vallesianus Braasch, Schönfeld, Polomski, & Burgermeister, 10. B. nuesslini Rühm, 19564 2004 RYSS et al. | 9 of 16 (Continues) 2002; d'Errico et al., 2015 (review). 2015 2002; al., d'Errico et Braasch et al., 2001; Braasch & Philis, 2002; Ryss et al., 2005 (review); Cermak et al., 2013; Prospero, Polomski, & Rigling, 2015. 1980; Tenkácová Ryss 1991; & Mituch, et al., 2005 1987, Carletti,(review); 2008. Braasch, Tomiczek, 1996; 2000; Penas, Bravo, Pires, & Mota, 2002; Penas, Correia, Bravo, Mota, & Tenreiro, 2004; Escuer, Arias, & M., Bello, A., 2002; Arias, Escuer, & Bello, 2004; Karageorgos, & Skarmoutsos, Kalapanida, , Michalopoulos, 2004; Ryss et al., 2005 (review); Carletti, 2008; d'Errico, 2015 (review). 1987, 1988 & Mituch, Vilagiova 1987; & Mituch, Ryss 1991; et al., 2005 (review) Penas et al., 2004; Penas et al., 2006; Akbulut, Vieira, et al., 2008a; Han, Chung, & Shin, 2009; Tomalak, 2010; Cermak et al., 2013; Vieira & Mota, 2013; Prospero et al., 2015 et al., Ryss 1999; et al., 2005 (review); Calin et al., 2013, 2015; 2015 al., Prospero et Kolossova, 1998. Kruglik & Eroshenko, 2004; Calin et al., 2015; Braasch & Philis, Rühm, Kakulia, 1956; Kurashvili, 1971; Kakulia, & Devdariani, Korentchenko, 1980; Braasch, Metge, & Burgermeister, 1999; References Calin et al., 2013; Dayi et al., 2014 Ruhm, Gu 1956, et al., this 2017; paper Rühm, Kurashvili 1956; et al., 1980; Ryss & Mokrousov, 2014. Baujard, 1980; & Ambrogioni, 1994 Philis, 11994; Philis 1996; & Rühm, Kakulia 1956; & Shalibashvili, Tenkácová & Mituch, 1976; Baujard, 1980; Brzeski & Baujard, 1997 Fuchs, 1937, Rühm, KurashviliFuchs, 1956; 1937, et al., 1980; Tenkácová Brzeski & Baujard, Braasch, 1997; 2001; Penas et al., 2002; Kanzaki et al., 2008; Nguyen, & Lin, Ye, Tan, 2016 Fuchs, 1937, Rühm, TenkácováFuchs, 1956; & Mituch, 1937, Braasch 1987; Switzerland; Czech Republic (European part) (European part) Portugal; Spain, Germany; Austria; Austria; Germany; Spain, Portugal; Russia Slovakia Portugal; Turkey; Republic of Korea; Republic Czech Brunswick); Vietnam Romania Russia (Far East) (AsianRussia part); Romania, Cyprus Germany; Slovakia; Italy; Georgia, Italy; Slovakia; Germany; Russia (Asian part); Cyprus; Germany; Country, region Romania Turkey; Germany; Poland; Belarus; Russia Belarus; Russia Germany; Poland; Germany; Georgia, Russia (European (European Russia Georgia, Germany; France; Cyprus; Greece; Italy; Germany; Georgia; Slovakia Georgia; Germany; France; Poland Germany; The Netherlands; Georgia; Georgia; Netherlands; The Germany; Poland; Germany; Switzerland; USA (Alaska); Canada (New Germany; Switzerland; Slovakia; Slovakia; Switzerland; Germany;

;

Pinus Pinus ; ; spp. , (Lamb.); spp. (Lamb.) sosnowskyi sp. (L.); Larix brutia; P. Ten. L.; P. excelsa L. spp., Pinus Sieb. & Zucc.; Pinus ; Pinus halepensis Pinus ; ; Pinus nigra J.F. Aiton Aiton; sylvestris Pinus sp., Larix Ten.; Picea (Bong.); orientalis Picea Mayer. (Antoine & Kotschy); L. L. sp. group. Nematode vectors, plant hosts and regions of records (L.); P. Mill. Turcz. spp.,koraiensis Pinus ; Pinus radiata D. Don Ten. , ; Picea; spp.; Picea spp.; abies Picea spp., excelsa Picea spp. spp. Pinus sitchensis sylvestris brutia P. Pinus brutia Pinus Arnold Pinus pinea Pinus Nakai; Abies (L.); Pinus cedrus dahurica P. Mill. L; nigra Pinus koraiensis Pinus Plant species Plant Abies cilicica sibirica Pinus Fraxinus excelsior Fraxinus abiesPicea Pinus Abies Pinus pinaster Pinus Abies alba Abies Pinus spp. Pinus Pinus Pinus pinaster Pinus Pinus Picea

