i28 journal o] Nematoiogy, Volume 13, No. 2, April 1981 choidea (Nematoda). Indian J. Nematol. 1:25-43. 19. Triantaphyllou, A. C., and H. Hirschmann. 18. Tarjan, A. C. 1973. A synopsis of the genera 1980. Cytogenetics and morphology in relation to and species in the Tylenchorhynchinae (Tylen- evolution and speciation of plant-parasitic nema- choidea, Nematoda). Proc. Helm. Soc. Wash. 40:123- todes. Ann. Rev. Phytopathol. 18:333-359. 144.

Description and SEM Observations of marylandicus n. sp. with a Key to Species of Dolichodorus ~ Stephen A. Lewis and A. Morgan Golden -° Abstract: Dolichodorus marylandicus n. sp. is described and illustrated from grass (Zoysia japonica) in College Park, Maryland. Specimens have also been collected from perennial blue- grass ( pratensis) pasture at Beltsville, Maryland, and from pine (Pinus sp.) in North Caro- lina. This new species is related to D. heterocephalus Cobb, D. similis Golden, and D. aestuarius Chow g: Taylor. Females are distinct in having a longer styler and shorter body length than D. aestuarius. The excretory pore is at the level of the isthmus or slightly posterior and the tail is abruptly reduced in diameter, tapering to an acuminate terminus. The tails of D. similis and D. heterocephalus conically taper to a median point, with D. similis having an especially long tail. D. marylandicus does not possess the rounded, sclerotized accessory structures adjacent to the vulva observed in lateral views of D. similis and D. heterocephalus. SEM observations of D. heterocephalus and D. marylandicus revealed details of the head of males and females, and species difference in shape of the oral disc. Key words: , morphology, Doliehodorus, awl nema- tode, new species, SEM ultrastructure, grass, Zoysia japonica, Poa pratensis, Pinus, pine.

The genus Dolichodorus was established , weeds, and grasses. Dolichodorus by N. A. Cobb in 1914 from specimens ob- spp. are commonly collected from moist tained primarily from Silver Springs, Flor- habitats, but they have also been found 1.2 ida, when he described D. heterocephalus m deep in sandy soil lying 20-40 cm above (6). No other species were described for the water table (15), in dry, shale soil at the more than 40 yr, until D. obtusus Allen in top of a mountain (9), and in coastal sand 1957 (1) and D. similis Golden in 1958 (10). dunes in New Zealand (5). Since then, additional species have been de- Dolichodorus heterocephalus is impor- scribed and placed in this genus. However, tant in Florida agriculture (4,16). In 1975 in 1976 Andr;issy (2) established Neodoli- D. minor Loof g: Sharma was associated with chodorus to accommodate the species with galled roots of diseased cocoa and rubber bluntly rounded tails and four lines in the trees (13), and D. silvestris Gillespie & lateral field; and at the same time trans- Adams was found in 1962 surrounding un- ferred D. obtusus to tiffs new genus as the thrifty white pine tree roots (9). The type and only species. In 1977 Siddiqi (17) Dolichodorus species described herein was shifted five other species from Dolichodorus found (with other associated plant-parasitic to Neodolichodorus. Presently, there are ) in sparse, chlorotic stands of eight Dolichodorus spp. associated with zoysia and perennial bluegrass. The turf varied hosts including corn, celery, turf, stands improved in vigor after nematicide white pine, cocoa, rubber, pepper, clove, application (8).

