(subgenus Heterodera), a cyst-forming nematode

Cone-top morphology of the white females and cysts of the genus Heterodera (subgenus Heterodera), a cyst-forming nematode ROLANDH. MULVEY

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Entomology Research Institute, Research Branch, Agriculture Canada, Ottawa, Canada Received June 18, 1973 R. H. 1974. Cone-top morphology of the white females and cysts of the genus Heterodera MULVEY, (subgenus Heterodera), a cyst-forming nematode. Can. J. Zool. 52: 77-81. Comparative studies with the scanning electron and interference contrast microscopes of white females and cysts provided new information for identification of species of the genus Heterodera (subgenus Heterodera). Most significant was the differences found in the development of the underbridge and bullae of several economically important species. Differences in vulval cone and fenestral structures of the white female provided additional information for the separation of species.

MULVEY,R. H. 1974. Cone-top morphology of the white females and cysts of the genus Heterodera (subgenus Heterodera), a cyst-forming nematode. Can. J. 2001.52: 77-81. Des examens wmparatifs au microswpe klectro~~iqve h balayage et au microscope interfdrentiel de femelles gravides et de kystes d'Heterodera (sous-genre Hererodem) ont permis de dtcouvrir des caractkres additiomels pour l'identification des espkes. I1 s'api~sunout de diffbrences dans i e dCveIoppement des bullae et des structures situkes sous le pont, chez pl~;sieursespbes &importance fcooomique. La dicouverte de diffbrences dans l'aspect du cane vulvaire el dcs struciures de la reni:trechez Iesfemelles gravides [Traduit par le journal] aide 9. la skparation des espkces.

Introduction Differences in cone top and associated cyst structures of Heterodera (Heterodera)A. Schmidt, 1871 are frequently used in the separation of species. However, the development and relationships of these structures have received very little attention. Hesling (1965) and Thorne (1961) reviewed and discussed the information available to that time. Mulvey (1972, 1973) added to this information. The present paper is a second in a series of morphological studies of Heterodera cysts and white females using a scanning electron and an interference contrast light microscopes. Mulvey (1972) assigned the various species of Heterodera into fairly well-defined groups based

SPECIES AND GROUP Group 2 Heterodera (Heterodera) betulae Hirschmann and Riggs, 1969 Heterodera (Heterodera) estonica Kirjanova and Krall, 1963 Heterodera (Heterodera) weissi Steiner, 1949 Group 3 Heterodera (Heterodera) avenue Wollenweber, 1924 Heterodera (Heterodera) iri Matthews, 1971 Hererodera (Heterodera) latipons Franklin, 1969 Heterodera (Heterodera) mani Matthews, 1971

on differences of cone-top structures* in mature cysts. Since differences of taxonomic value are also present in the mature white female, the present work was undertaken to evaluate the taxonomic significance of the variability of these structures and their development during the time the white female undergoes transformation into a mature cyst. Materials and Methods White females and cysts (arranged by groups, Mulvey 1972) used in the study were obtained from the following sources: *Four morphologically distinct groups within the genus Heterodera (s.g. Heterodera) were recognized on the basis of differences in vulval slit length, fenestral length, underbridge structures and bullae.

Dr. R. D. Riggs, Fayetteville, Arkansas, United States Dr. E. Krall, Estonian SSR Dr. A. M. Golden, Beltsville, Maryland, United States

Betula nigra L. (river birch)

Collected by author, Sarnia, Ontario

Avena sativa L. (oats)

Dr. H. J. P. Matthews, Draperstown, Northern Ireland Mr. G. H. Lewis, Prince Edward Island, Canada Dr. H. J. P. Matthews, Draperstown, Northern Ireland

Agrostis tenuis Sibth. (brown top grass) Soil from cultivated land

Possibly Polygonurn aviculare L. (pigweed) Polygonurn pennsylvanicurn L. (knotweed)

Lolium perenne L. (perennial rye grass)


