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25Russian Journal of Nematology, 2011, 19 (2), 121 - 129
Description of Bursaphelenchus mazandaranense sp. n. (Nematoda: Parasitaphelenchidae)
from Iran
1 12 3
Majid Pedram , Ebrahim Pourjam , Weimin Ye , Mohammad Reza Atighi , Robert T. Robbins4 and Alexander Ryss5
'Department of Plant Pathology, College of Agriculture, Tarbiat Modares University, Tehran, Iran 2North Carolina Department of Agriculture, Nematode Assay Section, Raleigh, NC27607, USA;
e-mail: [email protected] 3Department of Plant Protection, Faculty of Agricultural Sciences and Engineering, University College of Agriculture and
Natural Resources,University of Tehran, Karaj, Iran 4Department of Plant Pathology, University of Arkansas, Fayetteville, AR 72701, USA 5Zoological Institute RAS, Universitetskaya Naberezhnaya 1, St Petersburg 199034, Russia
Accepted for publication 22 July 2011
Summary. Bursaphelenchus mazandaranense sp. n., recovered from an old dead fallen trunk of a beech tree, Fagus orientalis, is described and illustrated based on molecular and morphological characters. A combination of morphological characters, such as having three incisures, seven caudal papillae in males, a conoid and ventrally bent female tail, the presence of a small vulval flap, and the excretory pore posterior to the metacorpus, all indicate that the new species is a member of the B. hofmanni group. The placement of the new species in this group is corroborated by molecular phylogenetic analysis of ribosomal DNA sequences. The new species has unique molecular sequences of SSU and the LSU D2D3 expansion segments, separating it from all known species of the genus, but place it close to B. hofmanni and B. pinasteri. Bursaphelenchus mazandaranense sp. n., is characterized by females 457-701 ^m in length, a stylet length of 11.5-13.0 ^m, a pointed ventrally bent tail, a small vulval flap, the body smooth at the vulva region, and post uterine sac 1.5-2.5 longer than body width at vulva region. Males of the new species have spicules 12.0-13.5 ^m long rounded dorsally, 1 ^m long posteriorly bent condylus, 2 ^m long rostrum and a distinct cucullus. This new species can be separated from B. hofmanni by its 1 ^m long posteriorly bent condylus (vs almost parallel to shaft axis, and 3 ^m long), a flat vulval region (vs dome shaped) and spicules rounded dorsally (vs angular). It can be separated from B. pinasteri by a posteriorly located excretory pore (73-91 vs 52-70 ^m distance from the anterior end), females with anterior and posterior lip of anus at the same level (vs with a sudden constriction after anus) and posteriorly bend condylus (vs prominent and rounded end). Differences with other members of hofmanni group namely B. ratzeburgii, B. sachsi, B. corneolus and B. curvicaudatus are also discussed. This is the first report of the occurrence of the genus Bursaphelenchus in Iran.
Key words: Mazandaran province, molecular, morphology, new species, Parasitaphelenchidae.
To study the presence of Bursaphelenchus spp. in Iran, we collected several wood and bark samples from dead or weakened Pinus spp. trees from natural regions and parks in north and northwestern Iran during 2008. Because of the economic significance of the pine wilt nematode, Bursaphelenchus xylophilus (Steiner & Buhrer,
1934) Nickle, 1970, identifying the tentative causal
agent of these dying Pinus spp. trees for the possible presence of pine wilt nematode, prompted
us to perform this survey. No species of the genus Bursaphelenchus Fuchs, 1937 was recovered as a result of this initial survey.
During more recent surveys from January 2010 to March 2011, we collected more than 100 wood samples from forest and pine trees from natural regions, parks and forests of north and northwestern Iran from which a new species of Bursaphelenchus was recovered from samples with bark beetle galleries on a dead beech tree (Fagus orientalis Lipsky) collected
Fig. 1. Bursaphelenchus mazandaranense sp. n. A&B: Entire body; A: Female, B: Male, C: Anterior region of female, D: Reflexed portion of testis, E: Posterior region of female, F: Anterior end in detail, G&H: Caudal region; G: Male, H: Female, I&J: Ventral view of male tail, showing number and arrangement of papillae, K&L: Spicule, M: Cross section of female showing three incisures in each lateral ala.
