Nernatol. rnedit. (2004), 32: 147-154 147

SOME BELONGING T0THE GENUS TIZENCHORHUVCHUS COBB, 1913 (NEMATODA: ), FROM

M. Ciobanul, G. Karssen2 * and I. Popovicil

Iizstitute ofBiologica1 Research, Department of Taxonomy and Ecology, 48 Republicii Street, 3400 Clzi-Napoca, Romania Plant Protectioiz Service, P.O. Box 9102, 6700 HC Wageningen, the Netherland.7

Summary. Specimens belonging to three known species of (T. agri, T. dubiz~sand T. maximus) collected from various localities in Romania were studied by light microscopy. Additional morphometrics, illustrations and data referring to their habitat are provided. The geographical distribution of T. dubius and T. maximtis in Romania is broadened. A description and il- lustration of a previously undescribed species, based upon a single temale, is given.

The nematodes belonging to the genus Tylen- is broadened. chorhynchus Cobb, 1913 are ectoparasites of roots and One undescribed species was identified in the Ro- common in various soil types. According to Brzeski manian material; the measurements and illustration are (1998), the genus is very heterogeneous, therefore requir- provided based upon a single female. ing taxonomic revision. Data on the presence and distribution of the species Six species of Tylenchorhynchus have been reported have been included in the Romanian fauna so far from Romania: T agri Ferris, 1963 from Maliuc database. The paper is also a contribution towards an (located in the Danube Delta) (Popovici, 1992); T clarus inventory of the species belonging to the genus Tylen- Allen, 1955 from Maliuc (forest) and Enisala (wet soil) chorhynchus in Romania. also located in the Danube Delta (Popovici, 1992); T cylindricus Cobb, 1913 from grasslands near the locality Cluj-Napoca (Popovici, 1973, 1974); T dubius (Butschli, MATERIALS AND METHODS 1873) Filip'ev, 1936 from severa1 localities: soil around Mentha sp. roots at Plavisevica (Andrhssy, 1959), grass- Soil samples were collected by two of us (M.C. and lands near Cluj-Napoca (Popovici, 1974), in the I.P.) during an ecologica1 survey carried out between Gorges (Trasc'ai Mountains), at Piatra Closani (Mehed- 1991 and 1997. Seven sites, including grasslands, vege- inti Mountains) (Popovici, 1998) and from vegetated tated cliffs, one Populus sp. plantation and one techno- cliffs in various locations in the Romanian Carpathian genic soil, were investigated, and information on their Mountains (Popovici and Ciobanu, 1997); T maximus altitude, geographical position, plant association and Allen, 1955 from grassland at Piule (Retezat Mountains) soil type are given in Table I. Nematodes were extracted (Popovici, 1993, 1998); and T obscurisulcatus Andrassy, using the centrifuga1 method of de Grisse (1969), killed 1959 from soil around Artemisia sp. roots, Cerna, Do- and preserved in a 4% fornlaldehyde solution heated to brudscha (Andrhssy, 1959). 65 "C, mounted in anhydrous glycerin (Seinhorst, 1959) Apart from the paper of Andrassy (1959) on T ob- and examined by light microscopy using an Olympus scurisulcatus, in which he provided good description BH-2 light microscope with differential interference and illustration, basic measurements and illustration contrast. Photographs were taken using a digital Leica were given for T cyli~zdricus(Popovici, 1974) but only DC 3 00 camera. basic measurements and short descriptions for 'I: agn The following papers were used for the identification and T clarus (Popovici, 1992). of the species: Tarjan (1973); Fortuner and Luc (1987); Specimens of Tylen~horh~nchusnot yet identified at Anderson and Potter (1991); Brzeski (1998) and Han- the species level, as well as some individuals that have do0 (2000). undergone preliminary identification and are stored in Al1 measurements in the tables are in pm; average the nematode collection of the Institute of Biological values and range were calculated for each measurement Research, were studied. More morphometrics, illustra- if more than two specimens were collected from a single tions and data referring to their habitats are provided site. for T agri, T dubius and T maximus and the geographi- Plant associations' classification was used according cal distribution of T dubius and T nzaximus in Romania to Coldea (1991). Soil types were classified according to the Romanian System of Soil Classification (Conea et " Corresponding author: [email protected]. al., 1980). DESCRIPTIONS Tylen~horh~nchusdubiw (Butschli, 1873) Filip'ev, 1936 Tyenchorhynchzts agri Ferris, 1963 (Table 111; Fig. 2) (Table 11; Fig. 1 A-C) Female body ventrally arcuate, cuticle variously wrin- Female body more or less ventrally arcuate, cuticular kled anterior and/or posterior to vulva (observed in pre- annulation distinct. Lateral field with outer margin cre- served and living specimens). Spores of Pasteuria sp. at- nated. Head slightly off-set, with four annules, first neck tached to the cuticle were observed in some specimens. annule posterior to head slightly reduced. Stylet 19.5- Lateral field with outer margin crenated. Head from 20.5 pm long, basal knobs set-off from the shaft. Median slightly to well off-set, with five-six annules. Stylet 16.5- bulb ovoid,basal bulb elongate pyriform, not overlap- 20.0 pm long, basal knobs sloping bacliwards. Median ping the intestine. Epiptygma observed in one of the two bulb ovoid, basal bulb elongate pyriform, not overlap- females. Spermatheca relatively small, ovoid, either emp- ping the intestine. Body posterior to cardia with distinct ty or filled with ovoid sperm, about 2.0-2.5 pm in diame- fasciculi. Vulva clearly expressed outside the body con- ter. Tail sub-cylindrical with eighteen-nineteen annules, tour in some females. Epiptygma observed in some fe- tail terminus sub-hemispherical, smooth. Post-anal in- males. Spermatheca rounded, empty, (except in one fe- testinal sac occupying 53.8-58.3% of the tail length. male, in which it was filled with ovoid sperm, about 2.5- Phasmid located at the leve1 of the seventh and the thir- 3 .O pm in diameter). Tail cylindrical or sub-cylindrical (a teenth annule posterior to anus. tail slightly clavate was observed only in one female) Male not found. with 36-66 annules, tail terminus hemispherical or sub- Distribution: soil collected from Populus sp. planta- hemispherical, annulated. Phasmid located between the tion from Maliuc (Danube Delta), site no. 7 [(TableI). ninth and the twenty-first annule posterior to anus. Remarks: T. agri was originally reported from soil col- Male slightly larger than female, with broad bursa, lected from a field cropped with corn for 85 years locat- spicules 21 pm long, gubernaculum 7 pm long. ed in Urbana, Illinois, U.S.A. It was also reported in Distribution: four locations, sites nos 1-4 (Table I), Southern Europe from soil collected from a barley field including three grasslands and one technogenic soil. located at Misrah Suffara, Malta (Larizza and Lamberti, Remarks: Tylen~horh~nchusdubius was previously re- 1995) and from the rhizosphere of Persea americana ported from several locations from Romania (Andrissy, Mill. (avocado) from Greece (Koliopanos and Kalyvio- 1959; Popovici, 1974, 1998) and even from vegetated tis-Gazelas, 1979). Tylenchorhynchus agri was only cliffs of the Romanian Carpathians (Popovici and recorded in Romania from Maliuc (in the Danube Ciobanu, 1997). One of the habitats from which the spec- Delta) (Popovici, 1992). imens were collected was a technogenic soil under biore- It is probably a very rare species in Romania. mediation at Rodna Veche (Rodnei Mts.), characterized

