Nematol. medito (1988). 18: 93-102

Istituto di Nematologia Agraria, C.N.R. 70126 Bari, ltaly

SYNCYTIA DEVELOPMENT IN GERMPLASM PEA ACCESSIONS INFECTEDWITH HETERODERA GOETTINGIANA

byBLEVE-ZACHEO, M. T. MELILLO and G. ZACHEO

Summary. The ultrastructure of syncytial development in roots of pea accessions attacked by Heterodera goettingiana Liebscher is described4 and lO days after nematode invasion. Four days after inoculation a feeding plug was evident where the stylet had penetrated the wall ofthe initial syncytial celI. In the celI the fine structure was modified. Nonmembrane-bound materials, which appeared to be nematodesecretions, were evident near the stylet terminus and within the syncytial cytoplasm. Syncytia continued to develop in susceptible peaaccessions lO days after nematode invasion. In the modified cells proliferation of actively synthetising cytoplasm indicate that the syncytialcomponents function as secretory ~ystem. At the same time the content of syncytial cells of the resistant pea accessions is strongly disin-tegrated. The hypersensitive response in terms of physiological celI death is discussed.

Many investigations bave been made on the ultrastruc-turaI changes induced in plant hosts by cyst nematodes.Second-stage juveniles enter the roots, moving intracellu-lary through the cortical cells and then usually fix their lipregion on ODe endodermal celI tram which they can stim-ulate a syncytium. The maturation of the nematode is di-rectly related to the development and persistence of syn-cytia (Acedo et al., 1984). Many authors bave givendetailed descriptions of the ultrastructural features of syn-cytia induced in different susceptible and resistant hostsby Heterodera spp. (Hoopes et al., 1978; Kim et al., 1986;Wyss et al., 1984; Rice et al., 1985; Bleve-Zacheo and Za-cheo, 1987) following the progressive changes in the rootcells starting tram 42 h after nematode inoculation (Riggset al., 1973)': In affected cells juveniles were observed tostimulate syncytial formation about two days after initialpenetration of the roots and no differences were detectedbetween susceptible and resistant roots during the follow-ing two days apart tram more extended necrosis aroundthe outer limits of syncytia in the resistant hosts. Sevendays after penetration the syncytial components of the sus-ceptible roots had expanded and contained dense andgranular cytoplasm in contrast to those in resistant rootswhere the cytoplasmic contents were extremely vacuolatedand reduced to a thin layer around the celI walls (Rice etal., 1985; Bleve-Zacheo et al., 1990; Melil1o et al., 1990a).

rea (Pisum sativum L.) is the principal host far the peacyst nematode Heterodera goettingiana Liebscher. Little in-formation is, however, available on the morphological re-sponse of pea roots to nematode invasion. In this paper wecompare the ultrastructural changes induced by the pea

cyst nematode in germplasm pea accessions. In previousstudies they were selected as susceptible or resistant to H.goettingiana through the analysis of syncytial developmentand enzyme activities in roots (Melillo et al., 1990b; Za-cheo et al., 1981, 1986).

The results reported bere follow the course of struc-turaI changes at intervals after nematode invasion of thishost by H. goettingiana with a view to elucidating themechanism associated with resistance or susceptibility.

Materials and methods

Juveniles of Ho goettingiana were obtained tram cystscollected tram infected pea plahts that were grown in claypots containing field soil infested with the pea cyst nema-todeo Seeds of six accessions of germplasm pea: MG101877a and MG 101877b (Pisum arvense Lo, collected inEthiopia), MG 101956a and MG 101956c (Po elatiusStevo, obtained tram Netherland collection), MG 101748(P. arvense, collected in South Hungary) and MG 103738(P. sativum spo transcaucasicum, obtained tram the Gater-sleben collection), collected and multiplied at the Istitutoof Germoplasma, Bari, were germinated under sterile con-ditionso When root initials appeared the seedlings weretransplanted irito clay pots containing 10 mI of sterilizedsand and simultaneously a suspension of 50 second-stagejuveniles of Ho goettingiana was added to each poto The in-oculated plants were maintained at 17°C in growth cham-berso At four and ten days after nematode inoculation,roots were removed and washedo Segments of infestedroots were excised and fixed in 3% glutaraldehyde in 0005

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M sodium cacodylate buffer pH 7.2 far 4 h, rinsed in thesame buffer, post-fixed in 2% osmium tetroxide far 4 h at4°C, then stained in 0.5% uranyl acetate, dehydrated inSpurr's medium (1969). Sections 2IJ.m thick were cut withan LKB ultratome IV, stained with 1 % toluidine blue andobserved under a light microscope to verify the syncytiallocation. Ultrathin sections were cut in that region andstained with uranyl acetate and lead citrate and examinedunder a Philips 400 T transmission electron microscope at80 kV.

