Description of Bursaphelenchus populi sp.n.(Nematoda ...2363,20-bursa-populi-2010.pdfThe molecular phylogenetic status of B. populi sp. n. both within the genus and within the xylophilus

Nematology, 2010, Vol. 12(3), 399-416

Description of Bursaphelenchus populi sp. n. (Nematoda:Parasitaphelenchidae), a new member of the xylophilus group

from aspen, Populus tremula L., in EuropeMarek TOMALAK ∗ and Anna FILIPIAK

Department of Biological Pest Control and Quarantine, Institute of Plant Protection – NationalResearch Institute, Wladysława Wegorka 20, 60-318 Poznan, Poland

Received: 20 May 2009; revised: 7 October 2009Accepted for publication: 8 October 2009

Summary – Bursaphelenchus populi sp. n. is described from dying and dead aspen trees, Populus tremula, in Poland. The nematodewas found in trees infested with a long-horn beetle, Saperda perforata, and is vectored in the insect haemocoel. The characteristicmorphology of male spicules, extended anterior vulval lip, lateral fields with four incisures, and number and arrangement of malecaudal papillae, place B. populi sp. n. in the xylophilus group. Bursaphelenchus populi sp. n. can be separated from all other species inthat group by the distinctive vulval flap, which is always bent with its distal half sunken in a conspicuous, sharp depression posterior tothe 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 statusof the new species is confirmed by the unique molecular profile of the ITS region (ITS-RFLP). DNA sequencing and phylogeneticanalysis of 28S rDNA region placed the new species close to B. fraudulentus in the xylophilus group. In vitro cross-breeding of thenew nematode with B. xylophilus, B. mucronatus, B. fraudulentus and B. doui revealed full reproductive incompatibility between thesespecies. In laboratory experiments on 2-year-old seedlings B. populi sp. n. was specific to aspen. It did not develop on pine or oakand reproduced only in weakened or dead aspen seedlings. No pathogenicity to the tree host or vector insect was observed. In Botrytiscinerea PDA cultures, body dimensions were significantly altered in both sexes and female tail morphology differed when compared toindividuals extracted from aspen wood.

Keywords – ITS-RFLP, molecular, morphology, morphometrics, phylogeny, Poland, Saperda perforata, taxonomy.

The economic significance of the pine wilt nematode,Bursaphelenchus xylophilus (Steiner & Buhrer, 1934)Nickle, 1970 (Nematoda: Parasitaphelenchidae), as a dev-astating pest of pine in Far East Asia and its recent spreadto new localities in Portugal (Mota et al., 1999) togethercreate a serious threat to coniferous forests in Europeand in other susceptible areas worldwide. The morpho-logical similarity of this nematode to other species fromthe genus Bursaphelenchus, and particularly to those fromthe xylophilus group, has rendered its taxonomic identifi-cation difficult for quarantine services. However, in thelast decade, the combined use of both morphological andmolecular methods has improved diagnostic precision andas a consequence has facilitated the description of a num-ber of new taxa within the genus. So far, ten species havebeen placed into the xylophilus group (Braasch, 2008),

* Corresponding author, e-mail: [email protected]

namely B. xylophilus, B. fraudulentus Rühm, 1956, B.mucronatus Mamiya & Enda, 1979, B. kolymensis Ko-rentchenko, 1980, B. conicaudatus Kanzaki, Tsuda & Fu-tai, 2000, B. baujardi Walia, Negi, Bajaj & Kalia, 2003,B. luxuriosae Kanzaki & Futai, 2003, B. doui Braasch,Gu, Burgermeister & Zhang, 2004, B. singaporensis Gu,Zhang, Braasch & Burgermeister, 2005 and B. macromu-cronatus Gu, Zheng, Braasch & Burgermeister, 2008. In-terestingly, besides B. mucronatus and B. fraudulentus,which are considered native to Europe, and B. xylophi-lus, which has been recently introduced to this region, allnewly described species from the xylophilus group havebeen isolated only from Asia (Kanzaki et al., 2000; Kan-zaki & Futai, 2001, 2003; Walia et al., 2003; Braasch etal., 2004; Gu et al., 2005, 2008). Also, the European typeof B. mucronatus could also have its origin in Asia as, ac-

© Koninklijke Brill NV, Leiden, 2010 DOI:10.1163/138855409X12559479584963Also available online - www.brill.nl/nemy 399

M. Tomalak & A. Filipiak

cording to recent suggestions by Braasch (2008), it seemsto be identical to B. kolymensis.

Both B. mucronatus and B. fraudulentus are relativelycommon in Poland on pine (Pinus silvestris L.) and oak(Quercus robur L.), respectively. Our survey of nematodeparasites and associates of wood-inhabiting insects onother tree species revealed an interesting population ofB. fraudulentus-like nematodes in wood and bark samplestaken from dead aspen (Populus tremula L.). Surprisingly,individuals of that population, which was designatedas Bursaphelenchus sp. PL-03, were unable to produceoffspring in diallelic cross-breeding with other isolatesof B. fraudulentus. Our subsequent detailed study onindividuals of Bursaphelenchus sp. PL-03 reared both inaspen and on B. cinerea/PDA cultures revealed a seriesof morphological, molecular and reproductive differencesbetween this isolate and B. fraudulentus. The researchreported here provides the account of our taxonomicand bionomic studies on the Bursaphelenchus sp. PL-03isolate, which we consider as a new species, proposedherein as B. populi sp. n.

Materials and methods

ISOLATION AND MORPHOLOGICAL EXAMINATION

OF NEMATODES

The research was conducted on the Bursaphelenchussp. PL-03 population of a B. fraudulentus-like nematodeoriginally isolated in 2001 and periodically re-isolatedon other occasions from dying or dead aspen trees (P.tremula) growing in a small stand within an urban forestin Poznan, Poland. Trunks of these trees were usuallyconcurrently infested with long-horn beetles, buprestidsand, occasionally, with other wood-inhabiting insects,mainly bark beetles. Samples of wood and bark withphloem were collected separately, chopped into smallpieces, and subjected to nematode extraction in wateron 18 cm diam. sieves with a mesh size of 0.2 mm.Subsamples of live males and females were transferredto laboratory cultures of Botrytis cinerea on PDA mediafor further rearing, while the remaining individuals wereprocessed for detailed morphological analysis.

For the morphological analysis, adult nematodes werepicked with a fine pipette or mounted eyelash, placed on aglass slide, killed with gentle heat over a flame and exam-ined morphologically in water mounts or processed fur-ther by fixation in TAF and gradual dehydration to pureglycerin (Seinhorst, 1959) for subsequent examination in

permanent mounts. Morphological observations and allmeasurements were done on an Olympus BX50 micro-scope with Nomarski differential interference contrast op-tics. Micrographs were taken with an Olympus C7070digital camera. Forty randomly picked individuals wereexamined for each morphological category. Male spiculelength was measured along the arc (Ryss et al., 2005).

MOLECULAR CHARACTERISATION

The DNA samples of B. populi sp. n. were extractedfrom nematodes reared both in aspen wood and on B.cinerea PDA cultures following the method described byIwahori et al. (1998), with minor modifications, namely,the composition of lysis buffer was 100 mM Tris, pH8.5, 100 mM NaCl, 50 mM EDTA, 1% SDS, 1% β-mercaptoethanol, 100 μg ml−1 proteinase K per 100 μlbuffer. Before incubation the mixture was frozen at−80◦C for 40 min.

Different sets of primers were used for PCR reactionsof each examined region of rDNA. For amplification ofthe ITS1 and ITS2 rDNA regions the forward primerF194 5′-CGT AAC AAG GTA GCT GTA G-3′ (Ferriset al., 1993) and reverse primer 5368r 5′-TTT CACTCG CCG TTA CTA AGG-3′ (Vrain, 1993) were used.Amplification of the D2/D3 region of 28S rDNA was donewith the forward primer D2A 5′-ACA AGT ACC GTGAGG GAA AGT TG-3′ and reverse primer D3B 5′-TCGGAA GGA ACC AGC TAC TA-3′ (De Ley et al., 1999).

