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Description of Sheraphelenchus parabrevigulonis n. sp. (Nematoda: Aphelenchoididae) in pine wood packaging from Italy and redescription of S. sucus in onion bulbs from South Korea, isolated at Ningbo, China

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  • 1 1Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, P.R. China
  • | 2 2Technical Centre, Ningbo Entry-Exit Inspection and Quarantine Bureau, 9 Mayuan Road, Ningbo 315012, Zhejiang, P.R. China
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Two Sheraphelenchus populations were isolated from pine wood packaging from Italy and onion bulbs from South Korea at Ningbo Port, China, respectively. The former, on the basis of morphology and molecular sequencing, is described as S. parabrevigulonis n. sp., which is characterised by a stylet length of 11 μm, three lines in the lateral field, excretory pore at level with, or slightly posterior to, nerve ring, V = 93.1, and spicules with long condylus and membrane-like rostrum in a shallow triangular shape. The species from onion, also on the basis of morphology and sequence data, was identified as S. sucus, which is characterised by a stylet length of 13 μm (erroneously stated to be ca 6 μm in the type description), excretory pore at level of, or posterior to, median bulb, and V = 80.9. The phylogeny trees based on rDNA 18S, 28S D2-D3 and ITS1/2 sequences revealed that S. parabrevigulonis n. sp. and S. sucus clustered into a clear clade and grouped with Bursaphelenchus species. The study enriched the taxonomic information of Sheraphelenchus and confirmed its status as an independent genus.

Abstract

Two Sheraphelenchus populations were isolated from pine wood packaging from Italy and onion bulbs from South Korea at Ningbo Port, China, respectively. The former, on the basis of morphology and molecular sequencing, is described as S. parabrevigulonis n. sp., which is characterised by a stylet length of 11 μm, three lines in the lateral field, excretory pore at level with, or slightly posterior to, nerve ring, V = 93.1, and spicules with long condylus and membrane-like rostrum in a shallow triangular shape. The species from onion, also on the basis of morphology and sequence data, was identified as S. sucus, which is characterised by a stylet length of 13 μm (erroneously stated to be ca 6 μm in the type description), excretory pore at level of, or posterior to, median bulb, and V = 80.9. The phylogeny trees based on rDNA 18S, 28S D2-D3 and ITS1/2 sequences revealed that S. parabrevigulonis n. sp. and S. sucus clustered into a clear clade and grouped with Bursaphelenchus species. The study enriched the taxonomic information of Sheraphelenchus and confirmed its status as an independent genus.

Sheraphelenchus Nickle, 1970 belongs to the Aphelenchoidinae and is distinguished from other genera in the subfamily by the well-developed stylet without basal knobs or swellings, vulva posterior with a V value larger than 80, and tail forming a digitiform spike with a finely pointed terminus (Hunt, 1993). The genus includes three nominal species: S. brevigulonis (Massey & Hinds, 1970) Hunt, 1993, S. entomophagus Nickle, 1970 (the type) and S. sucus Kanzaki & Tanaka, 2013. To date, S. sucus is the only species with both a detailed morphological description and molecular data, Kanzaki & Tanaka (2013) revealing the first view of the phylogenetic status of this rare genus and an especially close relationship with Bursaphelenchus.

During inspections of imported wood packaging and plant materials at Ningbo Port, China, two Sheraphelenchus isolates were obtained. According to the morphological and molecular characters, one isolate obtained from pine wood packaging from Italy in May 2012 was described as a new taxon, S. parabrevigulonis n. sp. Another isolate, obtained from onion bulbs from South Korea in September 2011, was identified on molecular data as S. sucus. However, due to its variation in stylet length from the original description, this S. sucus isolate was redescribed using morphological characterisations and phylogenetic analyses in order to provide a better interpretation of the genus and its relationships.

Materials and methods

Culturing of nematode and morphological observations

Sawn wood packaging samples and onion bulbs were cut into small pieces and nematodes were extracted by a modified Baermann funnel technique for 24 h. Nematodes failed to culture on Botryotinia fuckeliana. Measurements were performed on permanent slides made by heat-killed nematodes fixed with FA 4:1 and ethanol-glycerin dehydration according to Seinhorst (1959) as modified by De Grisse (1969). The light micrographs were made by a Zeiss Imager Z1 microscope equipped with a Zeiss AxioCam MRm CCD camera.