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Heterodera (Heterodera) ustinovi Kirjanova, 1969 Group 4 Heterodera (Heterodera) cajani Koshy, 1967 Heterodera (Heterodera)fici Kirjanova, 1954 Heterodera (Heterodera)galeopsidis Goffart, 1936 Heterodera (Heterodera) glycines Ichinohe, 1952 Heterodera (Heterodera)lespedezae Golden and G. S. Cobb, 1963 Heterodera (Heterodera) leuceilyma Di Edwardo and Perry, 1964 Heterodera (Heterodera) oryzae Luc and Brizuela, 1961 Heterodera (Heterodera) rosii Duggan and Brennan, 1966 Heterodera (Heterodera) sacchari Luc and Merny, 1963 Heterodera (Heterodera)salixophila Kirjanova, 1969 Heterodera (Heterodera)schachtii Schmidt, 1871 Heterodera (Heterodera) trifolii Goffart, 1932 Heterodera (Heterodera) vigni Edward and Misra, 1968 Heterodera (Heterodera) zeae Koshy, Swarup and Sethi, 1971 Group 5 Heterodera (Heterodera)cruciferae Franklin, 1945 Heterodera (Heterodera)cyperi Golden, Rau and G. S. Cobb, 1962 Heterodera (Heterodera) goettingiana Liebscher, 1892 Heterodera (Heterodera)graminis Stynes, 1971 Heterodera (Heterodera) graminophila Golden and Birchfield, 1972 Heterodera (Heterodera) humuli Filipjev, 1934 Heterodera (Heterodera) morhi Khan and Husain, 1965 Heterodera (Heterodera) urticae Cooper, 1955

CAN. J.

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VOL. 52, 1974

SOURCE Dr. E. Krall, Estonian SSR

Agrostis tenuis Sibth. (brown top grass)

Dr. P. K. Koshy, New Delhi, India Collected by author, Vacaville, California, United States Dr. H. Stelter, Rostock, Germany Dr. J. N. Sasser, Raleigh, North Carolina, United States Dr. D. I. Edwards, Urbana, Illinois, United States Dr. V. G. Perry, Gainesville, Florida, United States Dr. D. G. Merny, Abidjan, Ivory Coast Dr. J. J. Duggan, Dublin, Ireland Dr. D. G. Merny, Abidjan, Ivory Coast Dr. E. Krall, Estonian SSR Collected by author. Sarnia, Ontario

Cajanus cajani (L.) Millsp. (pigeon pea) Ficus eastica Roxb. (rubber plant)

Collected by author, Lardner, Ontario

Trifolium repens L. (white Dutch clover) Vigna sinensis Savi (cowpea)

Soil Glycine max Merr. (soybean) Lespedeza striata Hook. (Kobe lespedeza) Stenotaphrum secwuiatwn (Walt.) Kuntze (St. Augustine grass) Oryza sativa L. (rice) Rwnex crispus L. (curled dock) Saccharum oficinale L. (sugarcane) Salix purpurea L. (willow) Beta vulgaris L. (red beet)

Dr. J. C. Adward, Allahabad, U.P., India Dr. P. K. Koshy, New Delhi, India

Zea mays L. (maize)

Dr. R. D. Winslow, Rothamsted, England Dr. A. M. Golden, Beltsville, Maryland, United States

Coronopus squamatus (Forsk.) Aschers (wart cress) Cyperus esculentus L. (yellow nut grass)

Dr. R. D. Winslow, Rothamsted, England Dr. B. A. Stynes, New South Wales, Australia Dr. A. M. Golden, Beltsville, Maryland, United States

Pisum sativum L. (garden pea)

Collected by the author, Sardis, British Columbia Dr. A. M. Khan, Aligarh, U.P., India Dr. H. J. P. Matthews, Draperstown, Northern Ireland

Cvsts and white females used in the study were preservid in 5% form01 and some subsequently processed to lactophenol. For examination by the interference contrast microscope, the cone tops (posterior end of the cyst) were prepared as described by Mulvey (1972); and for the scanning electron microscope (Cambridge Mk. 2A Stereoscan), the white gravid fanales and cysts were dehydrated in absolute alcohol, stuck to a microscope stub (with cone top upwards) with silver paint, and then gold-plated in a vacuum (Edwards "Speedivac").

Cynodon dactylon (L.) Pers. (pasture grass) Echinochloa colonwn (L.) Link (barnyard grass) Humulus Iupulus L. (hops) Cyperus rotundus L. (nut grass) Urrica dioica L. (stinging nettle)

M O- ~ D ~O~O~V Development of the Underbridge and Bullae (Figs. 28-35) Mulvey (1959) investigated and illustrated the development of the cone-top structures and underbridge of H, (H.) trifolii from the white the This in newly formed cysts is distinctly different between