Table 1. Morphometries of Bursaphelenchus mazandaranense sp. n. (All measurements in цт and in the form: mean ± s.d. range.)
n 20 ÇÇ 15 SS Holotype female
L 558.0±55.5(457-701) 496±44(415.5-581.5) 662
a 25.0±1.5(22.3-28.0) 26± 1(24-27) 27.5
b 7.8±0.8(7-10) 6.8±0.5(6.2-7.2) 8.5
c 15.3±2.0(12.0-19.3) 15.0±1.3(12.5-17.5) 17.5
c' 3.5±0.3(2.5-4.0) 2.3±0.1(2.1-2.5) 3.2
M 32±2(30.5-33.5) 32±2(30.5-33.5) 38
T or V 75±1(72.5-77.0) 58.0±3.5(51.5-63.0) 75
Lip diam. 8.0±0.2(8-9) 7.5±0.5(7-8) 8
Lip height 3.0±0.1(3.0-3.5) 3.0±0.0(3-3) 3.5
Stylet conus 4.0±0.3(3.5-4.5) 3.7±0.3(3.5-4.0) 4.5
Stylet length 12.0±0.5(11.5-13.0) 11.0±0.5(11.5-12.0) 13
Median bulb length 16.5±1.0(13-18) 15.5±0.8(14-17) 18
Median bulb diam. 13.8±1.2(12-18) 12.5±1.5(11-17) 15
Median bulb length/diam. 1.2±0.1(0.9-1.3) 1.2±0.1(1.0-1.4) 1.2
Oesophagus length 72±2.0(69.0-76.5) 71.0±2.5(68.5-75.0) 76.5
Anterior end -to oesophageal gland base 127.0±8.5(107-141) 123±7(112-139) 139
- to middle of median bulb 60.5±3.0(55-65) 60.5±2.5(55-65) 64
- to nerve ring 74.0±3.5(68-81) 74.5±3.0(70-80) 78.5
-to excretory pore 81.0±5.5(73-91) 78.5±4.5(73-83) 91
-to hemizonid 87.0±3.5(82-94) 86.0±3.5(81.5-90.0) 94
Body diam. at middle of median bulb 16.5±1.2 (15-19) 15±1 (13-15) 18
- at vulva 21.0±2.5(16-27) - 24
-at anus 10.7±1.0(10-13) 14.0±0.7(13-15) 11
Maximum body width 22.5±3.0(17-30) 19.5±1.0(18-21) 24
Testis or ovary length 260±50(192-343) 289±25(255-321) 230
Post uterine sac length 44.5±4.5(38-57) - 55
Vulva-anus distance 96±17.5(63-128) - 122
Tail length 36.5±4.0(30-44) 32.5±2.0(30-36) 38
Rectum length 17±3(15-25) - 18
Spicule length (arc line) - 12.5±0.5(12.0-13.5) -
Spicule (rostrum to condylus) - 7.0±0.5(6.5-7.5) -
Spicule maximum width - 3.8±0.3(3-4) -
Ratio of spicule length (along arc) to width (Ryss et al., 2005; C15) - 3.5±0.5(3-4) -
Ratio of spicule length (along arc) to capitulum width (Ryss et al., 2005; C19) - 1.8±0.1(1.6-2.0) -
Ratio of capitulum depth to width (Ryss et al, 2005; C20) - 0.1±0.0(0.1-0.2) -
from Mazandaran Province, north of Iran. Molecular analysis of this species revealed it was an undescribed species most closely related to some species of the B. hofmanni group sensu Braasch et al, 2009. Detailed study revealed diagnostically shaped spicules and in
females a uniquely shaped vulval region. This species, is described herein as B. mazandaranense sp. n. We believe this is the first record of the presence of Bursaphelenchus in Iran and also the first report of the occurrence of the genus in Iran.
