Virus Group-specific and Virus-specific Cytological Alterations induced by Members of the Tymovirus...

Phytopath. 2., 90, 315—336 (1977)@ 1977 Verlag Paul Parey, Berlin und HamburgISSN 0031-9481/ASTM-Coden: PHY2A3

AHS der Biologischen Bundesanstalt fiir Land- und Forstwirtschaft,Institut fur Viruskrankheiten der Pflanzen, Braunsdoiveig

Virus Group-specific and Virus-specific Cytological Alterationsinduced by Members of the Tymovirus Group

By

D.-E. LESEMANN

With 40 figures

Received March 24, 1977

Introduction

Tymoviruses represent a well defined group of plant pathogenic viruses(HARRISON et al. 1971). Some of the data known about this group have beenreviewed and discussed by KOENIG and GIVORD (1974). Relationships betweenmembers of the group as determined by serological methods range from veryclose to very distant or not detectable. A classification sdieme based on sero-logy has been worked out by KOENIG and GIVORD (1974) and KOENIG (1975).It shows that tymoviruses whidi are distantly related to one another, werestepwise interconnected by other viruses which were more closely related.Cytopathological data of turnip yellow mosaic (for review see MATTHEWS1973), wild cucumber mosaic (ALLEN 1972), belladonna mottle (HARRISON andROBERTS 1969, MOLINE 1973), and eggplant mosaic (GIBBS and HARRISON1973) viruses suggested that small double membrane-bounded vesicles formedat the periphery of host cell chloroplasts are a typical alteration induced bymembers of the tymovirus group (ALLEN 1972, MATTHEWS 1973). Recently innuclei of infected cells accumulations of electron lucent material have beenlound with some group members (MOLINE 1973, HATTA and MATTHEWS 1976).The present study has been undertaken to compare cytopatbological data ofadditional members of the group in order to define group specific characterson a broader basis. It was also intended to evaluate characters allowing to dif-ferentiate between group members on the basis of cytopathology.

316 LESEMANN

Material and Methods

Fifteen isolates of tymoviruses were used In this work: andean potato latent, isolateGibbs (APL.V-Gibbs), isolate AY (APLV-AY), isolate Caj (APLV-Caj), Isolate HU (APLV-HU), belladonna motile (BMV), clitorea yellow vein (CYVV), cacao yellow mosaic (Co-YMV), desmodium yellow motile (DeYMV), dulcamara mottle (DMV), eggplant mosaic(EMV), ej gplant mosaic-isolate from Abelia (EMV-AL), okra mosaic (OKMV), ononis yellowmosaic (OYMV), .scrophularla mottle (ScrMV), and wild cucumber mosaic (WCuMV) viruses.All isolates were kindly provided by Dr. RENATE KOENIG, and ihe origin of most of themhas been given by KOENIG and GIVORD (1974), and KOENIG (1975). APLV-isoIates AY, Caj,and HU were recently isolated in Peru by Dr. C. E. FRIBOURG and Dr. R. JONES (Inter-national Potato Center, Lima). CYVV was isolated in Kenia by Dr. K. R. BOCK (BOCK et al.1977) and EMV-AL in the USA by Dr. H. E. WATERWORTH (WATER-OCORTH et al. 1975).

Ultrathin sections were studied with most of the viruses from infected plants ofDatura stramonmm L. and Nicotiana clevelandii Gray, CYVV and EMV-AL only from N.cle-velandii. OYMV, WCuMV, and DeYMV did not infect these two species and were studiedin Pisum sativum L., Cucurbita pepo L., and Phaseolus vulgaris L., respectively. Additionallyto D. stramonium and N. clevelandii, with APLV-Gibbs Nicotiana glutinosa L. was used, withBMV Atropa belladonna L., with DMV N. glutinosa, with CoYMV Chenopodium quinoaWilld., and with OKMV Cucumis sativus L. and Hibiscus esculentus L.

Small pieces mostly from systemically infected leaves with fully developed symptomswere embedded in Epon as described by LESEMANN and HUTH (1975). Ultrathin sections werecut with glass knives and after staining with lead citrate (REYNOLDS 1963) examined with aSiemens Elmiskop 1A electron microscope. Leaves of healthy plants were embedded for control.

Results

Alterations of fine structure of infected cells varied very much in degreeof severeness with the different virus isolates studied here. Tn general, heavydamage of cell structure, e. g. of chloroplasts was observed concurrently withsevere macroscopic symptoms like necrosis or heavy dilorosis. Cytoplasmicvirus concentrations were very high with eadi virus studied here. But severedamage of cell structures was not always following the production of virus.For example with DeYMV on P. vulgaris, or ORMV on N. clevelandii andD. stramonium the fine structure of organelles did not seem to be heavilydisturbed, although the cytoplasm was thickly inflated by virus particles.

