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83Russian Journal of Nematology, 2010, 18 (2), 95 - 102
A new stem nematode, Ditylenchus weischeri sp. n. (Nematoda: Tylenchida), a parasite of Cirsium arvense (L.) Scop. in the Central Region of the Non-Chernozem Zone of Russia
1 1 12 Vladimir N. Chizhov , Boris A. Borisov and Sergei A. Subbotin '
'Center of Parasitology of A.N. Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences, Leninskii
Prospect 33, Moscow, 117071, Russia e-mail: chizhov@list.ru
2Plant Pest Diagnostics Center, California Department of Food and Agriculture, 3294 Meadowview Road, Sacramento, CA
95832-1448, USA
Accepted for publication 14 June 2010
Summary. A new species of the stem nematode, Ditylenchus weischeri sp. n. parasitising creeping thistle, Cirsium arvense (Asteraceae), is described from material collected in the Moscow region, Russia. Ditylenchus weischeri sp. n. differs from D. dipsaci by shorter tails in adults, larger c index, shorter spicules, longer vulva-anus distance, larger vulva-anus distance to tail length ratio and longer posterior sac. Ditylenchus weischeri sp. n. differs from other Ditylenchus in ITS-rRNA and hsp90 gene sequences and chromosome numbers. Ditylenchus weischeri sp. n. did not develop and complete its life cycle in onion and garden strawberry seedlings, which are typical hosts for D. dipsaci. Diagnostic PCR-ITS-RFLP profiles are given for D. weischeri sp. n. and D. dipsaci. Key words: hsp90 gene, Ditylenchus dipsaci, onion, PCR, RFLP, strawberry.
Creeping thistle, Cirsium arvense (Asteraceae), has been considered as a host-plant for the stem nematode Ditylenchus dipsaci and several experimental studies showed that different host races of D. dipsaci can infect and develop on this plant (Salentiny, 1957; Nolte, 1959; Anon, 2008). Cirsium arvense is a weed, which is widely distributed in the territory of the European region of Russia. The creeping thistle plants infected by the stem nematode, which causes stem swellings, were sometimes found at the edges of agricultural fields, abandoned agricultural fields, along roadside ditches and banks of small rivers and streams, where regular grass cutting does not occur and the plant forms continuous brushwood for many years. However, several studies showed that populations of stem nematode from Cirsium plants found in natural conditions are different from the races of D. dipsaci commonly infecting agricultural crops, or D. dipsaci sensu stricto, in morphology, karyology, biology (Ladygina, 1978; Barbashova, 1979) and the sequences of the ITS rRNA gene (Subbotin et al., 2005). The results of several studies showed that the
stem nematode parasitising C. arvense should not be considered as a race of D. dipsaci but independent species. In this paper we provide the description of this stem nematode as a new species.
MATERIAL AND METHODS
Nematodes were extracted from the stem swellings of Cirsium arvense, garden strawberry and infected bulbs of onion collected in the Moscow region, Russia. Crushed plant tissue was placed in Petri dishes with water for 2-4 h and then extracted nematodes were fixed in hot 4% TAF. Nematodes were processed in glycerol and embedded on permanent slides using standard methods (Seinhorst, 1959). All measurements of nematodes were made from permanent slides using Axio Image A1 objective of a Carl Zeiss microscope.
To evaluate the ability to infect onion (Allium cepa L.) and garden strawberry (Fragaria x ananassa), seedlings of these two crops were sprayed in field condition with a suspension of nematodes from C. arvense. The inoculum density was 300-350 nematodes of different life stages per
Fig. 1. Ditylenchus weischeri sp. n. A: Anterior region of male; B: Lateral field; C: Region of cristaformeria; D: Head region of male; E: Posterior end of female; F: Posterior region of male; G: Spicules.
Table 1. Morphometries of Ditylenchus weischeri sp. n. and two populations of D, dipsaci sensu stricto (all measurements in |am).