95
98
99
60
100
55
95
100
96
AY508022 B. mucronatus AY508023 B. mucronatus AY508020 B. mucronatus — AY284648 B. mucronatus AM397015 B. mucronatus AY508021 B. mucronatus — AB097864 B. luxuriosae AY508034 B. xylophilus AM397022 B. xylophilus AB067760 B. xylophilus GU206792 B. xylophilus FJ768947 B. xylophilus AB067759 B. mucronatus AB067758 B. fraudulentus 84 HQ699855 B. populi AY508015 B. fraudulentus AY508014 B. fraudulentus FJ501985 B. doui AB299223 B. doui AB299224 B. doui AM397012 B. doui |_i AB067757 B. conicaudatus 100 AM397011 B. conicaudatus
001- HQ727727 B. sp.
1-JF317267 B. sp.
-JF317266 B. africanus
66
74
100
100
AB358983 B. okinawaensis
-AB430445 B. sp.
-AY753531 B. kevini
100PI
100 I—
100
J00.
AY508025 B. anatolius AY508029 B. seani AY508030 B. seani AY508016 B. fungivorus
100 I- GQ845409 B. braaschae
1- AM397021 B. willibaldi
-AM397019 B. thailandae
96
GU337997 B. sp. HQ599188 B. sp.
AY508026 B. platzeri AY509153 B. cocophilus
100
73
53
97r .
Hi'
100T
100 r AM397023 B. yongensis L AB299221 B. clavicauda 100r AY508033 B. tusciae '"Hr AY508013 B. eggersi
1-AM397013 B. hildegardae
AY508028 B. poligraphi AY508012 B. borealis AY508031 B. sexdentati AM397020 B. vallesianus AY508032 B. sexdentati 100— AY508019 B. hylobianum L AM279709 B. antoniae AY508024 B. gerberae AB368529 B. rufipennis — AB232162 B. sinensis HQ407406 B. corneolus
100
97
- AB218829 B. parvispicularis
GQ421483 B. paraparvispicularis AY284649 B. sp. AY284650 B. sp. AF037369 B. sp. AY508018 B. hofmanni
JN153102 B. mazandaranense sp. n. AM397016 B. pinasteri — AY508027 B. paracorneolus
100|- AY508017 B. hellenicus
76 68 56 00
1001- '
-P-'
I- aM^Q7i
AY508011 B. abietinus AM397017 B. rainulfi
100
_I AB067756 B. abruptus
1 AY508010 B. abruptus AB368535 Aphelenchoides stammeri
— 5 changes
Fig. 2. Bayesian 10001th tree inferred from 18S under GTR+I+G model (lnL=8336.5986; AIC=16693.1973; freqA=0.2628; freqC=0.1842; freqG=0.2604; freqT=0.2925; R(a)=1.2988; R(b)=2.7482; R(c)=1.5795; R(d)=0.5118; R(e)=6.0082; R(f)=1; Pinvar=0.5388; Shape=0.5298). Posterior probability values exceeding 50% are given on appropriate clades.
MATERIAL AND METHODS
One sample from a dead beech tree had many bark beetles galleries, which yielded a population of an aphelenchid nematode having a bursa at the tail tip of males and clearly belonging to the genus Bursaphelenchus. Nematodes were isolated from the wood samples by soaking a small amount of wood in water for 48 h and were handpicked under a Nikon stereomicroscope model SMZ1000. To observe the shape of bursa and number and arrangement of caudal papillae, male specimens were transferred to a small drop of water on a clean slide and killed by gentle heat. A cover slide was placed on the nematodes and using stereomicroscopy the slide was moved by hand very gently to obtain a ventral view of the tail. Drawings of the bursa and male caudal papillae were made using a drawing tube attached to a Nikon E600. For culturing nematodes, Botrytis cinerea was grown on PDA plates. After 5 days, a small drop of water was placed on the fungus mycelia and 5-10 females and males of freshly extracted nematodes were transferred to it using a nematode pick. After 20-25 days, the plate was rinsed to collect the nematodes. The nematodes were heat killed by adding boiling 4% formalin solution and then transferred to anhydrous glycerin and processed to permanent slides according to De Grisse (1969). Drawings were made through a drawing tube attached to a Nikon E600 light microscope and were redrawn using CorelDRAW® software version 12.