For ITS-RFLP analysis, a ribosomal DNA section con-taining the ITS1 and ITS2 regions was amplified by PCRas described by Burgermeister et al. (2005). The obtainedPCR products were purified according to the protocol pro-vided by the manufacturer (Qiaex II Gel extraction kit,Qiagen, Hilden, Germany). Suitable aliquots of the am-plified DNA were digested with 3 units of the restric-tion endonucleases RsaI, HaeIII, MspI, HinfI and AluI.Restriction fragments were resolved by electrophoresis in2% agarose gel using TBA buffer, stained with 1 μg ml−1

ethidium bromide and photographed under UV light.The ITS-RFLP profiles of B. populi sp. n. were calcu-

lated based on the DNA base sequence of the ITS regionby using the restriction site/fragment lengths analysis ofthe computer program BioEdit Sequence Alignment Edi-tor v. 7.0.5.3.

For rDNA partial sequences of B. populi sp. n., PCRproducts were cloned into pGEM-T easy vector (Promega,Madison, WI, USA) and used to transform into DH5α

Escherichia coli cells for further sequencing. Nucleotide

400 Nematology

Bursaphelenchus populi sp. n. from Populus tremula

sequences were determined (IBB, Warsaw, Poland) andcontigs assembled using the SeqMan program from theDnastar software package (DNASTAR, Madison, WI,USA).

The molecular phylogenetic status of B. populi sp. n.both within the genus and within the xylophilus groupwas determined using the program ClustalX for multi-ple sequence alignments. Automatic alignment was thenmanually improved in order to eliminate improper phy-logenetic signals. Phylogenetic trees were generated us-ing Phylip by neighbour-joining (NJ) and maximum parsi-mony algorithms. The maximum likelihood tree was cal-culated with the PhyML program and the Bayesian treewith MrBayes. The substitution model for Bayesian andML analysis was chosen by the Findmodel program. Alltrees were used to create a consensus phylogenetic treeusing a consensus program from the Phylip package. Thespecies names and GenBank accession numbers of the se-quences compared to B. populi sp. n. are shown in thephylograms.

REARING OF NEMATODES ON B. CINEREA ON PDAAGAR PLATES

Depending on the ultimate purpose, three types ofin vitro cultures were used. For examination of themorphological trait stability of the nematode the cultureswere initiated by individual, mature females extractedfrom wood samples. Rearing was conducted in 7 cmdiam. Petri dishes half-filled with a sterile 2% agar. Aftersolidification of the agar, a 5 × 5 mm piece of matureB. cinerea culture on PDA was added to the centre ofthe agar surface. A single female nematode was thentransferred with a mounted eyelash to each plate andallowed to develop and reproduce at 20 or 25◦C for at least14 days. In this method the piece of B. cinerea cultureserved as a food source for nematodes, while the agarbase provided necessary moisture (the pure agar does notsupport good growth of the fungus). Thus, the partiallytransparent media allowed us to conduct observationof nematodes during their development. As all femalenematodes originated from the crowded wild population,at least a proportion was assumed to be impregnatedand ready to produce a new generation offspring. Thecultures were periodically examined under a dissectingmicroscope and, when justified, individual nematodesremoved for detailed examination under a compound lightmicroscope. At least 30 individual cultures were set foreach examined batch of the extracted nematodes.

Identical rearing plates were used for intra- and inter-specific cross-breeding of the nematodes. In this part ofthe research, pairs of juveniles (J3/J4), i.e., one eachfrom each parental population, were transferred with themounted eyelash to the agar surface in individual plates.In the cross-breeding experiments performed, individualsof the Bursaphelenchus sp. PL-03 isolate were crossedwith four B. fraudulentus isolates from Poland (PL-01 andPL-07) and Germany (Helmstedt and H26), two isolatesof B. mucronatus (Mdz-01 and UKR-02 – both of Eu-ropean genotype) from Poland and Ukraine, respectively,and with a single isolate of B. xylophilus (China) origi-nating from Nanjing, China. Since our molecular analy-sis revealed a close phylogenetic relationship of B. po-puli sp. n. and B. doui, a series of similar diallelic crosseswas also performed between these species in order to ex-amine their potential reproductive compatibility. The iso-lates of B. xylophilus (China), B. fraudulentus (Helmst-edt and H26), and B. doui (Ne9/06) were kindly providedby Dr T. Schröder from the Julius Kühn Institute, FederalResearch Centre for Cultivated Plants (formerly FederalBiological Research Centre for Agriculture and Forestry(BBA)), Braunschweig, Germany. The B. xylophilus iso-late had been reared in our laboratory for over 4 years. Atleast 30 individual crosses were performed for each com-bination of the nematode parental lines.

En masse rearing of nematodes required for subse-quent inoculation of tree seedlings was done in 7 cmdiam. Petri dishes with a 7-day-old culture of B. cinereagrown on PDA. Initially, about 30 wild adult individualsof the nematode were picked from the aspen wood ex-tract and, after additional microscopic examination, trans-ferred to growing plates. The cultures were then incu-bated at 20-25◦C for 14-21 days. For inoculation of sub-sequent cultures the nematodes were washed off the orig-inal plate and 100-200 adult individuals were transferredwith a pipette onto fresh B. cinerea/PDA plates. The cul-tures were incubated as previously. Before experimentaluse, all nematodes were washed off the plates with dis-tilled water and the suspension concentrated by sedimen-tation.

IN VIVO REARING OF NEMATODES IN TREE SEEDLINGS

In order to examine the ability of the Bursaphelenchussp. PL-03 isolate to develop on various species of hostplants, the nematodes were transferred to 2-year-oldseedlings of aspen (P. tremula), oak (Q. robur), or pine(P. silvestris) grown in 18 cm pots. Initially, two separatepilot trials were conducted with B. populi sp. n. obtained

Vol. 12(3), 2010 401


from en masse rearing in vitro on B. cinerea/PDA platesand also from freshly extracted nematodes from aspenwood samples. For nematode inoculation an upside-downU-shaped wound was made with a scalpel in the bark ofthe seedling’s stem, ca 10 cm above ground level. Circa1000 nematodes at various developmental stages, and in30-50 μl of distilled water per plant, were pipetted into asmall cotton plug inserted into the wound. After injectionthe wound was sealed with parafilm to protect water inthe cotton plug from evaporation. In pilot experimentsthe wound was made as small as possible and coveredless than one-third of the stem circumference. However,after the initial series of unsuccessful inoculations ofvigorous seedlings, the main experiment was conductedon mechanically weakened seedlings with a trimmedcrown and relatively large inoculation wound, whichextended to over three-quarters of the stem circumference.In each experiment, five seedlings of pine, oak and aspenwere used for inoculation with B. populi sp. n. and thesame numbers used for the control where seedlings weresubjected to a identical treatment with the exception of thenematode inoculum. After 1 month of incubation at 20-22◦C the stems were cut into 4 cm sections, each sectionbeing separately chopped into 5 mm pieces and immersedin distilled water for nematode extraction. The presenceof living nematodes and their position in the stem wererecorded after 24 h extraction. In addition, juveniles andadults of the new nematode generation extracted fromthe seedlings were examined morphologically under acompound microscope equipped with Nomarski optics.

As all subpopulations of Bursaphelenchus sp. (PL-03)reared in vitro showed similar changes in morphologywhen compared with the original population extractedfrom wood samples, additional, detailed observationswere done on the morphology of the in vitro-rearednematodes after their return and single passage throughseedlings of aspen – the original host tree species.

IDENTIFICATION OF B. POPULI SP. N.NATURAL VECTORS

As nematodes from the genus Bursaphelenchus requireeffective vectors for successful colonisation of a new treehost, it was of interest to learn which of the insect speciesassociated with the nematode-infested trees could actuallyperform this function. In order to identify the vector(s)of B. populi sp. n., all adult beetles, which had emergedfrom field-collected (and subsequently stored in the in-sectary) aspen logs, were dissected. After decapitation,the insects were individually dissected in M9 buffer (Sul-

ston & Hodgkin, 1988). All body parts which could poten-tially serve as hiding sites for the nematodes, i.e., thoraxand abdomen surface, internal surface of elytra, trachea,haemocoel and genitalia, were inspected for the presenceof dauer juveniles with characteristic structures, such asaphelenchoid bulbus and head shape. All nematode ju-veniles extracted from the dissected insects were trans-ferred to B. cinerea/2% agar plates (as described above)and reared at 25◦C for at least 2 weeks to initiate dauerrecovery and development of adults to assist identifica-tion.

