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Electron microscopic studies of plant tissue cultures infected with the aster yellows disease
Printed in Sweden Copyright © 1974 by Academie Press, Inc. All rights of reproduction in any form reserved
34
J. ULTRASTRUCTURE RESEARCH
46, 34-42 (1974)
Electron Microscopic Studies of Plant Tissue Cultures Infected with the Aster Yellows Disease G. G. JACOLI a n d W. P. RONALD
Canada Agriculture, Research Station, 6660 N. IV. Marine Drive, Vancouver 8, B.C., Canada Received February I, 1973, and in revised form April 13, I973 Electron microscopic observation showed the occurrence of unusual structures in the phloem of carrot root and aster stem tissue cultures, infected with the aster yellows disease. The first structures to appear in the growing callus were spherical "bodies" of dimensions between 400 and 1 000 nm. The bodies were surrounded by a double membrane and contained smaller vesicles with diameters of ca. 100 rim. As the tissues aged, the bodies lost their distinct outlines, became enlarged, and their membranes gave rise to layered structures which enclosed round, viruslike particles with diameters of ca. 25 nm. Intra- and extranuclear inclusions were also seen. None of these structures was found in the phloem of healthy tissue cultures.
I n p l a n t tissue cultures, m a i n t a i n e d by us in a n effort to preserve a n d propagate the disease agent of aster yellows, we observed u n u s u a l bodies in the p h l o e m of infected preparations. We describe these bodies a n d discuss their analogies with similar structures observed by other authors in different conditions (1, 5-7, 13, 16, 19, 20, 27, 28).
MATERIALS AND METHODS
Tissue cultures. From roots of carrot (Daucus carota) and stems of aster (Callistephys chinensis) infected by leafhoppers (MacrosteIes fascifrons) with aster yellows disease, sterile explants were isolated and prepared according to Gautheret (15). Similar explants from healthy carrot roots and aster stems were used as controls. The explants were grown in the dark at 25°C in 10-cm petri plates on a modified Murashige and Skoog (26) medium in high humidity. Electron microscopy. For the sections a vertical slice through the core of the callus was removed, the slice was horizontally subdivided, and the resulting strips were embedded and sectioned, starting from the outer surface toward the core of the callus. Each strip was fixed for 1 hour in 5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). After two 15-minute washing with the same buffer, the tissues were post fixed in 1%
ASTER YELLOWS IN PLANT TISSUE CULTURES
35
Palade-OsO4 solution for 1 hour, dehydrated by 15-minute transfers through a graded ethanol series, transferred to propylene oxide, and embedded in Epon 812 (24). The sections were cut in a LKB Ultrotome and collected on collodion-coated carbonreinforced copper grids. The mounted sections were stained with uranyl acetate and lead citrate, and examined in a Philips 2130electron microscope using an accelerating voltage of 60 kV. OBSERVATIONS Our observations were confined to phloem cells. In infected tissues we observed clusters of spherical bodies, varying from 400 to 1 000 nm diameter (upper right, Fig. 1). Each was surrounded by double membranes and contained structures that appeared to be vesicles (Vs, Fig. 1), apposed to or mainly oriented toward the inner membrane of the spherical body. The vesicles often contained filamentous, strandlike material, and were also bounded by double membranes. It is not clear whether the two double membranes consisted of two-unit membranes with intermembranous spaces or single, inflated membranes. As the phloem cells aged, the spherical bodies lost their distinct contours and became enlarged. In some instances, one or more large internal vacuoles appeared, which were also bounded by double membranes (Vc, Fig. 2). In other instances, the membranes of the spherical bodies broke and the vesicles migrated toward the opening (arrow, Fig. 3). Clusters of vesicles could be seen outside the perimeter of the bodies (Vs, Fig. 5). Enlargement of the bodies was also sometimes associated with migration of the vesicles toward the center, away from the bounding membrane (Vs, Fig. 4). The apparent degeneration of the bodies coincided with the appearance of spherical viruslike particles (VLP), having diameters of ca. 25 nm. These were associated usually with layered structures (VLP, Figs. 2 and 3). The phenomenon is evident also in Fig. 5, and in Figs. 6 and 7, where the structures indicated by the arrows i n Fig. 5 are shown separately and in more detail. These structures appeared to be arranged in a linear (Fig. 6) or in a spiral multilayer formation (Fig. 7). The arrows (Figs. 6 and 7) indicate portions of the membrane of two spherical bodies which appear to be in process of incorporation into newly formed layered structures; there appears to be close association between the layered material and the membranes. The transition from spherical body membranes to layered structures is noticeable also in Fig. 2, which shows the membranes of three adjacent bodies densely stained and about to enclose a cluster of VLP (arrow). Fig. 7 shows VLP arranged in a spiral with the lowest layer in process of formation. The figure demonstrates again that the layered material may originate from the membrane of the spherical body (arrow). The VLP were found nearly always outside the bodies but some VLP arranged in a crystal-like array inside a spherical body are shown in Fig. 4 (VLP).