For molecular analysis, nematodes were put in 50 ^l of AE buffer (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0) and crushed into multiple pieces on a microscope slide with a pipette tip. Each DNA sample was stored at -20°C until used as a PCR template. Different sets of primers were used in the PCR reactions. Primers for 18S amplification were forward primer 18S-G18S4 (5' GCT TGT CTC AAA GAT TAA GCC 3') and reverse primer 18S-18P (5' TGA TCC WKC YGC AGG TTC AC 3') (De Ley et al, 2002; Dorris et al., 2002), forward primer SSUF07 (5' AAA GAT TAA GCC ATG CAT G 3') and reverse primer SSUR26 (5' CAT TCT TGG CAA ATG CTT TCG 3') (Floyd et al., 2002), forward primer 18s965 (5' GGC GAT CAG ATA CCG CCC TAG TT 3') and reverse primer 18s1573R (5' TAC AAA GGG CAG GGA CGT AAT 3') (Mullin et al, 2005). Primers for 28S D2/D3 amplification were forward primer D2a (5' ACA AGT ACC GTG AGG GAA AGT 3') and reverse primer D3b (5'TGC GAA GGA ACC AGC TAC TA3') (Nunn, 1992).
The 25 ^l PCR contained 12.5 ^l 2* GoTaq DNA polymerase mix (Promega Corporation,
Madison, WI, USA), 1 ^l each of 0.4-^M forward and reverse primers and 1 ^l of DNA template. The thermal cycling programme was as follows: denaturation at 95°C for 6 min, followed by35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 1 min. A final extension was performed at 72°C for 10 min. PCR products were cleaned by Montage™ PCR Centrifugal Filter Devices (Billerica, MA, USA). PCR primers were used for direct sequencing by dideoxynucleotide chain termination using an ABI PRISM BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems) in an Applied Biosystems 377 automated sequencer (Applied Biosystems) by the Genomic Sciences Laboratory in North Carolina State University (Raleigh, NC, USA). The sequences were deposited in the GenBank database. DNA sequences were aligned by Clustal W (http://workbench.sdsc.edu, Bioinforma-tics and Computational Biology group, Dept. Bioengineering, UC San Diego, CA). The DNA sequences were compared with those of the other nematode species available at the GenBank sequence database using the BLAST homology search program. The top matched Bursaphelenchus species were selected to run phylogenetic analysis. The model of base substitution was evaluated using MODELTEST (Posada & Crandall, 1998; Huelsenbeck & Ronquist, 2001). The Akaike-supported model, the base frequencies, the proportion of invariable sites and the gamma distribution shape parameters and substitution rates were used in phylogenetic analyses. Bayesian analysis was performed to confirm the tree topology for each gene separately using MrBayes 3.1.0 (Huelsenbeck & Ronquist, 2001) running the chain for 1 * 106 generations and setting the 'burnin' at 1,000. We used the Markov Chain Monte Carlo (MCMC) method within a Bayesian framework to estimate the posterior probabilities of the phylogenetic trees (Larget & Simon, 1999) using the 50% majority rule.
DESCRIPTION
Bursaphelenchus mazandaranense sp. n.
(Fig. 1)
Measurements: Table 1.
Female. Body slender, slightly curved when heat-killed. Cuticle finely annulated, the annuli are more visible anterior to the vulva and about 1 ^m thick. Lateral fields with three incisures, 9-14% of corresponding body width. Lip region offset, separated from the rest of the body by a sharp constriction and 2.6-2.7 times as high as wide. Stylet well developed, 11.5-13.0 ^m long, slightly
100
59
97
98
100
100
AY508085 B. hylobianum AY508095 B. paracorneolus AY508104 B. tusciae
100 H- AM396569 B. hildegardae
AY508078 B. eggersi AM396568 B. eremus
AY 100 |l_
99
AY
100 |~ AM396581 B. yongensis '— AB299222 B. clavicauda 100 AY508100 B. sexdentati
100
100 ' 100 p
AM396578 B. vallesianus AY508102 B. sexdentati
6L
9^ AY508075 B. borealis AY508096 B. poligraphi - AB368530 B. rufipennis 100 j EU752257 B. sinensis
100
82
82
AB368538 B. sinensis AM279710 B. antoniae ■ EU107359 B. chengi
AY508092 B. gerberae
100_^ AM396566 B. corneolus
100
86
95
100
100
56 89 Pi
HQ407405 B. corneolus GQ429010 B. paraparvispicularis AB368537 B. parvispicularis FJ643489 B. anamurius FJ768949 B. anamurius
-AY508084 B. hofmanni
- JN153103 B. mazandaranense sp. n.