Species Host Characters D. wischeri sp. n. D. di psaci
Circium arvence Allium сера Fragaria x ananassa
Holotype female Paratype females Paratype males Females Males Females Males
n 1 23 25 23 22 21 22
L 1575 1371-1619(1545+72.8) 1281-1578 (1433+78.8) 1250-1708 (1392+98.3) 1201-1473 (1362+69.6) 1203-1480 (1340+62.1) 1182-1390 (1264+55.6)
a 38.9 35.5-44.8 (40.6±2.7) 50.1-60.4 (54.3+2.9) 31.3-45.5 (40.0±3.6) 39.7-53.5 (44.4±3.6) 30.2—47.1 (38.0+4.1) 35.4-52.7 (46.1+4.3)
b 8.4 7.0-9.0 (8.2±0.5) 6.3-8.2 (7.4+0.6) 6.4-8.2 (7.1±0.5) 6.3-7.8 (6.9±0.4) 6.0 8.8 (6.9+0.6) 5.5-6.4 (6.0+0.3)
c 22,6 18.3-28.1 (23.2*2.3) 18.4-26.4 (23.4+2.4) 13.5 19,5 (15.1+1.5) 13.9 16.3 (14.9 ±0.6) 14.1 18.7(16.3+1.4) 14.0-17.3(16.1+1.1)
c' 4.0 2.9—4.8 (3.7+0.4) 3.5-5.0 (4.2+0.5) 4.2-5.5 (4.8±0.4) 4.7-6.2 (5,3±0.4) 3.7-6.0 (4.9±0,6) 4.0-6.4 (5.1+0.7)
e* 2.8 2.4-3.8(3.0 ±0.3) - 1.4-2.1 (1.7±0.1) - 1.6-2.4 (2.0±0,2)
Stylet 10 9-13 (11+0.7) 9-11 (10±0.4) 10-12 (11±0.5) 10-12 (11±0.4) 9-11 (10+0.3) 9-11 (10+0.4)
V% 83 81-85 (83±1.0) - 80-86 (82±1.5) - 79-84 (81± 1.2) -
Spicules - - 20-24 (21 ±1.4) - 22-28 (26 ±1.6) - 20-27 (24+0.7)
Gubernaculum - - 6-9 (7±1.3) - 8-11 (9+1.2) - 8-10(9+0.6)
Body width 40 32^14 (37±3.7) 23-29 (26± 1.8) 31—45 (35±3.5) 24-34 (28±2.8) 27^13 (36+4.3) 24-35 (28+3.1)
Oesophagus length 188 170-203 (184+8.5) 176-214 (194+10.6) 175-208 (194±8.3) 159-207 (189± 11.9) 153-245 (196+21.6) 193-230(213+11.4)
Tail length 69 54-84 (65+7.0 ) 50-73 (61 ±6.2) 85-103 (93±5.5) 80-97 (87±4.3) 75-105 (83+7.6) 70-93 (79+6.7)
Anal body diameter 17 14-19 (17±1.1) 13-16 (14±1.0) 17-23 (20±1.2) 14-18 (16±1.1) 14-22(17+2.5) 12-18(16+1.6)
Anterior end to:median bulb 69 65-75 (71+2.7) 67-86 (75±4.5) 67-78 (73±3.0) 68 85 (73±4.3) 67-80 (72+4.2) 70-82 (75+3.1)
Anterior end to nerve ring 107 101-119 (108±6.2) 95-124 (U2±7.3) 112-130 (119±5.8) 94-134(116+9.4) 102-138(116+10.7) 95-135 (112+11.1)
Anterior end to hemizonid 139 127-145 (137±6.8) 128-145 (139+6.5) 127-158(144±7.8) 131-151 (142+5.7) 130-155 (142+7.5) 125-158 (142+8.6)
Anterior end to excretory' pore 144 133-150 (143±4.3) 130-150 (143+4.8) 140-168 (153+6.5) 136-162 (151±5.6) 137-165 (149+8.5) 135-165 (151+8.9)
Anterior end to vulva 1305 1132-1308 (1240+56.3) - 1020-1475 (1144+97.2) - 1025-1212 (1097+53.3) -
Vulva-anus distance 201 172-240(194±21.2) - 132-175 (150-113.5) - 145-188(166+11.8) -
Post-vulval uterine sac 114 101-150 (121±14.7) - 70-100 (83±9.2) - 63-118 (80+12.3) -
Bursa length - - 48 78 (65±9,8) - 62-97 (82±11.3) - 50-93 (73+9.1)
Tail part length without bursa - - 12-30(21+5.7) - 25-34 (29+2.8) - 18^0 (29+8.6)
*e - vulva anus distance to tail length ratio.
