Nematology, 2008, Vol. 10(3), 335-346

Bursaphelenchus chengi sp. n. (Nematoda: Parasitaphelenchidae)isolated at Nanjing, China, in packaging wood from Taiwan

Hongmei LI 1,2, Phap Quang TRINH 2,3, Lieven WAEYENBERGE 4 and Maurice MOENS 2,4,∗1 Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, China2 Laboratory for Agrozoology, Ghent University, Coupure Links 653, 9000 Ghent, Belgium

3 Institute of Ecology and Biological Resources, VAST, 18 Hoang Quoc Viet, Hanoi, Vietnam4 Institute for Agricultural and Fisheries Research, Burg. Van Gansberghelaan 96, 9820 Merelbeke, Belgium

Received: 20 August 2007; revised: 25 October 2007Accepted for publication: 26 October 2007

Summary – Bursaphelenchus chengi sp. n. is described and illustrated. Dauer juveniles were isolated from imported wood packagingmaterials from Taiwan to Nanjing Port, China. Bursaphelenchus chengi sp. n. was reared and maintained on Petri dish cultures of thefungus Botrytis cinerea. The new species is characterised by the medium body size in both sexes, the presence of only two incisures inthe lateral field and the robust and strongly curved spicules. The spicule lamina is angular distally, the rostrum digitate and the condylusrounded. The tail is arcuate with a pointed terminus. The bursa is usually truncate with the posterior margin indented in some specimensor rounded with a fine axial point. Females have a small vulval flap formed by a short extension of the cuticle of the anterior lip, anda conical tail that gradually tapers to an almost straight or slightly recurved, pointed or rounded terminus. Because of the presence oftwo lateral lines, similar spicule shape, tapering female tail and the presence of a small vulval flap, B. chengi sp. n. should be groupedin the abietinus-group sensu Braasch. together with B. abietinus, B. antoniae, B. hellenicus, B. hylobianum and B. rainulfi. ITS-RFLPprofiles support the proposal of the new species, and phylogenetic analysis of the 28S rDNA D2/D3 domain sequence places it close toB. antoniae and other species of the abietinus-group.

Keywords – ITS-RFLP, molecular, morphology, morphometrics, phylogeny, SEM, sequence analysis, taxonomy.

With booming international trade in China, the threat ofintroducing non-indigenous nematode diseases via com-modities and their packaging is increasing. Most of theshipping containers are produced from unprocessed lowerquality hardwood or coniferous wood. These woods aremore likely to harbour pests such as the pine wood ne-matode, Bursaphelenchus xylophilus (Steiner & Buhrer,1934) Nickle, 1970, and its vector species of the genusMonochamus (Braasch et al., 2004). La et al. (1999)demonstrated that the pine wood nematode was intro-duced into Korea through wood that contained both thenematode and the vector beetle.

Since pine wilt disease was first discovered in 1982 inthe Purple Mountains of Nanjing, China (Cheng et al.,1983), the damage caused by B. xylophilus to Chinese for-est stands is still increasing (Yang, 2004). Gu et al. (2006)reported the interception of 15 Bursaphelenchus spp. byChinese plant quarantine services in recent years in im-ported packaging wood materials originating from Japan,

∗ Corresponding author, e-mail: maurice.moens@ilvo.vlaanderen.be

Korea and Taiwan. Bursaphelenchus mucronatus Mamiya& Enda, 1979, B. xylophilus, B. fungivorus Franklin &Hooper, 1962, B. rainulfi Braasch & Burgermeister, 2002and B. thailandae Braasch & Braasch-Bidasak, 2002 werethe most frequently detected species. Border or post-border inspections of packaging wood or wood importsare considered the most effective way to control the ex-ecution of measures for preventing the introduction anddistribution of the pine wood nematode (Braasch et al.,2004). Currently, almost all imported packaging wood isinspected by the Chinese Entry-Exit Inspection and Quar-antine Bureaux. During our participation in this inspec-tion at Nanjing Port, we detected several Bursaphelen-chus spp. in wooden packaging material originating fromEast Asian countries (unpubl.). One of these species wasdetected in packaging wood imported from Taiwan. Al-though its morphology, morphometrics and genetics weremore or less similar to B. antoniae Penas, Metge, Mota& Valadas, 2006 and B. hylobianum (Korenchenko, 1980)

© Koninklijke Brill NV, Leiden, 2008 335Also available online - www.brill.nl/nemy

H. Li et al.

Hunt, 1993, enough characteristics allowed the conclusionthat this population belonged to an undescribed species. Itis herein described as Bursaphelenchus chengi sp. n. inmemory of Dr Hurui Cheng who was the first to reportthe pine wilt disease in China.