Table I. Site locations, vegetation and soil types of a nematological survey in Romania

Site Locality Altitude Geographical Plant association Soil type nr. (m) position

1 Fanatele Clujului 350 46"45'N-2355'E Jurineo traizssilu~izicae-Stipetumpulcherimoe Chernozem (Somesan Plateau) 2 Suatu 370-450 46"46'N-23"58'E Saluio nutantis-Festucetur~zrupicolae Not available () 3 Tureni Gorges 400 46"301N-23"4l'E Melico-Phleetum montani Lithic rendzina (TrascBu Mts.') 4 Rodna Veche 525 4795'N-24'26'E - Technogenic soi12 (Rodnei Mts.)

5 Suhardu Mic 1450 46"43'N-2556'E - Undeveloped soil (HZsrnas Mts.) 6 Muntele Rosu 1500 45"26'N-25"52'E 17iolodeclinatae-Nardetur~z Cambic rendzina (Ciuca2 Mts.) 7 Maliuc 12 45"13'N-29"04'E - Not available (DanubeDelta)

' Mts.=Mountains Soil from mining spoil containing Pb and Zn impurities under bioremediation. Ciobanu et al. 149

Fig. 1. Tylenchorhynchz~sagri: fernale; A, anterior end; B, reproductive system; C, tail. Tylenchorhynchus sp.: female; D, anterior end; E, reproductive system; F, tail. Table 11. Measurements and diagnostic features of T3,1enchorhynchusagrz, Tylenchorhynchus maximus and Tylenchorhynchus sp. (al1 measurements in pm).