Observed alterations were not found to be qualitatively different in dif-ferent host plants of one individual virus isolate. But differences in the degreeof severeness of the alterations were present even within one piece of tissue.In the following mainly maximal qualitative effects are described in order todefine the individual effects of the isolates. All observed alterations are sum-marized in Figure 1 for a comprehensive view, and illustrated and describedbelow.

a) Alterations of chloroplasts

Alterations of chloroplasts were most conspicuous and were found withall virus isolates. Small double membrane-bounded vesicles occurred con-sistently at the chloroplast peripheries (Fig. 2). Such vesicles were already

Virus Group-specific and Virus-specific Cytological Alterations 317

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Fig. 1. Cytological alterations observed with 16 members of the tymovirus group. E.\p!anationof different diaracters see text. ='" Data for TYMV were taken from the literature {GEROLAL-i al. 1966, CHALCROFT and MATTHEWS 1966, USHIYAMA and MATTHEWS 1970, citations secMATTHE'S'S 1973; HATTA and MATTHEWS 1974). '•••' With TYMV, EMV, and WCuMV viruscrystals have not been observed in the present material, but have been reported in the litera-ture (USHIYAMA and MATTERS 1970, ALLEN 1972, GIBBS and HARRISON 1973, HATTA and

MATTHEWS 1974)

known as earliest events detectable in ultrathin sections of TYMV-infectedtissues (MATTHEWS 1973, HATTA and MATTHEWS 1974). Vesicles of apparentlythe same type had also been reported for BMV, EMV, and WCuMV (HARRI-SON and ROBERTS 1969, MOLINE 1973, ALLEN 1972), and they occurred in thepresent material with these viruses and additionally with the different APLV-isolates, CoYMV, CYVV, DMV, DeYMV, EMV-Al, OKMV, OYMV, andScrMV (Fig. 1). The vesicles were always bounded by a double membranewhich seemed to be continous with the chloroplast double membrane. They are

318 LESEMANN

Fig. 2. Small, double membrane-bounded, peripheral vesicles in diloroplast of Atropa bella-donna infected by belladonna mottle virus. • Fig. 3, 4, 5. "Sunken regions" in chloroplastsof Nicotiana glutinosa (3, arrows, 4) and N. clcvelandii (5) infected by andean potato latentvirus (isolate Gibbs). All magnification bars without specification represent one micrometer


thought, therefore, to represent invaginations into the diioroplasts from thecytoplasmic space. However, they do not contain normal cytoplasmic materialbut more or less electron transparent material, often with some irregularstrings. The latter resemble double-stranded nucleic acid material (HATTA andMATTHEWS 1974). The thin neck of the invaginations appeared to be pluggedby electron dense material. Sometimes in the neck region and in the neigh-bourhood of the vesicles the two membranes of the chloroplast envelope wereadhering to one another on some distance, showing only one heavily stainedcontour (Fig. 7, arrows).

Chloroplasts of C. quinoa, N. glutinosa, and D. stramonium infectedwith APLV-Gibbs showed a specific modification in the arrangement of peri-pheral vesicles (Eigs. 3, 4, 5). A similar arrangement was only very seldomseen also with CoYMV. Parts of the peripheral vesicles did not originatedirectly at the outer chloroplast periphery as with the other viruses, but frommore or less deeply sunken invaginations of the periphery (Fig. 3). These in-vaginations contained cytoplasmic material and had wide openings to thecytoplasm. Small double membrane bounded vesicles as described above wereoften found clustered along the internal parts of the invaginations (Figs. 4and 5). With TYMV, HATTA and MATTHEWS (1974) found similar structuresand designated them "sunken regions". These occurred only very early, aftersystemic infection of a cell had begun, and disappeared lateron. The sunken

Fig. 6. Rounded chloroplasts witb vacuoies of different sizes in cell of Hibiscus esculentusinfected by okra mosaic virus. Note enlarged osmiophilic globules (arrows). In the lower cell

chloroplasts appear normal

320 LESEMANN

Virus Group-specific and Virus-specific Cytologica! Alterations 321

regions In APLV-Gibbs-infected cells, were found in the three different hostsand in material embedded at different times, but always late after inoculation.So with APLV-Gibbs, these structures seem not to be transient as with TYMV.