Received for publication 29 July 1980. 1Contribution No. 1800 of the South Carolina Agricul- MATERIALS AND METHODS tural Experiment Station. 2Respectively, Assoicate Professor of Plant Pathology and Specimens used in this description were Physiology, Clemson University, Clemson, SC 29631; and Nematologist, Nematology Laboratory, Plant Protection In- obtained from a zoysia lawn and from cul- stitute, USDA SEA AR, Beltsville, MD 20705. This study tures originating from the lawn and grown was performed while the senior author was on sabbatic leave in the laboratory of Dr. Golden. Appreciation is ex- on zoysia in an isolated area of a green- tended for the financial support of the USDA and to Bruce house. Ingber of the Electron Microscope Laboratory for SEM tech- nical assistance. Males, females, and larvae were collected Dolichodorus Description and SEM Observations: Lewis, Golden 129 from soil by commonly used methods, heat area (labial rib) on each side of the open- relaxed, and fixed in 3% formaldehyde. ing (Figs. 12, 13, 18, 21, 22). Cavities or The procedures used in measuring, drawing, clefts appear under the oral disc in the and preparing specimens were those used dorsal and ventral sectors. Stylet with conus by Golden and Birchfield (11). Living speci- longer than shaft and with slightly pointed mens were prepared for scanning electron knobs sloping posteriorly (Fig. 1). Guides microscope (SEM) observation by fixing in for both the stylet shaft and extension very 3% glutaraldehyde in 0.01 m phosphate faint. Esophagus typical from the genus with buffer pH 6.7, dehydrating in ethanol, the nerve ring encircling the basal portion critical-point drying, and sputter coating of tile isthmus. Excretory pore clearly visible with gold-palladium. and located slightly anterior to the basal bulb but occasionally located from the level of tile upper half of the basal bulb to the SPECIES DESCRIPTION anterior of tile isthmus. Hemizonid 17.4- Dolichodorus marylandicus n. sp. 26.1 /zm and hemizonion 23.2-34.5 /~m be- Females (30): Length 1646-2489 ~tm low excretory pore, respectively. Anterior (average 2120.2 tzm, standard deviation [SD] cephalids opposite, 4-5 annules posterior to 245.7/zna); width 39.2-54.6/~m (45.3 ~tm, SD constriction at lip region. Vulva a transverse 4.0); a = 36-54 (45.5, SD 5.0); b = 6.0-10.1 slit (Fig. 6) without sclerotized accessory (8.5, SD 0.8); c = 30.2-51.4 (38.8, SD 4.9); pieces observed in lateral views of D. V = 50-61 (55, SD 5.0); total styler 83.5- heterocephalus and D. similis. Vagina 92.4 /~m (88.8 /zm, SD 3.0); stylet conus strongly sclerotized near vulva, with ser- 46.6-51.3 /zm (49.3 ttm, SD 1.9); dorsal rated opening and with well-developed, esophageal gland orifice (DGO) 3.8-5.8/~m convoluted cuticular lining extending to (4.9 /zm, SD 0.8) from base of stylet; center almost half the body width (Fig. 7). Am- of median bull) 134.8-154.8 ~tm (147.9 ttm, phidelphic, with o6cytes in several rows in SD 8.7) from anterior end; excretory pore germinal zone. Proximal end of oviduct 156.6-193.9 #m (180.9/xm, SD 11.0) from an- with conspicuous spermatheca. Cuticle terior end; anal body width 29.9-41.8 ~m finely striated with areolated lateral field having three lines, occupying about 1/5 (36.6 t~m, SD 2.9). Holotype (female): Length 2038.4 t~m; body width (Fig. 3). Center line more 40.1 t~m; a = 50.8; b = 8.8; c = 34.8; V = prominent than outer lines and extending 52; stylet 86.7 /zm; stylet conus 47.5 t~m; from a point adjacent to anterior 1/3 of DGO 4.4 /~m from base of stylet; center of stylet to a deeply-cut line just anterior to median bulb 137.2 ttm from anterior end; the phasmid after which tile line becomes excretory pore 181.4 t~m from anterior end; uneven. Outer lines not as distinct, extend- phasmids 46.6 tzm from tail terminus; anal ing from the level of the procorpus to a body width 35.6 ttm. point just anterior to the level of the anus, Description of [emales: Body long and and appearing more faint posterior to the cylindrical, slightly arcuate. Lip region vulva. Phasmids opposite, pore-like, averag- prominent, markedly offset from body (Fig. ing 46.2 ~tm from tail tip