Species: Tylenchorhynchus agri Tylenchorhynchusnzaximus Tylenchorhynchussp. Habitat : Populzts sp. Cliffs vegetated by Sedum sp. Grassland plantation and Gentiana sp. Locality: Maulic Suhardu Mic Muntele Rosu (Danube Delta) (H%jmas Mts.) (Ciuca3 Mts.) n L a b

C C' V% Head width Head height Conus Shaft Pharynx Ant. part. pharynx3 Post. part. pharynx4 Ant. part.% pharynx Excretory pore Head - vulva Tail Body width Lateral field width Anal body width

Present study Popovici (1992) ' Measured from anterior body end to the posterior end of median bulb. Measured from the posterior end of median bulb to pharyngeo-intestina1 junction.

by low nutrient resources and by high concentrations of and the U.S.A. (Esser, 1980). Pb and Zn impurities (Pasca et al., 1997).These data sug- It is probably the most common species of the genus gest that T dubius has a high capacity to withstand heavily Tylenchorhynchus in Romania. polluted environments. Acording to Brzeski (1998), it is a very common species in arable and meadow soils, especiafiy frequent Tylen~horh~nchusmaximus Allen, 1955 and numerous in acidic, coarsely textured soils. (Table 11; Fig. 3) Spores of Pasteuria sp. attached to the cuticle were observed in two females (one collected from site no. 3 Female body C- to spiral-shaped, cuticular annula- and one from site no. 4) (Fig. 2C). Such associations be- tion distinct. Lateral field regularly areolated, outer tween the Pasteuria penetrans group and T dzlbius have margin strongly crenated. Head almost continuous, with been reported from Belgium (Coomans, 1962), Ger- five-six annules. Stylet 23.5-24.0 pm long, slender, basal many (Sturhan, 1985), the Netherlands (Kuiper, 1958), knobs relatively sia11 compared to stylet, sloping back- Scotland (Prasad, 1971 quoted in Sayre and Starr, 1988) wards. Median bulb oval, basal bulb pyriform, not over- Ciobanu et al. 15 1

Fig. 2. Tylenchorhynchus dubius: female; A, anterior end; B, vulva region; C, spores of Pasteuria sp. attached to cuticle; D, E, pos- terior body region (Scale bar = 10 pm).

Fig. 3. Tylenchorhynchus maximus: female; A, Anterior end; B, vulva region; C, posterior body region (Scale bar = 10 pm). Table 111. Measurements and diagnostic features of Tylenchorhynchus dubius (all measurements in pm).

Habitat : Grassland Grassland Grassland Technogenic soil under bioremediation Locality : Fanatele Clujului Suatu Tureni Gorges Rodna Veche (Somesan Plateau) (Transylvanian Plain) (Trascau Mts.) (Rodnei Mts.)

a 34.6+3.3(30.7-36.5) 37.3 3 1.711.6(28.9-33.2) b 6.210.2(6.0-6.4) 7.0 5.8+0.8(5.0-7.5)

C 17.2+_1.4(16.2-18.8) 17.0 14.1+_1.6(11.3-16.3)

C' 3.1iO.4(2.7-3.5) 2.6 2.9+0.3(2.6-3.4) V% 54.811.7(53.0-56.3) 55.011.9(5 1.9-58.7) Head width 6.810.3(6.5-7.0) 7.0 7.4+_0.5(7-9) Head height 3.410.4(3-4) 4 3.6+0.4 (3-4) Conus 8.6+_0.4(8-9) 10.0 9.910.5(9-11) Shaft 8.411.0(7.5-9.5) 9 8.8&0.6(8.0-10.0) Pharynx 121.3,1.9(119-123) 121 120.9111.2(106-137) Ant. part. ~harynx' 66.411.3(65-68) 62 65.2+_6.4(58-73) Post. part. pharynxz 54.912.7(52-57) 59 55.7+5.8(47-64) Ant. part.% pharynx 54.7i1.6(53.8-56.6) 51.2 53.912.0(50.8-57.5) Excretory pore 96.315.5(92-102) 109 97.5+_7.9(85-107)

Head - vulva 413.9128.7(381-432) 381.8133.1(333-438) Tail 44.0+_3.5(40-47) 5 O 49.4+4.1(44-55) Body width 21.9&1.3(21-23) 23 21.9+1.3(21-24) Lateral field width 6.5+1.5(6-8) 6 6.0+0.7(5-7) Anal body uridth 14.51(13-16) 19 16.9+1.6(15-20)

l Measured from anterior body end to the posterior end of median bulb. Measured from the posterior end of median bulb to pharyngeo-intestina1 junction.