36
JACOLI AND RONALD
Spherical bodies, vesicles a n d V L P were f o u n d only in the callus of infected c a r r o t r o o t s a n d aster stems, p r e d o m i n a n t l y in carrots. N o n e of the structures described was f o u n d in healthy c a r r o t a n d aster tissues. A n o t h e r feature which characterized the infected cultures was the f o r m a t i o n of inclusions ( / / a n d Ie, Fig. 8). These were m a i n l y f o u n d outside the nuclear m e m b r a n e , b u t in a few cases they were seen within the nuclei of p h l o e m cells (Ii, Fig. 8). A c o m p a r i s o n between these inclusions a n d P - p r o t e i n or slime bodies (P, Fig. 8) shows that, a l t h o u g h the inclusions s o m e w h a t r e s e m b l e d P-protein, they differed in having a stronger affinity for stain (Fig. 8) a n d in lacking the typical fibrillar or t u b u l a r structure (8, 9, 14) (Fig. 9). I t is possible t h a t these inclusions represent precursors or m o d i fications of the slime bodies. W e f o u n d it difficult to establish a clear sequence of events in the a p p e a r a n c e of the spherical bodies, vesicles, a n d VLP. H o w e v e r , these structures a p p e a r e d , as a function of their l o c a t i o n in the growing callus, in the o r d e r listed here. W e d i d n o t find these three types of structures in the original tissues f r o m which the explants were taken, b u t large n u m b e r s of m y c o p l a s m a - l i k e bodies ( M L B ) have been seen in the infected c a r r o t tissues a n d smaller n u m b e r s in aster.
DISCUSSION A t present, it is difficult to establish the identity of the described structures which were f o u n d only in sections of callus infected by the aster yellows disease. W e have n o t i c e d structural analogies between our vesicles a n d vesicular structures which other investigators have f o u n d associated with chloroplasts (1, 5-7, 16, 27, 28), m i t o c h o n d r i a (19, 20), nuclei (12, 13), a n d the e n d o p l a s m i c reticulum (13) of virus-infected plants.
FIG. 1. Cross section through the phloem of carrot callus infected by the aster yellows disease. Note the cluster of spherical bodies in the upper right of the picture (arrow) with vesicles lined up along their inner membranes (Vs). x 37 600. FIG. 2. A spherical body within the phloem of infected carrot callus. Note the incipient degeneration of the body with the formation of two large vacuoles (Vc) and the appearance of round, viruslike particles (VLP). The preliminary phase of converting the membrane of a spherical body into layered structures is indicated by the arrows, x 43 000. F~G. 3. Cross section through the phloem of infected carrot callus showing the migration of vesicles toward an opening in the body (arrow). The arrangement of the VLP inside the incipient layered structures is also apparent (VLP). x 37 200. FIG. 4. A cluster of spherical bodies from infected aster stem callus. Note the array of VLP inside the central body (VLP) and the large vacuole in the body on the right side of the picture (Vc). The vesicles have lost their membrane-oriented position (Vs). x 155 603. FIG. 5. Cross section through the phloem near the core of infected carrot callus. Note the linear and spiral arrangement of the VLP within the layered structures (arrows). A cluster of vesicles is shown outside the perimeter of a spherical body (Vs). x 39 700. FIGS. 6 and 7. Enlargements of the layered structure indicated by the arrows in Fig. 4. x 66 300.
