Description of a new species (Nematoda, Aphelenchoididae) isolated from wood packaging material

2005·0.88 MB·

Acta Zootaxonomica Sinica, 30 (2) : 314- 319 (Apr. , 2005) ISSN 1000 0739
动物分类学报
DESCRIPTION OF A NEW SPECIES (NEMATODA , APHELENCHOIDIDAE)
ISOLATED FROM WOOD PACKAGING MATERIAL
WANGJin Cheng, YU Ben Yuan, LIN Mao Song
Plant Pathology Departmentof NanjingAgricultural University, Nanjing　210095, China; E mail: [email protected]
Abstract 　Bursaphelenchuscurvicaudatussp. nov. is describedandillustrated. Specimenswereextractedfromtheintercepted
packagingwoodfromLianyungang Port, Lianyungang City, China. Bursaphelenchuscurvicaudatussp. nov. is characterized
by relativelylong bodylengths (female767 960μm; male663 831μm) ,shortstylets (female13 9 17 4μm; male13 9 16 5
μm) with weak basal thickening, smallspicules (16 5 21 6μm) with obscure cucullus, distinguishingly bent female tails and
six male caudalpapillae. Lots of featuressuch asspiculeshape, female tailshape, bodylengthsupported Bursaphelenchuscur
vicaudatussp. nov. distinguishesfrom B. hofmanni, B. abietinus, B. fungivorus, B. hellenicus, B. hylobianum, B.
rainulfi, B. eggersi and B. corneolus. The PCR ITS RFLPpattern also provided further evidence that this isolate is a new
species.
Key words　Nematoda, Aphelenchoididae, Bursaphelenchus, newspecies, ITS RFLPpattern, packagingwood.
　　Up to now, there are about 60species under genus measurement were mounted in permanent slides.
Bursaphelenchus Fuchs (1937) described (Yin, 1988; The PCR rDNA RFLPanalysis as auxiliaryidenti
Braasch, 2001; Kolossova, 1998; Kanzaki, 2000; fication technique was applied. The method of DNA
Braasch and Braasch Bidasak, 2002; Braasch and extraction refers to the published protocol with some
Burgermeister, 2002 ). Due to the devastating modification (Subbotin et al. , 2000).
pathogenicity of Bursaphelenchus xylophilus to Firstly, 2 or 3 nematodes were picked into 15μl
conifer, nematodes in genus Bursaphelenchus have ddH O and each was cut into 3 segments using No. 3
2
been studied worldwide. However, to our knowledge, scalpel. Then all the nematode segments with 12μl
most of Bursaphelenchus species merely inhabit in ddH O were transferred into 200μl PCR tubes. 1 5μl
2
dead, dying or weak host trees and unrelated to para 10 × PCR reaction buffer and 1 5μl proteinase K
sitism. (1000μg/ml) were added into each PCR tube to com
In 2001, a piece of packaging wood, intercepted plete total volume of 15μl. The tubes were frozen at
in Lianyungang port, China from a Mexican cargo - 80℃for at least 30 min. At last, the tubes were in
ship , wassent to our labfor confirmation of pine wood cubated at 65℃for 60 min and at 95℃for 10 min.
nematode (PWN) existence. However, no PWN but a The PCR reaction system (50μl) contained 0 6
strange species was found. In this paper we gave a de μmol/L of each primer, 2 mmol/L of MgCl , 0 1
2
scription to the species despite little information on its mmol/L dNTPs and 2 units of Taq polymerase (Hoyer
biology, pathogenicity, embrology was known. et al. , 1998). The forward primer is 5′CGTAA
1　Materials and Methods CAAGGTAGCTGTAG3′(Ferris et al. , 1993) and
the reverse primer is 5′TTTCACTCGCCGT
Nematodes were isolated by Baermann funnel
TACTAAGG 3′(Vrain, 1993). The PCR reaction was
method from the cut slices of the packaging wood.
carried out as the following procedure: 94℃for 2 5
Then ca 100 nematodes withsimilar shape were picked
min; 40 cycles of 94℃for 40 sec, 55℃for 40 sec,
into the mycelium of Botritiscinerea on the PDA medi
72℃for 90sec; 72℃for 5min. 5μl of the PCR am
um for further culture at 25℃. In 2002, we further
plified product was used for electrophoresis to check if
purified the isolate by just breeding one pregnant fe
the concentration of the amplification product is
male in a petri dish with fresh mycelium of Botritis
enough for the enzyme cutting. Suitable tube of am
cinerea. Then purified specimens were killed by incu 2
plification product was chosen and digested by restric
bating at 65℃for 90 sec. , fixed in TAF for further
tion endonuclease Rsa Ⅰ, HaeⅢ, Msp Ⅰ, Hinf Ⅰand
investigation. All thespecimens used for morphological
2
Theproject wassupportedbythe“863 Project”programmeof Ministryof Scienceand Technology, China.