77
■ AM396574 B. pinasteri AY508083 B. hellenicus
AY508073 B. abruptus
100
100
96
99
63
100
k,
1001 1 100 A
AY508091 B. mucronatus |00 GU206791 B. xylophilus JF317243 B. xylophilus AY508108 B. xylophilus AY508079 B. fraudulentus 100 | FJ998281 B. populi HQ699856 B. populi
73
100
AM396571 B. luxuriosae
100
86
100
AB299228 B. luxuriosae AB299227 B. conicaudatus 85J DQ899733 B. doui EU295505 B. doui EU295501 B. doui AB358982 B. okinawaensis
_r AY508076 B. cocophilus
L AY508077 B. cocophilus
100
100
AY508094 B. platzeri
100 r AM396577 B. thailandae ' DQ497184 B. thailandae -AM396579 B. willibaldi
100
AM396564 B. arthuri AY508098 B. seani AY508082 B. fungivorus
AY508093 B. anatolius — AY508109 Aphelenchoides besseyi
— 10 changes
Fig. 3. Bayesian 10001th tree inferred from 28s under GTR+I+G model (lnL=9892.8789; AIC=19805.7578; freqA=0.1477; freqC=0.1962; freqG=0.3414; freqT=0.3146; R(a)=0.5897; R(b)=3.3139; R(c)=1.3181; R(d)=0.42; R(e)=4.7727; R(f)=1; Pinvar= 0.3719; Shape=0.9756). Posterior probability values exceeding 50% are given on appropriate clades.
swollen at base and without well developed basal knobs. Median bulb more or less oval, 13-18 x12-18 ^m sized or 0.9-1.3 times longer than wide with almost centrally located valve plates. Positions of pharyngeal gland orifice and pharyngo-intestinal junction not clearly seen. Latter usually located 3-5 ^m posterior to median bulb. Pharyngeal gland lobe 1.4-2.2 times body diam. long, dorsally overlapping intestine. Nerve ring located immediately posterior to median bulb. Excretory pore about a half median bulb length posterior to median bulb, anterior to hemizonid. Vulva at 339-531^m distance from anterior end, with its anterior lip modified into a small 2.5-3.0 ^m long flap directed posteriorly. Vagina 5-8 ^m long, directed somewhat anteriorly and surrounded with 4-5^2.0-2.5 ^m sclerotized pieces. Ovary outstretched, spermatheca oval and quadricolumella visible. Post uterine sac 1.5-2.5 times longer than body width at vulva region, occupying 40-70% of vulva to anus distance, filled with 5-6x3 ^m diam sperm. Tail conical, ventrally bent, with a pointed terminus and rounded tip.
Male. Body ventrally arcuate with tail region strongly curved ventrally after fixation. Testis expanded anteriorly and reflexed in distal end. Anterior region similar to that of female. Spicules paired, not strongly arcuate, with rounded dorsal side, 1 ^m long posteriorly bent condylus, 2 ^m long rostrum and a distinct but not prominent cucullus at terminus. Seven preanal and postanal papillae present (Fig. 1 I, J); one preanal papilla (P1) in ventral midline about 2-3 ^m above cloacal opening, one pair of subventral preanal papillae (P2) almost in line with P1 papilla, one subventral pair of postanal papillae (P3) at about 46-57% of a the tail length behind cloacal opening and one small ventral pair of papillae (P4) about 11-12 ^m from the tail terminus at the base of bursa. Tail conical with a sharp terminus in lateral view. Bursa rounded end in ventral view.
