Phytopath. Z., 84, 97—104 (1975)© 1975 Verlag Paul Parey, Berlin und HamburgISSN 0031-9481 / ASTM-Coden: PHYZA3

Faculty of Agricultural Sciences of the University of Zagreband Rudjer Boskovic Institute, Zagreb, Yugoslavia

Fine Structure Changes in Different Host Plants Inducedby Grapevine Fanleaf Virus

By

ANA SARIC and MERCEDES WRISCHER

With 10 figures

Received May 12,

The grapevine fanleaf virus (GFV) has already been studied at the ultra-structural level by GEROLA et al. (1969) in roots and leaves of its natural host,grapevine, and in leaves of meehanically infected Nicotiana clevelandii andChenopodium amaranticolor. In two cases, in roots of Vitis vinifera and inmesophyll cells of Chenopodium amaranticolor, these authors observed virus-like particles aligned in rather short rows. In addition to that they noticed inChenopodium amaranticolor a membrane ridi area with many electron denseparticles scattered among the membrane elements.

PENA and RUBIO-HUERTOS (1971) found in Chenopodium qmnoa virusparticles arranged in rows inside the tubules. The tubules were observed in thecytoplasm, and occasionally also in the nucleoplasm, as well as in someplasmodesmata. The presence of large cytoplasmic inclusions, consisting ofvesicles and curled membranes with files of virus particles in parallel rows,were noticed by SARIC and WRISCHER (1972).

This paper presents some new findings concerning the intraccllular behav-iour of GFV in Nicotiana clevelandii and Petunia hybrida, as weil as cyto-pathological changes induced by the virus in Chenopodium amaranticolor andChenopodium quinoa at different stages of infection.

Material and Methods

All herbaceous plants (Chenopodium amaranticolor, Chenopodium quinoa, Nicotianuclevelandii and Petunia hybrida) were infected by medianical inoculation with an isolate of

Phyropaili. 2., Bd. K4. Heft 2 7

98 SARIC and WRISCHER

Figs. 1—3. Chenopodium amaranlicotor, leaf with systemic symptoms. Fig. 1. Linear aggre-gates of virus particles in the cytoplasm surrounded by masses of membraneous structures(MS) and Hpid globules (Lt. X 62,500. Fig. 2. Part of figure I showing virus particles in rows.

X 125,000. Fig. 3. Virus particles lining the tonoplast. X 125,000

Fine Structure Changes in Different Host Plants Induced by Grapevine Fanleaf Virus 99

GFV originating from naturally infected grapevine from Daltnatia. Uitrathin sections forelectron microscopy were prepared from both locally and systemically infected leaves andfrom apical meristems of Chenopodium amaranticolor and Chenopodium quinoa, and fromsystemically infected leaves of Nicotiana clevelandii and Petunia hybnda. Small pieces ofleaves were cut from mottled areas together with some green surrounding tissue. They werefixed in 1 "/, glutaraldchyde in 0.1 M cacodylate buffer (pH 7.2) — both at 2—4-C and atroom temperature — and postfixed in 1 % osmium tetroxide. After dehydration the sampleswere embedded in Araldite. The uitrathin sections were stained with uranyl acetate and leadcitrate (REYNOLDS 1963) and examined in a Siemens Elmiskop I.

Results

Chenopodium amaranticolor and C. guinoa

No visible dianges in fine structure of the cells were observed in inoculat-ed leaves before the appearance of symptoms. The first detectable cyto-pathological changes at the ultrastruetural level occurred immediately afterthe appearance of local symptoms. Large inclusion bodies, usually located nearthe nucleus, were visible in some of the mesophyl! cells of the leaves with localsymptoms. They consisted of masses of membraneous material mostly in theform of vesicles. Endoplasmic reticulum, ribosomes and large lipid globuleswere interspersed among the membranes. Virus particles packed in parallelrows were found only in a few inclusion bodies. In systemically infectedleaves, on the contrary, numerous cells contained inclusion bodies eadi withmany virus particles aligned in rows (figs. 1 and 2). Virus particles have neverbefore been found outside the inclusion bodies, except in one case where theylined the tonoplast (fig. 3). The plastids in some cells contained several un-usually large grana, showing the structural changes whidi have already beenreported for the virus-infected plant tissue (e.g. SJOLUND and SMITH 1974).

Neither virus particles nor any changes in the fine cell structure werenoticed in apical meristems.

Nicotiana clevelandii

The cytoplasm of this host showed virus particles arrayed in long parallelrows, but they were never surrounded by membraneous structures (fig. 4).Some peroxysomes were usually visible in the vicinity of these virus inclusions.

The most striking feature induced by GFV in N. clevelandii was thepresence of large agglomerations of virus particles in the central vacuole.These virus particles preserved the same arrangement in rows as they had inthe cytoplasm (figs. 5 and 6). Small vacuole-like structures, dictyosomes andendoplasmic reticulum were abundant in the cytoplasm surrounding thevacuole (fig. 5).