70 mkm 70 mkm 50 mkm
A, K B-F G-J
Fig. 2. Ditylenchus weischeri sp. n. - A: Anterior region of female; C-E: Posterior region of female; G, H: Tail region of male; J: Posterior region of male with testis; K: Region of uterus. Ditylenchus dipsaci - B, F: Posterior region of female; I: Tail region of male; an - anus.
seedling. After this treatment the seedlings were five plants of each crop were included in the test. covered by polyethylene caps overnight. At least The plant tissues were examined 1 month after
inoculation. The life cycle of the new species was studied in a naturally infected area. The samples were taken in 2 weeks intervals.
DNA extraction, PCR, PCR-RFLP cloning and sequencing were made as described by Subbotin et al. (2005) and Mundo-Ocampo et al. (2008). The ITS-rRNA and hsp90 gene (Mundo-Ocampo et al, 2008) were amplified using TW81 and AB28, U831 and L1110 primers, respectively. Newly obtained sequences were submitted to the Genbank under the accession numbers: HM778133-HM778140. Sequences were aligned with ClustalX 1.83 and then analysed using Bayesian inference using MrBayes 3.1.2 as described by Mundo-Ocampo et al. (2008).
DESCRIPTION
Ditylenchus weischeri sp. n. (Table 1; Figs. 1 & 2)
Holotype female. See Table 1. Females. Body shape is typical for parasitic nematodes of the genus Ditylenchus and became straight, sometimes slightly curved ventrally in posterior region, after killing by heating. Lip region offset body, 6-8 (7) |m wide and 2-3 (2.5) |m height. Stylet small, short with stylet knobs 2 |m in width. The orifice of dorsal pharyngeal gland in 1-2 |m from stylet basis. Cuticle with fine annulation, which is uniform throughout the entire length of body. Annules 0.7 to 0.9 |m wide. Lateral field indistinct, with four incisures, occupies 15-30% of body width, or 4-6 |m, two internal incisures poorly visible. Distance between incisures almost equal, but sometimes it is larger between internal than between external incisures. Procorpus cylindrical, narrowing slightly toward the median bulb. Median bulb oval with developed valve, which is situated in a bulb center or slightly displaced anteriorly. Median bulb 17-23 (30) |m long and 10-16 (13) |m wide. Isthmus narrow and relatively long. Basal bulb well developed, occupies nearly 75% of body width. Basal bulb with a slight overlap over intestine. Nerve ring at the level of posterior half of isthmus. Hemizonid and excretory pore at the level of anterior half of basal bulb. Posterior edge of hemizonid within few annules, or 1-10 |m anterior to excretory pore. Ovary outstretched, 80-188 (130) |m long, sometimes reaching basal or median bulb, with one flexure. Spermatheca sac-shaped. Crustaformeria in form of quadricolumella consists of 16-20 cells arranged in 4 rows, per 4-5 cells in a row. Oocytes and ooginia arranged in 2-3 rows. Post-vulval uterine sac well developed, more than 50% of vulva to anus length. Anus well observed. Tail relatively short, elongate conical with pointed terminus.
Males. Body elongated and slender. Structure of anterior region is similar to that for females. Testis
outstretched, reaching basal bulb basis, with one flexure. Spermatozoa oval shape, 7-10 (9) |m. Bursa does not enclose a tail tip. Spicules tylenchoid type. Tail elongate-conical, tapers to pointed tip.
Eggs (n=15): L = 69-91 (78) |im, W = 26-40 (32) |im.
Biology. In Moscow region, three-four generations occur during a vegetation season. Symptoms on infected plants can be observed from the second half of summer and become clearly visible at the beginning of flowering. Stem swellings or galls are formed at a distance of nearly 1 m and higher above soil level (Fig. 3). The galls were characterised by extensive hypertrophy and hyperplasia, differentiation of nutritive tissue, nuclear modification, and a central cavity containing nematodes (Watson & Shorthouse, 1979). At the end of flowering during the second half of August, when stem swellings become black and start cracking, the majority of nematodes start leaving the plant tissues and move into soil, where the fourth-stage juveniles overwinter. The fourth-stage juveniles infect seedlings of creeping thistle plants during early spring. Nematodes can survive in plant tissues in an anhydrobiotic condition for at least up to three years.
The results of the host tests revealed that D. weischeri sp. n. did not develop and complete its life cycle in onion or garden strawberry seedlings.