Molecular studies

DNA samples were prepared according to Li et al. (2008). Three sets of primers were used in the PCR analyses to amplify the partial 18S region, the 28S D2-D3 region and the ITS1/2 region of rDNA. The 18S region was amplified with the forward primer K4f, 5′-ATG CAT GTC TAA GTG GAG TAT TA-3′, and the reverse primer K1r, 5′-TTC ACC TAC GGC TAC CTT GTT ACG ACT-3′ (Penas et al., 2006); the 28S D2-D3 region was amplified with the forward primer D2A and the reverse primer D3Br (De Ley et al., 1999), and the ITS1/2 region was amplified with the forward primer F194 (Ferris et al., 1993) and the reverse primer 5368r (Vrain, 1993). PCR conditions were as described by Li et al. (2008). PCR products were separated on 1.5% agarose gels and visualised by staining with Gelred (Biotium). PCR products of sufficiently high quality were purified and the cloning and sequencing was performed by Invitrogen.

The partial 18S, 28S D2-D3 and ITS1/2 sequences of S. parabrevigulonis n. sp. and S. sucus were compared with those of other Aphelenchoidea species available in GenBank using the BLAST homology search tool. The best-fit model of DNA evolution was obtained using Modeltest v.3.7 (Posada & Crandall, 1998). Phylogenetic analyses were carried out by implementing the Akaike-supported model, the base frequency, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates in the AIC. The tree topology for each gene was determined separately using MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003). The running programs of the trees were as described by Fang et al. (2014). The consensus trees were selected to represent the phylogenetic relationships with branch length and support level, and visualised using TreeGraph2 (Stöver & Müller, 2010).

Results

Sheraphelenchus parabrevigulonis* n. sp. (Figs 1-3)

Measurements

See Table 1.

Fig. 1.
Fig. 1.

Sheraphelenchus parabrevigulonis n. sp. A: Entire female; B: Entire male; C: Head region; D: Stylet; E: Posterior part of young female gonad; F: Lateral view of mature female tail; G: Lateral lines; H: Male tail; I: Ventral view of male tail. (Scale bars = 10 μm.)

Citation: Nematology 17, 2 (2015) ; 10.1163/15685411-00002864

Fig. 2.
Fig. 2.

Light photomicrographs of Sheraphelenchus parabrevigulonis n. sp. male. A: Entire male; B-E: Head region; F, G: Caudal papillae indicated by arrows; H-O: Male tail. (Scale bars = 10 μm.)

Citation: Nematology 17, 2 (2015) ; 10.1163/15685411-00002864

Fig. 3.
Fig. 3.

Light photomicrographs of Sheraphelenchus parabrevigulonis n. sp. female. A: Entire female; B: Head region; C: Lateral lines; D, E: Uterus with embryonated eggs; F-L: Lateral view of female tail; M: Ventral view of vulval region. (Scale bars = 10 μm.)

Citation: Nematology 17, 2 (2015) ; 10.1163/15685411-00002864

Table 1.

Morphometrics of Sheraphelenchus parabrevigulonis n. sp. All measurements are in μm and in the form: mean ± s.d. (range).

Table 1.

Description

Male

Body slender, cylindrical, ventrally arcuate when heat-relaxed. Cuticle thin with fine annulations. Lateral field with three incisures. Lip region convex, distinctly offset from body. Stylet with small basal swellings. Procorpus cylindrical, ca 1.5 body diam. long. Metacorpus (median bulb) round, oval or pear-shaped, conspicuous valve plates situated almost centrally. Pharyngo-intestinal junction immediately posterior to end of metacorpus. Pharyngeal gland lobe ca 3-4 body diam. long, overlapping intestine dorsally. Nerve ring located ca 1-2 body diam. posterior to metacorpus. Excretory pore at level with or slightly posterior to nerve ring. Hemizonid at ca 1.5 body diam posterior to nerve ring. Gonad single, outstretched. Spermatocytes arranged in 2-5 rows for most of testis length and in a single row in posterior part. Tail region almost straight or slightly arcuate ventrally. Anterior half part of tail broadly conoid, then narrowing abruptly to form a digitiform spike tapering to pointed tip. Spike ca one anal body diam. long. Cloacal lips distinctly protruding. Spicules paired, separate. Condylus long with a rounded end, membrane-like rostrum shallow triangular in form. Dorsal limb slightly arcuate ventrally, tapering smoothly to a pointed distal end. Three pairs of subventral caudal papillae: one pair located just posterior to cloacal aperture, two pairs adjacent to one another and located just anterior to spike-like projection. Bursa absent.

