Phytopath. Z., 78, 134—140 (1973)© 1973 Verlag Paul Parey, Berlin und HamburgISSN 0031-9481 /ASTM-Coden: PHYZA3

Departamento de Virologia, Instituto "Jaime Ferrdn" de Microbiologia, C. S. 1. C.Madrid (Spain) •

Ultrastructure of Cells infected with Carnation Mottle Virus

By

SERAHNA CASTRO ROBLEDA

With 8 figures

Received October 31, 1971

Introduction

Carnation mottle virus (CaMV) was described by KASSANIS (1955), andwas later studied by HOLLINGS and STONE (1964) and KEMP (1964). In Spain,CaMV IS frequently found infecting 'Sim' Carnation varieties.

In the present paper, we describe the localization and distribution of thevirus particles of CaMV in the host cells and the relationships between virionsand cell organelles as observed by electron microscopy.

Materials and MethodsI

Virus source and test plants

The virus was isolated from Dianthus caryophyllus var. 'Sim' from cultivars in theMaresma near Barce ona With thc samples taken we inoculated the following plants: Dianihuscaryophyllus, Dianthus barbatus, Dianthus chmcnsis. Lychnis dioica, Chenopodium quinoaChenopodium amaranticolor, Atriplex hortensis, Gomphrena globosa and Celosia cristata. Theplants were mediamcaliy inoculated by infected sap, using carborundum 400 mesh as abrasive.

Electron microscopy

Leaf tissue of plants showing symptoms after inoculation was cut into small pieces and

buticr pH 6.9, and postfixed m 2% osmium tetroxidc in Veronal acetate buffer pH 6 9 Thetissue pieces were dehydrated in a graded series of acetone and embedded in "Durcupan"

M S ^ ^ " " V " ' " ' " ^ " T ' '^°"" ^"""^ ' '^' ^° '' dehydration, according to HILLS and PLASKIT(1968) and DE ZOETEN and GAARD (1969).

Ultrastructure of Cells infected with Carnation Mottle Virus 135

Fig. 1, Electron micrography of a purified preparation of CaMV. X 136000Fig.2. Section through cytoplasm of the infected cells showing great amount of virus particles.

X 46 000Fig. 3. Virus particles near the external membrane of the diloroplasis. X 60 000

136 ROBLEDA

Sections were made with a LKB ultramicrotome and were stained with lead citrate.Observations were tnade with a Siemens I electron microscope.

_ Dip preparations from infected leaves were made in phosphotungstic acid for negativestaming, HITCHBORN and HILLS (1965).

The virus was purified by Steere's method (1959),

Results

TTie following plants were Infected with mechanical inoculation and show-ed the indicated symptoms;

Dianthus barhatus — mosaic.Dianthus caryophyllus — mosaic.Chenopodium amaranticoior — (necrotic lesions),Atriplex hortensis — chlorotic spots and later chlorosis of the whole plant.Dianthus chinensis, Lychnis dioica, Gomphrena globosa, Chenopodium

qmnoa, and Celosia cristata were not infected. Negative staining of crude sap inall the mfected plants showed isometric particles 27 to 29 nm. Negative stainingof purified virus particles from Dianthus caryophyllus, Dianthus barbatusChenopodium amaranticoior and Atriplex hortensis also showed the same kindof isometnc particles of a similar size, according to B. KASSANIS (1955) andHoLLiNGS and STONE (1964) (fig. 1).

Ultrathin sections from infected leaves of Dianthus barbatus, Dianthuscaryophyllus, Atriplex hortensis, and Chenopodium amaranticoior, showedisometnc particles in the cytoplasm, nucleus and vascular tissues.

The cytoplasm of the infected cells was destroyed and a great amount ofvirus particles were observed at random in it (fig. 2). Sometimes the virions werelocated around and close to the external membrane of the diloroplasts (fig. 3) orm ineal form along the cytoplasmic membrane (fig. 4). The tonoplast in severalcells was broken and virus particles were observed in the vacuole.