Received6Sep. 2004, accepted20Dec. 2004.
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Apr. , 2005 王金成等: 木质包装材料中线虫一新种 (线虫门, 滑刃科) 记述 315 　

Figs 1 8. Bursaphelenchuscurvicaudatus sp. nov. 1 Anterior part. 2 Lateral lines. 3 Whole bodies of female and
male. 4 Female tail. 5 Vulvalflap. 6 Male tail (ventral view). 7 Male tail (lateral view). 8 Spicules. Scale bars
=10μm.
AluⅠwith each enzyme cutting 7μl of the PCR prod slender, cylindrical, slightly ventrally curved when
uct. Enzyme cutting fragments were resolved on 2% heat killed. Lip region high with six lips of apparently
agrose gel andstained with 1%ethidium bromide. The equal size, anteriorly flat, offset with body by distinct
details of constructing the ITS RFLP profile refer to constriction; labial annules present (Fig 20) ; the cir
the published protocol (Hoyer et al. , 1998; Braasch cular oral aperture surrounded by a nearly rectangular
et al. , 1999) depression. Stylet 13 9 17 4μm long, slender, with
2　Result weak basal thickening. Stylet cone less than half of to
tal stylet length. Median bulb oval, occupying ca 3/4
Bursaphelenchus curvicaudatus sp. nov. (Figs 1
of body diameter, with distinct valve situated central
20)
ly. Excretory pore very faint, ca 1 0 1 5 body dia
Description (unit:μm, Table 2). Female. Body 　　　　　　
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31　6　　　　　　　　　　　　　Acta Zootaxonomica Sinica动物分类学报　　　　　　　　　　　　　　　　Vol. 30　No. 2
Figs 9 17. Light microscope observations of Bursaphelenchus curvicaudatus sp. nov. 9 Anterior part. 10 Head. 11, 12, 14
Spicules. 13 Female vulvalflap. 15 Male tail (heat killed). 16 Female tail (heat killed). 17 Bursa.
meters behind median bulb. Dorsal oesophageal gland Gonad outstretched, developing oocytes in multiple
strong, ca 3 to 4 body diameters long, overlapping in files (2 to 3 files) except at the posterior end. Vulval
testine. Reproductive system single and prodelphic. flap short andsmall. Postuterinesac beyond 1/2 of the
1 2 1 1 1
1 1 2 1 2 1
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Apr. , 2005 王金成等: 木质包装材料中线虫一新种 (线虫门, 滑刃科) 记述 317 　

Figs 18 20. Scanningelectronic microscopeobservationsof Bursaphelenchuscurvicaudatussp. nov. 18 Maletail. 19 Lateralfield.
20 Head. Scale bars=2μm.
Table 1. Restrictionfragments of the amplified PCRprod cent shape. Six caudal papillae present. One pair of
uct of the new species B. curvicaudatus sp. nov. adanal ones ca. 1μm above the cloaca, one preanal
PCR Restrictionfragments papilla on the ventral midline ca. 0 3μm higher than
production RsaⅠ HaeⅢ MspⅠ HinfⅠ AluⅠ
the adnal pair, and three postanal papillae arranged in
1150 500 550 380 620 920
triangle form, i. e. one subventral pair located at the
430 390 260 290 230
proximal end of the bursa, another midventral one
220 220 190 240
about 3μm lower (Figs 6, 18).
Diagnosis and relationship. Bursaphelenchus cur
vicaudatus sp. nov. is characteristic by its small
spicule with very weak cucullus, big rostrum and tall
condylus, small female vulval flap , slim and distinctly
ventrally curved female tail. According to the shape of
the male spicule, the female tail and some other fea
tures, B. curvicaudatus resembles B. hofmanni
(Braasch, 1998 ) , B. abietinus ( Braasch &
Schmatzenhofer, 2000) , B. fungivorus (Franklin &
Hooper, 1962) , B. hellenicus (Skarmoutsos, Braasch
& Michalopoulou, 1998) , B. hylobianum ( Ko
renchenko, 1980) , B. rainulfi (Braasch &Braasch
Bidasak, 2002) , B. corneolus (Massey, 1966) and
Fig 21. The ITS RFLPpattern of Bursaphelenchuscurvicau
B. eggersi (Rühm, 1956).
datussp. nov. Lane M: maker. Lane1: PCRproduct of ITS
B. curvicaudatus sp. nov. distinguished from
regions of Bursaphelenchus curvicaudatus sp. nov. Lane 2,
B. hellenicus by its bigger“a”ratio (female: 30 45
3, 4, 5, 6: the enzyme cutting fragments of PCR product
digested with Rsa Ⅰ, Hae Ⅲ, Msp Ⅰ, Hinf Ⅰ, Alu Ⅰrespec μm vs 22 36μm; male: 32 40μm vs 22 38μm) and
tively bigger spicules (16 5 21 6 vs 11 5 17 5 v). The fe
male tails of B. hellenicus look almost straight, which
vulva anus distance. Tail sharply concoid with termi
also differ from the distinctly curved ones of the new
nus distinctly curved ventrally. Three lateral lines (i.