ASTER YELLOWS IN PLANT TISSUE CULTURES
37
38
JACOLI AND RONALD
ASTER YELLOWS IN PLANT: TISSUE CULTURES
39
40
JACOLI AND RONALD
Nevertheless, the vesicles in our callus preparations were not associated with any of these cellular structures. The strong similarity between VLP and true virions, and their peculiar arrangement in layered structures which have already been described for other diseases in the literature (10, 22, 29), is noteworthy. We do not believe that the VLP are clusters of ribosomes. They differ from ribosomes by their larger size, better defined outlines, and occasional hollow structure. Furthermore, the occurrence of these particles in infected preparations only, suggests some correlation between these entities and the aster yellows disease. There is a vast body of published evidence which supports the concept that mycoplasmaqike bodies (MLB) rather than viruses are the causative agents of many yellows diseases (1I, 18, 23). Nevertheless, the occurrence of viruslike particles associated with MLB has been described in different plant diseases (2, 4, 17, 2I), but a definite association between MLB and viruses has yet to be demonstrated. Atanasoff's hypothesis (3) that mycoplasma are symbionts acting as vectors of viruses, in the manner of nematodes, bacteria, or fungi, is still to be disproved. In our studies, we saw a few structures which resembled mycoplasma, but these were associated with neither the vesicles nor the VLP. The origin of the bodies and vesicles and the purification of the VLP were not considered at this stage in our studies, neither was their relation to the etiology of the yellows disease. Thus, the question whether these structures are causative of the aster yellows disease or are part of a secondary infection, remains to be answered. There is a possibility that the bodies may be degenerated organelles such as mitochondria or plastids. Careful examination of our infected preparations show few intact mitochondria. Intact plastids which were in good number in the phloem of healthy explants, appeared to be rare in the phloem of infected explants. Several plant hosts susceptible and not susceptible to aster yellows were mechanically inoculated with our preparations, but they failed to show any recognizable symptom. We were unable to transmit the aster yellows disease from our cultures with leafhopper vectors. It is possible that the conditions used in our cultures might have induced the need for an abnormally long acquisition time for the vector or an abnormally long incubation period to establish infectivity as suggested by Mitsuhashi and Maramorosch (25). Another explanation for the lack of infectivity might be that some components of the cultural medium inhibited the causative agent of the disease. We think this is unlikely, although it could explain why the structures discussed here were not~found in peripheral cells of our infected preparations. @ork is in progress to isolate and identify the viruslike particles described above. To our knowledge, this is the first time that these structures have been reported in plant tissue cultures.
ASTER YELLOWS IN PLANT TISSUE CULTURES
41
Fro. 8. LongitudinaI section of the phloem of infected carrot callus showing intra- and extranuclear inclusions (Ii and le) and P-protein (P). × 13 800. FIo. 9. Enlargement of the inclusion (Ii) of Fig. 8. The nonfilamentous structure is very apparent at this magnification, x 42 000.
42
JACOLI AND RONALD
We thank Miss Esther Lo for electron microscopy, Mr. F. E. Skelton for infected materials and infectivity tests, and Mr. Howard Severson for photography.