With all virus isolates studied here the diloroplasts of infected cells show-ed a tendency to increase in volume and to assume a round shape (Fig. 6).With two exceptions they were also found to form more or less conspicuousclumps (Fig. 7). In these clumps the chloroplasts adhered closely together, buttheir double membranes did not fuse or toudi each other.

Another diaracteristic of infected cells was the formation of large,vacuole-like vesicles in the chloroplast stroma (Figs. 6, 7, 8, 9). Only withDeYMV these were not observed. With the other isolates they were formedto very varying extents. In some instances they remained relatively small(Figs. 6, 7, 8), whereas in others they grew extremely large, leading to veryabnormal structures and shapes of the diloroplasts (Fig. 9). Thus with APLV-Gibbs, APLV-Caj, EMV, BMV, OYMV, ScrMV, OKMV, and CoYMV thevacuoles protruded from the chloroplasts and formed large vesicles in thecytoplasms or in the central vacuoles of the cells (Fig. 9). Sometimes the wholespace of the central vacuoles was filled by the swollen diloroplast vacuoles.The contents of these vacuoles normally was unstained. Only membranaceousstructures were sometimes seen in them as single layers, loosely parallel to thebounding membrane (Fig. 6), or as several concentric layers in myelin-likearrangement (Fig. 9). Sometimes also irregularly shaped membrane-sacs areiound in them. Virus particles were not detected inside the vacuoles.

Sometimes at the chloroplast peripheries a series of vesicles with increasingdiameters occurred. The smallest of these represented typical double-membranebounded peripheral vesicles, the larger ones typical vacuoles in the stroma(Fig. 8). Both types were interconnected morphologically by intermediate sizedvesicles, in which partly a loose membrane was visible whidi could possiblyrepresent a deformed remnant of the inner membrane of the double membranevesicle-envelope after swelling of the whole vesicle to form a vacuole. Sucharrangements suggested a possible origin of the chloroplast vacuoles from thesmall peripheral vesicles.

In advanced stages of chloroplast alterations generally the number ofthylakoid membranes and the size of grana appeared reduced, concurrentlyosmiophilic globules were more conspicuous and were increased in number(Fig. 9) or size (Fig. 6), compared to healthy chloroplasts. Some different typesof affection were found whidi might represent different ways of diloroplast

Fig. 7. Three adjacent diloroplasts from a clump in a cell of Nicotiana clevelandii infectedby cacao yellow mosaic virus. Note parts of diloroplast double membranes whidi are fusedto give one electron dense contour (arrows). • Fig. 8. Sequence of vesicles at the peripheryof a diloroplasc of Datura stramonium infected by eggplant mosaic virus. At the left a typicaldouble membrane-bounded peripheral vesicle is seen, at the right typical diloroplast vacuoles,intermediate structures in between. • Fig. 9. Extremely hypertrophied chloroplast vacuoles,protruding into the central celt vacuole in a cell of Datura stramonium infected by eggplant

mosaic virus. Note groups of many small osmiophilic globules (arrows)

Phytopath. 2., Bd. 90, Hefi 4 21

LESEMANN


disorganization under the influence of the tymovirus infections. 1) Vacuoliza-tion proceeds until the individual chloroplast structure can barely be identified(Fig. 9). 2) The stroma of chloroplasts can swell and remnants of thylakoidsand other membranes are seen in an abnormally electron transparent stroma(Fig. 10). 3) Parts of the stroma are divided by membranes into small, sausage-shaped pieces thus leading to a fragmentation of the stroma within the outermembrane of the chloroplast envelope (Fig. 11). 4) With many of the viruses,extreme membrane production occurred in the chloroplasts leading to theformation of myelin-Iike bodies (Fig. 12). 5) In final stages chloroplasts losttheir enveloping membranes and were completely desintegrated (Figs. 13, 14).Grana-elements and osmiophilic globules were then found freely in the cyto-plasm, and were finally difficult to recognize as chloroplast remnants (Fig. 14).

The complete desintegration of diloroplasts did obviously not lead tolethal conditions of the cells. It could often be observed that infected cellscontained chloroplasts in very different stages of affection. Thus, in the samecell remnants of completely desintegrated chloroplasts occurred side by sidewith chloroplasts showing only some peripheral vesicles (Fig. 13). The effect ofvirus infection seems to be individual for each chloroplast. The reason for thisis not clear. It could be caused by differences in the duration of the infectionof individual chloroplasts within one cell or it could be caused by differentgenetic equipment of the individual chloroplasts, leadmg to differing sus-ceptibility for the influence of the virus.

