An unique host-parasite relationship between HyZonema ivorense .(Nematoda : Heteroderidae) and the roots of a tropical tree

Donald P. TAYLOR,Patrice CADET and Michel Luc Laboratoire de Nématologie, ORSTOM, B. P. 1386, , Sénégal; Laboratoire de Nématologie, ORSTOM, B. P. V 51, , Côte d’Ivoire; Muséum national d’Histoire naturelle, Laboratoire des Vers, 43 rue Cuvier, 75 008 .

SUMMARY Roots of young avodire (Turraeanfhus africana), a member of the used as a source of , were observed in to have slight swellings frequently with a small necrotic depression. Dissection of thesc swellings revealed the presence of a.necrotic tunnel leading from the necrotic depression to a saccate female of Hylonema ivorense usually located near the vascular system. Histological examination of infected roots showed that each female incited production of only one giant cell, each giant ce11 m‘as uninucleate, each nucleus was greatly enlarged-up to 200 times the volume of a normal nucleus, and each nucleus contained an enlarged nucleolus itself usually larger than a healthy nucleus. No discrete ce11 wall was observed surrounding the giant cell. Xylem tissue near infection sites was abnormal. Eggs, and occasionally juveniles, were lound throughout the tunnel. Although a suitable host-parasite relation exists, infected tissueis characterized by considerable necrosis. Giant ce11 formation and nuclear enlargement were detected in ten-day-old material. Enlargementof these structures continued through 40 days. Two additionalhosts, Neuropeltisprevosteoides and Khayaivorensis, exhibitedthe same symptoms. Tabernaemontana crassa was also observed to be naturally infected.

RÉSUMÉ Un mode unique de relations parasitaires chez Ilylonema ivorense (Nematoda : Heteroderidae) attaquan.t les racines d’un arbre de la forêt tropicale humide. ’ Les racines de jeunes avodirés ( africana, Meliacées), essence exploitée en Côte d’Ivoire pour l’ébénis- terie, montraient de légers renflements souvent accompagnés en surface d’une dépression nécrotique.A la dissection, ces renflements ont révélé la présence d’un tunnel nécrotique conduisant de la dépression à une femelle renflée de Hylonemaivorense, généralement localisée près du cylindre central. L’étude histologique des racines infestées a montré que chaquefemelle induit la formation d’une cellule géante unique, possédant un seul noyau anormalement développé (plus de 200 fois le volume d’un noyau normal) contenant lui-même un nucléole hypertrophié, d’une taille supérieure à celle d’un noyau normal. Aucune paroi, même discrète, n’a été observée autour des cellules géantes. Les tissus du xylème sont perturbés autour des sites d’infestation. Des ceufs et occasionnellement des juvénilesont été observés à l’intérieurdu tunnel. Bien qu’un modederelation hôte/parasite efficace paraisse exister, les tissus infestés sont caractérisés par l’étendue considérable des zones nécrotiques. La formation des cellules géantes et l’hypertrophie de leur noyau ont pu observéesêtre sur du matériel végétal n’ayantpas plus de dix jours, ces phénomènes se poursuivant pendant 40 jours. Deux autres plantes hôtes, Neuropeltisprevosteoides et Khayaivorensis, présententles mêmes symptômes. Tabernaenzontanacrassa estégalement infesté en milieu naturel.

Cellular responses of to -parasitic effectsof parasitismvary greatly depending nematodeshave been thoroughly reviewed upon the species of nematodeinvolved. [Dropkin, 1969), from which it is obvious that Members of theHeteroderidae (Filipjev & SchuurmansStekhoven, 1941) Skarbilovich, 1947 andthe Meloidogynidae(Skarbilovich, (l) A portion of thispaper was presented at the XIIIthInternational Nematology Symposium, 1959) Wouts,1973 induce the most profound Dublin,Ireland, 5-11 September 1976. anatomical changeshost. thein Members of