Virus particles were observed within the nucleoplasm near the nucleolusof the nucli, and sometimes within the perinuclear space (figs. 5 and 6).

In the vascular tissues, a high concentration of particles was present (fig. 7);the particles frequently formed short chain patterns in the destroyed cytoplasm.No crystalline arrays of particles were observed.

Vims particles were not observed associated to mitochondria or Golgiapparatus although the structure of some mitochondria showed concentric bandsand swelhng such as those described by RUBIO HUERTOS and VELA (1966) inAmaranthus virus (fig. 8).

Discussion

Virus particles appeared as uniformly stained isometric bodies with adiameter of 27 to 29 nm. Their identification as CaMV virus particles wassuggested not only by comparison with other isometric plant viruses seen in thinsections by DE ZOETEN and GAARD (1969), WEINTRAUB and RAGETLI (1970),RUBIO HUERTOS and GARCIA HIDALGO (1971), MARTELLI and Russo (1972)!

Ultrastructure of Cells infected with Carnation Mottle Virus 137

Fig. 4. Lineal formation of virui pariitjlci >iloiig c> topl.i,smic membrane. 50 000.g

Detail of the lineal formation. X 10 000Fig. 5. Nucleus showing virus particles in the nuc!eoplasm. X 50 000

Fig. 6. Detail of a nucleus showing virus particles in the nucleoplasm, near the nucleolus.X 60 000

138 ROBLEDA

rii;. 7. Virus particles of CaMV in vascuLir tissues. ••'.. 44 200Fig. 8. Mitochondria showing concentric bands and swelling. X 50 000

Ultrastruciurt' of Cells infected with Carnation Mottle Virus 139

among others but also by their absence in the healthy controls. The particleswere also differentiated from the cellular ribosomes by their size and uniformityof staining with uranil acetate.

The presence of virus particles in the vascular tissues seems to indicate thatlong-distance translation of smalt isometric viruses can be achieved through thevascular tissues as BENNSTT (1956) and SCHNEIDER and WORLEY (1959) pointedout. It seems to be also a general feature of small icosahedral plant viruses thatthey occur in the nucleus of the host cell and CaMV seems to be no exception(WEINTRAUB and RAGETLY 1970, Russo and MARTELLI 1972, ESAU andHoEFERT 1972). Longer isometric viruses have been also recently reported asappearing in the nucleoplasm of the host cell, RUBIO et al. (1972). In the presentwork, we have not found experimental support in determing whether intra-nuclear or intracytoplasmic virus synthesis occurs or if perhaps such synthesisoccurs in both, the nucleous and the cytoplasm. Vesicles containing RNA havebeen described by ESAU and HOEFERT (1972) and DE ZOETEN et al. (1972). Thevesicles seem to be involved in viral replication and also seem to play a role inviral nucleic acid transportation to the nucleus or viceversa. In our observationson CaMV similar vesicles were not observed.

Summary

Ultrathin sections of CaMV infected plants contained isometric 27 to29 nm 0 particles in the cytoplasm, vascular tissues and within the nuclei. Thevirus particles in the cytoplasm were frequently associated with cytoplasmicmembranes.

Zusammenfassung

Ultrastruktur von mit Nelkensdiedtungsvirus (CaMV) infizierten Zellen

Ultrafeine Schnitte infizierter Pflanzen zeigten isodiametrische Teildien von27 bis 29 nm Durchmesser in Cytoplasma, Cefafien und in den Kernen. Die imPlasma vorhandenen Partikeln lagen haufig den Plasmamembranen an.

Our sincere thanks are due to Dr. M. RUBIO-HUF.RTOS and R. MORENO San Martinfor their most valuable help in this work.

Literature

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140 RoBLEDA, Ultrastructure of Cells infccu-d with Carnation Moitle Virus

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Address of the author: Departamento de Virologia, Instituto "Jaime Ferran" de Micro-biologia, Joaquin Costa 32, Madrid 6 (Spain).