lapping the intestine. Body posterior to cardia with dis- ences, not only to meadow soils as mentioned by Brzeski tinct fasciculi observed in one female. Vulva expressed (1998) but also to the "just born habitats" (Popovici and outside the body contour, without epiptygma. Sper- Ciobanu, 1997), with primary soils in which the early matheca relatively small, rounded and empty. Tail cylin- stage of natura1 succession is occurring. drical with 42 annules, tail terminus hemispherical, an- Tylencborynchus maximus was also collected from nulated. Phasmid located at the level of the seventeenth soil aroimd roots of herbaceous plants and grasses on annule posterior to anus. the slopes of Malika Parbat, surrounding Lake Saiful- Male not found. muluk, located at 3,200 m above sea level in Pakistan Distribution: undeveloped soil on cliffs vegetated by (Maqbool and Shahina, 1987). These data confirm that Sedurn sp. and Gentinna sp. located at Suhardu Mic the species is not restricted to lowland habitats and (Hiismas Mts.) (site no. 5 in Table I). grass vegetation. Remarks: Tylenchorynchus maxinzus was previously reported from subalpine grassland on rendzinic lithosol at 1,850 m above sea level located at Piule (Retezat Tylenchorhynchus sp. Mountains) (Popovici, 1993, 1998). By reporting T mnx- (Table SI; Figs. l D-F, 4) imus from an undeveloped soil around roots of Sedum sp. and Gentinna sp., we broaden its trophic prefer- Female body C-shaped, cuticular annulation coarse Ciobanu et al. 153

Fig. 4. Tylenchorhynchus sp.: female; A, anterior end; B, irregular cuticular annulation in the neck region; C, lateral field; D, wlva region; E, posterior body region (Scale bar = 10 pm).

and irregular over entire body; annules about 2.5 pm at Discussion: the measurements of the single female posterior part of neck, 1.5 ym at midbody and 1 pm on collected come closest to T siccus Sauer et Annells, 1981 ventral part of tail. Lateral field regularly areolated, out- and T velatus Nobbs, 1989, both species being reported er margin strongly crenated. Head almost continuous, from arid regions of Australia (Sauer and Annells, 1981; rounded, with six annules. Stylet slender, 25.5 ym long; Nobbs, 1989). basal knobs rounded, sloping backwards, about 2.5 pm The specirnen has the following morphological and in diameter, remarkably smaU. compared to the stylet. morphometrical characteristics different from T siccus Median bulb oval, basal bulb pyriform and rather short, and T. velatus: head almost continuous, wlva with sin- not overlapping the intestine. Body with distinct fasci- gle non-protruding epiptygma, shorter tail, and differ- culi from cardia to tail. Post-anal intestina1 sac absent. ences in shape with reference to head offset, and vulva Vulva with distinct epiptygma. Vagina relatively deep, with double protruding epiptygma in T siccus and T ve- 12 pm long, extending inwards to more than half latus. The Romanian specimen possess remarkably small (54.2%) of the corresponding body diameter. Sper- basal knobs in comparison to its stylet and a relatively matheca rounded, filled with ovoid sperm, about 2.5- deep vagina. 3.0 pm in diameter. Tail cylindrical with twenty-two an- nules, tail terminus hemispherical, irregularly annulated and indented. Phasmid located at the leve1 of the sev- ACKNOWLEDGEMENTS enth annule posterior to anus. Male not found. The work would not have been possible without the Distribution: grassland located at Muntele Rosu financial support offered to the first author by the (Ciuca? Mts.), site no. 6 (Table I). Netherlands Ministry of Agriculture, Nature Manage- ment and Fisheries through the International Agricul- Larizza A. and Lamberti F., 1995. Some (Nemato- tura1 Centre in Wageningen, which is gratefully ac- da) from Malta and Gozo. Nematologia Mediterranea, 23: knowledged. 153.165. The Plant Protection Service in Wageningen, the Maqbool M.A. and Shahina F., 1987. Nematodes of northern Netherlands is thanked for offering al1 the necessary fa- areas in Pakistan. Description of Nagelus saifulmulukensis cilities to carry out this study. n. sp. and Merlinius montanus n. sp. (Nematoda; Merlini- The authors are grateful to Dr. A.C. van Aelst (Wa- inae) with notes on three species of Tylenchorhynchus geningen University) for his help in processing the digi- Cobb, 1913. Revue de Nématologie, 10: 289-294. tal photographs of nematode specimens. Nobbs J.M., 1989. Four new species of plant parasitic nema- todes (Nematoda: Tylenchida) from the arid region of South Australia. 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Accepted for publication on 28 Apri1 2004.