species. The morphometrics of B. fungivorus are
e. 2 ridges) in the midbody (Figs 2, 19).
very similar to the new species, whereas, its spicules
Male. Body, J shaped after heat killed, anterior
lack cucullus, its incisures in the midbody are more (4
region similar to the females. Lateral region with three
vs 3). The spicules of B. eggersi are relatively longer
incisures in the midbody. Testis reflexed or not, ex
(18 24μm vs 17 22μm) with no cucullus, and the
tending for ca 50% of body length. Spicule is small,
ventral sides are almost straight distinguishing the
16 5 21 6μm long, with a triangle rostrum and a
smoothly bent ones of the new species. The spicules
high condylus. Cucullus hardly visible unless observed
and female tails of B. hofmanni is morphologically al
under 1000×magnification. Tail claw like, terminus
most the same as those of the new species, and yet its
pointed, the distal edge of the terminal bursa in cres
stylets are shorter (female: 11 14μm vs 14 17μm;
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31　8　　　　　　　　　　　　　Acta Zootaxonomica Sinica动物分类学报　　　　　　　　　　　　　　　　Vol. 30　No. 2
male: 11 13μm vs 13 9 16 5μm) , its“a”ratio is The molecular analysis also showed that the
muchsmaller (female: 23 31μm vs 30 45μm; male: rDNA PCR RFLP pattern of the new species (Fig 21)
2231μm vs 32 40μm) and its spicules are shorter differs from those of B. abietinus, B. hellenicus, B.
(11 17 vs 17 22μm). The measurements of B. abiet hylobianum and B. rainulfi (Braasch and Burger
inus are distinctly from the new species, meanwhile, meister, 2002) , and from B. fungivorus, B. hof
B. abietinus has two lateral lines in contrast to three manni and B. eggersi (Braasch et al. , 1999).
ones of the new species. B. rainulfi has a extremely
Table 2. Measurements of Bursaphelenchus curvicaudatus
similar spicule and female tail to the new species, and sp. nov. (unit:μm)
yet the distal edge of the bursa of the new species is
Females Males
cresent which differs from the irregular triangle shape Holotypeparatypes 　　Paratypes
of B. rainulfi; the incisures of B. rainulfi are two, n 1 20 1 20
less than the new species. B. hylobianum has two in L 779 894±54(767960) 830 750±52(663831)
a 36 6 35 4±4 0(30 245 1) 36 7 35 0±2 4(31 540 2)
cisures. In addition, the terminal bursa of B. hylo
b 10 7 11 2±0 7(10 112 6) 8 4 9 2±0 9(8 010 4)
bianum is three or four pointed inshape different from
c 15 6 17 2±1 6(15 020 2) 21 4 19 2±1 5(16 722 9)
the crescent form of the new species. Comparing with V 74 5 71 9±1 5(69 774 8) — —
the new species, B. corneolus is much shorter (fe st 15 2 15 1±0 8(13 917 4) 14 4 15 5±0 9(13 916 5)
male: 650 700μm vs 767 960μm; male: 570 700μm sp — — 18 1 18 9±1 6(16 521 6)
vs 663 831μm) and has smaller spicules (13μm vs 　　St:stylet; Sp:spicule.
17 22μm) , smaller stylets (female: 12μm vs 13 9 Type host and locality. The interceptedpackaging
17 4μm; male: 12μm vs 14 17μm) , and the stylets wood from a Mexican cargo ship, which can only be
of B. corneolus is devoid of basal swellings. More de identified as conifer.
tails on the morphometrics of thepublishedspeciessim Type material. Now all the permanent slides are
ilar to the new species are given in the paper (Table deposited in the Nematology Laboratory of Nanjing
3). Agricultural University, Nanjing, China.