Materials and methods

NEMATODE ISOLATION AND CULTURE

Samples were sawn from solid wooden packagingmaterial originating from Taiwan and consisted of about10 cm long pieces, which were subsequently cut intosmaller pieces of no more than 1 cm. Nematodes wereextracted using the modified Baermann funnel techniquefor 48 h at 25◦C. Extracted nematodes were maintainedin fungal cultures of non-sporulating forms of Botrytiscinerea for further studies.

MORPHOLOGY AND MORPHOMETRIC OBSERVATIONS

For morphological observations, nematodes were col-lected from the B. cinerea cultures, mounted in waterand heat-relaxed on temporary slides, observed under anOlympus BX-51 light microscope, and photographed withan Olympus U-TV 0.5×C-3 digital camera. Measure-ments were made on permanent slides by heat-killed ne-matodes fixed with FA 4 : 1 and ethanol-glycerine dehy-dration according to Seinhorst (1959) as modified by DeGrisse (1969).

For scanning electron microscope (SEM) observations,the specimens were prepared as described by Eisenback(1985) and viewed and photographed using a JEOL35 scanning electron microscope. The spicules wereexcised and prepared for SEM observations by the methoddescribed by Penas et al. (2006).

DNA EXTRACTION

DNA was isolated from nematodes collected from theBotrytis cultures. The procedure used for DNA extractionwas as described in He et al. (2003) with some modi-fication. Ten nematodes were transferred into 30 µl 2×worm lysis buffer (WLB: 20 mM Tris-HCl pH 8.0, 100mM KCl, 3.0 mM Mg2Cl, 2.0 mM DTT, 0.9% Tween20) and cut into two or three fragments with a sterilisedscalpel. Ten µl WLB, along with all nematode fragments,was pipetted into 8 µl ddH2O with 2 µl proteinase K (60µg/ml) in an Eppendorf tube, which was then briefly spunand stored at −70◦C for at least 10 min. Subsequently,

the Eppendorf tube was incubated at 65◦C for 1-2 h andthe proteinase K was denatured at 95◦C for 10 min. Fi-nally, the DNA suspension was cooled to 4◦C and storedat −20◦C for further applications. No additional purifica-tion was required for the following PCR procedure.

ITS-RFLP PROFILES

ITS-RFLP analysis was done as described by Braaschand Burgermeister (2002). A fragment of nematode rDNAcontaining the internal transcribed spacer regions ITS1and ITS2 was amplified by PCR using the forward primer5′-CGT AAC AAG GTA GCT GTA G-3′ (Ferris et al.,1993) and the reverse primer 5′-TTT CAC TCG CCGTTA CTA AGG-3′ (Vrain, 1993). The PCR mixture(50 µl) contained 0.6 µM of each primer, 2 units TaqDNA polymerase (Promega, Leiden, The Netherlands),10 mM Tris-HCl pH 8.8, 50 mM KCl, 2 mM MgCl2, 0.1mM dNTP’s and 2 ng DNA template. Amplificationwas carried out using a PTC-100/200 thermocycler (MJResearch, San Diego, CA, USA) employing an initialdenaturation step at 94◦C for 2.5 min, 40 reaction cyclesof 94◦C for 1 min, 55◦C for 1 min and 72◦C for 2 min, anda final extension step at 72◦C for 5 min. After PCR, 5 µlof amplified product was analysed by electrophoresis ina 1% agarose gel and DNA fragments were visualised bystaining in 1 µg/ml ethidium bromide with a 100 bp DNALadder (Promega, Madison, WI, USA) as a molecular sizemarker.

Suitable aliquots of the amplified DNA were digestedfor at least 3 h at 37◦C using 10 U of each of the fiverestriction endonucleases (RsaI, HaeIII, MspI, HinfI andAluI) (Promega) following the manufacturer’s instruc-tions. Fragments were resolved by electrophoresis in a2.5% agarose gel and stained with ethidium bromide.Species-specific ITS-RFLP profiles for Bursaphelenchuswere generated using these five restriction enzymes.