Female

Body habitat similar to male when heat-killed. Cuticle and anterior body region similar to male. Gonad outstretched, occasionally reflexed. Developing oocytes present in multiple rows in anterior part of ovary. Several well-developed oocytes in a single row at posterior end. Oviduct tube-like, occasionally occupied by a well-developed oocyte. Spermatheca slightly irregular oval shape, sometimes filled with well-developed sperm. Crustaformeria not conspicuous. Uterus short, with thick wall, usually containing one or two embryonated eggs. Vagina slightly inclined anteriorly. Vulva located very posteriorly and close to anus, vulval flap lacking. Vulval lips protruding slightly. Post-uterine sac absent. Rectum and anus visible. Body abruptly narrowing posterior to vulval opening, tapering to a digitiform spike with a finely rounded or bluntly pointed terminus.

Type habitat and locality

Isolated from packaging wood of Pinus sp. exported from Italy and inspected at Ningbo Port, P.R. China. Nematodes failed to culture on a lawn of Botryotinia fuckeliana.

Type material

Holotype male, 26 paratype males and 36 paratype females isolated from pine wood packaging from Italy were deposited in the nematode collection of Ningbo Entry-Exit Inspection and Quarantine Bureau (NBCIQ) (slides no. 1516-1 to 1516-15); four paratype males and five paratype females were deposited in Nanjing Agricultural University (NAU), P.R. China (slide nos N04-1 to N04-3); and three paratype females and two paratype males (slide nos T546a and T546b) were deposited in the Canadian National Collection of Nematodes (Ottawa, ON, Canada).

Diagnosis and relationships

Sheraphelenchus parabrevigulonis n. sp. is characterised by body length of the male at 545 (387-790) μm and female at 539 (433-620) μm. The cuticle is weakly annulated with three incisures in the lateral field. The stylet is 11.5 (10.2-12.6) μm long with small basal swellings. The excretory pore is located at the same level as, or slightly posterior to, the nerve ring. The male tail has a 9.8 (8.0-10.6) μm long digitiform spike with a finely pointed terminus but lacks a bursa. Three pairs of subventral caudal papillae are present. The female vulva is located very posteriorly, V = 93 (92-94).

Sheraphelenchus parabrevigulonis n. sp. is close to S. brevigulonis, a poorly known species with a rather brief description, but apparently differs by the number of incisures in the lateral field (three vs not mentioned) and the number of male subventral caudal papillae (three pairs vs two pairs). Of course, it is possible that these differences merely reflect shortcomings in the original description but, until S. brevigulonis can be re-isolated from the type locality and sequenced, we feel it is more prudent to describe our isolate, which is complete with all morphological and molecular data, as a new taxon in order to provide a firmer platform from which to view the genus. It differs from S. entomophagus by the excretory pore position (slightly posterior to nerve ring vs anterior to nerve ring), V = 93.1 (92.0-94.0) vs 82.9 (77.8-88.1), male tail length (21.4 vs 38 μm), female tail length (25.6 vs 73 μm) and female c ratio of 21.7 (16.3-31.2) vs 8.7 (7.5-10.2). It differs from S. sucus by the excretory pore position (slightly posterior to nerve ring vs anterior to nerve ring), V = 93.1 (92.0-94.0) vs 82.3 (80.3-85.1), male tail length of 21.4 (15.9-25.7) vs 49 (41-63) μm, female tail length of 25.6 (19.9-36.4) vs 82 (63-93) μm, male ratio c = 25.5 (20.4-34.0) vs 10.8 (8.5-13.8) and female ratio c = 21.7 (16.3-31.2) vs 7.6 (6.6-9.4).