MULVEY: CONE-TOP MORPHOLOGY IN HETERODERA


and within the Heterodera groups. H. (H.) trifolii (Fig. 28) and H. (H.) schachtii (Fig. 30), which are typical of species with strongly developed underbridge and bullae, have a large underbridge attached to the inner wall of the cyst by strong muscular bands running parallel to the vulval bridge. In addition to this, some newly formed cysts of H. (H.) trifolii, H. (H.) glycines, and H. (H.) oxiana (reported by Kirjanova 1962) have muscles running at right angles from the underbridge. The bullae in these species are closely associated with the underbridge but develop independently of it. In H. (H.) oryzae (Fig. 31), H. (H.) leuceilyma, and H. (H.) sacchari, the underbridge arms are connected to several areas of the inner cyst wall (Fig. 3 l), and the bullae appear to be part of this complex. As the cyst matures, the dorsal and ventral arms separate from the cyst wall and form fingerlike projections (Fig. 33) extending out from the underbridge. The underbridge of H. (H.) cajani (Fig. 34) and closely related species H. (H.) zeae and H. (H.) vigni is much smaller than that of H. (H.) trifolii (Fig. 32), but the bullae are longer and more closely associated with the underbridge. The underbridge of H. (H.) latipons (Fig. 3 3 , group 5, is similar to H. (H.) trifolii but the bullae, when present, are few and small. Development of the underbridge in H. (H.) grarninophila (Fig. 29) and H. (H.) graminis is similar to that of H. (H.) trifolii. The ends of the underbridge arms of H. (H.) graminophila and H. (H.) graminis branch several times and are strongly developed. Golden and Birchfield (1972) describe these in H. (H.) graminophila as furcations of the massive underbridge which appear to have the same texture as the underbridge. In addition, they only occur at either end of the underbridge. The underbridge is either weakly developed or absent in the remaining species of group 5.

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genus Heterodera. The structure of the vulval cone of H. (H.) betulae also differs significantly from that of all species of Heterodera and is therefore of taxonomic importance. The ovalshaped vulval aperture (Fig. 10) has a length of 6-8 pm, and the fenestra (Fig. 11) completely surrounds it. Below the fenestra are three distinct circular cores of varying density; an inner core enclosing the vagina, a middle core, and an outer core (Fig. 12). Radiating out from the inner core are at least 10 bands of muscles (Figs. 12, 13) which extend beyond the outer core. Several of these bands have furcated ends which appear to be attached to the inner wall of the vulval cone. These structures deteriorate as the cyst matures (Fig. 14). The long anal aperture is encircled by a prominent parapet (Fig. 15).

Anal-Vulva1 Distance and Basin The anal-vulva1 distance of the 30 species examined ranged from 30 to 70 pm with most from 40 to 60 pm. H. (H.) belulae had a range of 90-100 pm for mature cysts. The anal aperture of H. (H.) betulae (Fig. 15) and H. (H.) cyperi (Fig. 5) is long while that of the other species is generally short and obscure. The basin which surrounds the fenestra is distinct in many species including H. (H.) glycines (Fig. 22), H. (H.) schachtii (Fig. 23), and H. (H.) jici (Fig. 24). In group 4, all species have a distinct basin with the exception of H. (H.) cajani, H. (H.) leuceilyma, H. (H.) oryzae, H. (H.) sacchari, and H. (H.) salixophila. The basin of H. (H.) glycines and closely related species has a rather flattened arch while that of H. (H.) jici is highly arched. In group 5, H. (H.) humuli, H. (H.) graminis, and H. (H.) graminophila (Fig. 25) have a distinct basin which is much narrower than that of species in group 4. H. (H.) cruciferae and H. (H.) cyperi have obscure basins. None of the species in groups 2 and 3 including H. (H.) iri (Fig. 26) and H. (H.) latipons (Fig. 27) Development of Vulva1 Cone in H. (H.) betulae have a basin. (Figs. 10-15) The white females of H. (H.) betulae are Cuticular Pattern and Vulva1 Bridge (Figs. 1-9, mostly spherical in shape with a very short 16-21) posterior protuberance (vulval cone). According The prominent wavy striae extending from the to Hirschmann and Riggs (1969) the anal-vulva1 base of the vulval cone to the vulval aperture in distance of the female is 60-150 micrometers H. (H.) graminophila (Figs. 1, 2, 6, and 17) are (pm). In the white females which I examined, this characteristic of this species only, and is best distance was 90-120 pm, which is significantly demonstrated using a scanning electron microgreater than that of any other species in the sub- scope. These striae were not found in any of the

CAN. J. ZOOL. VOL. 52. 1974

TABLE 1, Comparison of cone-top structures of cysts of Hererodera groups; measurements in micrometers Grou~ 2

3 4


5

Bullae

Present in few s ecies; few and smalf Present in most species; strongly developed Present in all species ; strongly developed Present in few species; few, small

Underbridee

Anal-vulva1 distance

Basin Absent

Absent

30-70;

Present in several species; weak to strongly developed Present in all species ; bullae attached in three species Present in most s ecies; weak to stron& developed