SEQUENCING

The 28S rDNA D2/D3 domain region was amplifiedwith the forward primer D2A 5′-ACA AGT ACC GTGAGG GAA AGT TG-3′ and the reverse primer D3B5′-TCG GAA GGA ACC AGC TAC TA-3′ (De Leyet al., 1999). After purification, the PCR product wasligated to a pGEM-T vector (Promega) using T4 DNAligase (Promega) and transformed in Escherichia colistrain JM109 according to the manufacturer’s instructions(Promega) (Sambrook et al., 1989). The white colonieswere picked and recultured in fresh LB with ampicillin

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(100 µg/ml) overnight. Plasmids were purified using aQiagen Miniprep kit (Qiagen, Hilden, Germany) andsequenced by the ABI 377TM automated sequencer usinga Big-dye terminator cycle reaction (Applied Biosystems,Foster City, CA, USA). The fragment was sequenced inboth directions to obtain overlapping sequences of theforward and reverse DNA strand.

The 28S rDNA D2/D3 sequence data of B. chengisp. n. were compared to sequences of Bursaphelenchusspecies from different groups (abietinus-, eggersi-, fun-givorus-, sexdentati- and xylophilus-group) deposited inthe GenBank database. Aphelenchoides besseyi Christie,1942 was used as an outgroup. The sequences werealigned using ClustalW in BioEdit 7.0.4.1 (Hall, 1999)and the pairwise sequence divergences were calculated.The phylogenetic trees were generated by minimum evo-lution (ME) and maximum parsimony (MP) algorithms inPAUP* 4.0b10 (Swofford, 1998).

Bursaphelenchus chengi* sp. n.(Figs 1-3)

MEASUREMENTS

See Table 1.

DESCRIPTION

Male

Displaying all the features of Aphelenchoidoidea ac-cording to Hunt (1993). Body slender, cylindrical. Distalpart of body curved and J-shaped when killed by gen-tle heat. Cuticle with fine annulations. Lateral field withtwo distinct incisures (i.e., with a single ridge or band).Lip region high, rounded, offset by constriction, labialannules lacking. SEM en face view of head showing sixequal, closely arranged, lips. Oral disc distinct, oral aper-ture circular, surrounded by six inner papillae. Amphidialapertures pore-like, cephalic papillae obscure. Stylet long,basal knobs absent, but very slight basal swellings present.Median bulb elongate-oval. Secretory/excretory pore lo-cated 1.0-1.5 body diam. posterior to nerve ring. Hem-izonid located 5-6 µm posterior to excretory pore. Pha-ryngeal glands overlapping intestine for 2-3 body diam.,mostly on dorsal side. Testis monorchic, usually ante-riorly outstretched, occasionally reflexed, occupying ca

*Named after Dr Hurui Cheng who was the first to report pinewilt disease in China.

one-third to half of body length; cells initially arrangedin single row and then in two rows. Spicules paired, ro-bust, rosethorn-shaped, strongly curved; rostrum promi-nent, not sharply pointed, condylus high, rounded andwell developed; distal end truncate with broad and bluntcucullus offset slightly more from dorsal limb than ven-tral (SEM). Distal third of dorsal limb laterally expandedforming flattened wing-like alae. Tail arcuate, terminuspointed; bursa usually truncate with posterior margin in-dented in some specimens or more rounded to a fine, moreor less axial, point. Copulatory papillae comprising singleprecloacal ventromedian papilla, one pair of precloacalsubventral papillae at same level, one pair of post cloa-cal subventral papillae at middle of tail; gland openingsappearing as two pairs of small papillae.

Female

Body slightly curved ventrad when heat-relaxed. Gen-ital tract monoprodelphic, outstretched, cells initiallyarranged in single row, thereafter in two rows. Spermath-eca differentiated, roundish, irregular rectangular, filledwith rounded sperm. Quadricolumella visible. Postuterinebranch extending for ca 60% of vulva-anus distance, of-ten containing sperm. Vulva inclined anteriad at ca 45◦to body axis. Vulva with anterior lip slightly extendedto form a small, but distinct, flap. Tail medium length,conoid, gradually tapering to bluntly rounded or acute ter-minus with thickened cuticle forming a hyaline region,terminal region bearing irregular, flap-like, cuticular folds(SEM), sometimes with a finely pointed or irregular mu-cron.

Juvenile stages

Juveniles with conical tail, that of J3 and J4 (female)tail slightly ventrally curved and J4 male tail also ventrallycurved. Developing gonad visible in posterior region of J4male.

TYPE HABITAT AND LOCALITY

Isolated from packaging wood arriving at Nanjing(China) from Taiwan with other commodities and culturedon a lawn of Botrytis cinerea growing on PDA.