Molecular phylogenetic relationships. For molecular analysis, two fragments of ribosomal DNA were sequenced including the 1518-bp nearly-full-length of the SSU (GenBank accession number JN153102) and 618-bp LSU D2D3 (JN153103). A Blastn search of SSU and LSU sequences revealed the highest match as B. Hofmanni Braasch, 1998 collected from Picea abies in Georgenthal/Eisenach, Germany and B. pinasteri Baujard, 1980 from Pinus sylvestris L in Brandenburg, Trebitz, Germany. Sequence alignment of SSU between B. mazandaranense sp. n. and B. hofmanni yielded 1521 total characters with 23 variable nucleotides including 3 insertions/deletions. Sequence alignment of SSU between B. mazandaranense sp. n. and
B. pinasteri yielded 1521 total characters with 25 variable nucleotides including 3 insertions/deletions. A Blastn search of LSU revealed the highest match also as B. hofmanni and B. pinasteri. Sequence alignment of LSU between B. mazandaranense sp. n. and B. hofmanni yielded 726 total characters with 126 variable nucleotides including 69 insertions/deletions. Sequence alignment of B. mazandaranense sp. n. and B. pinasteri yielded 735 total characters with 122 variable nucleotides including 73 insertions/deletions. The large sequence disparity in SSU and LSU supported B. mazandaranense sp. n. as a distinct unique species when compared with all Bursaphelenchus species in GenBank. These data revealed that the LSU is more variable than the SSU.
Figure 2 presents a phylogenetic tree based on the nearly-full-length SSU from a multiple alignment of 1564 total characters, which includes all available sequences of aphelenchoidids. This dataset has 1132 constant characters (72.38%). The average nucleotide composition was as follows: 26.28% A, 18.42% C, 26.04% G and 29.25% T. Using Aphelenchoides stammeri as an outgroup, this tree inferred many highly supported monophyletic groups. This tree included more recently sequenced species of Bursaphelenchus than the paper in Ye et al. (2007), but the SSU trees had similar tree topology. Bursaphelenchus mazandaranense sp. n. is unique and a putative sister to B. hofmanni and B. pinasteri. This 100%-supported monophyletic clade also included three unidentified Bursaphelenchus species.
Figure 3 presents a phylogenetic tree based on LSU D2D3 sequences from a multiple alignment of 759 total characters. This dataset has 338 constant characters (44.53%). The average nucleotide composition was as follows: 15.14% A, 20.04% C, 33.88% G and 30.94% T. Rooted by A. besseyi, B. mazandaranense n. sp. is unique and remains sister to B. hofmanni and B. pinasteri. The branch length of B. mazandaranense n. sp. in both Figures 2 and 3 was significantly different from the other closely related species, which supported the new species status based on the molecular data.
Diagnosis and relationships. Bursaphelenchus mazandaranense sp. n. is characterized by a body length of 457-701 ^m in females and 415.5-581.5 ^m in males, three incisures in the lateral fields, the excretory pore posterior to the metacorpus, seven caudal papillae in males, spicules 12.0-13.5 ^m long dorsally rounded, 1 ^m long posteriorly bent condylus, 2 ^m long rostrum and a cucullus. Females of the new species have a ventrally bent and pointed tail end, a small vulval flap, smooth
body in the vulval region, and post uterine sac1.5-2.5 times the vulval body diam.