S. sucus Kanzaki & Tanaka, 2013 (Figs 4-6)

Measurements

See Table 2.

Fig. 4.
Fig. 4.

Sheraphelenchus sucus. A: Entire female; B: Entire male; C: Head region; D: Stylet; E: Lateral lines; F: Posterior part of young female gonad; G: Lateral view of mature female tail; H: Male tail; I: Ventral view of male tail. (Scale bars = 10 μm.)

Citation: Nematology 17, 2 (2015) ; 10.1163/15685411-00002864

Fig. 5.
Fig. 5.

Light photomicrographs of Sheraphelenchus sucus male. A: Entire male; B, C: Head region, secretory-excretory pore indicated by arrow; D: Lateral lines; E, F: Caudal papillae (arrows); G, H: Male tail; I-N: Spicules. (Scale bars = 10 μm.)

Citation: Nematology 17, 2 (2015) ; 10.1163/15685411-00002864

Fig. 6.
Fig. 6.

Light photomicrographs of Sheraphelenchus sucus female. A: Entire mature female; B: Entire young female; C: Vulval region; D: Female tail; E, F: Uterus without eggs; G-J: Uterus with embryonated eggs. (Scale bars = 10 μm.)

Citation: Nematology 17, 2 (2015) ; 10.1163/15685411-00002864

Table 2.

Morphometrics comparison of Sheraphelenchus sucus isolates. All measurements are in μm and in the form: mean ± s.d. (range).

Table 2.

Description

Male

Body slightly ventrally arcuate when heat-relaxed. Cuticle marked by fine transverse striations, ca 1 μm wide. Lateral field ca 2 μm wide, with three equally spaced lines at mid-body. Lip region convex, not offset. Stylet ca 13 μm long, without basal knobs but with slight swellings. Procorpus cylindrical, very short. Median bulb round, oval or pear-shaped, conspicuous valve plates situated almost centrally. Pharyngo-intestinal junction immediately posterior to end of metacorpus. Pharyngeal gland lobe ca 3-4 body diam. long, overlapping intestine dorsally. Nerve ring located ca 1-2 body diam. posterior to metacorpus. Excretory pore located at level of, or posterior to, metacorpus, ca 40-65 μm from anterior end. Hemizonid ca 84-108 μm from anterior end. Gonad single, outstretched, occupying ca three fifths of body length. Spermatocytes arranged in 2-5 rows for most of testis length and in a single row in posterior part. Tail region almost straight or slightly arcuate ventrally. Anterior one third part of tail broadly conoid, narrowing sharply to form a digitiform spike tapering to pointed tip. Spike ca three anal body diam. long. Cloacal lips distinctly protruding. Spicules paired, separate. Condylus long with a rounded end, rostrum low and triangular, membrane-like. Dorsal limb slightly arcuate ventrally, smoothly tapering to a pointed distal end. Three pairs of subventral caudal papillae present: one pair located immediately posterior to cloacal opening, two pairs adjacent to one another and just anterior to spike-like projection. Bursa absent.

Female

Body habitat similar to male when heat relaxed. Cuticle and anterior body region similar to male. Gonad outstretched, occupying ca three fifths of body length. Developing oocytes present in multiple rows in anterior part of ovary, several well-developed oocytes in a single row at posterior end. Oviduct tube-like, occasionally occupied by a well-developed oocyte. Spermatheca oval, sometimes filled with well-developed sperm. Crustaformeria formed of small, rounded cells, not conspicuous. Uterus short and wall thickened, usually containing one or two embryonated eggs. Vagina slightly inclined anteriorly. Vulva located very posteriorly, vulval flap lacking. Vulval lips slightly protruding. Post-uterine sac absent. Rectum and anus visible. Body gradually narrowing posterior to vulval opening, tapering smoothly to a conical tail with a finely rounded or bluntly pointed terminus.

Voucher material

Sixteen permanent slides of S. sucus isolated 10984 from onion bulbs from South Korea were deposited in NAU (slide nos R01-1 to R01-4) and NBCIQ (slide nos 10984-1 to 10984-12). Each slide contained several adults and juveniles.