TYPE MATERIAL

Holotype male (UGMD 104113), ten female paratypes(UGMD 104111) and ten male paratypes (UGMD 104112and UGMD 104113) were deposited at the nematode col-lection of the Institute of Zoology, Ghent University, K.L.

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Fig. 1. Bursaphelenchus chengi sp. n. A: Entire female; B: Entire male; C: Female anterior region; D: Female tails; E: Spicules; F:Male bursa; G: Ventral view of male tail and position of papillae; H: Lateral view of male tails; I: Lateral view of female vulva regionand reproductive tract.

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Fig. 2. Light micrographs of Bursaphelenchus chengi sp. n. A-C: Variation in female tails; D: Tail of J4 (female); E: Vulval region; F,G: Male tails; H-J: Male bursa. (Scale bar = 20 µm.)

Ledeganckstraat 35, 9000 Ghent, Belgium. Six female pa-ratypes and five male paratypes were deposited in thenematode collection of Nanjing Agricultural University,China.

DIAGNOSIS AND RELATIONSHIPS

Bursaphelenchus chengi sp. n. is characterised by themedium body size in both sexes, the presence of only twoincisures in the lateral field and the robust and stronglycurved spicules. The spicule lamina is angular distally,the rostrum digitate and the condylus rounded. The tailis arcuate with a pointed terminus. The bursa is usuallytruncate with the posterior margin indented in somespecimens or rounded with a fine terminal point. Femaleshave a small vulval flap formed by a short extension of thecuticle of the anterior lip, and a conical tail that graduallytapers to an almost straight or slightly recurved, pointedor rounded terminus.

Braasch (2001) grouped the species of Bursaphelen-chus on the basis of the number of lateral lines. Ryss etal. (2005), however, classified the species into six groupsmainly based on the shape of the spicules. Based on thislatter classification, B. chengi sp. n. would be classifiedwithin the piniperdae-group, i.e., a group of species withstout spicules, concave capitulum, elongated condylus,lamina smoothly curved or angular at midpoint, cucullususually absent although small cucullus sometimes present.However, the piniperdae-group sensu Ryss et al. is an ar-tificial conglomerate of species, members of which mayhave two, three or four lateral lines and various arrange-ments of caudal papillae.

Recent studies on the phylogeny of the genus haveused a combination of molecular data with morphologicalcharacters, including the number of lateral lines andspicule shape (Lange et al., 2007; Ye et al., 2007). Inthese recent classifications B. chengi sp. n. clusters in the

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Bursaphelenchus chengi sp. n. in packaging wood from Taiwan

Table 1. Morphometrics of Bursaphelenchus chengi sp. n. All measurements are in µm and in the form: mean ± standard deviation(range).

Male Female

Holotype Paratypes Paratypes

n – 20 20L 709 723 ± 51.0 742 ± 45.5

(646-825) (661-828)a 34.3 29.8 ± 2.0 30.0 ± 2.0

(25.4-34.3) (25.1-33.9)b 7.6 8.1 ± 0.8 9.0 ± 1.0

(6.7-9.5) (7.3-10.8)b′ 5.6 5.2 ± 0.8 5.8 ± 0.9

(3.3-6.4) (4.2-5.8)c 17.5 18.4 ± 1.7 14.8 ± 1.3

(14.8-21.7) (12.7-17.2)c′ 3.1 2.6 ± 0.3 4.4 ± 0.4

(2.2-3.2) (3.6-5.1)V – – 73.2 ± 2.0

(69.5-77.0)Lip region diam. 7.3 7.7 ± 0.5 7.7 ± 0.4

(6.9-8.7) (6.8-8.5)Lip constriction diam. 6.8 6.8 ± 0.4 7.0 ± 0.3

(6.0-7.5) (6.5-7.6)Lip region height 3.3 3.6 ± 0.3 3.6 ± 0.2

(3.2-4.6) (3.2-4.0)Stylet length 15.5 15.8 ± 0.9 15.6 ± 1.4

(14.1-16.9) (10.3-17.1)Median bulb length 18 18 ± 0.7 18 ± 0.7

(16-19) (17-20)Median bulb diam. 12 13 ± 0.8 14 ± 0.9

(12-15) (12-16)Median bulb length/diam. 1.5 1.4 ± 0.1 1.3 ± 0.1

(1.2-1.5) (1.2-1.5)Maximum body diam. 20.7 24 ± 1.7 25 ± 1.7

(21-27.0) (22-29)Body diam. at middle of median bulb 16 17 ± 1.0 18 ± 0.8

(15-20) (16-19)Anterior end to excretory pore 95.1 93 ± 6.1 91 ± 4.1

(80-103) (84-97)Anterior end to hemizonid 99 98 ± 6.5 96 ± 4.1

(85-110) (88-104)Anterior end to median bulb base 72 71 ± 4.0 67 ± 2.8

(64-82) (62-71)