STATISTICS

Differences in individual morphometric characters be-tween adult B. populi sp. n. extracted from wood samplesand those reared in vitro in B. cinerea cultures were exam-ined using Student’s t-test (for all direct measurements)and the Kruskal-Wallis test (for ratios: a, b, c, c′ and V%)at the significance level 0.05. Data obtained in experi-ments on nematode reproduction in pine, oak, and aspenseedlings were normalised through logarithmic transfor-mation and subjected to ANOVA using Tukey’s multiplerange test (P = 0.05).

Results

ISOLATION AND MORPHOLOGICAL EXAMINATION

OF NEMATODES

So far, the newly described nematodes have beenisolated only from aspen, P. tremula. In the examinedforest stand they are widely distributed in the trunksof dying and recently dead older trees. The nematodescould be periodically re-isolated from the same host treesfor at least 1 year following the tree’s decline or death.A mixture of all developmental stages of B. populi sp.n. was present in samples of both the bark with phloemand wood, with no clear preference to any of these niches.Most nematodes were extracted from samples taken inthe vicinity of larval galleries of Saperda perforata Pall.(Coleoptera: Cerambycidae). Despite earlier records ofaspen and black poplar infestation with B. fraudulentus(Rühm, 1956), no B. fraudulentus was found in theexamined trees, although this species was frequentlypresent in neighbouring oaks.

The characterised population was also successful incontinuous rearing on B. cinerea/PDA plate cultures at20-25◦C, provided that the nematodes were periodically

402 Nematology


transferred to fresh rearing medium. The whole matureculture or nematodes extracted to distilled water couldalso be stored from a few weeks up to 8 months at 1-4◦C.

Detailed morphological and molecular analyses of ne-matodes isolated from the aspen wood and bark samples,and subsequently reared on PDA/B. cinerea cultures, re-vealed that the isolate designated as Bursaphelenchus PL-03 represented a new species. We propose to name thisspecies B. populi sp. n.

Bursaphelenchus populi* sp. n.(Figs 1, 2A-L)

MEASUREMENTS

See Table 1.

DESCRIPTION

Female

Body elongate, cylindrical, ventrally arcuate whenheat-killed. Cuticle finely annulated. Lateral fields withfour incisures. Cephalic region high, offset by constric-tion. Stylet well developed, 15-17 μm long, slightlyswollen at base. Median bulb oval. Pharyngeal gland lobe4-5 body diam. long, dorsally overlapping intestine. Nervering located at 10-30% of body diam. posterior to medianbulb. Position of excretory pore variable from level withanterior end of median bulb to just anterior to nerve ring.Vulva postmedian. Anterior vulval lip elongated, directedposteriorly to form a 9-12 μm long flap (i.e., 50-65% ofbody diam. as measured just anterior to vulva). Flap al-ways bent with distal half sunken in a conspicuous, sharpdepression located immediately posterior to vulva. Vaginaat right angle to body axis or directed somewhat poste-riorly. Ovary monoprodelphic, outstretched. Developingoocytes in two rows. Spermatheca elongated ovoid. Post-uterine sac extending for 57-76% of vulva-anus distance.Ratio of post-uterine sac length to body diam = 7.1 ±0.8 (6.0-8.1). Tail subcylindrical to slightly conoid. In in-dividuals extracted from aspen wood, terminus broadlyrounded or occasionally finely rounded with a small pro-jection of variable shape, but in females reared in vitro onB. cinerea/PDA cultures, tail conoid, gradually tapering tofinely rounded terminus, frequently elongated into a short,1-2 μm projection.

* The specific epithet is derived from the genus of the tree host.

Male

Body ventrally arcuate with tail region sharply curvedventrally when heat-killed. Anterior region similar tothat of female. Testis expanded anteriorly. Tail conoid,terminus pointed. Small terminal bursa present, broadlyrounded in ventro-dorsal view. Spicules paired, large,26-37 μm along arc, arcuate, with capitulum almostparallel to shaft axis. Ratio of spicule length along arcto its width measured posterior to rostrum = 6.6-8.1.Angle subtended between lines along capitulum andextending spicule = 54-65◦. Capitulum 8.8-10.9 μm long,slightly concave with small depression in middle region.Condylus rounded, rostrum elongated, pointed. Distinctdisc-like cucullus present at spicule terminus, 1.5-2.0 μmdiam. Distal fourth of spicule dorsal contour usuallystraight. Distinct short depression present slightly beforecucullus. Seven caudal papillae arranged as follows: onepair of adanal ventrosublateral papillae, a single preanalventromedian papilla just anterior to cloacal opening,two postanal pairs located ventrally and ventrosublaterallynear base of bursa and almost at same level.

TYPE HOST AND LOCALITY

Isolated from a dead aspen tree, P. tremula, found in anurban forest at the Eastern edge of Poznan, Poland (GPS52◦24′49′′N, 17◦0′50′′E).

TYPE MATERIAL

Holotype female (slide no. MIZ 216138), 15 femaleparatypes (slide no. MIZ 216139) and 15 male paraty-pes (slide nos MIZ 216137 and MIZ 216140) depositedin the nematode collection of the Museum and Instituteof Zoology, Polish Academy of Science, 00-679 Warsaw,Wilcza 64, Poland. Two slides, one with 15 female para-types and one with 15 male paratypes of B. populi sp. n.,deposited in the nematode collection of the Julius KuehnInstitute (JKI), Federal Research Centre for CultivatedPlants, Messeweg 11/12, 38104 Braunschweig, Germany,under the accession number Ne 11/09. Plate cultures ofthe nematode are available upon request from the authorsat the Institute of Plant Protection – State Research Insti-tute, Wladysława Wegorka 20, 60-318 Poznan, Poland.

DIAGNOSIS AND RELATIONSHIPS

Bursaphelenchus populi sp. n. is distinctive by thebody length of 1020 (909-1111) μm in female and 850

Vol. 12(3), 2010 403


Fig. 1. Bursaphelenchus populi sp. n. A: Female; B: Male; C: Anterior region (female); D: Head with stylet (female); E: Vulva; F:Mid-body region (female); G: Male tail with spicules (lateral view); H: Male tail with bursa and positions of caudal papillae (ventralview, somewhat flattened); I-K: Female tail (individuals from wood samples); L-N: Female tail (individuals from in vitro culture).

404 Nematology


Fig. 2. Light micrographs of Bursaphelenchus populi sp. n. Adults. A: Head region; B-D: Variable position of excretory pore; E: Lateralfield with four incisures; F: Vulva region; G, H: Female tail (individuals from wood samples); I, J: Female tail (individuals from invitro culture); K: Male tail with bursa (ventral view); L, M: Comparison of spicules with straight and concave distal fourth of dorsalrim in B. populi sp. n. (L) and B. fraudulentus (M), respectively. Dauer juveniles. N: Anterior region; O: Tail.

Vol. 12(3), 2010 405


Table 1. Morphometrics of Bursaphelenchus populi sp. n. All measurements are in μm and in the format: mean ± s.d. (range).

Character Type population from wood In vitro

Female Male Female Male

Holotype Paratypes Paratypes Paratypes Paratypes

n – 40 40 40 40L 1105 1020 ± 61 850 ± 88 657 ± 47∗ 631 ± 40∗

(909-1112) (756-1055) (564-738) (548-674)a 46.5 45.4 ± 3.0 42.0 ± 3.5 33.6 ± 2.0∗ 33.8 ± 2.2∗

(40.2-52.4) (36.1-49.3) (29.3-36.9) (30.9-36.1)b 14.3 13.3 ± 0.8 11.5 ± 1.3 9.5 ± 0.7∗ 9.3 ± 0.8∗

(11.9-14.7) (9.5-14.6) (8.4-10.5) (8.0-10.7)c 27.3 23.2 ± 1.7 25.2 ± 2.8 21.8 ± 1.2 20.2 ± 1.5∗

(20.9-27.3) (21.9-31.4) (19.4-24.4) (17.6-22.5)c′ 3.2 3.9 ± 0.2 2.2 ± 0.1 3.1 ± 0.4∗ 2.2 ± 0.2

(3.2-4.2) (1.8-2.5) (2.6-3.9) (2.0-2.4)V 73 72.9 ± 1.2 – 72.7 ± 0.7 –

(70.4-75.3) (71.8-73.9)Max. body diam. 23.8 22.6 ± 2.1 20.3 ± 1.4 19.7 ± 2.0 18.7 ± 1.0