The lytic process of syncytia in resistant roots started inthe incorporated cells furthest tram the nematode. Inthese cells there were vacuoles with indented tonoplastcontaining granular material or myelinic structures.Around each vacuole endoplasmic reticulum was presentand a dark matrix appeared to be apposed to the tonoplast(Figs. 3a, 3b). This suggests that the ER, as the principalsite of synthesis of digestive enzymes, could be involved inthe deposition of vacuole contents. In the degenerating cy-toplasm a tangle of membranous structures were associatedwith the lytic vacuoles (Fig. 3c).

Ten days after nematode inoculation the syncytia in thefour resistant pea accessions had degenerated (Fig. 4). Thecells appeared to be necrotic, with the typical hypersensi-tive response. Cytoplasmic contents were disintegratedand nuclei destroyed, a process referred to as karyolysis(Fig. 4b). The feeding tubes, which are stable structures,remained in the cytoplasm of the syncytial cells (Fig. 4c).

Ten days after nematode inoculation in susceptible peaaccessions, the modified cells increased in number. Mostof the celI walls, particularly in the initial cells of the struc-tute, were digested and the communal cytoplasm was freeto move within syncytium (Fig. 5a). The ground cytoplasmwas granular with small vacuoles and occupied alI the sur-tace of the syncytium. Mitochondria were well preservedand similar to those in young syncytia. Endoplasmic retic-ulum, arranged in parallel arrays, was present in bothsmooth and rough forms (Fig. 5b). Nuclei were amoeboidin profile so that in section a single nucleus could appear asmultinucleate. The amoeboid shape increases the capacityfar nuclear-cytoplasm exchange. This features of nuclearcontent is typical of the reticulate nucleus reported inPisum by Lafontaine (1974) (Fig. 6a). Accumulation ofprotein within rough endoplasmic reticulum profiles ap-pears to be associated with Golgi cisternae as seen in Fig.6b. Free polyribosomes and a large quantity of filamentousdark material (proteins?) were scattered in the cytoplasm(Fig. 6c). Wall ingrowths, typical of transfer cells, havebeen found adjacentto the xylem elements in both suscep-tibIe pea accessions (Fig. 6d).

Results

CelIs directly fed upon by the juveniles of H. goettingi-ana were easily identified because of a plug like deposit inthe celi walI in both susceptible (MG 101956 c) (Fig. la)and resistant (MG 101748, 103738) rea roots (Figs. 1b,1c). The electron dense deposits forming the plug closethat part of the celi walI through which the nematode hasinserted its stylet to establish its feeding site. In the samesections amphidial channels containing a matrix of densematerial were detected, some of which appeared to be con-tinuous with the plug material, suggesting that they maybe related (Endo, 1978). In a 4-day infection of rea rootsthe feeding plug extended along the thickened wall of theinitial celi of the syncytium. The odontostyle passedthrough the plug, pushing the plasma membrane deeplyinto the celi (Fig. la). Tubule-like material, which ap-peared as smalI vesicles when cut in transverse section, wasaggregated around the stylet and was distributed through-out the cytoplasm (Figs. la, 1b). Similar material was ob-served around the feeding tube (Fig. 1b).

The modifications to the fine structure in the celIs of101956c consisted of numerous ribosomes, either as freeparticles in the cytoplasm or as polysomes, many mito-chondria with dense matrices and enlarged cristae and en-doplasmic reticulum in smooth and rough form (Fig. la).In MG 103738 roots the cytoplasmic ground substance ap-peared to be less dense (Fig. 1b) and in MG 101748 rootswhorls of rough endoplasmic reticulum and smalI irregularvacuoles were present (Fig. 1c). WalI fragments inside thesyncytia were thickened, and nuclei were amoeboid andthe nucleolus highly vacuolated (Figs. 2a, 2b). The cyto-plasm of celIs incorporated into the syncytium in 101956 ahad many smalI vacuoles, ali containing protein storage.Some of them were fused to form an irregular larger vac-uole (Fig. 2a). Numerous plastids were widely distributedwithin the syncytial cytoplasm of 101877 b. Their previ-ously regular shape was distorted because of starch grainsthat they had accumulated (Fig. 2b).

Discussion

The results extend our previous observations (Melillo etal., 1990b) and confirm that there is a striking differencein reaction to R. goettingiana between the susceptible MG101956 c and MG 101877 a and the resistant MG101748, MG 103738, MG 101877 b, MG 101956 a, peaaccessions. It appears to be a common feature that Rete-rodera spp. readily invade roots of resistant hosts and in-

Fig. 1 (Frant page) - Feeding of Heterodera goettingiana on a susceptible (MG 101956 c) (a) and resistant pea accessions (MG 103738, MG101748) (b, c). Longitudinal sections through a syncytium 4 days after nematode inoculation. The stylet has penetrated the celI wallstimulating the formation of syncytium (sy). The plug (pg) in b and c indicates the site of stylet perforation. Vesicle-shaped nematodesecretions (arrows) and a feeding tube (head arrow) appear in the cytoplasm, stili well preserved in the three accessions (Ne: nematode; ER:endoplasmic reticulum) (a x 7,000, b x 10,600, c x 5,900).