in a cleft formed 1), bearing seven very fine annules (Figs. by center line in lateral field (Fig. 3). Tail 10, 13). Lip region en [ace with four lobes 54.9 /~m (50.6-63.8), abruptly tapering to and with an offset oral disc (Figs. 2, 9, 12, an acuminate, often spikate, terminus with 13). Anterior portion of head with strong more reduction in diameter ventrally than cephalic framework and massive basal plate. dorsally or laterally. Tail shape generally Observations of the smooth elongate lips appearing as in Figs. 3, 4, 5, 14, 17 but also with a transmitted-light microscope revealed occurring as in Figs. 15, 16. Juveniles with curved lines in the lateral sectors which tails conically tapering to a long median could not be definitely identified. However, point. SEM micrographs revealed the presence of Males (25): Length 1685.6-2114.1 t~m wide (0.8/zm) amphid openings 2.9-3.3 #m (1950.4 /~m, SD 130.3); width 35.5-44.4 #m long. The shape and width of the openings (42.3 /~m, SD 3.3); a = 41.1-55.8 (46.3, SD may be partially determined by a raised 4.2); b = 6.7-9.4 (8.3, SD 0.6); c = 47.1- i30 .Journal o/ Nematology, Volume 13, No. 2, April 1981

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Figs. 1-8, Drawings of Dolichodorus marylandicus n. sp. 1) Anterior region of female (note location of excretory pore and massive basal plate of sclerotized labial framework). 2) En face view of head. 3-5) Tail shapes of females (note reduction in tail diameter and location of anus). 6) Ventral view of vulva. 7) Lateral view of vulva (note lack of sclerotized accessory pieces adjacent to vulva, the strongly sclerotized distal vagina, and convoluted proximal vagina). 8) Male tail, lateral view (note sclerotized structure between gubernaculum and spicule). Dolichodorus Description and SEM Observations: Lewis, Golden 131

Figs. 9-17. SEM micrographs of D. marylandicus n. sp. females. 9) En face view (note amphidial secre- tion covering amphid openings). 10) Labial region with offset oral disc. 11) Lateral field just anterior to vnlva. 12-1~) View of cephalic region (note amphidial opening in Fig. 12). 14-17) Variation in tail shape. 132 Journal of Nematology, Volume 13, No. 2, April 1981 Dolichodorus Description and SEM Observations: Lewis, Golden 183 64.8 (56.2, SD 5.1); total stylet 82.9-92.4 ttm zoysia and perennial bluegrass in an isolated (87.3/~m, SD 3.1); stylet conus 45.8-51.6 izm greenhouse area. Slide T-325t, USDA Nema- (48.6 ttm, SD 1.8); DGO 3.7-5.8 ~m (4.7 tode Collection (USDANC), Beltsville, tzm, SD 0.5) from base of stylet; center of Maryland, USA. median bulb 134.8-152.8/~m (143.1 /~m, SD Allotype (male): Slide T-326t. Same data 6.7) from anterior end; spicules 41.4-51.0 as holotype. USDANC, Beltsville, Maryland, /zm (45.6 tttn, SD 2.0); gubernaculum 24.4- USA. 29.0 /~m (27.0 /~m, SD 1.1); tail 30.1-39.4 Paratypes (males, females, juveniles): t~m (34.9 tLm, SD 2.6); phasmids 17.4-23.7 USDANC, Behsville, Maryland, USA; De- /zm (19.2 /~m, SD 2.1) from tail terminus; partment of Plant Pathology and Physiol- anal body width 22.0-28.2/~m (25.1 t~m, SD ogy, Clemson University, Clemson, South 1.9); excretory pore 157.5-207.9 /~m (182.0 Carolina, USA; Nematology Department, /~m, SD 13.5) from anterior end. Rothamsted Experimental Station, Harpen- Allotype (male): Length 1920.8 ttm; a = den, Herts, England; Laboratoire des Vers, 49.0; b = 8.9; c = 57.5; total stylet 83.2 tzm; Mus6um National d'Histoire Naturelle, styler conus 46.2/~m; DGO 5.1/~m from base Paris, France; Institute voor Dierkunde, of stylet; center of median bulb 139.2 t~m Laboratorium voor Morfologie en Sys- from anterior end; spicules 41.6/~m, guber- tematiek der Dieren, Ledeganckst, 35, B-900, naculum 24.4 tzm; tail 33.4 /~m; phasmids Gent, Belgium; and Laboratory voor Nema- 19.4 /~m from tail terminus; anal body tologie, Binnehaven 15, Wageningen, Neth- width 22.0 t~m; excretory pore 160.3 ~m erlands. from anterior end. Type host and locality: Soil around roots Description of males: Body similar to of Zoysia japonica in a lawn in College female in head shape (Fig. 18), annulation, Park, Maryland, USA. and location of excretory pore; but