Diagnosis and relationships

The original specimens of S. sucus described by Kanzaki & Tanaka (2013) were isolated from a sap flow of Quercus serrata. Compared to morphological and morphometric data of the original isolate, our redescribed onion isolate of S. sucus was very similar to the type description, the major apparent differences being the male stylet length of 13.5 (12.8-14.0) vs 6.5 (5.5-7.0) μm, the female stylet length of 13.2 (12.3-13.9) vs 6.5 (5.5-7.0) μm, male b = 7.6 (6.7-9.7) vs 12.6 (11.5-14.4) and female b = 8.2 (6.6-10.8) vs 14.5 (10.9-16.7). The extremely short stylet length recorded by Kanzaki & Tanaka (2013) may be an underestimate, all other known records of the genus having a stylet about 12 μm long. Indeed, in Figure 3 of Kanzaki & Tanaka (2013), the LM may indicate that the conus and shaft have become detached from one another and slightly overlap, the procorpus being pushed well anteriad, possibly as a result of the fixation process. If this is the case, by measuring both putative parts, the total length of the stylet can be calculated to be in excess of 10 μm (the tip of the conus is not clearly visible could therefore be several microns longer).

The onion isolate of S. sucus differs from S. brevigulonis (Massey & Hinds, 1970) by the excretory pore position (anterior to nerve ring vs slightly posterior to nerve ring), V = 80.9 (79.0-82.8) vs 91 (91-93), male tail length of 53 (48-59) vs 23 μm, female tail length of 80 (57-116) vs 29 μm, male c ratio = 10.8 (9.3-12.8) vs 23.8 (16.4-27.7), and female c ratio = 7.5 (6.5-9.3) vs 22.0 (19.9-22.8). It differs from S. entomophagus (Nickle, 1970) by the male tail length of 53 (48-59) vs 38 μm and the number of incisures in lateral field (three vs not mentioned). It differs from S. parabrevigulonis n. sp. by the excretory pore position (anterior to nerve ring vs slightly posterior to nerve ring), V = 80.9 (79.0-82.8) vs 93.1 (92.0-94.0), male tail length of 53.3 (47.9-58.6) vs 21.4 (15.9-25.7) μm, female tail length of 80.0 (57.4-116.3) vs 25.6 (19.9-36.4) μm, male c ratio = 10.8 (9.3-12.8) vs 25.5 (20.4-34.0), and female c ratio = 7.5 (6.5-9.3) vs 21.7 (16.3-31.2).

Molecular characterisation and phylogeny

The sequences of S. parabrevigulonis n. sp. isolate 1516 for the partial 18S (1707 bp, GenBank accession number KC875226), 28S D2-D3 (780 bp, KC875232), ITS1/2 (1050 bp, KC875227), of S. sucus isolate 10984 for the partial 18S (1708 bp, KC875229), 28S D2-D3 (780 bp, KC894755) and ITS1/2 (1059 bp, KC875230 and 1060 bp, KC875231) were determined. Alignment of the sequences with Clustal W resulted in datasets of 1791 characters for 18S, 807 characters for 28S D2-D3 and 1177 characters for ITS. Phylogenetic relationships among the isolates were determined separately for each dataset using Bayesian inference (BI) with Aphelenchus avenae as outgroup. The 50% majority rule consensus phylogenetic trees were generated from 18S and ITS1/2 dataset alignment by BI analysis under the GTR + I + G model, and from 28S D2-D3, under TrN + I + G model.

Fig. 7.
Fig. 7.

Phylogenetic relationships of Sheraphelenchus and aphelenchid nematodes based on 18S. The 10 001st Bayesian tree inferred from 18S under GTR + I + G model (lnL=15710.5410; freqA = 0.2507; freqC = 0.1940; freqG = 0.2593; freqT = 0.2960; R(a) = 1.2691; R(b) = 2.8781; R(c) = 1.1276; R(d) = 0.8350; R(e) = 4.2440; R(f) = 1.0000; Pinvar = 0.2839; Shape = 0.5036). Aphelenchus avenae served as the outgroup species. Posterior probability values exceeding 50% are given on appropriate clades.