‐

(L.), L., I. am sp. ‐ (Eichhoff) Motschulsky; species belonging to the Sexdentati the to belonging species Fabricius gatus autographus Dryocoetes Ratzeburg itinus (Kugel.) Ratzeburg (Germar) Blastophagus (Herbst) (L.) (Kirby) Orthotomicus erosus Unknown crenatus Hylesinus typographus Ips Unknown Vector species Vector subelon Ips Dendroctonus micans Dendroctonus Dryocoetes autographus Dryocoetes Pityokteines curvidens Pityokteines Tomicus piniperda Tomicus Unknown Pityogenes bidentatus Pityogenes Polygraphus poligraphus Polygraphus Dendroctonus rufipennis Dendroctonus crenati eidmanni eroshenkii leoni nuesslini piniperdae pinophilus rufipennis andrassyi B. fuchsi Busaphelenchus B. borealis Nematode species Nematode B. incurvus B. B. B. B. B. naujaci B. B. B. B. poligraphi B. TABLE 4 BursaphelenchusTABLE 10 of 16 | RYSS et al.

Comments to the species list:

1. Bursaphelenchus eidmanni, B. leoni and B. silvestris are not mo‐ lecularly characterized and classified by Braasch, Burgermeister, and Gu (2009) in the Leoni group, which was combined by Ryss and Subbotin (2017) with the Sexdentati group. 2. Bursaphelenchus eroshenkii is not molecularly characterized and con‐ sidered by Kanzaki and Giblin‐Davis (2018) in the Sexdentati group. 3. Bursaphelenchus fuchsi, B. incurvus, B. naujaci and B. piniperdae are not molecularly characterized and classified by Braasch et al. (2009) in the Sexdentati group. 4. Bursaphelenchus nuesslini and B. sutoricus are not molecularly characterized and considered by Braasch et al. (2009) and Kanzaki and Giblin‐Davis (2018) in the Sexdentati group. 5. Bursaphelenchus talonus is not molecularly characterized and con‐ sidered by Braasch et al. (2009) as three‐incisure ungrouped spe‐ cies. It was included by Ryss and Subbotin (2017) in the Sexdentati 2006; Polomski et al., 2006; Akbulut, Vieira, et al., 2008a; Akbulut, Elekcioglu, & Keten, 2008b; Anon, 2010; Dayi & Akbulut, 2012; & Elekes, Toth 2013; Marek et al., 2014; d'Errico et al., 2015. Carletti, 2008;, d'Errico et al., 2015 (review); Nguyen et al., 2016 al., 2002; Penas et al., 2002; Penas et al., 2004; Palmisano & 2004; Michalopoulos al., et Carletti,Ambrogioni, 2008; 1994; Ryss & Mokrousov, Slonim 2014; et al., 2018 Braasch et al., 2004; Gu, Braasch, Burgermeister, & Zhang, Zhuo et al., 2007 al., et Zhuo Lieutier & Laumond, Baujard, 1978; 1980; Polomski et al., 2006; Tomalak et al., 2017; Rühm, 1960; Kurashvili et al., 1980; Tomiczek, 2000; Escuer et References Thorne, Kaisa, Massey, 1935; 1974; 1956, 2003 Devdariani 1974 Devdariani group, because of its similarity with B. rufipennis indicated by Kanzaki et al. (2008). 6. Bursaphelenchus uncispicularis is not molecularly characterized and considered by Zhuo et al., 2007 in the Borealis group sensu Ryss, Vieira, Mota, and Kulinich (2005) and by Braasch et al. (2009) in the Eremus group, and in the present article in the Sexdentati group.