slightly Diagnosis: Dolichodorus marylandicus smaller, with posterior slightly hooked ven- n. sp. is related to D. heterocephalus Cobb, trally, and with tail terminus bifurcate 1914 (6), D. aestuarius Chow g: Taylor, (Figs. 19, 20). Lateral field about 1/4 of 1979 (3), and D. similis Golden, 1958 (10). body width; with three lines beginning an- It differs from these species by, among teriorly as for the female and extending, other characters, tail shape and lack of areolated, to the posterior portion of the rounded sclerotized structures (18) ad- body. One outer line fades near the prox- jacent to the strongly sclerotized distal por- imal portion of the caudal alae and the tion of the vagina. It also differs from D. other two lines extend to a level adjacent similis by the location of the excretory pore. to the middle of the spicules or beyond. Characters useful in distinguishing species Caudal alae typical for the genus, with within the genus are provided in the key small projection on each distal portion and given below. small, pore-like phasmids located about Distribution: D. marylandicus has also half the distance from anus to tail tip (Fig. been found in soil around roots of perennial 8). Testis outstretched with globular sperm bluegrass (Poa pratensis) pasture in Mary- in seminal vesicle. In lateral view, guber- land and from pine (Pinus sp.) in North naculum slightly curved with proximal part Carolina. enlarged, often forming a hook. Guber- naculum with accessory structure interposed Key to Genera of Dolichodorinae with adjacent spicule (Fig. 8). Holotype (female): Collected in June 1. Stylet very strong, much less than 60/~m; 1971 by A. Morgan Golden at College Park, female tail greatly attenuated .... Brachy- Maryland, and subsequently grown on dorus De Guiran ~ Germani, 1968 (7).

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Figs. 18-23. SEM micrographs of D. marylandicus n. sp. males and D. heterocephalus females. 18) Cephalic region of D. marylandicus n. sp. 19-20) Caudal area of D. marylandicus n. sp. 21-22) D. hetero- cephalus cephalic region (note large amphidial opening and asymmetrical oral disc). 23) Caudal area of D. heterocephalus. 134 Journal o[ Nematology, Volume 13, No. 2, April 1981 Styler greater than 60 t~m; female tail Observations on the identity of D. not greatly attenuated ...... 2 heterocephalus: The male in the original 2. Female tail conically pointed; lateral description of D. heterocephalus was re- fields each with three incisures ...... ported by Cobb to have come from Douglas ...... Dolichodorus Cobb, 1914 (6). Lake, Michigan (6). Examination of his Female tail bluntly rounded, lateral original pencil drawings indicate that a fields each with four incisures _ male from Silver Springs, Florida, and one _Neodolichodorus Andr~issy, 1976 (2) from Michigan were drawn. Measurements (= Plesiodorus Siddiqi, 1976 (17,18). included in his publication are of the Michigan male, but the drawings are of the male from Florida. Key to Species of Dolichodorus Cobb, 1914 Thorne reported stylet lengths of 83-95 1. Excretory pore opposite, or anterior to ~m for females of D. heterocephalus (19). median bulb ...... 2 The stylet lengths of collections of D. Excretory pore posterior to median bulb heterocephalus gathered for this study and ...... 5 of the drawing of the female in the type de- 2. Excretory pore opposite procorpus or scription (6) were at least 99 /zm. In addi- base of stylet ...... D. pulvinus Khan, tion, Loo[ ~: Sharma reported stylet meas- Seshadri, Weischer, 8e Mathen, 1971 (12). urements of at least 99 ttm for this species Excretory pore opposite median bulb __ 3 03). 3. Female T/ABW 3; body less than 2 mm The accessory piece, later called the long ...... D. nigeriensis gubernaculum, pictured in Cobb's drawing Luc g: Caveness, 1963 (14). of the male D. keterocephalus is composed Female T/ABW 2 or less; body greater of two sclerotized parts. There is the nar- than 2 mm long ...... 