REFERENCES 1. ALLEN, T. C., Virology 47, 467 (1972). 2. - - - - ibid. 47, 491 (1972). 3. ATANASOFr,D., Biol. Zentralbl. 88, 571 (1969). 4. BANTTARI,E. E. and ZEYEN, R. J., Phytopathology 60, 1282 (1970). 5. Bov~, J. M., Bull. Soc. Fr. Physiol. VOg. 12, 307 (1966). 6. CARROLL,T. W., Virology 42, 1015 (1970). 7. CHALCROFT,J. and MATTHEWS,R. E. F., Virology 28, 55 (1966). 8. CRONSHAW,J. and ESAU, K., J. Cell Biol. 34, 801 (1967). 9. - - - - ibid. 38, 292 (1968). 10. DAVIDSON,E. M., Virology 37, 694 (1969). 11. DAVIS, R. E. and WHITCOMB,R. F., Annu. Rev. Phytopathol. 9, 119 (1971). 12. DE ZOETEN, G. A., GAARD, G. and DIEZ, F. B., Virology 48, 638 (1972). 13. ESAU, K. and HOEFERT,L. L., Virology 48, 724 (1972). 14. EVERT, R. F. and DESHPANDE,B. P., J. Cell Biol. 44, 462 (1970). 15. GAUTt~ERET,R. J. (Ed.), La Culture des Tissus V6g6taux, p. 92, Masson, Paris, 1959. 16. GEROLA,F. M., BASSI, M. and G1USSANI,G., Caryologia 19, 457 (1966). 17. GIANNOTTI,J., DEVAtJCHELL~:,G. and VAGO, C., Proe. Int. Congr. Mierobiol. lOth p. 222, Mexico City, 1970. 18. HAMPTON,R. O., Annu. Rev. Plant Physiol. 23, 389 (1972). 19. HARRISON,B. D., STEFANAC,Z. and ROBERTS,I. M., J. Gen. Virol. 6,127 (1970). 20. HATTA,R., NAKAMOTO,T., TAKAGI,Y. and USHIYAMA,R., Virology 45, 292 (197l). 21. HmUMI, H. and MARAMOROSCH,K., Phytopathol. Z. 75, 9 (1972). 22. HITCHBORN,J. H. and HILLS, G. J., Virology 35, 50 (1968). 23. HULL, R., Rev. Plant Pathol. 50, 121 (1971). 24. LUI~T,J. H., J. Biophys. Bioehem. Cytol. 9, 409 (1961). 25. MITSUHASHI,J. and MARAMOROSCtt,K., Virology 23, 277 (1964). 26. MURASHIGE,T. and SKOOG,F., Physiol. Plantarum 15, 473 (1962). 27. RUBIo-HUERTOS,M., VELA, A. and LOeEz-ABELLA,D., Virology 32, 438 (1967). 28. USmYAMA,R. and MATrHEWS, R. E. F., Virology 42, 293 (1970). 29. WALK,Y, D. G. A. and WEBB, M. J. W., J. Gen. Virol. 7, 159 (1970).
34
J. ULTRASTRUCTURE RESEARCH
46, 34-42 (1974)
Electron Microscopic Studies of Plant Tissue Cultures Infected with the Aster Yellows Disease G. G. JACOLI a n d W. P. RONALD
Canada Agriculture, Research Station, 6660 N. IV. Marine Drive, Vancouver 8, B.C., Canada Received February I, 1973, and in revised form April 13, I973 Electron microscopic observation showed the occurrence of unusual structures in the phloem of carrot root and aster stem tissue cultures, infected with the aster yellows disease. The first structures to appear in the growing callus were spherical "bodies" of dimensions between 400 and 1 000 nm. The bodies were surrounded by a double membrane and contained smaller vesicles with diameters of ca. 100 rim. As the tissues aged, the bodies lost their distinct outlines, became enlarged, and their membranes gave rise to layered structures which enclosed round, viruslike particles with diameters of ca. 25 nm. Intra- and extranuclear inclusions were also seen. None of these structures was found in the phloem of healthy tissue cultures.
I n p l a n t tissue cultures, m a i n t a i n e d by us in a n effort to preserve a n d propagate the disease agent of aster yellows, we observed u n u s u a l bodies in the p h l o e m of infected preparations. We describe these bodies a n d discuss their analogies with similar structures observed by other authors in different conditions (1, 5-7, 13, 16, 19, 20, 27, 28).