Fig. 3. Scanning electronic micrographs of Bursaphelenchus chengi sp. n. A: En face view of male (aa = amphidial aperture;ilp = inner labial papilla); B: Male anterior region, lateral view; C: Male tail, lateral view (p1 = single precloacal papilla,p2 = precloacal subventral papillae, p3 = postcloacal subventral papillae, p4 = two gland papillae); D: Male tail tip, subventralview; E: Spicules (arrow shows the flattened wing-like structure in the distal third of the dorsal limb); F, G: Variation in tail tip offemales; H, I: Variation in female tail shape; J: Lateral field in midbody of female; K: Vulva in ventral view.

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Table 1. (Continued).

Male Female

Holotype Paratypes Paratypes

Anterior end to pharyngo-intestine junction 93 89 ± 6.7 83 ± 4.7(72-102) (75-95)

Anterior end to posterior end of pharyngeal glands 126 142 ± 21.5 130 ± 20.3(118-207) (84-176)

Anterior genital branch 375 348 ± 46.7 227 ± 31.6(287-478) (183-285)

Posterior genital branch – – 93 ± 15.4(59-123)

Body diam. at vulva – – 22 ± 1.1(20-23.6)

Vulva to anus distance – – 148 ± 13.0(114-167)

Anterior end to vulva – – 544 ± 44.2(467-638)

G1 – – 30.6 ± 4.4(22.3-38.8)

G2 – – 12.6 ± 2.0(8.6-17.3)

Anal/cloacal body diam. 13 15 ± 1.0 11 ± 0.8(13-17) (10-13)

Tail length 41 39 ± 2.7 50 ± 2.8(36-44) (46-55)

T 52.9 48.3 ± 6.8 –(36.5-63.0)

Spicule (condylus to distal end) 21.3 22 ± 1.0 –(20-24)

Spicule (rostrum to distal end) 10.5 11.4 ± 0.7 –(10.1-12.7)

Spicule (curved median line) 17.7 18 ± 1.2 –(16-20)

Spicule (rostrum to condylus) 11.8 12.5 ± 0.9 –(10.4-14.3)

Spicule width 5.1 5.6 ± 0.5 –(4.8-6.6)

abietinus-group (B. abietinus Braasch & Schmutzenhofer,2000, B. antoniae Penas, Metge, Mota & Valadas, 2006,B. hellenicus Skarmoutsos, Braasch & Michalopoulou,1998, B. hylobianum and B. rainulfi). The species in thisgroup have two lateral lines, a similar spicule shape, atapering female tail and the presence of a small vulvalflap. The new species can be easily differentiated fromthe xylophilus-group and the sexdentati-group (sensuBraasch, 2001), both of which have four lateral lines, andfrom the leoni-group which has three lateral lines.

Comparing with species from the abietinus-group,therefore, the new species differs from B. abietinus by

larger and stouter spicules (20-24 vs 11-14 µm), spiculeswith broad and blunt cucullus vs with distinct cucullus,one precloacal pair and a single ventromedian precloacalcaudal papilla vs two precloacal pairs, and a longer malestylet (14-17 vs 11-13 µm).

Bursaphelenchus chengi sp. n. differs from B. anto-niae by a longer male body (646-825 vs 476-660 µm), abroader male lip region (diam. 6.9-8.7 vs 5.5-6.0 µm) anda longer male stylet (14-17 vs 11-14 µm). Bursaphelen-chus chengi sp. n. also has longer spicules (as measuredbetween the condylus and the distal end) of 20-24 vs 15-20 µm, and a longer distance from the distal tip of the ros-

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trum to the condylus (10-14 vs 6.5-10 µm). The a value isalso different, being 25.4-34.3 vs 34.2-44.0. The spiculesof B. chengi sp. n. resemble those of B. antoniae in shapebut are broadly truncate and lack a well-defined, disc-likecucullus.

The new species differs from B. hellenicus by thetruncate distal end of the spicule which bears a broad andblunt cucullus vs a distinct cucullus in B. hellenicus, the cvalue of 18.4 (14.8-21.7) vs 24 (19-30) in males and 14.8(12.7-17.2) vs 21 (17-31) in females, and the lateral fieldwith two distinct incisures vs three (occasionally two)incisures.