Type host-plant. Creeping thistle, Cirsium arvense (L.) Scop.
Type locality. Village Maikovo, Pushkin district, Moscow region, Russia.
Other localities. Ditylenchus weischeri sp. n. was found in other north and north-west districts of the Moscow region and in several regions of Russia: Jaroslavl, Tver, Ivanov and Vladimir regions. Watson and Shorthouse (1979) reported the stem nematodes from C. arvense collected in a pasture near Regina, Saskatchewan, Canada, which, perhaps, belongs to D. weischeri sp. n. Janezic (1994) also found the stem nematode from Cirsium oleraceum leaves in Croatia.
Differential diagnosis. Ditylenchus weischeri sp. n. differs from D. dipsaci by shorter tails in adults (females = 55-84 |m vs 70-108 |m; males = 50-73 |m vs 80-97 |m), larger c index (18-28 vs 1122), shorter spicules (20-24 | m vs 20-30 | m), longer vulva-anus distance (172-240 |m vs 132-188 |m), larger vulva-anus distance to tail length ratio (2.4-3.8 vs 1.4-2.1), longer post-vulval uterine sac (101-150 vs 70-100 |im) (Table 1; Metlitskii, 1968; Brzeski, 1998).
Chromosome numbers. Barbashova (1979) reported 2n = 56 for the stem nematode from Cirsium and 2n=24 for D. dipsaci sensu stricto.
Etymology: This species is named after Prof. Dr Bernhard Weischer.
Fig. 3. Ditylenchus weischeri sp. n. A-D: Symptoms of infection on Cirsium arvense. Ditylenchus dipsaci. E, F: Symptoms of infection on strawberry.
Fig. 4. PCR-ITS-RFLP profiles. A: Ditylenchus weischeri sp. n., B: D. dipsaci. M - 100 bp DNA marker (Promega); U -unrestricted PCR product; 1 - Bsh 1236I; 2 - Hinfl; 3 - MspI; 4 - RsaI; 5 - TaqI.
Table 2. Restriction fragment lengths (bp) for PCR-ITS-RFLP of two plant-parasitic Ditylenchus species.
Restriction enzymes D. weischeri sp. n. D. dipsaci
Unrestricted PCR product 750 752
Bsh 1236I 750 467, 285
Hinfl 262, 214, 111, 88, 46, 29 374, 289, 89
MspI 315, 140, 139, 133, 23 316, 140, 139, 134, 23
RsaI 450, 300 300, 264, 127, 61
TaqI 358, 242, 65, 51, 34 359, 208, 65, 51, 35, 34
100
100
71
96
100
-D. dipsaci {Fragaria, CD279, cl1)
- D. dipsaci (Allium cepa, CD278, cl2)
— D. dipsaci (Fragaria sp., CD279, cl2)
L D. dipsaci (Allium cepa, CD278, cl1) ■ D. weischeri sp. n. (CD276, cl2)
D. weischeri sp. n. (CD276, cl1) Ditylenchus sp. G. (Plantago maritima, CD275, cl1)
Ditylenchus sp. G. (Plantago maritima, CD275, cl2) Cephalenchus hexalineatus (EU915495) - Ditylenchus sp. (AY603512)
100
Fig. 5. Phylogenetic relationships of plant-parasitic Ditylenchus species as inferred from the analysis of hsp90 gene sequences using Bayesian analysis.
Slides. Holotype and paratypes of D. weischeri sp. n. were deposited under the number 98/33-40 in the Nematological collection of the Center of Parasitology of A.N. Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences, Moscow, Russia.
Molecular characterisation. The analysis of the ITS-rRNA of plant-parasitic Ditylenchus species revealed that D. weischeri sp. n. (or Ditylenchus sp. C) was different from D. dipsaci sensu stricto and several other still undescribed Ditylenchus species and formed a separate clade (Subbotin et al., 2004, 2005). A similar picture of the phylogenetic relationships of plant-parasitic Ditylenchus species was obtained after analysis of hsp90 gene sequences using Bayesian analysis (Fig. 5). PCR-ITS-RFLP patterns for D. weischeri sp. n. and D. dipsaci sensu stricto are given in Fig. 4. The restriction of PCR products by enzymes Bsh1236I, Hinfl, Rsal and TaqI enables these two species to be distinguished from each other (Table 2).
