JOURNAL OF VIROLOGY, Feb. 1969, p. 261-264Copyright © 1969 American Society for Microbiology

NOTESElectron Microscopy of Rice Transitory

Yellowing VirusEISHIRO SHIKATA AND MOH-JIH CHEN'

Departmenftof Botany, Faculty of Agriculture, Hokkaido University, Sapporo, Japan

Received for publication 22 October 1968

Rice transitory yellowing disease from Taiwanwas first described in 1965. Severe outbreaks,occuring in the second rice crop in Taiwan, arecharacterized by yellowing of lower leaves andreduced tillering of the diseased plants (3). Itwas found that the disease symptoms wereinduced by the green rice leafhoppers, Nephotettixapicalis (Motsch.) and N. cincticeps Uhler (2).After having acquired the disease agent byfeeding, the vectors were able to inoculate plantsfor the duration of the insect's life (3). Thisobservation led to the conclusion that transitoryyellowing disease was caused by a virus. Mechan-ical transmission was successful when crudesap or clarified expressed juice from diseasedrice leaves was injected into healthy insects, andwas subsequently used to study physical propertiesof the virus (5, 6). Transovarian transmission ofthe virus in the insect vector does not occur.

Since no information was available on themorphology of the transitory yellowing virus, itwas decided to use diseased plants for an electronmicroscopic study of the disease agent in situ.This paper deals with the morphological charac-terization of rice transitory yellowing virus andwith its localization in plant cells.Young rice seedlings (Oryza sativa L. var.

Yukimochi) were inoculated at the two-leafstage by means of N. cincticeps leafhoppers thathad been injected with crude sap of diseasedrice plants. Leaves showing either characteristicwater-soaked specks and rusty flecks, or yellowingbuff on the later-developing leaves were used forelectron microscopic observations. Dip prepa-rations were prepared from young leaves ofdiseased rice plants, negatively stained with 2%phosphotungstic acid (PTA) and examined byelectron microscopy. Negatively stained, dilutedcrude extract was also examined. One loop ofextract of the diseased rice leaves was transferredby a wire loop to another drop of distilled water

I On leave from the Department of Plant Pathology, TaiwanProvincial Chung Hsing University, Taichung, Taiwan, TheRepublic of China.

and mixed. An equal volume of 2% PTA wasadded to the drop and the mixture was placedon supporting films for electron microscopicexaminations.

Small pieces of diseased leaves of rice plantswere fixed in 5% glutaraldehyde in 0.1 M phos-phate buffer, pH 7.0, in a cold room at 4 C for120 min, postfixed in 2% osmium tetroxide indistilled water for 90 min at 4 C, and stained in0.5% uranyl acetate-75% ethyl alcohol mixtureovernight. After dehydration in graded ethylalcohol, the preparations were poststained insaturated lead acetate in absolute ethyl alcoholfor 120 min, dehydrated again in absolute ethylalcohol for 60 min, subjected to two changes ofpropylene oxide for 60 min, and then embeddedin Epon 812. Ultrathin sections were cut on aPorter-Blum ultramicrotome, using diamond orglass knives. The sections were doubly stainedwith uranyl acetate and lead hydroxide. Observa-tions were made by a JEM-5Y electron micro-scope.

In the dip and diluted crude extract prepara-tions, numerous bullet-shaped particles weredetected (Fig. 1). At the flat end of the particles,a deep central core was noted. The bullet-shapedparticles measured 96 nm in diameter and 120to 140 nm in length, and were surrounded bytwo membranes with numerous outer projections.There were no bacilliform particles in the crudepreparations. The bullet-shaped particles wereonly found in sap from diseased leaves withtypical symptoms of transitory yellowing. Sapfrom healthy controls and from symptomlessleaves of infected plants were devoid of suchparticles.As shown in Fig. 2 and 3, numerous bullet-

shaped particles were seen in the cells of diseasedleaves. Most particles were bullet-shaped, butthere were also bacilliform ones. The size of thebullet-shaped particles was approximately 94nm in diameter and 180 to 210 nm in length andapproximated that of particles negatively stainedin crude preparations. The bacilliform particles