Petunia hybrida

Virus particles in Petunia hyhrida were found ui mesophyll cells ofsystemically infected leaves. They were observed in the cytoplasm, forminglinear aggregates or small crystalloids (fig. 7). Amassing of membraneous

100 SARIC and WRISCHER

Figs. 4—6. Nicotiana cievelandii, leaf with systemic symptoms, l-ig.4. Two long rows ofvirus particles in the cytoplasm. A peroxysome with a crystalline inclusion (P) lies in thevicinity. X 80,000. 1-ig. 5. Large aggregates of virus particles in the vacuole (arrow). Thecytoplasm surrounding the vacuole is without viruses. > 20,000. Fig. 6. Part of a vacuolar

inclusion containing virus particles in tightly apprcssed rows. -< 100.000

Fine Structure Changes in Different Host Plants Induced by Grapevine Fanlc.if Virus | 0 1

Figs. 7—10. Petunia hybridu, leaf with systemic symptoms. Fir;. 7. Linear aryre^ratcs rf virLi";particles lying in the cytoplasm in small groups. They are surrounded by the profiles of endo-plasmic reticulum (arrows). >: 64,000. Fig. 8. Virus particles in.sidc tubules crossing the cellwall in different directions (arrows). Several protrusions of the cell wall are observable(big arrows). '. 70,000. Fig. 9. Virus particles in a tubule inside the cell wall and its pro-truison. '• 80,000. Fig. 10. Virus particles in single or double linei in^idf tubules crossing

the ceil wall, '-" 80,000

102 SARIC and WRISCHER

structures was only occasionally noticed. In one case virus particles were alsofound inside the tubules — in single or double rows —, but always within thecell wall and its protrusions (figs. 8—10). Virus particles were not so abundantin P. hybrida as in other investigated hosts.

Discussion

Particles of GFV were found in the cytoplasm, usually in rows, and some-tmies forming small crystalloids. Viruses within the tubules, as noticed byPENA and RUBIO-HUERTOS (1971), could not be found in the cytoplasm. Virusparticles enclosed in tubules were observed in one material only — in youngsystemically infected leaves of Petunia hybrida. However, the tubules werenot situated in the cytoplasm but crossed the cell wall just in the same wayas described by JONES et al. (1973) for dierry leaf roll virus and by KIM andFULTON (1971) for bean pod mottle virus. Protrusions of the cell wall withvirus particles uiside the tubules have not been previously reported for GFV.PENA and RUBIO-HUERTOS (1971) reported findings of virus particles inplasmodesmata, but they did not find them in the protrusions of the cell wall.The presence of tubules with virus particles within the cell wall indicates amode of short distance spreading of GFV in its hosts.

In C. amaranticolor and C. quinoa virus inclusions were always sur-rounded by large membraneous structures called inclusion bodies (ROBERTSand HARRISON 1970). The fact that virus particles in this host have never beenfound outside the inclusion bodies points to the possible role of membraneousstructures in the formation of virus particles.

In the vacuole, virus particles in crystalline arrays were observed only inNicotiana clevelandii. This indicates that an active mechanism — probably thelysosomal activity (FINERAN 1971) — is involved in discarding virus particlesfrom the cytoplasm. The presence of virus particles in the vacuole is notuncommon. Similar crystalline aggregations of virus particles in the vacuolewere found by ROBERTS et al. (1970) in tobacco infected by another NFPOvirus — tobacco ringspot virus, and similar findings have also been reportedfor the artidioke mottled crinkle virus (MARTELLI and Russo 1973) and forsouthern bean mosaic virus (WEINTRAUB and RAGETLI 1970). The discardingof viruses into the vacuole is a possible way for the plants to get rid of virusparticles from the cytoplasm.

Summary

Cytopathological changes induced by GFV in locally and systemicallyinfected leaves of Chenopodium amaranticolor and in systemically infectedleaves of Nicotiana clevelandii and Petunia hybrida were studied. Ghanges inthe fine structure of infected C. amaranticolor leaves became visible with theappearance of local symptoms and were characterized by the presence of largemembraneous inclusion bodies localized in the cytoplasm. Virus particles

Fine Struciuru Changes in Different Host Plants Induced by Grapevine I'anleaf Virus \ 03

arranged in parallel rows were found inside these inclusion bodies. While inleaves with local symptoms such inclusion bodies were present only in a smallnumber of cells and only occasionally contained virus particles, m system-ically infected leaves a great number of cells contained inclusion bodies withvirus particles. Virus particles inside the tubules could not be observed in thishost.

The most prominent change in Nicotiana clevelandii was the presence ofagglomerations of virus particles in the central vacuole. In the cytoplasm, longparallel rows of virus particles were also found.