A tabular polytomous diagnostic key to species of the Sexdentati group is given in Table 3. List of vectors and plant hosts for species of the Sexdentati group is presented in Table 4. Greece; Turkey; Czech Republic; China Romania; Switzerland; Georgia; Israel; Portugal; Spain; Greece; Cyprus; Turkey; Croatia; Russia (European part) Germany; Switzerland; Hungary; Switzerland; Germany; China (Yunnan) China France; Italy; Switzerland; Vietnam Poland Germany; Austria; France; Italy; Country, region USA (Utah) Georgia ‐

; 3.6 | Molecular characterization and phylogeny

The D2‐D3 of 28S rRNA gene alignment included 32 sequences of Bursaphelenchus and was 720 bp in length. Sequences of the Abies alba ; Abies Belarusian and Russian populations of B. crenati were identical with L.; Pinus sosnow Franch. each other and with the Polish population. Phylogenetic analysis L.; Picea abies; Pinus L. (L.), pinaster Pinus Douglas ex Loudon ex Douglas L. resulted in the majority consensus BI tree presented in Figure 6.

sylvestris The partial 18S rRNA gene alignment included 11 sequences spp,

Nakai; Pinus nigra of Bursaphelenchus and was 788 bp in length. Sequences of the nigra Aiton; P. skyi Pinus Belarusian and Polish populations were identical. The majority con‐ Pinus sylvestris Pinus Pinus yunnanensis Pinus Picea orientalisPicea Plant species Plant sylvestris Pinus silvatica Fagus Unknown contorta Pinus sensus BI tree is given in Figure 7. Bursaphelenchus crenati formed a basal clade within the Sexdentati group.

L. The ITS rRNA gene alignment included 66 sequences of (Boerner), (Boerner), (Boerner) Bursaphelenchus and was 979 bp in length. Sequences of the Belarusian and Russian populations of B. crenati were identical with each other and with the Polish population. The majority consensus BI phylogenetic tree is presented in Figure 8. Bursaphelenchus crenati Orthotomicus erosus (Wollaston) (Herbst.). Hopk. Unknown unknown Ips sexdentatus Ips bicolor Taphrorychus Monochamus sutor Monochamus monticolae Dendroctonus Ips sexdentatus Ips Vector species Vector formed a basal clade within the Sexdentati group.

4 | DISCUSSION

The morphological characters of B. crenati corresponded to the orig‐ vallesianus unispicularis sexdentati taphrorychi sutoricus talonus silvestris inal description by Rühm (1956) and re‐description of the species B. B. B. B. Nematode species Nematode B. B. B.

TA B L E 4 (Continued) TA made by Gu et al. (2017). Bursaphelenchus crenati may be considered RYSS et al. | 11 of 16

FIGURE 6 Phylogenetic relationships within the Sexdentati group of the genus Bursaphelenchus as inferred from the Bayesian analysis of the D2‐D3 of 28S rRNA gene sequences. Posterior probability values more than 70% are given on appropriate clades. New sequences are indicated in bold. *—species identity of these sequences requires a confirmation

as a pest associate of ash dieback disease of ash Fraxinus spp. planta‐ results, the disease was caused by two agents: the ash dieback fun‐ tions in Central and South Europe, thus representing a perspective gus H. fraxineus (T. Kowalski) Baral et al. (Ascomycota) (= Chalara object of phytopathological research. Differences in morphology fraxinea T. Kowalski, = Hymenoscyphus pseudoalbidus Queloz et al.) of dauer juveniles from two studied populations may be explained and the emerald ash borer Agrilus planipennis Fairmaire (Coleoptera: by seasonal adaptations, and this fact needs to be studied in detail, Buprestidae). In Belarus, these agents were detected in five regions while the molecular results confirmed species identity of both geo‐ (Yaruk & Zviagintsev, 2015). The nematodes B. crenati were detected graphic isolates. in the inner bark and sapwood in all sampling points in areas infected The B. crenati nematodes were detected: (a) as dauer (J3D) with ash dieback. These facts may be explained by a possible syn‐ stage specimens under elytra of vector beetles Hylesinus crenatus, ergist role of nematodes and their vector beetle, causing additional (b) as well as the endoparasitic nematode populations consisting stress of ash trees and thus contributing to infection acceleration. In of different juvenile stages and adults of propagative generation in a stressed living tree, nematodes feed as the endoparasites in phloem inner beetle tunnels and in bark and sapwood of the living ash trees and sapwood tissues of plant host. The same host–parasite relations Fraxinus excelsior stressed by the ash dieback fungus Hymenoscyphus are known for the of pinewood disease: the first stage is the spread fraxineus (Ascomycota) in Belarus and the Voronezh region of Russia. of B. xylophilus nematodes into sapwood and resin canal destruction, According to the data of the state service “Rosselkhoznadzor,” ash ultimately causing tree death; later on, the pinewood nematodes trees in thirteen regions of Russia (eleven regions in European part feed upon the blue‐stain fungi (Futai, 2013). Similar host–parasite re‐ and two regions in Far East) are infected and partially devastated by lations were found for Bursaphelenchus ulmophilus in St. Petersburg. the ash dieback disease (Musolin, Selikhovkin, Shabunin, Zviagintsev, In all living elm trees (Ulmus glabra and U. laevis) dying from fungal in‐ & Baranchikov, 2017). According to the ash dieback monitoring fection caused by Ophiostoma novo‐ulmi vectored by the bark beetle 12 of 16 | RYSS et al.