Citation: Nematology 17, 2 (2015) ; 10.1163/15685411-00002864

Fig. 8.
Fig. 8.

Phylogenetic relationships of Sheraphelenchus and aphelenchid nematodes based on partial 28S. The 10 001st Bayesian tree inferred from 28S D2-D3 under TrN + I + G model (lnL=14229.2148; freqA = 0.1949; freqC = 0.1680; freqG = 0.3072; freqT = 0.3299; R(a) = 1.0000; R(b) = 3.0230; R(c) = 1.0000; R(d) = 1.0000; R(e) = 4.2819; R(f) = 1.0000; Pinvar = 0.1360; Shape = 1.0480). Aphelenchus avenae served as the outgroup species. Posterior probability values exceeding 50% are given on appropriate clades.

Citation: Nematology 17, 2 (2015) ; 10.1163/15685411-00002864

The trees based on sequences of 18S (Fig. 7) and 28S D2-D3 (Fig. 8) showed S. parabrevigulonis n. sp., S. sucus isolate 10984 and S. sucus isolate Chiyoda (Kanzaki & Tanaka, 2013) clustered into an independent clade among Bursaphelenchus species. Sheraphelenchus parabrevigulonis n. sp. shared a similar 18S sequence with S. sucus isolate Chiyoda (97.6% similarity) and for 28S D2-D3 similarity was 88.7%. Sheraphelenchus sucus isolate 10984 shared sequence similarity with S. sucus isolate Chiyoda for 18S (99.8%) and for 28S D2-D3 (99.8%). The low sequences divergence between these two S. sucus isolates revealed that intraspecific variation may exist and isolate 10984 is herein regarded as a geographical isolate and very similar to the type population of S. sucus isolate Chiyoda. The exceptionally large reported difference in stylet length is probably, as discussed above, the result of a fixation artefact in the original description.

Fig. 9.
Fig. 9.

Phylogenetic relationships of Sheraphelenchus and aphelenchid nematodes based on partial ITS1/2. The 10 001st Bayesian tree inferred from ITS1/2 under GTR + I + G model (lnL=24227.2969; freqA = 0.2634; freqC = 0.1887; freqG = 0.2329; freqT = 0.3150; R(a) = 1.1499; R(b) = 2.4916; R(c) = 1.3247; R(d) = 0.8831; R(e) = 3.0866; R(f) = 1.0000; Pinvar = 0.0814; Shape = 1.4797). Aphelenchus avenae served as the outgroup species. Posterior probability values exceeding 50% are given on appropriate clades.

Citation: Nematology 17, 2 (2015) ; 10.1163/15685411-00002864

The ITS1/2 tree (Fig. 9) indicated S. parabrevigulonis n. sp. and S. sucus isolate 10984 clustered into an independent clade. Furthermore, the topology of three trees (Figs 7-9) also revealed that Sheraphelenchus species have a close relationship with Bursaphelenchus species, which may infer that these taxa could have shared a most recent common ancestor.

Discussion

Based on the morphology and morphometric data, S. parabrevigulonis n. sp. is most similar to S. brevigulonis (Massey & Hinds, 1970), the number of male subventral caudal papillae (three pairs vs two pairs) being the only clear reported difference between these two species. However, the hosts of these two species are completely different; S. parabrevigulonis n. sp. was isolated from pine wood packaging imported from Italy, and S. brevigulonis was isolated from the necrotic tissues of Cenangium and Ceratocystis cankers of aspens in North America. As already mentioned, the original description of S. brevigulonis apparently lacks certain important information; the species has not been reported since its original description and molecular data are entirely lacking – hence the decision to name our isolate as a new taxon.