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Fig. 2 - Cross sections through syncytia of roots of resistant pea accessions (MG 101956 a and 101877 b) 4 days after nematode inoculation.Syncytial cells in MG 101956 ~ are filled with dense cytoplasm and numerous small ~acuol~s ~va) ~ontaining p~otein ~odies (a). No!e th~tthe syncytium has expanded nght up to the vessels and wall fragments (cw) are evldent inslde lt. The plasuds (p) in the syncyUum in101877 b contain large starch grains (b). Nuclei (N) are amoeboid with dense vacuolated nucleolus (a x 2,100, b x 4,500).

Fig. 3 - (a) Sections through part of a syncytium in MG 101956 a 4 days after inoculation. Note the irregular profiles of the tonoplast ofthe vacuoles containing lytic material and myelin-like structures. Portions of endoplasmic reticulum endose the vacuoles as rings (x 21,000).b) Same features of the vacuoles in a syncytium induced in MG 103738 accession (x 7,600). c) Section through a syncytial celi in MG101748 accession, dose to the xylem. Degeneration of syncytial celIs starts furthest tram the nematode head. Cytoplasmic content istransformed in a tangle of membranes (Me) occupying with the vacuoles, the most part of the degraded cytoplasm (x 10,400).

Fig. 4 - Sections through degenerated syncytia in resistant accessions M G 101877 b, M G 103738 and M G 101956a (a, b, C respectively) 10 days after inoculation. No cellular structures are evident and only fragments of preexisting cytoplasm and nuclear components remain. Note the well preserved feeding tubes (ft) (a x 1,180, b x 10,900, C x 6,600).

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I/fter nem'itodeFig. 5 - a) Section through a developed syncvtium £rom the root o£ the susceotible accession (MG 101956 c)inoculation. A large number o£ cells afe invopresent (x 1,250). b) Enlargement o£ a syncyt:.tubes present in the dense cytoplasm where 1

duce a syncytium, which disintegrated a few days later.The structures described in the resistant accessions aresimilar to those observed in other host cyst nematode in-teractions (Riggs etal., 1973; Wyss etal., 1984; Rice etal.,1985, 1987; Bleve-Zacheo et al., 1990). The changes oc-curring after the initiation of feeding sites can be directlyimplicated in the resistance mechanism. The developmentof a hypersensitive reaction enclosing the syncytium may

result either tram a component in the juvenile's saliva thatdirectly induces the resistant reaction, or tram a salivarycomponent that initiates a series of biochemical reactionsinducing the processo Rice et al. (1987) suggest that thesecond hypothesis is more likely because with the formeran immediate response within the initial syncytial cellsmight be expectedo We agree with this assumption becauseof the difference between necrotic cells (mechanically

Fig. 6 - a) Enlargement of an amoeboid nucleus within the syncytium of a susceptible root (MG 101956 c) lO days after inoculation. Thefeatures of the nucleoplasm and nucleolus afe typica! of reticulate nuclei of unaffected roots (x 3,000). b) ProfiIes of endoplasmic reticulumaggregated in parallel cisternae, containing synthetized products, evident as electron dense materia! inside the cytoplasm of a syncytium asin a (x 23,700). c) Aggregates of fiIamentous materia! (proteins?) in the cytoplasm of a developed syncytium lO days after inoculation inthe susceptible MG 101877 b accession. These aggregates appear to be associated with polysomes free in the cytoplasm (pr) (x 17,900). d)Transverse section of a syncytium in MG 101877 b showing darkly-stained wall ingrowths near the xylem (x). The ingrowths afe branchedand anastomosed and related to mitochondria (m) (x 4,800).

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from surrounding tissues (Jones and Gunning, 1976), mayindicate an enormous food reserve for the nematode.

We thank Mr. Lerario for bis technical assistance.

Literature cited

damaged by nematode penetration) and physiological celideath (host response). There is no single mechanism to ac-count for either the induction or the progression of celideath. Death may involve the activation of specific genes,with the synthesis of mRNA coding for novel protein tran-scripts. Such celi death in the hypersensitive reactionshould clearly be regarded as an active process, induced byphysiological factors to which particular celis are receptive.Physiological death of syncytial celis is at least, in its earlystages, a controlied process and is strikingly different fromthe traumatic death of cortical celis, due to the mechanicaldamage by nematode penetration and in which a celi issuddenly chalienged beyond its capacity to cope (Lockshinand Zakei-Milovanovic, 1984). Once the signal has beenreceived, the celis initiate a sequence of biochemicalevents. Foliowing minor cytoplasmic changes the syncytialcelis usually show some condensation of chromatin, vari-able development of lysosomes and autophagic vacuoles,and changes in the protein-synthesis machinery. This sug-gests that the celis are destined to die because they lose theability to respond to the specific needs indicated by thenematode. Thus, syncytial celi death must be invoked atthe level of the chromosome, where the synthesis of newcomponents capable of helping the celi adjust to its de-mand is blocked. This hypothesis envisages that the genesresponsible for initiating physiological celi death are re-sponsible for the synthesis of nascent degradative en-

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Accepted for publication on 17 Aprii 1990.

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