4 row, long, distally recurved portion shown 4. Lip region deeply offset, cushion-shaped in drawings of the different species. Also or flattened; anterior cuticle appearing present is a shorter piece, observed in lateral as rectangular plates; styler length less views of D. heterocephalus and D. mary- than 120 /ml ...... landicus n. sp., between the gubernaculum ...... D. pro[undus Luc, 1960 (15). proper and the adjacent spicule (Fig. 8). Lip region offset but rounded; anterior This portion has not been pictured in draw- cuticle not marked by rectangular ings of the gubernaculum and spicula since plates; stylet length greater than 130 tLm Cobb's 1914 article (6). All species of ...... D. silvestris Doliehodorus should be examined [or the Gillespie & Adams, 1962 (9). presence of this character, and if it is uni- 5. Excretory pore posterior to nerve ring formly present, the generic description and at a level with the middle of the should be amended. basal bulb __ D. similis Golden, 1958 (10). Excretory pore at level of isthmus ...... 6 LITERATURE CITED 6. Body length less than 2 mm long ...... I. Allen, M. W. 1957. A new species of the genus ...... D. minor Loof g: Sharma, 1975 (13). Dolichodorus from California (Nematoda:Tylen- Body length greater than 2 mm ...... 7 ehida), Proc. Helm. Soc. Wash. 24:95-98. 7. Stylet length 62-76/~m, unusually short 2. Andr~issy, I. 1976. Evolution as a basis for the systematization of nematodes. Pitman Publishing, in relation to a body length of 2.50-2.87 London. mm ...... D. aestuarius 3. Chow, F. H., and A. L. Taylor. 1978. Dolicho- Chow g~ Taylor, 1978 (3). dorus aestuarius n. sp. (Nematoda:). Stylet 83/~m or more ...... 8 J. Nematol. 10:201-204. 4. Christie, J. R. 1952. Some new nematodes of 8. Stylet 99 ttm or more, tail smoothly tap- critical importance to Florida growers. Proc. Soil ering to irregularly conoid, acute termi- Sci. Soc. Florida 12:30-39. nus (Fig. 23) ...... 5. Clark, W. C. 1963. A new species of Dolicho- ...... D. heterocephalus Cobb, 1914 (6). dorus (Nematoda:) from coastal sand dunes. New Zealand Journal of Science 6:531-534. Stylet 83-92 ttm, tail short, abruptly re- 6. Cobb, N. A. 1914. The North American free- duced in diameter, acuminate terminus living freshwater nematodes. Contributions to a often spikate ...... D. marylandicus n. sp. science of nematology. Tr. Am. Micr. Soc. 33:69-134. Dolichodorus Description and SEM Observations: Lewis, Golden 135

7. De Guiran, G., and G. Germani. 1968. Brachy- Dolichodorus minor n. sp. (Nematoda:Dolichodor- dorus tenuis n. g. n. sp. (Nematoda:Dolichodorinae), idae) with a key to the genus Dolichodorus. Revista associ6 ~t ravenala Madagascariensis sur la cote est Theobroma CEPEC, Itabuna, Brasil 5:35-41. Malgache. Nematologica 14:447-452. 14. Luc, M., and F. E. Caveness. 1963. Dolicho- 8. Feldmesser, J., and A. M. Golden, 1972. Con- dorus nigeriensis n. sp. (Nematoda:Dolichodoridae). trol of nematodes damaging home lawngrasses in Proc. Hehn. Soe. Wash. 30:297-299. two counties in Maryland. Plant Dis. Rptr. 56:476- 15. Luc, M. 1960. Dolichodorus profundus n. sp. 480. (Nematoda: Tylenchida). Nematologica 5:l-6. 9. Gillespie, "W. H., and R. E. Adams. 1962. An 16. Perry, V. G. 1953. The awl , awl nematode, Dolichodorus silvestris n. sp. from Dolichodorus heterocephalus, a devastating plant West Virginia. Nematologica 8:93-98. parasite. Proc. Helm. Soc. Wash. 20:22-27. 10. Golden, A. M. 1958. Dolichodorus similis 17. Siddiqi, M. R. 1977. Plesiodorus Siddiqi, (Dolichodorinae), a new species of plant nematode. 1976 (Nematoda:Dolichodoridae), a junior objective Proc. Helm. Soc. Wash. 25:17-20. synonym of Neodolichodorus Andr~issy, 1976. "Nema- 11. Golden, A. M., and W. Birchfield. 1972. tologica 23:265. graminophila n. sp. (Nematoda: 18. Siddiqi, M. R. 1976. New plant nematode ) from grass with a key to closely re- genera Plesiodorus (Dolichodorinae), Meiodorus lated species. J. Nematol. 4:147-154. (Meiodorinae subfam, n.), Amplimerlinius (Merlini- 12. Khan, E., A. R. Seshadri, B. Weischer, and inae) and Gracilancea (Tylodoridae grad. n.). Nema- K. Mathen. 1971. Five new nematode species asso- tologica 22:390-416. ciated with coconut in Kerala, India. Indian J. 19. Thorne, G. 1961. Principles of Nematology. Nematol. 1:116-127. McGraw-Hill, New York. 13. Loof, P. A. A., and R. D. Sharma. 1975.