cambium, bark, xylem and resin canals, in frames of the detritus food chain (Kuroda & Bergdahl, 2008; Mamiya, 2008). However, the only species, Bursaphelenchus xylophilus, changed its lifestyle for aggres‐ sive parasitism in living conifer host tissues: cambium, phloem, sap‐ wood xylem, resin canals and surrounding parenchyma. Nematode transmission takes place by two pathways: (a) during maturation feeding of the young adult beetle gnawing the needle bases and bark on host branches of living uninfected tree; (b) during oviposition of the beetle vector (Monochamus spp.) under bark of dead or dying tree (Aikawa, 2008). Therefore, it may be concluded that different species of myco‐ phagous bursaphelenchs vectored by saproxylic beetles may change their ecological detritivore role to a parasitic lifestyle. Most of the Bursaphelenchus spp. may be cultivated in the laboratory cultures of the fungi Monilinia fructicola (Winter) Honey and Botrytis cinerea Pers., without plant host tissues. B. xylophilus is a proved mycopha‐ gous parasitic nematode, having both obligatory phases: the plant parasitic and mycophagous ones. However, B. cocophilus, the other parasitic nematode of this genus, do not have a mycophagous phase or at least a known mycophagous phase (Ferreira, Mota, & Souza, 2018). In case of coinfection, these nematodes may be dangerous FIGURE 7 Phylogenetic relationships within the Sexdentati synergists which stress the infected tree by multiplying massively group of the genus Bursaphelenchus as inferred from the Bayesian in the cambium and nutrient pathway tissues. This possible danger analysis of the partial 18S rRNA gene sequences. Posterior probability values more than 70% are given on appropriate clades. from Bursaphelenchus spp. is to be taken into account during surveys New sequence is indicated in bold aimed at defining its pathogenic association with wilt and dieback symptoms observed in forest trees. Scolytus multistriatus, the nematodes B. ulmophilus were found in the Bursaphelenchus spp. combine mycotroph feeding (a type of phloem and sapwood, and their dauers were found under elytra and feeding in a detritus food web) and an endoparasitic lifestyle with around head of S. multistriatus (Ryss et al., 2015 and unpublished feeding in plant host tissues, which are responsible for transporting data). In the case of B. ulmophilus, it may be supposed that the nem‐ sap throughout the plant. These nematodes are associated with de‐ atode and its beetle vector additionally stressed the infected tree struction of host tissues that lead to decline and, sometimes, death destroying its cambium, phloem and active xylem (sapwood). In of forest trees. Therefore, bursaphelenchs may be characterized as laboratory experiments with B. ulmophilus and B. crenati, the host necrotrophic plant and fungal endoparasites. specificity was revealed. In experiments using tree branch cuttings Phylogenetic analyses of the Sexdentati group were given by grown in pots and phloem tissue in Petri plates, eightfold to 10‐fold Lange et al. (2007) using the ITS rRNA gene sequence and by Gu population increases were obtained for B. crenati in F. excelsior and et al. (2017) using combined alignment with 18S, ITS and D2‐D3 of B. ulmophilus in Ulmus glabra. In contrast, the final population level 28S rRNA of reference sequences for each species. In our analysis, was lower than inoculum in other deciduous and conifer hosts used we included all available rRNA gene sequences of species from this for an experiment with one exception of Populus nigra where both group. The position of the B. crenati in basal clade of the Sexdentati species of nematodes showed a moderate twofold to threefold pop‐ group is remarkable, and this fact may explain the lateral field struc‐ ulation increase (Ryss and Polyanina, unpublished data). Therefore, ture deviation of B. crenati (three incisures) from the typical number taking into account the ash dieback symptoms observed in infected for the group (four incisures). trees during field sampling, the localization of the nematode in the Since its earlier summary (Braasch et al., 2009; Ryss et al., 2005), destroyed nutrient pathway tissues and the host specificity to the many new species have been described or shifted to and from the native host, it may be concluded that the nematode B. crenati is Sexdentati group. In our emended diagnosis, the Sexdentati group highly likely to be a restrictive pathogen of F. excelsior. At least this includes the species of the former Leoni group diagnosed in intra‐ nematode may be a synergist of the two most important pathogens generic grouping of Braasch et al. (2009). It may be argued by the causing ash dieback in numerous regions of Belarus and Russia. fact of molecular phylogenetic and morphological closeness. The Similar processes of the transformation of saproxylic feeding to most common in the Leoni group is B. borealis. This species has a aggressive endoparasitic lifestyle are known in the Xylophilus group position in the clade of Sexdentati group (Ryss & Subbotin, 2017), within the genus Bursaphelenchus. The majority of the species of the while the morphological taxonomic characters of the Leoni group Xylophilus group are fungal feeders which participate with their detri‐ correspond to the characters of the Sexdentati group. The main dif‐ tivore longhorn beetle vectors in a destruction of dead tree tissues: ference between these groups, according to Braasch's et al. (2009) RYSS et al. | 13 of 16