(18.8-27.5) (17.5-22.5) (16.3-21.3) (17.5-20.0)Pharynx length1) 77.5 77 ± 2.7 74 ± 4.9 69 ± 2.8∗ 70 ± 3.4

(72-85) (65-84) (64-75) (62-74)Excretory pore position2) 83.4 75.2 ± 7.3 81.4 ± 3.9 65.8 ± 0.68∗ 68.6 ± 4.5∗

(59-84) (75-89) (57-73) (60-80)Nerve ring position2) 85.1 83.5 ± 2.4 73.2 ± 6.1 74.5 ± 2.5∗ 76.7 ± 3.7

(78-89) (64-81) (69-80) (69-83)Post-uterine sac length 183 158 ± 16.3 – 109 ± 17.7∗ –

(134-183) (83-139)Stylet length 16 16.1 ± 0.6 15.3 ± 0.7 14.1 ± 0.3∗ 13.8 ± 0.8

(15.1-17.2) (13.8-16.1) (13.5-14.4) (13.1-15.2)Tail length 40.5 44 ± 3.5 34 ± 2.2 30 ± 2.6∗ 31 ± 2.1

(40-53) (27-38) (26-35) (27-35)Anal body diam. 12.5 11.3 ± 0.8 15.4 ± 1.6 9.8 ± 0.8 14.4 ± 0.9

(9.8-12.6) (12.8-18.4) (8.8-11.3) (12.5-15.0)Spicule length (arc) – – 32 ± 4.3 – 28 ± 1.7

(26-38) (26-31)

All measurements of nematodes reared in vitro, marked with asterisks (∗), are significantly different (P = 0.05) from their counterpartsrecorded in individuals extracted from aspen wood samples.1) Distance from anterior end to base of median bulb.2) Distance from anterior end.

(756-1055) μm in male, its relatively slender body shape(a = 45.4 (40.2-52.4) and 42.0 (36.1-49.3) in femaleand male, respectively), lateral fields with four incisures,spicule length of 32.1 (25.7-37.0) μm and relatively large(i.e., 1.5-2.0 μm) cucullus at the spicule tips and thearrangement of the seven caudal papillae in the male(single papilla anterior to cloacal aperture, one pair adanaland two pairs at the base of the bursa). Females of B.populi sp. n. have a long post-uterine sac (158.2 ± 16.3

(134-183) μm) extending for over 57-76% of the vulva-anus distance. Females of B. populi sp. n. are also uniqueamong Bursaphelenchus species in the morphology ofthe vulva region, differing from all other species by thecharacteristic shape of the vulval flap, which is alwaysventrally bent with its distal half sunken in a conspicuous,sharp depression immediately posterior to the vulva. Thesurface of the flap is also irregular or roughened. Themorphology of B. populi sp. n. individuals obtained from

406 Nematology


aspen wood samples and those reared in vitro in B. cinereacultures clearly differ from each other by the shape offemale tail. As the physiological basis for this shift is notunderstood, we believe that it is necessary to consider thisfact in the diagnosis of this species.

Bursaphelenchus populi sp. n. is closely related tospecies of the xylophilus group. It can be easily separatedfrom all species in the other Bursaphelenchus groupsthat partially share the morphological characteristics ofthe xylophilus group. Species of the fungivorus grouphaving four incisures in the lateral fields differ from thexylophilus group by the lack of a vulval flap. Speciesof the sexdentati group, also with four incisures, mayhave a small vulval flap, but the arrangement of thecaudal papillae (i.e., a single papilla anterior to the cloacalaperture, one pair adanal and two pairs post-cloacal, oneof which is located just anterior to the bursa and theother at the bursa) is clearly different from the xylophilusgroup where there is a single papilla anterior to thecloacal aperture, one pair adanal and a double pair at thebase of bursa (see Braasch, 2001, 2008). The presenceof a cucullus on the spicule tips of some species ofthe sexdentati group may also make them similar to thexylophilus group. However, the two groups can be easilydistinguished from each other by the overall shape ofspicules, which in the xylophilus group are unique becauseof their large size, narrowly arcuate shape, contour ofdorsal lamina angular in posterior third of its length, smallrounded condylus and pointed rostrum vs much smallerand compact, contour of the dorsal lamina smoothlycurved or angular at midpoint and condylus and rostrumwell developed in the sexdentati group (see Braasch,2001; Ryss et al., 2005).

Female B. populi sp. n. extracted from wood samplescan be separated from most other species in the xylophilusgroup by the subcylindrical tail with broadly roundedterminus and absence of a distinct mucro although a smallprojection, which is irregular in form or shortly conicalrather than mucro-like, is sometimes present at the tailterminus. In these characters B. populi sp. n. resembles thenon-mucronate form of B. xylophilus, which has a broadlyrounded tail terminus, occasionally with a small conicalprojection (Mamiya & Kiyohara, 1972). By contrast, invitro-reared females of B. populi sp. n. have a conoid tailwhich gradually tapers to a finely rounded terminus andwhich is frequently elongated into a short, 1-2 μm long,projection vs the subcylindrical, broadly rounded tail withno mucro is present in both the wood- and in vitro-rearedfemales of B. xylophilus. Both types of B. populi sp. n.

can be distinguished from B. xylophilus by the positionof the excretory pore which is located anywhere betweenthe level of the anterior margin of the median bulb to justanterior to the nerve ring vs usually located at the level ofnerve ring or somewhat posterior to this organ.

In the subcylindrical form of the female tail extractedfrom wood samples, B. populi sp. n. is also similar to B.fraudulentus, B. doui, B. kolymensis and B. mucronatus.Bursaphelenchus populi sp. n. reared in vitro differs fromall of these species by the clearly conoid female tail.

Bursaphelenchus populi sp. n. extracted from woodsamples differs from B. fraudulentus by the lack of a tailmucro, which in the latter species is usually distinct andca 2 μm long. Bursaphelenchus populi sp. n. reared invitro can be separated from B. fraudulentus by the conoidtail, gradually tapering to a finely rounded terminus orto a short, 1-2 μm long terminal projection. It can alsobe separated from B. fraudulentus by the length of thespicules, which are 26-37 vs 21-22 μm long (as measuredalong the arc), respectively (Rühm, 1956).

Bursaphelenchus populi sp. n. differs from B. doui bythe lack of a tail mucro in females extracted from aspenwood and by the very short, centrally located terminalprojection on the tail of in vitro-reared individuals. Incontrast, the terminal mucro of B. doui is distinct, 2-4 μmlong, and ventrally located. The latter species has alsolarger spicules (34-43 μm long as measured along arc)(Braasch et al., 2004).

Bursaphelenchus populi sp. n. can be separated fromB. kolymensis by the presence of a distinct, 3.5-4.4 μmlong mucro on the female tail of the latter species(Korentchenko, 1980; Magnusson & Kulinich, 1996) andfrom B. mucronatus by the distinct, 3-7 μm long mucroin the latter species (Mamiya & Enda, 1979; Braasch,2008). The morphology of the female tail also separatesB. populi sp. n. from B. luxuriosae, which has a conoidtail gradually tapering to a narrowly rounded and ventrallybent terminus with an irregular dorsal surface (Kanzaki &Futai, 2003).

Bursaphelenchus populi sp. n. can be distinguishedfrom B. singaporensis by the smaller spicules, i.e., 26-37 vs 41-48 μm, as measured along arc (Gu et al., 2005)and from B. baujardi by the more slender body (a = 36-52 vs 25-36, respectively), short terminal projection intail of in vitro-reared females or lack of terminal mucroin females isolated from aspen wood vs distinct hair-likemucro, and presence vs absence of swellings at the baseof stylet (Walia et al., 2003).

Vol. 12(3), 2010 407


Bursaphelenchus populi sp. n. can be distinguishedfrom B. macromucronatus by the presence of a longconical tail with a distinct, pointed, 2.5-6.5 μm long,mucro in the latter species and by the more slenderbody (a = 36-52 vs 24-39, respectively) (Gu et al.,2008). Lastly, the new species differs B. conicaudatusby the more slender body (a = 36-52 vs 27-37), sub-cylindrical (wood-extracted individuals) vs conoid femaletail and which on average is shorter (c = 21-27 vs 13-25) and wider (c′ = 3.2-4.3 vs 3.6-5.0) than in the latterspecies and by the larger spicules (26-37 vs 23-28 μm, asmeasured along the arc) (Kanzaki et al., 2000).