In Petunia hybrida the virus particles were observed in the cytoplasm,forming small aggregates. However, they were also found inside the tubuleswithin the cell wall and its protrusions, indicating the mode of short-distance— cell to cell — movement of GFV.

Zusammenfassung

Veranderungen der Feinstruktur versdiiedener Wirtspflanzendurch das «Grapevine Fanleaf Virus"

Veranderungen der Feinstruktur in lokal und systemisch infizierten Blat-tern von Chenopodium amaranticolor, sowie in systemisch infizierten Blatternvon Nicotiana clevelandii und Petunia hyhrida wurden eingehend untersucht.

In Chenopodium amaranticolor entstehen Veranderungen m der Fein-struktur der Zellen gleidizeitig mit den lokalen Symptomen an den Blattern.Tm Cytoplasma bilden sich grofie membranose Finschlufikorper, in die Virus-teilchen, m parallel angeordncten Reihen, emgebettet sind. Solche Einschlufi-korper sind m den Blattern mit lokalen Symptomen recht seiten, in den syste-misch infizierten Blattern jedoch zahlreich; auch erhalten die letzteren in derRegel wesentlich mehr Virusteilchen. In C. amaranticolor wurden keine Virus-teilchen in den Tubuli gefunden.

In der Zentralvakuole der Blattzellen von Nicotiana clevelandii kommenauffallende Aggregate von Virusteilchen vor. Im Cytoplasma sind die Virus-teilchen in langen Reihen angeordnet.

In Petunia hyhrida bilden die Viriistcilchen nur kleine Aggregate imCytoplasma. Aufierdem kommen Virusteilchen in besonderen Tubuli vor, diejedoch immer in der Zellwand und deren Protuberanzen zu finden sind, wasman als eine VirusUbertragung von Zcllc zu Zelle deuten kann.

This investigation was partly supported by .i grant made by the USDA, AgriculturalResearch Service, authorized by PL 480.

Literature

FINERAN, B. A., 1971: Ultrastructure of vacuoLir inclusions in root tips. Protoplasma 72, 1 —18.GEROLA, F. M., M. BASSE, and G. BELLI, 1969: An electron microscope study of different plants

infected with grapevine fanleaf virus. Giorn. bot. Ital. 103, 271—290.

104 SARIC and WRISCHER, Fine Structure Changes in Different Host Plants

JONES, A. T., A. M. KINNINMONTH, and I. M. ROBERTS, 1973: Ultrastruetural dianges indifferentiated leaf cells infected with dierry leaf roll virus. J. gen. Virol. 18, 61—64.

KIM, K. S., and J. P. FULTON, 1971: Tubules with virus-like particles in leaf cells infectedwith bean pod mottle virus. Virology 43, 329—337.

MARTELLI, G. P., and M. Russo, 1973: Electron microscopy of artichoke mottled crinkle virusin leaves of Chenopodium quinoa Willd. J. Ultrastruct. Res. 42, 93—107.

PENA-IGLESTAS, A., y M. RUBIO-HUERTOS, 1971: Ultrastructura de hojas de Chenopodiumquinoa Willd. infectada con el virus entrenudo corto infeccioso de la vid. Microbiol.EspaS. 24, 183—191.

REYNOLDS, E. S., 1963: The use of lead citrate at high pH as an electron-opaque stain inelectron microscopy. J. Cell Biol. 17, 208—212.

ROBERTS, D . A., R. G. CHRISTIE, and M. C. ARCHERS, Jr., 1970: Infection of apical initials intobacco shoot meristems by tobacco ringspot virus. Virology 42, 217—220.

ROBERTS, I. M., and B. D. HARRISON, 1970: Inclusion bodies and rubular structures in Cheno-podium amaranticotor plants infected with strawberry latent ringspot virus. J. gen.Virol. 7, 47—54.

SJOLUND, R. D., and D. D. SMITH, 1974: Freeze-fracture studies of photosynthetically de-ficient "supergranal" diloroplasts in tissue containing virus-like particles. J. Cell Biol.60, 283—292.

SARIC, A., i M. WRISCHER, 1972: Ultrastrukturne promjene u biljkama zarazenim virusominfcktivne degeneracije loze. Mikrobiologija 9, 197—200.

WEINTKAUB, M., and H. W. J. RAGETLI, 1970: Electron microscopy of the bean and cowpeastrains of southern bean mosaie virus within leaf cells. J. Ultrastruct. Res. 32, 167—189.

Authors' addresses: Prof. Dr. ANA SARIC, Faculty of Agricultural Sciences of theUniversity, Simunska 25, Yu 41000 Zagreb (Yugoslavia). Dr. MERCEDES WRISCHER, RudjerBoskovic Institute, Bijenicka 54, Yu 41000 Zagreb (Yugoslavia).