The spicules of B. chengi sp. n. resemble those of B.hylobianum in shape but have a shorter condylus. Bursa-phelenchus hylobianum was described as having a cucul-lus by Braasch and Braasch-Bidasak (2002) although theoriginal description (Korenchenko, 1980) did not mentionthis feature. The new species also differs from B. hylo-bianum by having three postcloacal papillae pairs of papil-lae vs only one postcloacal pair according to Braasch andBraasch-Bidasak (2002), although Korenchenko (1980),in the original description, reported three pairs to bepresent.

Bursaphelenchus chengi sp. n. differs from B. rainulfiby longer spicules (22 vs 13 µm), a longer male styletlength (15.8 vs 12 µm) and the excretory/secretory porelocated posterior to the nerve ring vs at median bulb level.

ITS-RFLP PROFILES

The ITS-RFLP patterns of B. chengi sp. n. (Table 2;Fig. 4) are different from those of the closely related B.antoniae (Penas et al., 2006) and B. hylobianum (Braasch& Burgermeister, 2002), except for the pattern obtainedafter HaeIII digestion. The ITS-RFLP patterns of thenew species are also different from those of B. abietinus,B. hellenicus and B. rainulfi (Braasch & Burgermeister,2002).

SEQUENCE ANALYSIS

The sequence length of the 28S rDNA D2/D3 domainof B. chengi sp. n. is 741 bp (GenBank accession no.EU107359). The comparison of the pairwise sequencesimilarities between B. chengi sp. n. and other relatedspecies revealed that the sequence of the new species hasthe highest similarity value (93.5%) with B. antoniae (ac-cession no. AM279710). The sequence similarities be-tween the new species and B. hylobianum (AY508085),B. abietinus (AY508074), B. hellenicus (AY508083) and

Fig. 4. ITS-RFLP pattern of Bursaphelenchus chengi sp. n. Re-striction fragments were obtained by digestion of the amplifiedrDNA fragment (0) with RsaI (1), HaeIII (2). MspI (3), HinfI (4)and AluI (5). M: DNA marker (100 bp ladder).

B. rainulfi (DQ257624) are 85.7, 82.3, 80.9 and 76.3%,respectively. The new species has only 75.5 and 75.0%similarity with B. xylophilus (DQ364687) and B. mucro-natus (DQ364688), respectively. In the phylogenetic treesB. chengi sp. n. clusters within the abietinus-group and isthe closest species to B. antoniae (Fig. 5). The phyloge-netic analysis supports the conclusion that B. chengi sp. n.is a new species close to B. antoniae and other two-linedBursaphelenchus species.

Acknowledgements

This research was financially supported by a Ph.D.scholarship allocated by the Flemish Interuniversity Coun-cil of Belgium (VLIR) to the senior author. This paper ispart of the Ph.D. dissertation of the first author at GhentUniversity, Belgium. The authors kindly thank Mr PeiyinShen from Inspection and Quarantine for Exit and Entryin Nanjing for the collection of wood samples, and MsMarjolein Couvreur from Ghent University for the scan-ning electron microscope pictures.

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Fig. 5. Phylogenetic relationships of Bursaphelenchus chengi sp. n. and 17 Bursaphelenchus species. Aphelenchoides besseyi is theoutgroup. The global sequence alignments for tree constructions were calculated for 28S D2/D3 domain sequences by minimumevolution (top tree) and maximum parsimony (bottom tree) algorithms. Bootstrap values (%) are given for each node.

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Table 2. Restriction fragments of amplified ITS-rDNA from Bursaphelenchus chengi sp. n. and five morphologically similarBursaphelenchus species.

Species ITS-PCR product Restriction fragments (bp) Reference(bp)

RsaI HaeIII MspI HinfI AluI

B. chengi sp. n. 1150 610 790 460 340 810 This paper280 360 370 290 280160 310 21090 200

110

B. antoniae 1150 610 790 490 340 790 Penas et al. (2006)290 360 370 290 340230 250

220

B. hylobianum 1150 590 790 360 490 1150 Braasch & Burgermeister (2002)280 360 310 270

180 240120

B. abietinus 1070 610 670 530 530 580 Braasch & Burgermeister (2002)280 230 400 220 230180 170 160 120 50

B. rainulfi 1050 270 1050 690 510 340 Braasch & Burgermeister (2002)180 390 210 200160 200 100120 110

B. hellenicus 1080 610 530 690 520 340 Braasch & Burgermeister (2002)290 390 390 320 280180 160 220 180

120

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