Although not clearly stated in the original descriptions,several species of the xylophilus group have the dorsalrim of spicules concave in the distal fourth or third of itslength (Fig. 2M). This character was depicted in originaldrawings of B. fraudulentus (Rühm, 1956), B. kolymensis(Magnusson & Kulinich, 1996), B. singaporensis (Gu etal., 2005) and B. macromucronatus (Gu et al., 2008)and also in a micrograph of B. xylophilus (Mota etal., 1999). Close examination of large populations ofmales revealed that such a concave rim is also presentin all isolates of B. fraudulentus, B. mucronatus andB. xylophilus used in our study. By contrast, in B. populisp. n. the distal fourth of the spicule dorsal rim wasusually straight (Fig. 2L); a short depression is presentonly just before the cucullus. We therefore conclude thatthis character may also help to separate B. populi sp.n. from all the species listed above. Further research onother, geographically distant, isolates of B. xylophilusand B. mucronatus is, however, needed to confirm theuniversality of this character across populations of suchwidely distributed species.

Besides B. populi sp. n. and B. fraudulentus (Rühm,1956), three other Bursaphelenchus species, namely B.eremus Rühm, 1956, B. idius Rühm, 1956 and B. xe-rokarterus Rühm, 1956 have been recorded in relatedtrees belonging to the family Salicaceae (Ryss et al.,2005). Bursaphelenchus populi sp. n. clearly differs fromthese species by the presence vs absence of a dis-tinctive vulval flap in the female and presence vs ab-sence of a cucullus on the male spicules and by spiculeshape.

MOLECULAR CHARACTERISATION

Amplification of the ITS regions of B. populi sp. n. re-sulted in a PCR product of 980 bp. Restriction fragmentsproduced by digestion of the PCR product with RsaI,HaeIII, MspI, HinfI and AluI are listed in Table 2. Elec-

Table 2. Sizes of DNA restriction fragments obtained in ITS-RFLP analysis of Bursaphelenchus populi sp. n. and calculatedon sequencing results of the ITS region.

PCR product (bp) Restriction fragments (bp)

RsaI HaeIII MspI HinfI AluI

980 529 589 331 292 621429 297 260 232 34122 94 164 157 18

113 155112 64

50282

Fig. 3. ITS-RFLP profile of Bursaphelenchus populi sp. n. Lanes1, 8: DNA marker (100 bp ladder, MassRuler™, Fermentas);Lane 2: rDNA amplification product; Lanes 3-7: Restrictionfragments: Lane 3: RsaI; Lane 4: HaeIII; Lane 5: MspI; Lane6: HinfI; Lane 7: AluI.

trophoretic separation of restriction fragments resulted ina distinctive profile for the ITS-RFLP pattern (Fig. 3)which was different from those of other Bursaphelenchusspecies obtained with the same PCR primers and the sameset of restriction enzymes (see Gu et al., 2008; Burger-meister et al., 2009).

The new species can be distinguished from othersequenced Bursaphelenchus species by its characteristicsequence containing complete regions of ITS1, 5.8S andITS2, and partial 18S and 28S regions of rDNA. TheDNA base sequences of B. populi sp. n. are deposited inGenBank with the accession numbers FJ888483 for 18S,ITS1, 5.8S, ITS2 and 28S and FJ998281 for the D2/D3region of 28S rDNA.

Based on four molecular phylogenetic trees generatedfrom partial 28S rDNA with Bayesian, maximum likeli-hood, neighbour-joining and maximum parsimony algo-rithms (Fig. 4) and the consensus tree based on these fouralgorithms (Fig. 5), it can be concluded that B. populisp. n. clearly belongs to the xylophilus group. It is most

408 Nematology


closely related to B. fraudulentus, B. doui and B. conicau-datus.

REARING OF B. POPULI SP. N. ON

B. CINEREA/PDA-AGAR PLATES

Reproduction in vitro

Bursaphelenchus populi sp. n. can easily develop andreproduce on B. cinerea/PDA cultures. The new gen-eration nematodes maintained an excellent vigour andhigh reproduction rate on a series of subsequent in vitrocultures. A detailed study on individual in vitro rear-ing of freshly extracted round-tailed females from aspenwood revealed that 56-77% of the nematodes were al-ready impregnated and successfully produced new gen-eration offspring. As expected, the reproduction rate wastemperature-dependent and from a single female a popu-lation of 200±154 (57-364) nematodes developed at 20◦Cand 869 ± 221 (653-1196) nematodes at 25◦C, within 14days of rearing. In spite of continuous inbreeding, severalsubpopulations of B. populi sp. n., originally initiated byindividual females, have been reared in vitro for over tengenerations with no apparent negative effect on reproduc-tion rate.

However, when B. populi sp. n. is reared in vitro themajority of body measurements and their ratios changed,as compared with individuals extracted from aspen woodsamples. The measurements based on individuals ob-tained from in vitro culture, initiated by a population of30 wild females and males of B. populi sp. n. randomlytaken from wood extract, are given in Table 1.

All the new generation females had a conoid tail with afinely rounded terminus, frequently elongated into a shortprojection (Figs 1L-N; 2I-J). This character remainedrelatively stable in all subsequent nematode generationsreared on B. cinerea/PDA plates.

Diallelic cross-breeding

Diallelic cross-breeding of B. populi sp. n. with selectedisolates of B. fraudulentus (PL-01, PL-07, Helmstedt andH26), B. mucronatus (Mdz-01 and UKR-02), B. doui(Ne 9/06), and B. xylophilus (China) done in vitro onB. cinerea/PDA-2% agar plates did not produce anyoffspring. In contrast, 26-41% of intra-specific controlcrosses between individuals from the same isolate andbetween individuals from different isolates of the samespecies resulted in the production of viable offspring onthe experimental plates. In these plates, adults of the newgeneration offspring were recorded after 7-10 days at25◦C.

IN VIVO REARING OF NEMATODES IN TREE SEEDLINGS

In the pilot experiment, inoculation of vigorously grow-ing aspen, oak and pine seedlings with B. populi sp. n.obtained from either in vitro rearing on B. cinerea or withthose extracted from wood samples was unsuccessful. Thenematodes could not develop and establish in any of theexamined plants and most of the introduced individualsdied within 30 days of the start of the experiment. No liv-ing nematodes were found in inoculated pine seedlings af-ter the experiment. Only occasionally, a few B. populi sp.n. individuals were recovered from aspen, i.e., 7.8 ± 5.3(2-16) and 6.4 ± 3.5 (3-12) of in vitro- and in vivo-rearedindividuals per plant, respectively, and oak seedlings, i.e.,5.0 ± 4.1 (1-11) and 4.8 ± 2.6 (2-8) of in vitro- and invivo-reared individuals per plant, respectively. However,in these plants the nematodes remained in the site of theiroriginal placement (i.e., the cotton plug) with no furtherreproduction or dispersal. No apparent symptoms of anydamage to health of the host plants were recorded.

A similar lack of further development of B. populi sp.n. was observed in pine and oak when seedlings wereintentionally damaged by excessive wounding and crowntrimming before the main experiment. No nematodeswere recovered from pine, while in oak a few individuals,i.e., 6.8 ± 5.2 (0-14) per plant, were only extracted fromthe inoculation cotton plugs. Bursaphelenchus populi sp.n. did not penetrate into stems of these tree species. Incontrast, in heavily damaged aspen seedlings developmentand reproduction occurred. The resulting populationsdispersed outside the inoculation site. The nematodescould be recovered only from dead parts of the seedlingstem up to 8 cm above and 4 cm below the inoculationwound. The mean number of recovered nematodes was2210 ± 1594 (370-4680) per aspen seedling and theseresults significantly differed from those recorded in oak(F1,8 = 81.58; P < 0.001).

Interestingly, after 30 days of the experiment, mostof the nematode females extracted from aspen seedlingshad a broadly rounded tail terminus and resembled wildindividuals originally isolated from aspen wood samples.

IDENTIFICATION OF NATURAL VECTORS OF B. POPULI

SP. N.

Dissection of adult beetles that emerged from aspenlogs in the spring revealed the frequent presence of nema-tode juveniles under the elytra or inside the haemocoel.Based on morphology of buccal cavity and pharynx, ne-matodes extracted from Agrilus sp. (Buprestidae), and oc-

Vol. 12(3), 2010 409


Fig

.4.