The redescribed isolate 10984 of S. sucus only differs from the type isolate Chiyoda by the reported stylet length of the male (13.5 (12.8-14.0) vs 6.5 (5.5-7.0) μm) and female (13.2 (12.3-13.9) vs 6.5 (5.5-7.0) μm), the possible reason for this being discussed above. According to the high sequences similarity of 99.8% for both 18S and 28S D2-D3 genes between these two S. sucus isolates, we conclude that they are conspecific. Sheraphelenchus species are commonly associated with nitidulid beetles, i.e., S. brevigulonis was isolated from the necrotic tissues of Cenangium and Ceratocystis cankers of aspens associated with Epurea sp. (Massey & Hinds, 1970), S. entomophagus was discovered from Carpophilus mutilatus and Urophorus humeralis (Nickle, 1970), whilst S. sucus may be associated with nitidulid beetles found in association with the sap flow of Quercus trees (Kanzaki & Tanaka, 2013). In the present paper, S. parabrevigulonis n. sp. was isolated from pine wood packaging from Italy, which may have been incompletely treated according to ISPM15 (FAO, 2003). Before manufactured as packaging materials, the pine wood could have been infested by pine bark beetles, the eggs of which can be predated by a type of nitidulid beetle, Mimemodes japonus (Kishi, 1970). The redescribed isolate 10984 of S. sucus was obtained from onion bulbs from South Korea, which may have decomposed, thereby producing the volatiles that are known to attract nitidulid sap beetles (Nout & Bartelt, 1998). We may infer that both the S. parabrevigulonis n. sp. wood packaging isolate and S. sucus onion isolate were possibly, maybe probably, associated with nitidulid beetles. Attempts to culture these two species on B. fuckeliana failed and so re-isolation of the nematodes and observations on their insect associations and host preferences are necessary to elucidate the life history of Sheraphelenchus species.

Kanzaki & Tanaka (2013) thought that Sheraphelenchus was probably, based upon molecular phylogeny, a junior synonym of Bursaphelenchus, although they refrained from formally making the proposal. However, our phylogenetic analyses revealed that S. parabrevigulonis n. sp. and S. sucus clustered into an independent clade and grouped with Bursaphelenchus species, which indicates that Sheraphelenchus is a monophyletic group that may share a recent common ancestor with Bursaphelenchus. Among the species of Bursaphelenchus, only B. kiyoharai Kanzaki, Maehara, Aikawa, Masuya & Giblin-Davis, 2011 and B. posterovulvus Gu, Wang, He, Wang, Chen & Wang, 2014 showed a close similarity with Sheraphelenchus species in some similar morphological characters. For example, the male of B. kiyoharai possesses a similar genital papilla disposition and a spike-like tail as in Sheraphelenchus species, but the female, with V = 63.3-68.7, differs from that of Sheraphelenchus species where V is higher than 80. Bursaphelenchus posterovulvus is similar to Sheraphelenchus species only in the V value (82-86 vs >80). Furthermore, Hunt (1993) mentioned that Sheraphelenchus was characterised by the well-developed stylet lacking basal knobs or swellings. However, through the high resolution of photomicrographs, a stylet with slight basal swellings was clearly observed in S. parabrevigulonis n. sp. and both the S. sucus isolates 10984 and Chiyoda (Kanzaki & Tanaka, 2013). Sheraphelenchus parabrevigulonis n. sp. and the redescribed isolate 10984 of S. sucus have not only provided more detailed morphological and morphometric characters, but have expanded the molecular data available and confirmed the generic status of Sheraphelenchus.

*Specific epithet formed from the Latin word para = beside or near, and the species epithet brevigulonis, thereby reflecting its close similarity to S. brevigulonis.

Acknowledgements

The research was supported by the Natural Science Foundation of China (Grant No. 31471751), the National Science and Technology Support Program (2012BAK11B03) and Ningbo Natural Science Foundation (2014A610197).

References

  • De Grisse A.T. (1969). Redescription ou modifications de quelques techniques utilisées dans l’étude de nématodes phytoparasitaires. Mededelingen Rijksfaculteit Landbouw Wetenschappen Gent 34, 315-359.

    • Search Google Scholar
    • Export Citation
  • De Ley P., Félix M.A., Frisse L.M., Nadler S.A., Sternberg P.W., Thomas W.K. (1999). Molecular and morphological characterisation of two reproductively isolated species with mirror-image anatomy (Nematoda: Cephalobidae). Nematology 1, 591-612.

    • Search Google Scholar
    • Export Citation
  • Fang Y., Gu J., Wang X., Li H. (2014). Description of Aphelenchoides stellatus n. sp. (Nematoda: Aphelenchoididae) found in packaging wood from Japan. Nematology 16, 621-630.