With BMV and EMV in chloroplasts occasionally elongated, conspicouscrystals were found (Figs. 15 and 16). They did not occur with other tymo-viruses in the same host plants. The crystals were built up from thin layerswith a periodicity of about 10—12.6 nm (Fig. 15, inset). They often did not fitinto the normal chloroplast shape and thus led to very unusual shapes of theseorganelles. In shape and spacing of layers these crystals resembled those form-ed by fraction 1 proteins in tobacco protoplasts (WILLISON and DAVEY 1976).With BMV some crystal-containing chloroplasts additionally showed veryconspicous arrays of tubules (Fig. 15), and similar tubules but not aggregatedwere further found with EMV, BMV and DMV (Fig. 17), and to a lesserextent also with CoYMV.

b) Alterations of nuclei

Nuclei of cells infected with any tymovirus often showed conspicuousaccumulations of rather electron transparent material (Fig. 18). In severalinstances where virus particles could be directly recognized in ultrathin sec-

Fig. 10. Disorganized diloroplast with swollen and electron transparent stroma In a cell ofCurcubita pepo infected by wild cucumber mosaic virus. • Fig. 11. Stroma fragmentation ofchloroplast of Datura stramonium infected by eggplant mosaic virus. Cross sections ofsausage-shaped stroma fragments on both sides of the unfragmented central part of thediloroplast. • Fig. 12. Myelin-Iike membrane proliferations in diloroplasts of Datura stra-

monium infected by eggplant mosaic virus

21*

LESEMANN

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Figs. 13, 14. Desmiegration of diluroplasls. 13. Cell of Nicotiana glutinosa infected by dulca-mara mottle virus, 14. remnants of a diloroplast in cell of Nicotiana ctevelandii infected by

scrophularia mottle virus

Virus Group-specific and Virus-specific Cytotogical Alterations 325

Fig. 15. Part of chloroplast showing tubules in regular arrays, vacuoles, and crystals in cellof Nicotiana clevelandii infected by belladonna mottle virus. Inset shows enlarged part of acrystal. • Fig. 16. Crystals in diloroplast of Datura stramonium infected by eggplant mosaicvirus. • Fig. 17. Tubules in irregular arrangement in diloroplasts of Datura stramonium infected

by eggplant mosaic virus

tions as dark round dots, they could clearly be differentiated from circularprofiles representing empty protein shells (Fig. 19). In these instances theelectron transparent material in the nuclei could consistently also be seen to becomposed of protein shells, Hke those in the cytoplasm. Corresponding mate-rial has recently been studied by HATTA and MATTHEWS (1976) and theyfound it to contain viral antigen. Already MOLINE (1973) had suggested thatthis material in the nuclei represents viral particles. Thus it is clear now thatin addition to conspicuous alterations induced by tymoviruses in diloroplasts,ihe nuclei, too, are always strongly altered. They accumulate large quantitiesof protein shells of the virus. But the protein shells are not confined to thenuclei. Tliey occur in the cytoplasm, too, and occur also in mitochondria andvery seldomly in the space between chloroplast membranes (see below).

LESEMANN

"200 mil

Figs. 18, 19. Accumulation ot electron transparent material in nuclei of Nicotiana clevelandiiinfected by scrophularia mottle virus. 18. General view, 19. enlarged part showing ad-jacent nuclear (top) and cytoplasmic (bottom) region. In the nucleus only circular profiles arcseen, representing empty protein shells, in the cytoplasm dark dots and circular profiles are

seen, representing virus particles and empty protein shells, respectively

c) Alterations of mitochondria

Mitodiondria of infected cells often showed alterations like swelling indifferent degrees which are difficult to define exactly. This was seen withalmost any virus. About half of the studied virus isolate.s furtheron inducedthe appearance of irregular shaped vesicles in the centra! parts of the mito-chondria (Figs. 20, 21, 22), whidi most probably were formed by inflation ofsingle cristae. With EMV-Al, OYMV, OKMV, ScrMV, and CYVV these