MATERIALS AND METHODS
Tissue cultures. From roots of carrot (Daucus carota) and stems of aster (Callistephys chinensis) infected by leafhoppers (MacrosteIes fascifrons) with aster yellows disease, sterile explants were isolated and prepared according to Gautheret (15). Similar explants from healthy carrot roots and aster stems were used as controls. The explants were grown in the dark at 25°C in 10-cm petri plates on a modified Murashige and Skoog (26) medium in high humidity. Electron microscopy. For the sections a vertical slice through the core of the callus was removed, the slice was horizontally subdivided, and the resulting strips were embedded and sectioned, starting from the outer surface toward the core of the callus. Each strip was fixed for 1 hour in 5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). After two 15-minute washing with the same buffer, the tissues were post fixed in 1%
ASTER YELLOWS IN PLANT TISSUE CULTURES
35
Palade-OsO4 solution for 1 hour, dehydrated by 15-minute transfers through a graded ethanol series, transferred to propylene oxide, and embedded in Epon 812 (24). The sections were cut in a LKB Ultrotome and collected on collodion-coated carbonreinforced copper grids. The mounted sections were stained with uranyl acetate and lead citrate, and examined in a Philips 2130electron microscope using an accelerating voltage of 60 kV. OBSERVATIONS Our observations were confined to phloem cells. In infected tissues we observed clusters of spherical bodies, varying from 400 to 1 000 nm diameter (upper right, Fig. 1). Each was surrounded by double membranes and contained structures that appeared to be vesicles (Vs, Fig. 1), apposed to or mainly oriented toward the inner membrane of the spherical body. The vesicles often contained filamentous, strandlike material, and were also bounded by double membranes. It is not clear whether the two double membranes consisted of two-unit membranes with intermembranous spaces or single, inflated membranes. As the phloem cells aged, the spherical bodies lost their distinct contours and became enlarged. In some instances, one or more large internal vacuoles appeared, which were also bounded by double membranes (Vc, Fig. 2). In other instances, the membranes of the spherical bodies broke and the vesicles migrated toward the opening (arrow, Fig. 3). Clusters of vesicles could be seen outside the perimeter of the bodies (Vs, Fig. 5). Enlargement of the bodies was also sometimes associated with migration of the vesicles toward the center, away from the bounding membrane (Vs, Fig. 4). The apparent degeneration of the bodies coincided with the appearance of spherical viruslike particles (VLP), having diameters of ca. 25 nm. These were associated usually with layered structures (VLP, Figs. 2 and 3). The phenomenon is evident also in Fig. 5, and in Figs. 6 and 7, where the structures indicated by the arrows i n Fig. 5 are shown separately and in more detail. These structures appeared to be arranged in a linear (Fig. 6) or in a spiral multilayer formation (Fig. 7). The arrows (Figs. 6 and 7) indicate portions of the membrane of two spherical bodies which appear to be in process of incorporation into newly formed layered structures; there appears to be close association between the layered material and the membranes. The transition from spherical body membranes to layered structures is noticeable also in Fig. 2, which shows the membranes of three adjacent bodies densely stained and about to enclose a cluster of VLP (arrow). Fig. 7 shows VLP arranged in a spiral with the lowest layer in process of formation. The figure demonstrates again that the layered material may originate from the membrane of the spherical body (arrow). The VLP were found nearly always outside the bodies but some VLP arranged in a crystal-like array inside a spherical body are shown in Fig. 4 (VLP).
