Bursaphelenchus singaporensis, GU, JIANFENG, ZHANG, JIANCHENG, BRAASCH, HELEN & BURGERMEISTER, WOLFGANG, 2005

GU, JIANFENG, ZHANG, JIANCHENG, BRAASCH, HELEN & BURGERMEISTER, WOLFGANG, 2005, Bursaphelenchus singaporensis sp. n. (Nematoda: Parasitaphelenchidae) in packaging wood from Singapore-a new species of the B. xylophilus group, Zootaxa 988 (1), pp. 1-12 : 3-10

Description of Bursaphelenchus singaporensis sp.n. ( Figs 1–3 )

MEASUREMENTS See Table 1.

MORPHOLOGICAL DESCRIPTION

Female

Displaying all the features of the Aphelenchoidoidea according to Hunt (1993), heatrelaxed form slim and ventrally arcuate. Cuticle marked by fine annules. Lateral field 5 µm wide with four lines. Lip region convex, 4.2 µm high, 9.4 µm wide, and offset by a distinct constriction. Stylet slender with small basal swellings, shaft forming about two thirds of total stylet length. Procorpus cylindrical. Median bulb oval, sometimes almost rectangular. Valve plates slightly posterior to the middle of the median bulb. Oesophageal gland lobe extending dorsally for a considerable distance (two to three body widths long) down the body. Nerve ring located at ca. one quarter of body diameter posterior to the median bulb. Excretory pore position at the level of median bulb or slightly posterior to it. Hemizonid at the level of intestine, ca. 100 µm from anterior end. Reproductive system prodelphic, gonad outstretched, occupying about half of the body length. Developing oocytes arranged in multiple rows. Anterior vulval lip prolonged posteriorly forming a ca. 8 µmlong vulval flap. Vulva situated in a slightly sunken area, about as long as the vulval flap and alongside strengthened by two small ribs. Postuterine branch extending over two thirds of vulva to anus distance, acting as a seminal receptacle. Tail long and slender, tapering to a slightly ventrally bent and finely rounded terminus, sometimes with a very small mucro.

Male

Anterior body region and cuticle similar to that of female. Body bent ventrally when killed by heat. Testis outstretched, occupying about half of the body length. Spermatocytes arranged in multiple rows. Spicules paired, very large and arcuate with pointed rostrum, sometimes with slightly dorsally hooked condylus. Distal ends of spicules with small cucullus. Tail ventrally arcuate with a pointed, talon­like terminus bearing a distinct, ovalshaped terminal bursa, which can be seen in dorso­ventral position. There are three pairs of caudal papillae (one preanal and two pairs close to each other just anterior to the bursa) and a single mid­ventral papilla anterior to the anus. Their position is shown in Figures 1 and 3 .

DIAGNOSIS AND RELATIONSHIPS

Bursaphelenchus singaporensis sp. n. clearly belongs to the B. xylophilus group having males with the typically shaped spicules with a cucullus at their distal extremity, the typical number and position of caudal papillae (a pair and a single one preanal, two adjacent pairs postanal just before the bursa) and the anterior vulval lip of the females developed as a distinct flap. The new species is characterized by a body length of 792 (553– 950) µm and 850 (690–961) µm of males and females, respectively, robust body (a= 34 for males and 31 for females), 15–16 µm­long stylet, lateral field with four lines, long postu­ terine branch (mean 102 µm) occupying about two thirds of vulva to anus distance, and a strongly conoid female tail (c= 20) with a finely rounded, only slightly ventrally bent terminus, male with very strong spicules (41–48 µm long), distinct rostrum and small cucullus, and a dorso­ventral visible terminal bursa.

Having the typical characters of the B. xylophilus group, Bursaphelenchus singaporensis sp. n. is easily distinguishable from almost all other Bursaphelenchus species except species of this group. It is morphologically similar to B. abruptus Giblin­Davis , Mundo­ Ocampo, Baldwin, Norden & Batra, 1993 (not in the B. xylophilus group), B. xylophilus , B. mucronatus Mamiya & Enda, 1979 , B. kolymensis Korenchenko, 1980 , B. fraudulentus Rühm, 1956 , B. conicaudatus Kanzaki, Tsuda & Futai, 2003 , B. baujardi Walia, Negi, Bajaj & Kalia, 2003 and B. luxuriosae Kanzaki & Futai, 2003 . The differentiation within the B. xylophilus group is mainly based on the shape of the female tail, whereas the spicules of males in the group widely resemble each other. However, B. singaporensis sp. n. differs from all other species of the B. xylophilus group by having larger spicules.