200 nm

Figs. 20, 21, 22. Vacuole-like vesicles of mitochondria containing empty protein sliell-likeparticles, alt in Nicotiana clevelandii. 20. Eggplant mosaic virus (AL); 21. Scrophulariamottle virus; 22. Clitorea yellow vein virus. • Figs. 23—26. Small, double membrane-bounded vesicles in mitodiondria of Nicotiana clevelandii infected by clitorea yellow vein

virus

328 LESEMANN

2 0 0Figs. 27, 28, 29. Several affected mitodiondria of Nicotiana gluttnoia {17) and N. clevetandii(2S, 29), infected by andean potato latent virus (Gibbs), showing vacuole-tike vesicles,tendency to fragmentation, and an electron dense, homogeneous appearing matrix. • Fig. 30.Accumulation of empty virus-protein shells between inner and outer membrane of chloroplastenvelope in Nicotiana clevelandii infected by eggplant mosaic virus (AL). • Fig. 31. Particlesof scropbularia mottle virus escaping through breakage points of tonoplast (arrows) from the

cytoplasm into the central vacuole of an infected cell of Nicotiana clevelandii

mitochondrial vesicles contained small circular profiles like those of the emptyprotein shells in the cytoplasm and nuclei (Figs. 20, 21, 22). These empty shellswere only seen in tissue blodts in which cytoplasmic virus particles were clearlyresolved, but not with all virus isolates where the particles were resolved.

virus Group-specific and Virus-specific Gytological Alterations 329

Nucleo-protein particles were not found in mitochondrial vesicles. It shouldbe pointed out, that mitochondria were clearly recognized in these tissues bythe structure of their cristae. Membraneous structures like cristae did notoccur in adjacent chloroplasts and a confusion of these two organelles in ourmaterial seems very unlikely. Only once, with EMV-AL, empty protein shellswere also found accumulated in the space between chloroplast membranes(Fig. 30).

With CYVV, very unexspectedly, in some mitochondria small, round,double-membrane bounded vesicles were found, resembling very close thoseformed at the chloroplast peripheries (Figs. 23—26). They also sometimes con-tained stringy material resembling nucleic acids. Again, there was no doubt,ihat in fact these vesicles occurred in mitochondria and not in proliferationsof chloroplasts.

APLV-Gibbs and, less conspicuously, CoYMV induced heavy damage tohost mitochondria (Figs. 27—29). These organelles were often heavily swollen,showed an unusual homogenous and electron dense appearance of their matrixand contained small to large vacuole-like vesicles bounded by one membrane.Sometimes the mitodiondria appeared almost fragmented by these vesicles.The latter contained either cloudy material of medium electron density, orsmaller vesicles, or they were electron transparent. Empty protein shells couldnot be found in them. Like the chloroplasts also the mitochondria obviouslywere individually affected. In the same cell heavily affected, and not re-cognizably altered mitochondria were found side by side.

d) Alterations in cytoplasm and vacuoles

Infected cells often showed virus particles also in the central vacuoles.These particles seemed to be transported into the vacuoles from the cytoplasmthrough breakages of the tonoplast. Often at sites where the tonoplast wasbroken, the cytoplasm, full of virus particles, was seen to protrude into thevacuoie and to thin out in density from these points (Fig. 31). Thus there doesnot seem to exist a specific mechanism for transfer of virus particles intovacuoles like that described by MARTELLI and Russo (1972) with pelargoniumleaf curl virus and by Russo et al. (1968) with artichoke mottled crinkle virus.

With almost all virus isolates tissues were found that showed a dense,inflated cytoplasm due to an accumulation of virus particles in high concen-trations. Only rarely however, a spontaneous crystallization of virus particlesoccurred. No techniques were used to induce artificial crystallization (HATTAand MATTHEWS 1974). With ScrMV (Figs. 32 and 33) and BMV crystals ofthe same appearance were formed as described for TYMV, EMV, OKMV,.md DeYMV after artificial induction of crystallization (HATTA and MAT-THEWS 1976, HATTA 1976), and for BMV-Physalis strain without induction(MOLINE 1973). All these crystals represented densely padted arrays ofparticles. Similar arrays were in our material also formed by DeYMV (Figs.34 and 35), but only in the central vacuoles of infected cells. In the cytoplasmand nuclei, virus particles and empty protein shells, or protein shells alone.

LESEMANN


Figs. 32, 33. Different degrees of crystallization of virus particles in cells of Nicotiana cleve-landii infected by scrophuUria mottle virus. • Figs. 34—38. Virus crystals in cells of Pha-seolits vulgaris infected by desmodium yellow mottle virus. 34, 35. Crystals in central vacuoleswith dense arrays of particles, 36. crystals in cytoplasm and nucleus showing complex pat-terns, 37, 38. complex cytopiasmic crystals in different orientations, in 37 a square net-like

arrangement is visible

lespectively, formed apparently more complicated arrays (Figs. 36—38). Theseappeared in certain section planes like a regular net with square holes of twoparticles diameters in each direction (Fig. 37). The apparent periodicity ofthese crystals was about 73 nm, whereas the densely packed arrays showedperiodicities of about 30 nm.