    • Search Google Scholar
    • Export Citation
  • FAO (2003). Guidelines for regulating wood packaging material in international trade. International standards for phytosanitary measures (ISPM), No. 15. Rome, Italy, FAO.

  • Ferris V.R., Ferris J.M., Faghihi J. (1993). Variation in spacer ribosomal DNA in some cyst-forming species of plant parasitic nematodes. Fundamental and Applied Nematology 16, 177-184.

    • Search Google Scholar
    • Export Citation
  • Gu J., Wang N., He J., Wang J., Chen X., Wang X. (2014). Bursaphelenchus posterovulvus sp. n. (Nematoda: Parasitaphelenchidae) in packaging wood from Singapore. Nematology 16, 403-410.

    • Search Google Scholar
    • Export Citation
  • Hunt D.J. (1993). Aphelenchida, Longidoridae and Trichodoridae: their systematics and bionomics. Wallingford, UK, CABI Publishing.

  • Kanzaki N., Tanaka R. (2013). Sheraphelenchus sucus n. sp. (Tylenchina: Aphelenchoididae) isolated from sap flow of Quercus serrata in Japan. Nematology 15, 975-990.

    • Search Google Scholar
    • Export Citation
  • Kanzaki N., Maehara N., Aikawa T., Masuya H., Giblin-Davis R.M. (2011). Description of Bursaphelenchus kiyoharai n. sp. (Tylenchina: Aphelenchoididae) with remarks on the taxonomic framework of the Parasitaphelenchinae Ruhm, 1956 and Aphelenchoidinae Fuchs, 1937. Nematology 13, 787-840.

    • Search Google Scholar
    • Export Citation
  • Kishi Y. (1970). Mimemodes japonus Reitter (Coleoptera, Rhizophagidae), an egg predator of the pine bark beetle, Cryphalus fulvus Niijima (Coleoptera: Ipidae). Kontyu 38, 195-197.

    • Search Google Scholar
    • Export Citation
  • Li H., Trinh P.Q., Waeyenberge L., Moens M. (2008). Bursaphelenchus chengi sp. n. (Nematoda: Parasitaphelenchidae) isolated at Nanjing, China, in packaging wood from Taiwan. Nematology 10, 335-346.

    • Search Google Scholar
    • Export Citation
  • Massey C.L., Hinds T.E. (1970). Nematodes from aspen cankers in Colorado and New Mexico. Canadian Journal of Zoology 48, 97-108.

  • Nickle W.R. (1970). Description of Entaphelenchidae fam. n., Roveaphelenchus jonesi gen. n., sp. n., and Sheraphelenchus entomophagus gen. n., sp. n. (Nematoda: Aphelenchoidea). Proceedings of the Helminthological Society of Washington 37, 105-109.

    • Search Google Scholar
    • Export Citation
  • Nout M.J.R., Bartelt R.J. (1998). Attraction of a flying nitidulid (Carpophilus humeralis) to volatiles produced by yeasts grown on sweet corn and a corn-based medium. Journal of Chemical Ecology 24, 217-1239.

    • Search Google Scholar
    • Export Citation
  • Penas A.C., Metge K., Mota M., Valadas V. (2006). Bursaphelenchus antoniae sp. n. (Nematoda: Parasitaphelenchidae) associated with Hylobius sp. from Pinus pinaster in Portugal. Nematology 8, 659-669.

    • Search Google Scholar
    • Export Citation
  • Posada D., Crandall K.A. (1998). MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817-818.

  • Ronquist F., Huelsenbeck J.P. (2003). MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572-1574.

  • Seinhorst J.W. (1959). A rapid method for the transfer of nematodes from fixative to anhydrous glycerin. Nematologica 4, 67-69.

  • Stöver B.C., Müller K.F. (2010). TreeGraph 2: combining and visualizing evidence from different phylogenetic analyses. BMC Bioinformatics 11, 7.

    • Search Google Scholar
    • Export Citation
  • Vrain T.C. (1993). Restriction fragment length polymorphism separates species of the Xiphinema americanum group. Journal of Nematology 25, 361-364.

    • Search Google Scholar
    • Export Citation

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