Mol

ecul

arph

ylog

enet

icre

lati

onsh

ipof

Bur

saph

elen

chus

popu

lisp

.n.

and

22ot

her

Bur

saph

elen

chus

spec

ies

base

don

part

ial

28S

rDN

A.

Aph

elen

choi

des

frag

aria

ese

rved

asou

tgro

upsp

ecie

s.P

hylo

gene

tic

tree

sw

ere

gene

rate

dw

ith

1000

boot

stra

pre

plic

atio

ns.

A:

Bay

esia

nan

alys

isw

ith

subs

titu

tion

mod

elG

TR

;B

:M

axim

umli

keli

hood

(ML

)an

alys

isw

ith

subs

titu

tion

mod

elG

TR

;C

:N

eigh

bour

-joi

ning

(NJ)

anal

ysis

;D

:M

axim

umpa

rsim

ony

(MP

)an

alys

is.

410 Nematology


Fig

.4.(

Con

tinu

ed).

Vol. 12(3), 2010 411


Fig. 5. Consensus phylogenetic tree calculated from Bayesian,maximum likelihood (ML), neighbour-joining (NJ) and maxi-mum parsimony (MP) analysis.

casionally from Xyleborinus saxeseni Ratz. and Anisan-drus dispar F. (Curculionidae: Scolytinae), were identi-fied as rhabditids. The greatest morphological diversitywas observed among nematode juveniles extracted fromS. perforata. Besides rhabditids, which usually formedone to several separate aggregations under the elytra andbetween the thoracic tergites, numerous, morphologicallydistinct, juvenile nematodes were also dissected from thebeetle’s body cavity. Nematodes with typical aphelen-choid dauer juvenile head morphology and distinct, par-tially flattened bulbus (Fig. 2N-O) crowded inside the dis-tal part of the insect abdomen in the vicinity of the gen-ital organs. From a few to over 80 dauer juveniles couldbe extracted from an individual beetle. The morphologyof dauer juveniles was distinctive by the body length of559 ± 21.5 (535-600) μm, max. body diam. of 17.3 ± 1.4(13.9-19.0) μm, a = 32.2 ± 1.6 (30.3-34.5), head broadlyrounded, lip region only weakly offset and lips not dis-tinct. Stylet, bulbus, other internal organs and anus wereclearly visible only in some individuals but were other-

wise completely obscured by granular body contents. Thetail was narrowly conical and had a sharply pointed ter-minus. However, in all juvenile nematodes extracted fromthe wood samples or from in vitro cultures the lip regionwas distinctly offset and internal as well as external struc-tures, i.e., stylet, bulbus, intestine, nerve ring and anus,were clearly visible.

Transfer to B. cinerea/PDA-agar plates of dauer juve-niles dissected from the haemocoel of S. perforata re-sulted in only 3-12% dauer recovery and their further de-velopment. All of these nematodes developed into adult B.populi sp. n. Microscopic examination of genitalia and go-nads of the infested insects revealed no apparent effect ofthe nematode juveniles on development and morphologyof these organs. Development of both oocytes and spermwas comparable to that in immature non-infested beetles.

The nematodes apparently invaded the host insect afterits final moult from the pupal to the adult stage. Dissectionof mature larvae and pupae of S. perforata extractedfrom pupal chambers in the spring (April-May) revealedno nematodes in their haemocoel. Dauer juveniles of B.populi sp. n. could be found only in adult beetles justbefore and during their emergence from the host tree inMay.

Discussion

The xylophilus group comprises species which are ge-netically and morphologically most similar to the pinewilt nematode, B. xylophilus. As geographic distributionand economic significance of this pest is continuouslygrowing, the quarantine requirements and threat of poten-tial taxonomic misidentification has generated a substan-tial, worldwide, scientific interest for the whole group. Inthe last decade, the deployment of molecular methods hassignificantly improved taxonomic precision and has as-sisted in the identification of a number of new species inthe xylophilus group. Surprisingly, despite the presence ofBursaphelenchus-rich communities of tree nematodes andthe historical recognition of B. fraudulentus and B. mucro-natus as native to Europe (Rühm, 1956; Baujard, 1980;Brzeski & Baujard, 1997; Braasch, 1998; Skarmoutsos etal., 1998), all newly described species from the xylophilusgroup have been isolated only from Asiatic regions (Kan-zaki et al., 2000; Kanzaki & Futai, 2001, 2003; Walia etal., 2003; Braasch et al., 2004; Gu et al., 2005, 2008).

The results reported here provide evidence that B. po-puli sp. n. clearly belongs to the xylophilus group. Themorphologically- and reproductively-based taxonomic

412 Nematology


separation of B. populi sp. n. from other species of thegroup and genus is also supported by our results fromthe detailed molecular analysis. Bursaphelenchus populisp. n. presented a unique molecular pattern of the ITS re-gion (ITS-RFLP). Sequencing and construction of phy-logenetic trees based on the D2/D3 region of 28S rDNAgene clearly separated B. populi sp. n. from the remain-ing species of the xylophilus group and also from theother groups, and corresponded well with results obtainedin the morphological analysis. These findings also sup-port the view that the D2/D3 expansion region is anideal region for phylogenetic studies of nematodes (Li-Qin et al., 2007). Therefore, together with B. fraudu-lentus, B. xylophilus, B. mucronatus, B. kolymensis, B.conicaudatus, B. luxuriosae, B. baujardi, B. doui, B. sin-gaporensis and B. macromucronatus, B. populi sp. n. isthe 11th known species of the xylophilus group and, ifwe consider the recent synonymisation of B. mucrona-tus European type with B. kolymensis (Braasch, 2008),the fifth species of this group to be reported from Eu-rope. Three other species, which were earlier placed inthe xylophilus group, namely B. abruptus Giblin-Davis,Mundo-Ocampo, Baldwin, Norden & Batra, 1993 fromNorth America, B. crenati Rühm, 1956 from Europe, andB. eroshenkii Kolossova, 1998 from Asia (Ryss et al.,2005), have been recently removed from this group, basedon differences in specific morphological characters, suchas position of caudal papillae, number of incisures in lat-eral fields, and presence of a vulval flap (Braasch, 2008).In the case of B. abruptus the removal was further sup-ported by molecular and bionomic differences (Giblin-Davis et al., 1993, 2005; Braasch, 2008).

So far, B. populi sp. n. has been isolated only fromtrunks of dead or dying aspen, P. tremula. According toa previous report, aspen and the closely related blackpoplar, P. nigra, may be infested with another species,B. fraudulentus (Rühm, 1956). Interestingly, in the ex-amined forest stand B. fraudulentus was not found in as-pen, despite the frequent presence of this species in neigh-bouring oaks. These observations provoke the followingquestions: i) is the B. fraudulentus described by Rühm(1956) really the same species reported later by otherauthors from oak, beech, birch, cherry, alder and pine(Schauer-Blume & Sturhan, 1989; Braasch et al., 1995;Schönfeld et al., 2001)?; or ii) is Rühm’s B. fraudulen-tus the same species as the presently described B. po-puli sp. n. and is B. fraudulentus sensu Braasch a separate,undescribed, species? In our as yet unpublished study onpopulations of nematodes collected in the field and sub-