Rev. Nématol. 1 (1) : 99 à 108 (1978) 99 D. P. Taylor, P. Cadet & M. Luc the Meloidogyne Goeldi,1892 usually swellingswere cutand fixed inFAA for72 produce root galls characterized by hypertrophy hours or longer. They were then embedded in and hyperplasia of various tissues and by the paraffin, sectioned at 15 or 18 pm and stained presence of large,multinucleate giant cells, or with safranin and fast green. Photographs were syncytia (Dropkin, 1969). Members of the genus taken with a Leitz “Orthomat” photomicroscope. Heferodera Schmidt, 1871 normally do not Additional fixedroots were dissected by hand induce galls, but the histopathology of infection to further study the relationship. sitesis similar tothat of hleloidogyne. It is To studysymptom development, seeds of generally conceded that these syncytia, two to T. africana weregerminated and seedlings many perinfection, are formed by fusion of transplantedtosterile soil. Suspensions of cells at thefeeding site resulting in the formation H. ivorense juveniles were poured over the soil of abnormallyenlarged, multinucleate cells surfacearound the seedlings.One infected (Rohde & McClure, 1975). Mitoticdivision, plant wassacrificed every five daysand the frequentlyreported to be synchronous, con- infected roots were fixed, sectioned and stained tributesto the multinucleate condition ; in as described above. certain 1Weloidogyne infectionssyncytia have been reported tohave “hundreds of nuclei” (Dropkin,1969). These nuclei arefrequently enlarged, sometimes one hundred times normal Results (Krusberg & Nielsen, 1958 ; Dropkin & Nelson, 1960).Nucleoli are also frequently enlarged in OBSERVATIONS ON NATURALLY INFECTED Meloidogyne infections and have been reported ROOTS to beas large asnormal nuclei (Krusberg & Nielsen,1958). Thehost-parasite relations of Nucleus and nucleolus. Figure 1 illustratesa iVIeloidodera floridensis has been partially de- giant ce11 inducedby H. ivorense in a root of scribed byRuehle (1962), Who statedthat T. africana. The mostconspicuous feature of this “giant cells adjacent to the female nematode” ce11 is the enlargement of the single nucleus (N), are produced but no mention was made of their especiallywhen compared to the size of the nuclearcondition. The histopathology induced nucleus (n)in an adjacent unaffected cell. by othermembers of the Heteroderidae,i.e. When 25 nucleifrom similar giant cells were CryphoderaColbran, 1966, Afalodera Wouts & measuredtheir mean diameters were 78 x Sher, 1971, Sarisodera Wouts & Sher, 1971 and 59 pm ; normal nucleimeasured 11 x 10 Pm. Hylonema Luc, Taylor & Cadet, 1978, has not Caicuiation of the voiume of the iarger nuclei been described. indicate that the mass of thesenuclei can be Thehistopathology of roots of thetropical more than 200 times that of normalnudei. rainforest tree, avodiré, infected withHylonema Thesingle, deeply staining nucleolus (NU) is ivorense Luc,Taylor & Cadet,1978, is unlike also greatly enlarged, 17 x 15.5 pm, compared any known caused by a member of t8he Hetero- to a diameter of less than 1.6 in healthy cells. deridae and is, therefore, described in detail. In examining hundreds of giant cells, no case of multiple nuclei was observed. Giant cells. Examples of H. ivorense-induced Materials and Methods giant cells are shown in Figures 1-4. Giant cells werefound in thecortex (Fig. 1), adjacent to Soi1 and root sampleswere collected from the vascular tissue(= pericycle?) as in Figure3, avodiré,Turraeanthus africana Pellegr. , a and within the vascular tissue (Figure 4 illus- member of the Meliaceae, growing in a tropical trates a giant ce11 surrounded by xylem tissue). rainforestreserve at Banconear Abidjan, These cells areirregular in outline, although IvoryCoast. Nematodes were extractedby inthe xylem they are generallyelongated in standardtechniques. Small swellings,each the direction of the root axis (Fig. 4). They are usually with a small necrotic depression, were extremely variable in size and lengths of over notedon the roots.Root pieces exhibiting 350 pm have beenmeasured. Inaddition to

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Fig. 1-4 : Giant cells induced by Hylonema ivorense in roots of Turraeanthus africana (g = giant ce11 ; N = enlarged nucleus ; NU = enlarged nucleolus ; n = normal nucleus ; nu = normal nucleolus). Fig. 5-6 : Xylem tissue in roots of T. africana; 5 : non-infected root portion; 6 : root infected with H. ivorense.