A further alteration of cytoplasm in host cells occurred with CoYMVonly. TV. cle^velandii, D. stramonium (Fig. 40), and C. quinoa (Fig. 39) formedin infected cells conspicuous masses of medium to low electron density withirregular size and shape. No structural differentiation could be found in thismaterial and no structural relation to organelles of the host cells.

Figs. 39, 40. Amorphous cytoplasmic masses induced by cacau yellow mosaic virusin cells of Nicotiana clevelandii (39) and Datura stramonium (40)

Discussion

For comparison of cytological alterations as it is intended with this study,one has to differentiate between alterations of host cells that occur with manyviruses of different virus groups (nonspecific alterations), and alterationsassociated with one virus group or individual viruses (specific alterations).

332 LESEMANN

Nonspecific alterations can be characteristic consequences of virus infectionslike accumulation of isometric virus particles in the cytoplasm and concurrentinflation of the cytoplasm, or they can be a general expression of an alteratedphysiology of infected cells, e. g. high ribosome concentration, proliferationof membranes of endoplasmic reticulum, inflation of mitochondria, degrada-tion of thylakoid-membranes on chloroplasts, formation of hpid droplets indiloroplasts, degradation of tonoplasts and other membranes or finally dis-organization of the entire cell contents. Such nonspecific alterations were partlypresent in our material but will not be considered here furtheron.

The virusgroup- or virus-specific alterations are of interest in the follow-ing. From the presented data a definition of alterations specific for the tymo-virus group and of alterations that are specific for individual viruses will bediscussed.

Group specific alterations occur with all or at least most group members,but do not occur with viruses of other groups. These are with the tymovirusgroup alterations that were already known with some members (MATTHEWS

1973) and were now demonstrated with all group members studied here: smalldouble membrane bounded peripheral vesicles of chloroplasts, rounding andclumping of chloroplasts, formation of vacuoles in chloroplasts, and, possibly,formation of myelin-like structures and fragmentation of chloroplast stroma.The latter alterations were occasionally also observed in cells infected withother viruses than tymoviruses (unpublished), but they seemed to occur muchmore regularly in advanced stages of chloroplast alterations associated withtymoviruses. Additionally, the accumulation of material with low electrondensity in nuclei, possibly always empty virus protein shells, should be regardedas tymovirus group-specific alteration.

The studied material seemed to indicate that differences exist betweenindividual virus isolates in the degree of severeness of groupspecific alterations.Such quantitative differences would provide virus-specific characters. How-ever, great differences in severeness of these alterations are also known to beassociated with different tissues of a mosaic, with different developmentalstages of leaves and infections, and with different host plants. Thus from ourmaterial the suspected virus-specific quantitative differences in alterationscould not be clearly evaluated.

The material studied here is, however, apt to define qualitative differen-ces in the influence of the viruses on host cell structures. To make sure thatobserved effects were specific of the virus and not of the host plant, eadi viruswas studied on different host plants, where possible and, on the other side, thedifferent viruses were studied on the same host plants. There was no indica-tion, that individual virus isolates induced qualitatively different alterationson different host plants, although quantitative differences were present. Thusm cases where only one host was studied also the significance of qualitativeeffects should be assumed. The material studied here was embedded aftersymptoms were fully developed. In this stage the earliest events after virusinfection and possibly short-lived intermediate events are likely to be missed.But on the other side at this stage and with all the viruses high particle con-


centrations had been synthesized. So the cytological alterations accompanyingthis synthesis process should be comparable best at this stage independent fromdifferent velocities in infection establishment and development. For the com-parison the maximal induced effects were used to give the basis for a defini-tion of qualitative differences.

As can be seen in Figure 1 for some individual virus-isolates or small groupsof individual viruses specific characters can be defined. These are with TYMV,CoYMV, and APLV-Gibbs the sunken regions of the diloroplast peripheries,with BMV, DMV, EMV, and CoYMV the occurrence of tubules in chloro-plasts, BMV and EMV additionally induce formation of crystals in chloro-plasts, CoYMV induces the formation of amorphous masses in the cytoplasm,DeYMV is characterized by occurrence of complex virus crystals and by lackof chloroplast vacuolization, CoYMV, APLV-Gibbs, APLV-HU, ScrMV,EMV-AI, OYMV, OKMV, and CYVV induce formation of vacuole-likevesicles in mitochondria, ScrMV, EMV-Al, OYMV, OKMV, and CYVV formempty protein shells in these vesicles, and only with CYVV in the mito-chondria small double membrane bounded vesicles occur, similar to those inchloroplasts. The latter phenomenon is especially interesting in view of thespecific function of the similar vesicles at the chloroplast periphery (MATTHEWS1973). No virus specific structures apart from the group specific alterationswere detected with WCuMV, APLV-Cay, and APLV-Ay.