B. singaporensis sp. n. differs from B. abruptus by the degree of ventral curvature of the spicules (less bent in B. abruptus ), the larger spicules (41–48 vs. 23–30 µm) and the longer postuterine sac of females averaging 28% of the vulva­anus distance in B. abruptus (mean 57 µm) compared to 67% in B. singaporensis sp. n. (mean 102 µm).

B. singaporensis sp. n. differs from B. xylophilus mainly by the tapering female tail compared to the cylindrical tail with rounded tail terminus in B. xylophilus . It is more robust than B. xylophilus , B. mucronatus , B. kolymensis (a = 34/ 31 for males/ females vs. a averaging more than 40 in both males and females of the other three species). B. singaporensis sp. n. differs from the mucronate form of B. xylophilus and from B. mucronatus , B. kolymensis , B. fraudulentus , B. conicaudatus and B. baujardi by the lack of a distinct mucro at the female tail terminus. B. fraudulentus may have an indistinct mucro, but is reported as having spicules of 21–22 µm in length ( Rühm, 1956) vs. 41–48 µm in B. singaporensis sp. n. and a differently shaped tail. B. conicaudatus and B. baujardi , possessing a short mucro, also have shorter spicules of 23–28 µm and 25–28 µm in length, respectively.

B. singaporensis sp. n. is most similar to B. luxuriosae . The female tails of both species resemble each other in shape. B. singaporensis sp. n. differs morphologically from B. luxuriosae by the size of the spicules (41–48 vs. 27–30 µm), the vulva position (76 % vs. 71% of body length) and the position of the excretory pore, which is usually more anterior in B. singaporensis sp. n. (position at the level of median bulb or slightly posterior to it) than in B. luxuriosae (position ranging between centre of median bulb and hemizonid level).

MOLECULAR DIFFERENTIATION OF B. SINGAPORENSIS SP. N. FROM SIMILAR

SPECIES

The pattern of DNA restriction fragments obtained in the ITS­RFLP analysis of B. singaporensis sp. n. is different from the patterns of five other species of the B. xylophilus group and of the morphologically similar B. abruptus ( Fig. 4 and Table 2). It is also distinct from the ITS­RFLP patterns of 20 other Bursaphelenchus species summarized by Burgermeister et al. (2005).

As shown in the ITS­RFLP pattern of B. singaporensis , digestion of the amplified rDNA sequence (950 bp) with Hae III resulted in four restriction fragments of approximately 840, 550, 290 and 120 bp, respectively. With some DNA extracts of the nematode, only the restriction fragments of 840 and 120 bp were obtained which together correspond to the size of the undigested rDNA sequence. The appearance of the additional restriction fragments of 550 and 290 bp may be explained by the presence of a Hae III restriction site within the 840 bp fragment in part of the amplified DNA. This may be caused by microheterogeneity of rDNA either between different specimens or in the same specimen, as has been observed, for instance, in Meloidogyne spp. ( Zijlstra et al., 1995). The situation may be clarified by sequencing of amplified and cloned rDNA of B. singaporensis (experiments in progress).

TYPE LOCALITY AND HABITAT

Packaging wood of non­coniferous hardwood exported from Singapore and inspected in Ningbo Entry­exit Inspection and Quarantine Bureau, China, in 2003.

TYPES

Collected from a culture on Botrytis cinerea on malt agar, developed from a sample taken in China from packaging wood arriving from Singapore with other commodities. Slides in the nematode collection of Helen Braasch (holotype, allotype, paratypes 6 males and 6 females) , in the USDA Nematode Collection , Beltsville, Maryland, USA (paratypes 3 males and 3 females) and in the nematode collection of Ningbo Entry­exit Inspection and Quarantine Bureau, China (paratypes 6 males and 6 females) .

Figures

FIGURE 1. Bursaphelenchus singaporensis sp. n. A: Female; B: Male; C: Head region; D: Female tails; E: Male tail; F: Vulval region; G: Bursa; H:. Spicules. (Scale bars = 10 µm).

FIGURE 2. Light photomicrographs of Bursaphelenchus singaporensis sp. n. A, B: Head region; C, D: Female tail; E, F: Male bursa; G, H: Male tail; I: Vulval region. (Scale bars = 10 µm).

FIGURE 3. Scanning electron microscope (SEM) observations of Bursaphelenchus singaporensis sp. n. A: Head region; B: Female tail; C: Male tail showing caudal papillae, bursa and protruded tips of spicules; D: Female cuticle showing four lateral lines.

FIGURE 4. ITS­RFLP patterns of Bursaphelenchus singaporensis sp. n. and six morphologically similar species. Each picture shows (from left to right) DNA marker (100 bp ladder, Invitrogen), rDNA amplification product, restriction fragments obtained with Rsa I, Hae III, Msp I, Hinf I, Alu I, and again DNA marker.