30-70

Absent

30-70

Present in most species

30-70

Present in few species

H. betulae 90-100

other species examined including H. (H.) trifolii, H. (H.) glycines, H. (H.) zeae, H. (H.) salixophila, and H. (H.) cajani (Figs. 16 and 18-21, respectively). The cuticular pattern about the vulval area of H. (H.) betulae (Fig. 3) is significantly different from other species in the subgenus Heterodera, particularly from H. (H.) cyperi (Fig. 5) and H. (H.) trifolii (Fig. 4). The cuticle of H. (H.) cyperi (Fig. 7) is pitted and may assist in identification of this species. The vulval cone of H. (H.) betulae (Fig. 3) is very short compared with that of H. (H.) trifolii, H. (H.) graminophila, and H. (H.) cyperi (Figs. 4, 2, and 5, respectively). The vulval bridge of H. (H.) trifolii (Fig. 8) and closely related species is strongly developed while that of H. (H.) oryzae (Fig. 9) and similar species is weakly developed.

nomically important group), they are of doubtful taxonomic value. Significant differences were found between groups, especially between H. (H.) betulae (group 2), H. (H.) graminophila (group 5), and H., (H.) trifolii (group 4), and closely related species in this last group. The development of the underbridge and bullae in members of group 4 was significantly different. In the three closely related species of H. (H.) leuceilyma, H. (H.) oryzae, and H. (H.) sacchari the bullae eventually become an integral part of the underbridge, while the bullae in the other species of this group develop and remain free of the underbridge. In H. (H.) graminophila (group 5), H. (H.) graminis (group 5), and H. (H.) latipons (group 3), the bullae are absent but the ends of the underbridge are furcate and strongly developed.

Discussion Use of the stereoscan microscope has greatly facilitated the study of the fine structures in white females and cyst cone tops of species belonging to the subgenus Heterodera. Green (1971) and Mulvey (1973), using a stereoscan, provided new information of taxonomic significance for the subgenus Globodera Skarbilovich, 1959. Mulvey (1 972), using an interference contrast microscope, provided an illustrated taxonomic key to the species of the subgenus Heterodera based on differences in cone-top structures. The present work reveals new information on the development of the cone top during transformation of the white female into the cyst stage, and the morphological changes in the cone-top structures during this development (Table 1). Although there are recognizable differencesin the and prefenestral areas of the white females in group 4 (the largest and most eco-

Acknowledgments The author is grateful to Dr. A. M. Golden, Plant Nematology Laboratory, United States Department of Agriculture, Beltsville, Maryland, and Dr. R. V. Anderson, Entomology Research Institute, Research Branch, Canada Department of Agriculture, Ottawa, for valuable suggestions and criticisms. Special thanks to all those who provided cyst material as cited in this paper and to members of the Electron Microscope Centre, Canadian Department of Agriculture, Ottawa, especially to Mrs. E. Ford, for expertise in scanning electron microscopy. GOLD^, A. M., and W. BIR-wm.

1972. Hererodera ~rarnfnoplrilun. sp. lNernatoda:Heteroderidae) grass wlth a key to closely relaled spccies. J. Nernalol. 4: 137-155.

GREEN, C.D. 1971.T h e mo h o l o of ~ ~the terminal area of I h e round-c st nernatoxs., S. G . Hererodera rosrochiensis and aiied species. Nernatologica, 17: 34-46. HESLWG, J. J. 1965. Iffrerodera:morphology and identi-

MULVEY:

CONE-TOP MORPHOLOGY IN


fication. In Ptant nernatology. Edird by J. F. Southey. Tech. Bull. No. 7, Minist. Agric., Fish. Food, Lond. Engl. 2nd ed. pp. 103-130. WRSCHMANN. H.. and R . D. RIGGS.1969. Heterudera bcralae n. (Heterodcridae) a cyst-forming nematode from river Rirch. J. Nernaiol. 1: 169-1713. Krtrrmova, E. S. 1961. Hererod~rn oxiana sp. nov. Nematodes: Heteroderidae) from Kam-Kalpakra (Russian text). In Harmfu 1 nematodes or agrrcull~iralplants and their conlrol. I'roc. Fifrh A1I-Umon Synip. Phytone~nntol. Pub1. Univ, Ssmarkand, Uzhckstan SSR, pp. 122-131.

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HETERODERA

MULVEY,R. H. 1959. Investigations on the clover cyst nematode, Heterodera trifolii (Nematoda : Heteroderidae). Nematologica, 4: 147-156. 1972. Identification of Heterodera cvsts by terminal and cone top structures. Can. J: ZOO^. 50: 1277-1292. 1973. Morphology of the terminal areas of the white females and cysts of the genus Heterodera (s.g. Globodera) a cyst-forming nematode. J. Nematol. 5: 303-311. THORNE,G. 1961. Principles of nernatology. McGrawHill, New York.