In Figure 1 tymoviruses are arranged so, that similar combinations ofcytological alterations are neighbouring. The diaracters considered as groupspecific are not weighed as much for the arrangement as the virus specificcharacters. The order in this arrangement is roughly similar to that derivedfrom serological reactions (KOENIG 1975). But CoYMV and TYMV take adifferent position in this order because CoYMV shows "sunken regions" ofchloroplasts, tubules in diloroplasts, and vacuole-like mitochondrial vesicles,thus connecting between BMV-EMV-DMV and the APLV-isolates. TYMVhas been put between CoYMV and APLV-Gibbs because of the occurrence of"sunken regions". A more close relation than seen with serology betweenAPLV-Gibbs and CoYMV might also be indicated by the similarity in basecomposition of the RNAs (CIBBS et al. 1966, BRUNT 1970). But the discrepancybetween the data of GIBBS et al. (1966) and BRUNT (1970) for CoYMV shouldbe kept in mind. It should also be kept in mind that the use of cytopatho-logical observations for an arrangement as in Figure 1 could lead to a morelieterogenous result compared to arrangements based on serology. The reasonis that cytopathological alterations represent a number of different characterswhose individual weight is not known, whereas serological reactions can beconsidered as one set of diaracters that allow — to a certain extent — a quan-titative comparison.

From Figure 1 a clearcut subdivision of the tymovirus group cannot bededuced which would coincide with the subdivision into the andean potatolatent and the turnip yellow mosaic virus subgroups (GIBBS et al. 1966, GIBBS1969, HARRISON et al. 1971). Similarly, this subdivision was not evident fromthe most comprehensive serological data used by KOENIG and GIVORD (1974).

334 LESEMANN

All viruses designated APLV were isolated from potatoes of the Andeanlegion in South America. Although they can be clearly distinguished fromeadi other (FRIBOURG et al. 1977) they are closely interrelated. However, thepresent cytopathological observations show not only quantitative, but alsoqualitative differences between APLV-Gibbs and the other isolates. OnlyAPLV-Gibbs induces "sunken regions" in chloroplasts and vacuoHzation ofmitochondria. On the other hand, APLV-Ay and Caj do only induce a heaviervacuolization of chloroplasts than APLV-Gibbs. So these isolates from thesame natural host and the same geographical region show qualitative dif-ferences in cytopathology.

GiBBS and HARRISON (1973) consider APLV and EMV as strains of EMV,because of similarities in host range and serological reactions. The cytopatho-logy shows that in this case viruses are combined that are more dissimilar thanthe APLV-isolates, mentioned above. EMV did not induce "sunken regions" inchloroplasts and vacuolization of mitochondria as APLV-Gibbs did, but in-stead it did induce formation of crystals and tubules in chloroplasts whichwere not seen with any APLV-isolate, but which are similar to those inducedby BMV and DMV. Thus the present observations would suggest a groupingof EMV together with BMV and DMV. rather than with APLV.

It can de deduced from these examples that at least in specific cases thecytopathology is a very potent tool, allowing to visualize qualitative distinc-tions between closely related virus isolates that are not evident so clearly fromthe differences in serologica! reactions. On the other hand the variability ofcytopathology between closely related virus isolates should be studied withmore examples in detail in order to evaluate the significance of differences incytopathology for a classification within a virus group, i. e. for the definitionof individual viruses.

Summary

Gytological alterations induced in host plants by 15 different isolates oftymoviruses have been studied. The comparison revealed virus group specificdiaracters that were already known with some members of the group: forma-tion of small double membrane bounded peripheral vesicles of diloroplasts,rounding and clumping of chloroplasts, vacuolization and characteristicadvanced stages of chloroplast disorganization, like formation of myelin-likcstructures and fragmentation of stroma parts. Individual chloroplasts werecompletely desintegrated. A further virus group-specific alteration is the accu-mulation of material with low electron density, presumably masses of emptyprotein shells of the viruses, in the nuclei. Several distinct alterations, mostlynot described before, were induced only by individual virus isolates or smallgroups. One isolate of andean potato latent virus (APLV-Gibbs), cacao yellowmosaic virus (GoYMV), and turnip yellow mosaic virus (TYMV) induce"sunken regions" at the chloroplast peripheries. Tubules in the chloroplaststroma were found only with belladonna mottle (BMV), dulcamara mottle(DMV), eggplant mosaic (EMV) viruses, and GoYMV. BMV and EMV ad-