sequently reared in the laboratory, i.e., i) B. populi sp.n. (PL-03) from P. tremula; ii) Polish populations of B.fraudulentus from Q. robur (PL-01; PL-05; PL-06; PL-10); iii) Polish population of B. fraudulentus from Alnusglutinosa (PL-07); iv) five populations of B. fraudulen-tus obtained from the Federal Research Centre for Cul-tivated Plants, Braunschweig, Germany – (a) Austrian –Österreich; (b) Russian – DE10w; (c) German – Helm-stedt; (d) H26; and (e) Hungarian – Ungarn, as well as ondescription and drawings provided by Rühm (1956) for B.fraudulentus isolated from P. tremula, we have found that,in spite of different tree hosts, all nematodes listed here asB. fraudulentus, including those depicted in the originaldrawings by Rühm (1956), clearly present similar mor-phological characteristics that separate them from B. pop-uli sp. n. The differences are mainly in the morphologyof the female tail and vulva region and male spicules. Inthe spicules, all populations designated here as B. fraudu-lentus share a distinct elongate depression (concave dorsalrim of spicules at the final third of the spicule length) closeto a sharp depression present before the cucullus. In B.populi sp. n. the spicule border is almost straight in this re-gion. These characters are compared in Figure 2L and 2Mof the present paper. In B. populi sp. n., the female tail,which is subcylindrical with a broadly rounded terminusand lacking a mucro in individuals extracted from woodsamples, or conoid, gradually tapering to a finely roundedterminus in individuals reared in vitro, clearly differs fromthe subcylindrical tail bearing a distinct, ca 2 μm long,mucro present in all examined populations of B. fraudu-lentus, including that depicted in Rühm’s (1956) draw-ings. Another difference between both nematode groupslies in the position and shape of the vulval flap, which inmost individuals of all populations referred to as ‘B. frau-dulentus’ is almost parallel to the body line with no oronly a slight curvature of the distal third towards the bodywall. In B. populi sp. n., the vulval flap is always clearlybent with its distal half sunken in a conspicuous, sharplydefined depression posterior to the vulva (as shown in Fig-ures 1E, 1F and 2F of this paper). In B. fraudulentus, thevulva and flap may be somewhat deformed in older, egg-laying females, but in B. populi sp. n. this characteristicis constant and distinctive to all females, including youngadults.

In this context we conclude that B. populi sp. n. is there-fore a different species to Rühm’s B. fraudulentus from as-pen and the most probable mechanism that prevents dis-persion of B. fraudulentus to aspen trunks in the forestwe studied is the lack of an appropriate insect vector ca-

Vol. 12(3), 2010 413


pable of infesting both oak and aspen. However, in Eu-rope, there are several species of cerambycids, such asSaperda scalaris L., Leiopus nebulosus L. (Lamiinae) andPlagionotus arcuatus L. (Cerambycinae), that are rela-tively common in oak and which can also infest other treespecies, including poplar (Sama, 2002). We have foundthese insects associated with trees colonised by B. frau-dulentus, although further study is necessary to elucidatewhether they can transmit B. fraudulentus.

Abundance of the field-collected material allowed usto trace the association of B. populi sp. n. and its vectorinsect in nature. So far, the only confirmed vector of B.populi sp. n. is S. perforata, a long-horn beetle (Coleop-tera: Cerambycidae) from the subfamily Lamiinae. Inter-estingly, in cases when the insect vectors are known, bee-tles from this subfamily are frequent natural vectors ofnematodes belonging to the xylophilus group, i.e., B. xylo-philus (Enda & Mamiya, 1972), B. mucronatus (Mamiya& Enda, 1979), B. conicaudatus (Kanzaki et al., 2000), B.luxuriosae (Kanzaki & Futai, 2003) and B. doui (Kanzakiet al., 2008). Kanzaki and Futai (2001) suggest a close co-evolutional relationship between nematodes of this groupand long-horn beetles from the subfamily Lamiinae.

Bursaphelenchus populi sp. n. has a limited range oftree hosts and could not develop in pine or oak. Thespecies was not pathogenic to aspen, its original treehost. Only heavily injured aspen seedlings supportedfurther development of the nematode with reproductionand dispersion within the stem. These findings correspondwith observations made in the field, where the nematodenatural vector, S. perforata infests trunks of older dead ordying aspen trees only, and where B. populi sp. n. waspresent in the vicinity of the larval galleries of the beetle.

A wide range of variation has been reported in boththe form of particular characters and in morphometricsof several Bursaphelenchus species (Mamiya & Kiyohara,1972; Bolla & Boschert, 1993; Brzeski & Baujard, 1997;Kanzaki et al., 2008). In most cases this can be attributedto intra-specific genetic variation and, particularly in suchspecies as B. xylophilus and B. mucronatus, related totheir widespread geographic distribution. As documentedin B. seani, rearing conditions may also affect develop-ment and morphometrics of Bursaphelenchus (Giblin &Kaya, 1984). The distinctive morphological differencesobserved between individuals of B. populi sp. n. extractedfrom aspen wood and those reared in B. cinerea/PDA cul-tures clearly indicate that nematode morphology is en-vironmentally dependent. The tail morphology of subse-quent generations of females changed between the round

and mucronate forms with the nematode transfer from as-pen wood to B. cinerea/PDA cultures, and reversed to thewild form after their introduction into the tree seedlings.Moreover, most morphometric characters also changedand the overall body dimensions of nematodes reared inB. cinerea/PDA cultures were reduced when comparedwith those of wild individuals extracted from the host tree.These findings, supported by identical molecular (ITS-RFLP) patterns of conspecificity in individuals with bothrounded and pointed tails, suggest that rearing conditionsin the host tree (aspen) wood and in B. cinerea/PDA cul-tures are not really comparable for B. populi sp. n. Such adifferential morphological response to environment seemsto be more common among species of Bursaphelenchus.We have frequently observed similar effects when cultur-ing B. fraudulentus and B. mucronatus (Tomalak, unpubl.)and we therefore believe that, for taxonomic purposes, it isadvisable to obtain measurements of individuals extractedfrom the natural host tree and to support these with datafrom individuals reared in plate cultures on an appropriatefungus. If this is not done, then differences in morphologyand morphometrics may lead to misidentification.

Acknowledgements

The authors wish to thank Dr A. Jakubowska, the sci-entific staff of the Interdepartmental Laboratory of Mole-cular Biology and Department of Virology and Bacte-riology, Institute of Plant Protection, Poznan, and MissJ. Kasprzak from A. Mickiewicz University, Poznan, fortheir help and advice on the molecular part of the re-search, Mrs B. Plonka for her dedicated technical assis-tance and Mr D. Krawczyk for his help in digitising theoriginal drawings. Sincere thanks are also addressed toProf. M. Kozlowska, Poznan University of Life Sciences,for her advice on statistical analysis and to Dr T. Schröder,Federal Biological Research Centre for Agriculture andForestry, Braunschweig, Germany, for providing us witha number of live cultures of B. xylophilus, B. doui and B.fraudulentus isolates for comparative and cross-breedingstudies. The research was financially supported by thePolish Ministry of Agriculture and Rural Development,through the long-term Programme of the Institute of PlantProtection – National Research Institute, Project 1.1: Re-search on simplified methods of taxonomic identificationof the quarantine pest Bursaphelenchus xylophilus.

414 Nematology


References

BAUJARD, P. (1980). Trois nouvelles espèces de Bursaphelen-chus (Nematoda: Tylenchida) et remarques sur le genre. Re-vue de Nématologie 3, 167-177.

BOLLA, R.I. & BOSCHERT, M. (1993). Pinewood nematodespecies complex: interbreeding potential and chromosomenumber. Journal of Nematology 25, 227-238.

BRAASCH, H. (1998). Bursaphelenchus hofmanni sp. n. (Ne-matoda, Aphelenchoididae) from spruce wood in Germany.Nematologica 44, 615-621.

BRAASCH, H. (2001). Bursaphelenchus species in conifers inEurope: distribution and morphological relationships. EPPOBulletin 31, 127-142.

BRAASCH, H. (2008). The enlargement of the xylophilus groupin the genus Bursaphelenchus. In: Mota, M. & Vieira, P.(Eds). Pine wilt disease: a worldwide threat to forest ecosys-tems. Berlin, Germany, Springer, pp. 139-150.

BRAASCH, H., BURGERMEISTER, W. & PASTRIK, K. (1995).Differentiation of three Bursaphelenchus species by meansof RAPD-PCR. Nachrichtenblatt des Deutschen Pflanzen-schutzdienstes 47, 310-314.

BRAASCH, H., GU, J., BURGERMEISTER, W. & ZHANG, J.(2004). Bursaphelenchus doui sp. n. (Nematoda: Parasitaphe-lenchidae) in packaging wood from Taiwan and South Korea– a new species of the xylophilus group. Russian Journal ofNematology 12, 19-27.

BRZESKI, M.W. & BAUJARD, P. (1997). Morphology and mor-phometrics of Bursaphelenchus (Nematoda: Aphelenchoi-didae) species from pine wood of Poland. Annales Zoologici47, 305-319.

BURGERMEISTER, W., METGE, K., BRAASCH, H. & BUCH-BACH, E. (2005). ITS-RFLP patterns for differentiation of26 Bursaphelenchus species (Nematoda: Parasitaphelenchi-dae) and observations on their distribution. Russian Journalof Nematology 13, 29-42.