Table 3 Key morphometrics of Bursaphelenchus spp. morphologically similar to the new species. [data (unit:μm) from
published papers: Braasch, 2002b, Yin et al. , 1988]
L Stylet a c Spicule
B curvicaudatus F 767960 13 917 4 30 245 1 15 020 2
M 663831 13 916 5 31 540 2 16 722 9 16 521 6
B hellenicus F 680920 1317 2236 1731
M 640820 1317 2238 1930 11 517 5
B hofmanni F 450630 1114 2331 1321
M 489610 1113 2231 1631 1117
B eggersi F 8501122 1418 3739 20
M 450990 1416 2131 1928 1824
B abietinus F 540710 1114 2332 1626
M 530670 1113 2434 1830 1114
B rainulfi F 525750 1114 2340 1525
M 475750 1113 2544 1938 1215
B hylobianum F 541965 1417 2136 1425
M 473846 1416 2535 1526 1822
B fungivorus F 6101160 1418 2644 1017
M 5701030 1318 2645 1832 1523
B corneolus F 650700 12 29 18
M 570700 12 35 18 13
　　F:female; M: male spicules measuredalongthe middleline.
3　Discussion one nematode is enough for the PCR ITS RFLP analy
sis. However, in our studies we couldn t obtain the
Although the new species was isolated from the
ideal PCR ITS RFLP patterns. In fact, 2 or 3 nema
packaging material from the Mexican cargo ship, it s
todes are necessary. Sometimes, a faint DNA band
thoughtless to determine that the new species originates
2 2
(Fig 21) following the main PCR product (1 150 bp)
from Mex2ico. Theoretic1al2ly,1the DNA quantity of only 1 1 1 1 2 1 1 1 1 1 2 1
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Apr. , 2005 王金成等: 木质包装材料中线虫一新种 (线虫门, 滑刃科) 记述 319 　
could be visualized, which we think is the result of 140.
Ferris, V. R. , Ferris,J. M. andFaghihi,J. 1993. Variationsinspacer
nonspecific amplification. Anyway, it doesn t affect
ribosomalDNAinsomecystformingspeciesof plantparasiticnema
the ITS RFLPpattern of the new species, whichserves
todes. Fundamental andAppliedNematology, 16: 177184.
as an identification label. Generally, the male caudal Fuchs,A. G. 1937. Neue parasitische und halbparasitische Nematoden
papillae in genus Bursaphelenchus areseven, however, beiBorkenk fernundeinige andere Nematoden. Ⅰ. Teil Die Para
Bursaphelenchus curvicaudatus is different by disp siten der Waldg rtner Myelophilus piniperda L. und minor Hartig
unddie Genera RhabditisDujardin, 1845und AphelenchusBastian,
laying six ones, which is anotherspecialfeaturefor the
1865. Zoologische Jahrbücher Abteilung Für Systematik Oekologie
new species.
und Geographieder Tiere, 70: 291380.
Acknowledgements 　The first author is grateful for Hoyer, V. U. , Burgermeister, W. andBraasch, H. 1998. Identifica
tion of Bursaphelenchus species (Nematoda: Aphelenchoididae) on
the help of MA Yi Gui, Tianjin Entry exit Inspection
thebasisofamplifiedribosomalDNA (ITS RFLP). Nachrichtenblatt
and Quarantine Bureau, China, during preparation of desDeutschen Pflanzenschutzdienstes, 50: 273277.
the manuscript. Kanzaki, N. , Tsuda, K. andFutai, K. 2000. Descriptionof Bursaphe
lenchusconicaudatusn. sp. (Nematoda:Aphelenchoididae) ,isolated
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木质包装材料中线虫一新种 (线虫门, 滑刃科) 记述
王金成　余本渊　林茂松
南京农业大学植物病理系　南京　210095; Email: goldcitywang@yahoo com cn
摘　要　记述了伞滑刃属线虫1新种, 即弯尾伞滑刃线虫 道有7个尾乳突不同。交合刺形状、雌虫尾的形状及体长等
Bursaphelenchuscurvicaudatussp nov 。线虫样本自中国连云 特征能将新种与 B hofmanni、B abietinus、B. fun
港口岸截获木质包装材料中分离获得。新种体长相对较长 givorus、B. hellenicus、B. hylobianum、B. rainulfi 、B.
(雌虫 767～960μm; 雄虫 663～831μm) , 口针较短 (雌虫 eggersi 以及B. corneolus区分开来。新种特有的限制性酶切
13 9～17 4μm; 雄虫 13 9～16 5μm) , 口针基部略微加厚, 图谱 (PCR ITS RFLP 图谱) 是该分离物为1新种的分子证2
交合刺小 (16 5～21 6μm) , 末端盘状结构不明显, 雌虫尾 据。 2
明显向腹面弯曲。另外, 新种雄虫有6个尾乳突, 与以前报
(cid:190) 2
关键词　线虫门, 滑刃科, 伞滑刃属, 新种, ITS RFLP图谱, 包装材料. (cid:190)
中图分类号　Q959 17
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