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OF FIGURES EXPLANATION

FEG.1. Scanning micrograph of vulva[ area of white femaIe of Hererodera (H.) graminophila. A, wavy striae extending rrom outer area of cone top to vulva1 aperture; 3,vulval aperture. FIGS.- 5 . Scanning micrographs of the cone tops of mature while ltmales and cysts of H e f e d e r a (Heferod(,?'a)Npp- Fig. 2. H. {H.)~r(rn~i)~opl~iIu, wlrite female. Fig. 3. H . (If.)berrrloe, mature female showing unlque cuticular pattcrn and short cone top. I-ig. 3. H. (H.)rrijulri, cyst sbowrng vulva1 fenestra and cuticular pxttern. Fig. 5. H.( H . ) cypzri, cyst showing large anal aperiurc Wow vulva\ alxrture. iiros. 6-9. Scanning micrograph5 orcuticular ng_eesand Ienestra of ffeferoderu(Hereranera) spp. FIg.6. (H.) grarnino Iriln, wh~tcfemale sl~owrngwavy stnae. Fig.7. H . ( H . ) cyperi shorvlng charactenstic pitting In EYI~CIE. Fig. 11. ( K ) ~rqoiiicyst ~ l i o w m large ~ vulva1 brldgc, wlval aperture, and fmcaire. Flg. 9. If. (H.1 nrFne, cyst showing vulva1 aperture and teneslra. FIGS. 10-15. Pho~omicrographsof cone top of Heterodera (Hererodera)bcrulae (Figs. 10-13 and 15 of mature white female) a1 vanauq levels. F ~ E10. , Vulval aperture at top level. Fig. 11. SIighdy below (a prom rnaiaiy 6 pm) vulva{ aperture. h g . 12. L.owci level (approximately 12 p n bciow w i v a l apeflure) S!OW& serrated walls of the vagina and muscufar bands extending our from the walls. Fig. 13. Arca a b u t 20pm h10w vulval aperture. Fig. 14. Newly formed cyst showing fenestral area. Fig. 15. Anal aperture of whte female about 120 urn from vulva1 aperture. (AII f gures same mgnilicalion.) FIGS.16-21. Photomitrographs of the vulval aperturrsof wh~tefemales. Fig. 16. H. (H.) t+!ii. Fig. 17. If. (H.)~mnrinaplrilu.Fig. 18. H. (H.) r/ycime.r.Fig. 19. If. (H.) :for. Fig. 20. H. (H.) solixopkila. Frg. 21. H. (H.) cnjnni. (All iigures same rnagn~licatron.) FIGS.22-27. Photomicrographs of cone tops of Hererodera (Heferoderu]sp showin$ basin when resent. Fig. 22. K [N.),glyc~~.e., d~nlncr,wdc basin. Flg. 21. H. IH.1 .~~harhiii. ,IcE basin, ienestral wldtE about half that of b&rn,width. FIE. 24. H. ( H : ) $ c i , basin highly arched. T"ig.25. H . ( H . ) graminuphiln, narrow, rounded basin. Fjg. 26. H. ( H . ) Irt. Ims~nabsent. Fig. 27. H . ( H . ) laripons, widely spaced ienestra, basm absent. (‘Figs. 22-24 same mngdication; Figs. 15-27 same magnification.) FIGS.28-31. Photomicrographs of newly formed cys~sof f i e t ~ r ~ ~(Efererodera) ~eru sp .showing developing underbridge and b u l k in cont lop. Fig. 28. H. ( K )irfodi. Fig. 3.H. ( H . )gmrnino liiE heavy underbridge. bullae absent. Fig. 30. N. ( H . ) rcimciliii. Fig. 11. H. (H.) O!.Vx4P heavy undcrb&e 4 t h h g r i i k e bullae attached. (All figures same magnification.) FIGS.32-35. Photorn~crographsof mature cysts of Heterodera (Heterodera) spp. showing underbridge and hullae in cone top. Fir. 31. H. ( H . ) trifolir. Fig. 33. H. (H.) oryzae, very few, small peripheral bullae, underbridge massive with lingeriike projechons. Fig. 34. H. ( H . ) cajani, very long bullae and shoft underbridge. Flg. 35. H. (H.) Inirprttr.~,massive underbridge, bullae absent. (All figures same magnificabon.)

H-

&

.

NOTE: Figs. 1-35 follow.








PLATEVII