With the given characters, Bursaphelenchus mazandaranense sp. n. belongs to the B. hofmanni group sensu Braasch et al., 2009 and most closely resembles B. hofmanii and B. pinasteri, but is significantly different in sequences of ribosomal DNA LSU and SSU regions and is associated with a different host plant, Fagus orientalis vs Picea abies and Pinus sylvestris respectively. Furthermore, the new species could be separated from B. hofmanni by its short posteriorly bent condylus (vs almost parallel to shaft axis, 3 pm long), smooth vulval region (vs dome shaped), spicules rounded dorsally (vs angular). Compared with B. pinasteri, B. mazandaranense sp. n. has a posteriorly located excretory pore (73-91 vs 52-70 pm distance from anterior end), females with anterior and posterior lip of anus at same level (vs with a sudden constriction after anus) and posteriorly bent condylus (vs prominent and rounded end). The comparisons with other species in B. hofmanni group are as follow: Compared with B. ratzeburgii Rühm, 1956, the new species has a shorter body in females (457-701 vs 720-825 pm), smaller a (22.3-28.0 vs 39.3-41.1), smaller c (12.0-19.3 vs 20.6-22.0) and posteriorly bent condylus vs prominent rounded end. Compared to B. sachsi Rühm, 1956, the new species has different spicules (having small posteriorly bent 1 pm long condylus vs having big condylus and a constriction in distal end of spicules). Furthermore, the bursa shape differs for both species. Compared with B. corneolus Massey, 1966, the new species has differently shaped spicules (small posteriorly bent 1 pm long condylus vs as long as spicule width) and shorter post uterine sac (1.5-2.5 times corresponding body width long vs 4.5). Compared with B. curvicaudatus Wang, Yu & Lin, 2005, the new species has a shorter body length in females (457-701 vs 769-960 pm), smaller a (22.3-28.0 vs 30.2-45.1), smaller spicules (12.0-13.5 vs 16.3521.6 pm) with small posteriorly bent condylus (vs as long as spicules width and not posteriorly bent).
Type habitat and locality. Recovered from wood and bark samples with bark beetle galleries on a dead beech tree (Fagus orientalis Lipsky) in forests in Sisangan, Mazandaran province, north of Iran.
Type material. Holotype female and 10 paratype females and males deposited at Nematode Collection of Tarbiat Modares University, Tehran, Iran. Five paratype females and males are deposited in the USDA Nematode Collection, Beltsville; Department of Nematology, University of
California, Riverside and CABI Bioscience, UK
Centre, Surrey, UK.
Etymology. The specific epithet is derived from
the Mazandaran province of Iran, the region where it
was collected.
REFERENCES
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Резюме. Bursaphelenchus mazandaranense sp. n. описан по материалу из упавшего ствола бука Fagus orientalis. Дано иллюстрированное описание, основанное на морфологических и молекулярных признаках. Совокупность следующих морфологических признаков: три инцизуры, 7 папилл на хвостовом конце самца, конический, слегка загнутый вентрально хвостовой конец самки, наличие небольшой складки вульвы, экскреторная пора сзади от метакорпуса указывает на принадлежность нового вида к группе B. hofmanni. Отнесение нового вида к этой группе подтверждается и молекулярно-филогенетическим анализом последовательностей рибосомальной ДНК. Новый вид имеет последовательность для SSU (18S) и D2D3-сегмента LSU (28S), не отмеченную для других видов рода, и, в том же время, указывающую на родство с B. hofmanni и B. pinasteri. Самки Bursaphelenchus mazandaranense sp. n. характеризуется длиной тела 457-701 ^m, длиной стилета 11.5-13.0 мкм, заостренным, загнутым вентрально хвостовым концом, небольшой вульварной складкой, гладкой поверхностью тела в районе вульвы, мешковидной задней маткой превосходящей в 1.5-2.5 раза соответствующий диаметр тела. Самцы нового вида характеризуютися спикулами длиной 12.0-13.5 мкм, округло изогнутой дорсальной стороной, кондилюсом длиной 1 ^m, загибающимся в задней части, рострумом длиной 2 мкм и выраженным кукулюсом. Новый вид отличается от B. hofmanni загнутым сзади кондилюсом длиной 1 ^m (в отличие от кондилюса длиной 3 мкм почти параллельного оси основания спикулы), гладкой поверхностью тела у вульвы (в отличие от округло выступающей) и округло изогнутыми спикулами (в отличие от угловатых). Новый вид отличается от B. pinasteri задним расположением экскреторной поры (находящейся на расстоянии 73-91 мкм, а не 52-70 мкм от переднего конца), самками с одинаково выступающими передней и задней губами анального отверстия (без резкого сужения сразу за анусом) и изогнутым кондилюсом от других видов группы hofmanni, как-то B. ratzeburgii, B. sachsi, B. corneolus и B. curvicaudatus. Это первое сообщение о видах рода Bursaphelenchus в Иране.