Description of Trilineellus clathrocutis n.g., n.sp. (Tylenchorhynchinae: Tylenchida Thorne, 1949) with a Key to Species and Observations on Tylenchorhynchus sensu stricto 1 Stephen A. Lewis and A. Morgan Golden"- Abstract: TrilineeIlus clathrocutis n.g., n.sp. is described and illustrated. It was found as an associate of corn (Zea mays) in Stockton, Georgia, USA, and is related to a group of Tylen- chorhynchus sensu lato species having three lines in nonareolated lateral fields. This new species is closely related to Tylenehorhynehus divittatus Siddiqi 1961, T. scu[ptus Seinhorst 1963, and T. triglyphus Seinhorst 1963 (syn. T. chonai Sethi 8: Swarup 1968) Tarjan 1973. It differs from these species primarily by having longitudinal striae on the body. These four species are differentiated from Tylenchorhynchus sensu stricto by having three lateral lines instead of four. They differ from Uliginotylenchus Siddiqi 1971 by having nonareolated lateral fields, fewer than 25 annules on conoid rounded tails, differently shaped gubernacula, nonattenuated stylets, and other dis- tinctive characters. They differ from Triversus Sher 1973 by having the male tail enclosed by the bursa and by having rounded female tails. SEM observations of T. clathrocutis reveal a cuticle deeply cut by longitudinal and horizontal striae and bearing wide (> 2.0 tzm) annules. Trilineel- lus is proposed to accommodate the new species and the three-incisured species still within Tylenchorhynchus. Tylenchorhynchus is thereby the repository for species within Tylenchor- hynchinae having four lines in the lateral field, no conspicuous labial disc, and bursa enclosing the male tail. Key words: taxonomy, morphology, Tylenchorhynchus, Trilineellus, Uliginoty- lenchus, Quinisulcius, Triversus, stunt nematode, new species, new genus, ryegrass, corn, SEM ultrastructure, Lolium perenne, Zea mays.

The genus TTlenchorhynchus was estab- Received for publication 15 September 1980. 1Contribution No. 1844 of the South Carolina Agricul- lished by N. A. Cobb in 1913 when he de- tural Experiment Station. scribed T. cylindricus from brackish soil 2Associate Professor of Plant Pathology and Physiology, Clemson University, Clemson, S. C. 29631; and Nematologist, near a marine estuary in southern Cali- Nematology Laboratory, Plant Protection Institute, USDA fornia. The history of the genus since then SEA AR, Beltsville, MD 20705. This study was performed while the senior author v~-as on sabbatical leave in the lab- was discussed in a recent review by Hooper oratory of Dr. Golden. Appreciation is extended for the (6). Allen's (1) important review in 1955 financial support of the USDA; for the technical assistance of Donna M. S. Ellington, Support Scientist, and Paula established taxonomic criteria ~or the genus, Crowley, Laboratory Technician: and to Norman A. Minton, and a total of 95 species was described by USDA Coastal Plain Experiment Station, Tifton, GA 31794, for providing this nematode for study. 1970 (9). Many have now been placed in

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