FIGURE 8 Phylogenetic relationships within the Sexdentati group of the genus Bursaphelenchus as inferred from the Bayesian analysis of the ITS rRNA gene sequences. Posterior probability values more than 70% are given on appropriate clades. New sequences are indicated in bold classification, was a number of incisures of the lateral field: three in The homology of papilliform (P1–P3) and gland papillae (GP) is the Leoni group versus four in the Sexdentati group. But this pattern not evident. The detailed morphology and possible errors in quan‐ depends on the closeness of two bands of the lateral filed and may tification of both types of papillae were analysed by Kanzaki (2008) vary in species. In B. crenati, both patterns can be seen. Male caudal and Giblin‐Davis et al. (2006). The papilla forms seem to be func‐ papilla pattern in both groups is the same. Spicule structures, the tionally different: the papilliform ones are touch‐feeling organs, absence of the cucullus and lamina with two longitudinal inner lines, while gland papillae may combine sensitive and secretory functions. general outline of spicule, show no difference between both groups. Taking into view that a “bursa” is a supplementary organ adhering a Bionomics of both groups show no difference: their plant hosts are male on a female body during copulation, it seems logical that the mostly coniferous trees, and vectors are bark beetles (Curculionidae: combined function of sensory and glandular caudal papillae would Scolytinae). Therefore, the integration of the Leoni group into the also help to adhere the male tail end to female vulval region during Sexdentati group looks justified (Ryss & Subbotin, 2017). copulation. Thus, the nomenclature of the male papillae needs to be 14 of 16 | RYSS et al. revised to provide division into two separate groups of homologies Deutschland und ihre ITS‐RFLP‐Muster. Nachrichtenblatt Des with distinguishing numerals within each group. It may help in the Deutschen Pflanzenschutzdienstes, 51, 312–320. Braasch, H., & Philis, J. (2002). New records of Bursaphelenchus spp. in interpretation of the genus phylogeny (Ryss & Subbotin, 2017). Ryss Cyprus. Nematologia Mediterranea, 30, 55–57. et al. (2005) re‐established the forgotten term “gland papillae” orig‐ Braasch, H., Schönfeld, U., Polomski, J., & Burgermeister, W. inally proposed by Rühm (1956), who used vital staining of nema‐ (2004). Bursaphelenchus vallesianus sp. n. – a new species of the todes in temporary slides. Bursaphelenchus sexdentati group. Nematologia Mediterranea, 32, 71–79. Braasch, H., Tomiczek, C., Metge, K., Hoyer, U., Burgermeister, W., ACKNOWLEDGEMENTS Wulfert, I., & Schönfeld, U. (2001). 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