REFERENCES
Brzeski, M.W. 1998. Nematodes of Tylenchina in Poland and temperate Europe. Warszawa, Muzeum i Instytut Zoologii. Polska Akademia Nauk. 397 pp. Anon, (2008). Ditylenchus destructor and Ditylenchus
dipsaci. EPPO Bulletin 38, 363-373. Barbashova, V.N. 1979. [The karyotypes of stem nematodes of wild plants.] Parazitologiya 13: 257261.
BRZESKI, M.W. 1998. Nematodes of Tylenchida in
Poland and temperate Europe. Warsaw. 397 pp. Janezic, F. 1994. [Second contribution to the knowledge of zoocecidia in Croatia.] Zbornik Biotehniske Fakultete Univerze v Ljubljani, Kmetijstvo 63: 145152.
Ladygina, N.M. 1978. [The genetic and physiological compatibility of different forms of stem nematodes. VI. Cross-breeding of Ditylenchus from cultivated
plants and from weeds.] Parazitologiya 12, 349-353.
Metlitskii, O.Z. 1968. [Use of morphological parameters for recognizing different forms of stem nematodes]. Parazitogiya 2: 528-533.
Mundo-Ocampo, M., Troccoli, A., Subbotin, S.A., Del Cid, J., Baldwin, J.G. & Inserra, R.N. 2008. Synonymy of Afenestrata with Heterodera supported by phylogenetics with molecular and morphological characterisation of H. koreana comb. n. and H. orientalis comb. n. (Tylenchida: Heteroderidae). Nematology 10: 611-632.
Nolte, H.W. 1959. Weitere Beobachtungen uber eine Zwiebel-populatlon von Ditytenchus dipsaci (Kuhn 1858) Filipjev. Wissenschaftliche Zeitschrift der Martin-Luther-Universitat Halle-Wittenberg 8: 1123-1125.
Salentiny, T. (1957). Untersuchungen über den Wirtspflanzenkreis einer Rubenrasse von Ditylenchus dipsaci in Baden-Wiirttemberg. Nematologica 2: 382-386.
Seinhorst, J.W. 1959. A rapid method for transfer of nematodes from fixative to anhydrous glycerine. Nematologica 4: 67-69.
Subbotin, S.A., Krall, E.L., Riley, I., Chizhov, V.N., Staelens, A., De Loose, M. & Moens, M. 2004. Evolution of the gall-forming plant parasitic nematodes (Tylenchida: Anguinidae) and their relationships with hosts as inferred from Internel Transcribed Spacer sequences of nuclear ribosomal DNA. Molecular Phylogenetics and Evolution 30: 226-235.
Subbotin, S.A., Madani, M., Krall E., Sturhan, D. & Moens, M. 2005. Molecular diagnostics, taxonomy and phylogeny of the stem nematode Ditylenchus dipsaci species complex based on the sequences of the ITS-rDNA. Phytopathology 95: 1308-1315.
Watson, A.K. & Shorthouse, J.D. 1979. Gall formation on Cirsum arvense by Ditylenchus dipsaci. Journal of Nematology 11: 16-22.
Чижов В. Н., Борисов Б. А., Субботин С.А. Новая стеблевая нематоды Ditylenchus weischeri sp. n. (Nematoda: Tylenchida) - паразит Cirsium arvense (L.) Scop. в Центральном регионе Нечерноземной зоны России.
Резюме. По материалу, собранному в Московской области, описан новый вид стеблевых нематод Ditylenchus weischeri sp. n., паразитирующий на чертополохе Cirsium arvense (Asteraceae). Ditylenchus weischeri sp. n. отличается от D. dipsaci меньшей длиной хвостового конца, более высоким значением индекса «с», короткими спикулами, большим расстоянием от вульвы до анального отверстия, большим отношением этого расстояния к длине хвостового конца и большей длиной рудимента задней матки. Ditylenchus weischeri sp. n. отличается от других видов Ditylenchus по последовательностям ITS-rRNA и гена hsp90, а также по числу хромосом. Ditylenchus weischeri sp. n. не способен развиваться и завершать жизненный цикл в растениях лука и саженцах клубники, являющихся типичными хозяевами D. dipsaci. Приводятся характерные профили PCR-ITS-RFLP для D. weischeri sp. n. и D. dipsaci.