BURGERMEISTER, W., BRAASCH, H., METGE, K., GU, J.,SCHRÖDER, T. & WOLDT, E. (2009). ITS-RFLP analysis, anefficient tool for differentiation of Bursaphelenchus species.Nematology 11, 649-668.

DE LEY, P., FÉLIX, M.A., FRISSE, L.M., NADLER, S.A.,STERNBERG, P.W. & THOMAS, W.K. (1999). Molecu-lar and morphological characterisation of two reproduc-tively isolated species with mirror-image anatomy (Nema-toda: Cephalobidae). Nematology 1, 591-612.

ENDA, N. & MAMIYA, Y. (1972). [The effect of maturationfeeding of Monochamus alternatus contaminated with Bur-saphelenchus lignicolus on pine trees.] Transactions of theMeeting of the Japanese Forestry Society 83, 320-322.

FERRIS, V.R., FERRIS, J.M. & FAGHIHI, J. (1993). Variationin spacer ribosomal DNA in some cyst-forming speciesof plant parasitic nematodes. Fundamental and AppliedNematology 16, 177-184.

GIBLIN, R.M. & KAYA, H.K. (1984). Host, temperature andmedia additive effects on the growth of Bursaphelenchusseani. Revue de Nématologie 7, 13-17.

GIBLIN-DAVIS, R.M., MUNDO-OCAMPO, M., BALDWIN,J.G., NORDEN, B.B. & BATRA, S.W. (1993). Descriptionof Bursaphelenchus abruptus n. sp. (Nemata: Aphelenchoi-didae), an associate of a digger bee. Journal of Nematology25, 161-172.

GIBLIN-DAVIS, R.M., HAZIR, S., CENTER, B.J., YE, W.,KESKIN, N., TROP, R.W. & THOMAS, K. (2005). Bursaphe-lenchus anatolius n. sp. (Nematoda: Parasitaphelenchidae),an associate of bees in the genus Halictus. Journal of Nema-tology 37, 336-342.

GU, J., ZHANG, J., BRAASCH, H. & BURGERMEISTER, W.(2005). Bursaphelenchus singaporensis sp. n. (Nematoda:Parasitaphelenchidae) in packaging wood from Singapore –a new species of the B. xylophilus group. Zootaxa 988, 1-12.

GU, J., ZHENG, W., BRAASCH, H. & BURGERMEISTER, W.(2008). Description of Bursaphelenchus macromucronatussp. n. (Nematoda: Parasitaphelenchidae) in packaging woodfrom Taiwan and India – a new species of the ‘xylophilus’group. Journal of Nematode Morphology and Systematics 11,31-40.

IWAHORI, H., TSUDA, K., KANZAKI, N., IZUI, K. & FU-TAI, K. (1998). PCR-RFLP and sequencing analysis of ri-bosomal DNA of Bursaphelenchus nematodes related to pinewilt disease. Fundamental and Applied Nematology 21, 655-666.

KANZAKI, N. & FUTAI, K. (2001). Life history of Bursaphe-lenchus conicaudatus (Nematoda: Aphelenchoididae) in rela-tion to the yellow-spotted longicorn beetle, Psacothea hilaris(Coleoptera: Cerambycidae). Nematology 3, 473-479.

KANZAKI, N. & FUTAI, K. (2003). Description and phylogenyof Bursaphelenchus luxuriosae n. sp. (Nematoda: Aphelen-choididae) isolated from Acalolepta luxuriosa (Coleoptera:Cerambycidae). Nematology 5, 565-572.

KANZAKI, N., TSUDA, K. & FUTAI, K. (2000). Description ofBursaphelenchus conicaudatus sp. n. (Nematoda: Aphelen-choididae), isolated from the yellow-spotted longicorn beetle,Psacothea hilaris (Coleoptera: Cerambycidae) and fig trees,Ficus carica. Nematology 2, 165-168.

KANZAKI, N., AIKAWA, T., MAEHARA, N. & MATSUMOTO,K. (2008). Bursaphelenchus doui Braasch, Gu, Burgermeister& Zhang, 2005 (Aphelenchida: Parasitaphelenchidae), an as-sociate of Monochamus subfasciatus Bates (Coleoptera: Ce-rambycidae) and Pinus densiflora Sieb. & Zucc. Nematology10, 69-78.

KORENTCHENKO, E.A. (1980). [New species of nematodesfrom the family Aphelenchoididae, parasites of stem pests ofthe Dahurian Larch.] Zoologichesky Zhurnal 59, 1768-1780.

LI-QIN, J., XU-QING, L. & JING-WU, Z. (2007). First recordof Bursaphelenchus rainulfi on pine trees from eastern Chinaand its phylogenetic relationship with intro-genus species.Journal of Zhejiang University Science B 8, 345-351.

Vol. 12(3), 2010 415


MAGNUSSON, C. & KULINICH, O.A. (1996). A taxonomicappraisal of the original description, morphology and statusof Bursaphelenchus kolymensis Korentchenko, 1980 (Aphe-lenchida: Aphelenchoididae). Russian Journal of Nematology4, 155-161.

MAMIYA, Y. & ENDA, N. (1979). Bursaphelenchus mucronatusn. sp. (Nematoda: Aphelenchoididae) from pine wood andits biology and pathogenicity to pine trees. Nematologica 25,353-361.

MAMIYA, Y. & KIYOHARA, T. (1972). Description of Bursa-phelenchus lignicolus n. sp. (Nematoda: Aphelenchoididae)from pine wood and histopathology of nematode-infestedtrees. Nematologica 18, 120-124.

MOTA, M., BRAASCH, H., BRAVO, M.A., PENAS, A.C.,BURGERMEISTER, W., METGE, K. & COUSA, E. (1999).First report of Bursaphelenchus xylophilus in Portugal and inEurope. Nematology 1, 727-734.

RÜHM, W. (1956). Die Nematoden der Ipiden. ParasitologischeSchriftenreihe 6, 1-438.

RYSS, A., VIEIRA, P., MOTA, M. & KULINICH, O. (2005).A synopsis of the genus Bursaphelenchus Fuchs, 1937(Aphelenchida: Parasitaphelenchidae) with keys to species.Nematology 7, 393-458.

SAMA, G. (2002). Atlas of the Cerambycidae of Europe and theMediterranean area, Vol. 1. Zlín, Czech Republic, Naklada-telstvi Kabourek, 173 pp.

SCHAUER-BLUME, M. & STURHAN, D. (1989). Verkom-men von Kiefernholznematoden (Bursaphelenchus spp.)in der Bundesrepublik Deutschland? Nachrichtenblatt desDeutschen Pflanzenschutzdienstes 41, 133-136.

SCHÖNFELD, U., APEL, K.-H. & BRAASCH, H. (2001).Nematoden der Gattung Bursaphelenchus (Nematoda,Parasitaphelenchidae) in den Kiefernwälderndes LandesBrandenburg-Ergebnisse eines Monitorings. Nachrichten-blatt des Deutschen Pflanzenschutzdienstes 53, 180-184.

SEINHORST, J.W. (1959). A rapid method for the transfer ofnematodes from fixative to anhydrous glycerin. Nematologica4, 67-69.

SKARMOUTSOS, G., BRAASCH, H. & MICHALOPOULOU, H.(1998). Bursaphelenchus hellenicus sp. n. from Greek pinewood. Nematologica 44, 623-629.

SULSTON, J. & HODGKIN, J. (1988). Methods. In: Wood, B.(Ed.). The nematode Caenorhabditis elegans. Plainview, NY,USA, Cold Spring Harbor Laboratory Press, pp. 587-606.

VRAIN, T.C. (1993). Restriction fragment length polymorphismseparates species of the Xiphinema americanum group. Jour-nal of Nematology 25, 361-364.

WALIA, K.K., NEGI, S., BAJAJ, H.K. & KALIA, D.C.(2003). Two new species of Bursaphelenchus Fuchs, 1937(Nematoda: Aphelenchoididae) from pine wood and insectfrass from India. Indian Journal of Nematology 33, 1-5.

416 Nematology

Documents

Description of Bursaphelenchus populi sp.n.(Nematoda ...2363,20-bursa-populi-2010.pdfThe molecular phylogenetic status of B. populi sp. n. both within the genus and within the xylophilus