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being uninucleate, as previously mentioned, the intothe giant ce11 nearthe enlargednucleus giant ce11 ischaracterized bydense, deeply (Fig. 3). Acast juvenile cuticle can often be stainingcytoplasm. No discrete ce11Wall has seen surroundingthe lip region (Fig. 11, 12, been observed surrounding giant cells (Fig. 1-3). 13 and 15). Inmany casesobserved there Theapparent “wall” in Figure 4 was, in fact, appears to be another structure surrounding the composed of the ce11 walls of adjacentxylem anterior end of the nematode which, based upon tissue. It must beemphasized that in nocase stainingreaction, is presumed to be of plant was more than one giant ce11 associated with a origin(Fig. 10and 11). In Figure 12, the an- single nematode. When more than one giant ce11 terior end of thenematode (1) isobviously was observed in a root section, examination of withinan annulated juvenile cuticle (2) with adjacent serial sectionsalways demonstrated the a distinct lipregion ; however, theother presence of multiple infections.Cortical cells structure (3) has no distinct morphology to adjacent.to or nearthe giant cells frequently indicate if it is of nematodeor plant origin. appeared snlaller and more compact than cells In Figure 13, thestructure surrounding the in healthy tissue (Fig. 1 and 11). nematodeis clearly a castcuticle with the Abnormal xylern. Xylemtissues in non- anterior portion of the stylet (S) still attached. infectedportions of theroots of T. ufricana In contrastto that, Figure 14 shows the lip have a regular appearance (Fig. 5), in contrast region of H. iuorense closelyappressed tothe toheavily infected roots in which xylemis giant ce11 with neither a cuticle nor any other disorientedand cells are of atypical size and structure present. shape. Variability of this cellular configuration A common feature of thishost-parasite is shown in Figure 6. relationshipis the presence of necroticplant Tunnel. Anothercharacteristic of thishost- tissueadjacent tothe body of thenematode parasiterelationship is the presence of a andoften extending some distance beyond. “tunnel” extending from the posterior portion In Figure 14, an infection site of a root inoc- of themature female to theroot surface. In ulated 40 daysbefore, anamorphous necrotic unsectioned material fixed in FAA, the tunnel regionoccurs adjacentto the giant ce11 in appears as a reddish-purpletissue and upon which thenematode lip regionis embedded. dissection it usually renmins attachedto the In Figure17, a fifteen-day-old infection, a female (Fig. 7). The tunnel is harder in texture necrotic region extends to the left side of the thanadjacent root tissue. In sectioned roots photographsurrounding the anterior end of thetunnel Stains deepred andappears to be thenematode ; infact, this necrotic vascular composed of compact, dead ceiis and ceiiuiar tissueextends dongthe entirelength of the debris. In areas of thetunnel Wall where juvenile(Fig. 21) and beyond. It issuggested cellular integrity exists, ce11 walls are thickened that such a necrotic zone ultimately develops andstain a deepred and the cells are devoid intothe tunnel, the necrotic nature of which of protoplasm. In the central hollow portion of hasalready been mentioned. thetunnel, eggs and occasionallyjuveniles of Infected roots usually possess small swellings H. iuorense arefound (Fig. 8). Eggsoccur with a slightdepression in thecenter, cor- along thetotal length of thetunnel and fre- responding to , thetunnel opening.Sections quently a few are seen at the rootsurface in through such swellings indicate an increase in what appears as a small amount of gelatinous number of cortical cells but no general increase material. The entire aspect of this relationship intheir size (Fig. 9). Much of the volume of is illustratedin Figure 9. Soi1 fungireadily the swellingis occupied bythe tunnel. colonize the tunnelsas indicated by the frequent presence of mycelium amongstthe cellular debris. EARLYSTAGES OF SYMPTOMATOLOGY Relation of H. ivorense to giant cells and other tissues. The head of H. iuorense was frequently Penetrationby juveniles of H. iuorense was found closely appressed to the gianb ce11 (Fig. 10 observed in roots of T. africana five days after and 11) andsometimes apparently extending inoculation.Moderate (Fig. 22) to severe

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Fig. 7 : Three females of Hylonema iuorense (light) dissected from root showing attached tunnels (dark). Fig. 8 : Cross-section through tunnel shoming deeply stained wall andeggs in hollom portion. Note abnormal xylem. Fig. 9 : Longitudinal sec.tion through infected rootof Turraeanthus africana showing giant ce11 (G),nature female (F) and tunnel (T) extending to root surface.

Rev. Nématol. 1 (1) : 99 à 108 (1978) 103 D. P. Taylor, P. Cadet Le- M. Luc

Fig. 10-15 : Relationshipbetween anterior end of Hylonemaiuorense and giant cells of Turraeanthus africana. Explanation of markings is given in the text.