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were about 93 nm in diameter and 325 nm inlength, with occasionally longer ones observed.High magnification of the particles (Fig. 2)revealed their fine structure and numerous outerprojections on the surface. No such particleswere found in the healthy leaves. Cytologicalchanges were noted in the nuclei of infected cells.These changes were manifested by reducedchromatin substance and by the fine, uniform

appearance of the nucleoplasm (Fig. 4 and 5).The virus particles were frequently arranged atthe periphery of such abnormal nuclei (Fig. 5).The cells of leaves with water-soaked specks orrusty flecks were filled with virus particles. Duringearly stages of infection and during the yellowbuff stage, virus particles were always limited tothe phloem cells of diseased leaves.Our electron micrographs revealed the presence

FIG. 4. Plhloem cells from a diseased leaf showinig symptoms of yellow buff, which were fixed in glaitaraldelhydeanid osmic acid antd thent embedded in Eponz. Viruis accuimuilationis (V) are detected in some of the cells. X4,000.

FIG. 5. Cell in2fected with tranisitory yellowinig viruis. Note that the bacilliform anld buillet-shaped particles (V)are arraniged at the periphery of a iucleuells (N). The leaves were fixed in glutaraldehyde anid osmic acid anid thenembedded in Eponi. X 10,000.

FIG. 1. Tlhree baillet-shcaped particles int the negatively stainle?d (2' i PTA) dilutted criule extract /1roni trantsitoryyellowinig diseased leaves of rice planlts. Note the suirf ace projectionls at the periphery of the particles. X 100,000.

FIG. 2. Ultrathinl sectioni of ani inf1ected leaf Qf a rice planlt fixed in glutaraldehyde anid osmic acid, an2d theniembedded in Epon. Numerous surfcace projectionis are seeii at the periphery oJ the particles. Note that most of theparticles are bullet-shlacped. X 100,000.

FIG. 3. MCass of buillet-slhaped anid bacilliform viruts particles (V) in the cytoplasm of a cell trom diseased leafshlowinlg symptoms of water-soaked specks. (C) Disinitegrated chloroplast; (W) the cell wall. Th7e leaves werefixed in gluitaraldelhyde anid osmic acid anid embedded in Eponi. X30,000.

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of characteristic bullet-shaped and bacilliformparticles associated with the transitory yellowingdisease of rice plants in Taiwan. Therefore, it ismost likely that these particles are the causativevirus of this disease. Recently, several otherbullet-shaped viruses have been described:lettuce necrotic yellows virus (1, 4), potato yellowdwarf virus (10), Plantago virus (7), Gomphrenavirus (8), wheat striate mosaic virus (9), northerncereal mosaic virus (12), and Sowthistle yellowvein virus (11). These viruses resemble in shape alarge group of viruses infecting vertebrate andinvertebrate animals, such as vesicular stomatitisvirus and sigma virus of drosophila. Morpho-logically, transitory yellowing virus is similar tothese viruses.

ACKNOWLEDGMENTS

We thank Daiki Murayama, Department of Botany, Facultyof Agriculture, Hokkaido University, Sapporo, Japan, and KarlMaramorosch of the Boyce Thompson Institute, Yonkers, N.Y.,

J. VIROL.

for their valuable suggestions during the experiments and duringthe preparation of this manuscript.

LITERATURE CITED

1. Chambers, T. C., N. C. Crowley, and R. I. B. Francki. 1965.Virology 27:320.

2. Chiu, R. J., J. H. Jean, M. H. Chen, and T. C. Lo. 1968. Phy-topathology 58:740.

3. Chiu, J. G., T. C. Lo, C. L. Pi, and M. H. Chen. 1965. Botan.Bull. Acad. Sinica (Taiwan) 6:1.

4. Crowley, N. C., B. D. Harrison, and R. I. B. Francki. 1965.Virology 26:290.

5. Hsieh, S. P. Y. 1967. Plant Protect. Bull. (Taiwan) 9:21.6. Hsieh, S. P. Y., and S. C. Roan. 1967. Plant Protect. Bull.

(Taiwan) 9:23.7. Hitchborn, J. H., G. J. Hills, and R. Hull. 1966. Virology

28:767.8. Kitajima, E. W., and A. S. Costa. 1966. Virology 29:523.9. Lee, P. E. 1967. Virology 33:84.

10. Macleod, R., L. M. Black, and F. H. Moyer. 1966. Virology29:540.

11. Richardson, J., and E. S. Sylvester. 1968. Virology 35:347.12. Shikata, E., and Y. T. Lu. 1967. Proc. Japan Acad. 43:918.

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