ditionally induced the formation of crystals in chloroplasts. CoYMV inducedformation of amorphous masses in the cytoplasm. Desmodium yellow mottlevirus was diaracterized by formation of complex virus crystals and by lackof chloroplast vacuolization. Vacuole-like vesicles in mitochondria were foundwith CoYMV, APLV (isolates Gibbs and Hu), scrophularia mottle (ScrMV),EMV (isolate AL), ononis yellow mosaic (OYMV), okra mosaic (OKMV), andclitorea yellow vein (CYVV) viruses. In these vesicles empty protein shell-like structures were found with ScrMV, EMV-Al, OYMV, OKMV, andCYVV. With CYVV, only, small double membrane bounded vesicles occurredin mitodiondria, which were resembling those in chloroplasts.

ZusammenfassungVirusgruppen-spezifisdie und Virus-spezifisdie zytoiogisdie Veranderungen,

die von Vertretern der Tymovirus-Gruppe induziert werden

Die von 15 verschiedenen Isolaten von Viren der Tymovirus-Gruppe inden Wirtszellen induziertcn zytologischen Veranderungen wurden untersuditund vcrglichen. Als Virusgruppen-spezifische Veriinderungen sind anzusehen:die Bildung kleiner, doppelwandiger Vesikeln an der Chloroplastenperipherie,Abrundung und Verklumpung der Chloroplasten, Vakuolisierung des Chloro-plastenstromas und charakteristische Stadien einer fortgeschrittenen Disorgani-sation der Chloroplasten, wie die Bildung myelinahnlicher Membrankomplexeund Fragmentierung von Teilen des Chloroplastenstromas. Einzelne Chloro-plasten waren vollkommen desintegriert. Als weiteres Virusgruppen-spezi-fisches Merkmal infizierter Zellen mu(5 die Anhaufung von einem Material inden Zellkernen gelten, das hodistwahrscheinlich aus leeren Proteinhullen derViren besteht.

Verschiedene weitere Veranderungen werden im Zusammenhang mit ein-zelnen oder wenigen Viren beobachtet. Ein Isolat des andean potato latentvirus (APLV-Gibbs), sowie cacao yellow mosaic (CoYMV) und turnip yellowmosaic virus (TYMV) induzieren die Bildung von ,,eingesunkenen Regionen"an der Chloroplastenperipherie. Tubuli im Stroma der Chloroplasten werdennur mit belladonna mottle (BMV), dulcamara mottle (DMV), eggplant mosaic(EMV) virus gefunden, sowie mit CoYMV. BMV und EMV induzieren zu-satzlich die Bildung von Kristallen in den Chloroplasten. CoYMV verursachtdie Bildung von amorphen Massen im Zytoplasma. Desmodium yellow mottlevirus ist durch die Bildung komplex aufgebauter Viruskristalle ausgezeichnetund dadurch, dafi in den Chloroplasten keine Vakuolisierung auftritt. Vakuo-!en-ahnliche Vesikeln in den Mitodiondrien treten bei APLV (Isolate Gibbsund HU) auf, sowie bei scrophularia mottle (ScrMV), EMV (Isolat AL),CoYMV, ononis yellow mosaic (OYMV), okra mosaic (OKMV) und clitoreayellow vein (CYMM) virus. In diesen Vesikeln sind leere Virusproteinhiillenzu finden bei EMV-AL, CYVV, OKMV, OYMV und ScrMV. Nur bei CYVVzeigen sich in den Mitodiondrien kleine, doppelwandige Vesikeln, ahnlidi denenan der Chloroplastenperipherie.

336 LESEMANN, Virus Group-specific and Virus-specific Cytological Alterations

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I thank Dr. RENATE KOENIG for providing the virus isolates used in this study. Forreliable technical assistance I thank Mrs. URSULA HERZBERG, Miss CORA KAMPFER, and MissINGRID KiJHNAsT. The study has been supported by the Deutsche Forsdiungsgemeinsdiaft.

Author's address: Institut fiJr Viruskrankheiten dcr Pflanzen, Biologisdie Bundesanstalt,Messeweg 11/12, D-3300 Braunsdiweig.

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