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(Fig.23) necrosis, indicated with arrows, was addition, successful inoculationshave been present in tissues adjacent to the juveniles (J); madeon iuorensis Chev.(Meliaceae). however, no indication of giant ce11 formation Roots of N. preuosteoides and K. iuorensis were could be detected. examinedhistologically andthe samehost- Giant ce11 formation was first observed in parasiterelationships as descdbed for avodiré ten-day-oldmaterial. @ne ce11 (Fig. 16) werefound (Fig. 24 and 25). measured108 x 29pm witha nucleus 26 x 21 pm (normal nucleus = 11 pm) and a nucleolus 5.6 pm in diameter (normal nucleolus less than Discussion 1.6 pm). Necrosis near juveniles was conspicuous. Figure17 illustrates typicala giant ce11 On the basis of theobservations described fifteen days after inoculation. The ce11 is 160 x above, we consider thatthe combination of 75 pm with a nucleus 29 pm in diameter and a pathologicaleffects of Hylonema ivorense on nucleolus 6.4 pm indiameter. The largest its ‘hosts is unique among plant-parasitic nema- nucleus observed at this stage had a diameter todes. The combination of symptoms including of 32 pm. only one giant ce11 per parasite, a single greatly Aftertwenty days, further increase in size hypertrophied nucleus containing asingle hyper- of giant cells, nuclei and nucleoli was observed. trophied nucleolus, associated abnormal xylem, Thenucleus in Figure 18 is 33 x 32pm. Ab- andthe necrotic tunnel containing eggs, has normal xylem was observed near infection sites not been previously described. for the first time in material of this age. The single uninucleate giant ce11 most closely Figure19 shows agiant ce11 in 25-day-old resembles that inducedin soybean roots by material.The nucleus measures 42 x 35 pm Rotylenchulus rnacrodoratus (Cohn & Mordechai, and the nucleolus 11 x 8 pm. Examination of 1976 ; 1977). However, they reported t.hat the anothersection through the same nematode nucleuswas irregular in shape whereas that shown in Figure 14 revealed the presence of a inducedby H. ivorense isovoid to spherical. partially shed cuticle behind the head region - They also reported that the nucleus occupies up theearliest observation of molting of this to one-third of the ce11 area which is greater in nematode. relation to thesize of the ce11 than is the enlarged Retween25 and 40 days, development of nucleus of giant cells induced by H. ivorense. nematodesand affectedtissues continued. Anuneven thickened Wall surroundsthe ce11 Figure20 shows agiant ce11 40 daysafter induced by R. nmcrodoratus,whereas no such inoculation with a nucleus 51 x 35 pm having Wall has been demonstrated in Our material. a nucleolus9.6 pm indiameter. Another ce11 The validity of using the term, “giant cell”, inthe same root had a nucleus61 x 48pm. to describe thesestructures iscontrary to Many molting specimenswere presentin the general usage, eg. Endo (1971, p. 92) stated that 40-day-old material(Fig. 12 and 13). the term “giant cell”refers to a “multinuclear No subsequent developmentcould be observed unit”. Rather than propose a new term for an in the oldermaterial available for studyat uninucleatehypertrophied ce11 orunit, we thistime. preferto consider thatthe term “giant cell” includes any hypertrophied ce11 or unit induced in a host by a plant-parasitic nematode regard- OTHER HOSTS less of its nuclearcondition. Thus, in Meloi- dogyne-infections there are multinucleake giant Roots collected from various plants near the cells,whereas II. ivorense and R. nzacrodoratus typelocality wereexamined for Hylonema induceuninucleate giant cells.This point was ivorense. Roots of threeother specieswere considered at a discussion session at ‘he xmth found to be naturally infected with this nema- InternationalNematology Symposium (Nenza- tode : Neuropeltis preuosteoides Mangenot (Con- tologyNews : 30, 3, 1976)and the consensus volvulaceae) ; Tabermemontana crassa Benth. was that the term, giant cell, refers only to the (Apocynaceae) ; andan unidentified tree.In size of the ce11 and not to its nuclear condition.

Rev. Nématol. 1 (1) : 99 à 108 (1978) 105 D. P. Taylor, P. Cadet & M. Luc

Fig. 16-20 : Giant ce11 development in Turraeanthus africana; 16 : ten days after inoculation ; 17 : fifteen days ; 18 : twenty days ; 19 : twenty-five days ; 20 : forty days. Fig. 21 : Severe necrosis around juvenile fifteen days after inoculation.

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Fig. 22-23 : Necrosis, indicated by arrow near juveniles (J)of Hylonema ivorense in roots of Turraeanthus africana five days after inoculation ; 22 : moderate necrosis ; 23 : severe necrosis. Fig. 24-25 : Giant cells in other hosts of Hylonema ivorense; 24 : Neuropeltis prevosteoides; 25 : Khaya ivorensis.

Thisconcept has also been proposed by Cohn suggests that it may consist of a nalred mass of and Mordechai (1977). protoplasmbounded by adjacent cells. Confir- Theproduction of single,a uninucleate mation of this awaits electronmicroscopic exam- giant ce11 by H. ivorense appearsnematode- ination of infectedtissues. Similarly, no con- specifk rather than host-specific in that similar clusion can be made at present.regarding the histopathological effects were observed in Tur- chromosomal situation in the enlarged nucleus. raeanthusafricana (Meliaceae), Neuropeltis pre- Thelarge amounts of necrosisin infected vosteoides (Convolvulaceae) and Khaya ivorensis roots of T. africana, with or without associated (Meliaceae).Inoculations of T. africana with fungi,and the probable disruption of trans- juveniles of Meloidogyne spp.to observe the locationcaused bythe disorientedxylem nuclearcondition of giant cells inducedby suggest that Hylonema ivorense may adversely thesenematodes have repeatedly been unsuc- affect thegrowth and timber-producing po- cessful. tential of this tree. T. africana occurs in a belt Thenature andorigin of the Wylonema- in West Africafrom Liberia to Zaïre andits inducedgiant ce11 andits enlarged nucleus timber, possessingseveral desirable qualities, remainunknown. Our inability to discerna has been exploited commercially in and discrete ce11Wall surroundingthe giant ce11 Ivory Coast.Commercially importantdamage

Rev. Nématol. 1 (1) : 99 à 108 (1978) 107 D. P. Taylor, P. Cadet & M. Luc may also becaused toanother host, Khaya DROPKIN,V. H. (1969). Cellular responses of plants ivorensis (“Africanmahogany” or “acajou”), tonematode infections. Ann. Rev.Phytopathology 7 : 101-122. which grows over a much wider range in West DROPKIN,V. H. & NELSON, P. E.(1960). Thehisto- Africa and is of greater economic potential. pathology of root-knotnematode infections in soybeans. Phytopathology 50 : 442-447. ENDO,B. Y. (1971). Nematode-inducedsyncytia ACKNOWLEDGEMENTS (giant cells). Host-parasite relationships of Hetero- deridae. In : Zuclterman, B. M., Mai, W. F., Rohde, The authors express their appreciation to the fol- R.A. (Eds). PlantParasitic Nematodes. Volume lowing : B. Souchaud, ORSTOM, Dakar,for prepa- II. Academic Press, New York, pp. 91-117. ration of Figures 1-25 ; C. Netscher, ORSTOM, Dakar, KRUSBERG,L. R. & NIELSEN, (1958).Patho- for his comments the text ; T. Jones and his asso- L. W. on genesis of root-knotnematodes to the Porto Rico ciates, Centre for Overneas Pest Control, London, for variety of sweetpotato. 48 : 30-39. information on thetimber-producing trees of West Phytopathology Africa ; and D. Y. Alexandre, ORSTOM, Adiopo- Luc, M. TAYLOR,D. P. & CADET, P. (1978). Descrip- doumé, Ivory Coast, Who first observed the diseased tion of a new t.ropica1 Heteroderidae, Hylonema avodiré. ivorense n. gen., n. sp., and a new outlook on the familyHeteroderidae (Nematoda : Tylenc.hida). , Rev.Nematol. 1 : 73-86. ROHDE, R.A. & Mc CLURE, M. A. (1975). Autoradio- REFERENCES graphy of developing syncytiain Cotton roots infectedwith Meloidogyneincognita. J. Nematol. COHN, E. & MORDECHAI, M. (1976). Ultrastructure 7 : 64-69. of hypertrophied ce11 induced in soybean roots by RUEHLE,J. L. (1962).Histopathological studies of Rotylenchulusmacrodoratus. XIIIthInternational rootsinfected with lance andpine cystoid Nematology Symposium ; Abstracts.Dublin, Ire- nematodes. Phytopathology 52 : 68-71. land,September 5-11, 1976, 17-18. WOUTS, W.M. (1973). A revision of the family Hete- COHN, E. & MORDECHAI,M. (1977). Uninucleate giant roderidae (Nematoda : Tylenchoidea) 1. The family ce11 induced in soybean by the nematode Rotylen- Heteroderidae and its subfamilies. Nematologica 18 chulusmacrodoratus. Phytoparasifica 5(in press). . (1972) : 439-446.

Accepté pour publication le 31 octobre 1977.

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