36
JACOLI AND RONALD
Spherical bodies, vesicles a n d V L P were f o u n d only in the callus of infected c a r r o t r o o t s a n d aster stems, p r e d o m i n a n t l y in carrots. N o n e of the structures described was f o u n d in healthy c a r r o t a n d aster tissues. A n o t h e r feature which characterized the infected cultures was the f o r m a t i o n of inclusions ( / / a n d Ie, Fig. 8). These were m a i n l y f o u n d outside the nuclear m e m b r a n e , b u t in a few cases they were seen within the nuclei of p h l o e m cells (Ii, Fig. 8). A c o m p a r i s o n between these inclusions a n d P - p r o t e i n or slime bodies (P, Fig. 8) shows that, a l t h o u g h the inclusions s o m e w h a t r e s e m b l e d P-protein, they differed in having a stronger affinity for stain (Fig. 8) a n d in lacking the typical fibrillar or t u b u l a r structure (8, 9, 14) (Fig. 9). I t is possible t h a t these inclusions represent precursors or m o d i fications of the slime bodies. W e f o u n d it difficult to establish a clear sequence of events in the a p p e a r a n c e of the spherical bodies, vesicles, a n d VLP. H o w e v e r , these structures a p p e a r e d , as a function of their l o c a t i o n in the growing callus, in the o r d e r listed here. W e d i d n o t find these three types of structures in the original tissues f r o m which the explants were taken, b u t large n u m b e r s of m y c o p l a s m a - l i k e bodies ( M L B ) have been seen in the infected c a r r o t tissues a n d smaller n u m b e r s in aster.
DISCUSSION A t present, it is difficult to establish the identity of the described structures which were f o u n d only in sections of callus infected by the aster yellows disease. W e have n o t i c e d structural analogies between our vesicles a n d vesicular structures which other investigators have f o u n d associated with chloroplasts (1, 5-7, 16, 27, 28), m i t o c h o n d r i a (19, 20), nuclei (12, 13), a n d the e n d o p l a s m i c reticulum (13) of virus-infected plants.
FIG. 1. Cross section through the phloem of carrot callus infected by the aster yellows disease. Note the cluster of spherical bodies in the upper right of the picture (arrow) with vesicles lined up along their inner membranes (Vs). x 37 600. FIG. 2. A spherical body within the phloem of infected carrot callus. Note the incipient degeneration of the body with the formation of two large vacuoles (Vc) and the appearance of round, viruslike particles (VLP). The preliminary phase of converting the membrane of a spherical body into layered structures is indicated by the arrows, x 43 000. F~G. 3. Cross section through the phloem of infected carrot callus showing the migration of vesicles toward an opening in the body (arrow). The arrangement of the VLP inside the incipient layered structures is also apparent (VLP). x 37 200. FIG. 4. A cluster of spherical bodies from infected aster stem callus. Note the array of VLP inside the central body (VLP) and the large vacuole in the body on the right side of the picture (Vc). The vesicles have lost their membrane-oriented position (Vs). x 155 603. FIG. 5. Cross section through the phloem near the core of infected carrot callus. Note the linear and spiral arrangement of the VLP within the layered structures (arrows). A cluster of vesicles is shown outside the perimeter of a spherical body (Vs). x 39 700. FIGS. 6 and 7. Enlargements of the layered structure indicated by the arrows in Fig. 4. x 66 300.
ASTER YELLOWS IN PLANT TISSUE CULTURES
37
38
JACOLI AND RONALD
ASTER YELLOWS IN PLANT: TISSUE CULTURES
39
40
JACOLI AND RONALD
Nevertheless, the vesicles in our callus preparations were not associated with any of these cellular structures. The strong similarity between VLP and true virions, and their peculiar arrangement in layered structures which have already been described for other diseases in the literature (10, 22, 29), is noteworthy. We do not believe that the VLP are clusters of ribosomes. They differ from ribosomes by their larger size, better defined outlines, and occasional hollow structure. Furthermore, the occurrence of these particles in infected preparations only, suggests some correlation between these entities and the aster yellows disease. There is a vast body of published evidence which supports the concept that mycoplasmaqike bodies (MLB) rather than viruses are the causative agents of many yellows diseases (1I, 18, 23). Nevertheless, the occurrence of viruslike particles associated with MLB has been described in different plant diseases (2, 4, 17, 2I), but a definite association between MLB and viruses has yet to be demonstrated. Atanasoff's hypothesis (3) that mycoplasma are symbionts acting as vectors of viruses, in the manner of nematodes, bacteria, or fungi, is still to be disproved. In our studies, we saw a few structures which resembled mycoplasma, but these were associated with neither the vesicles nor the VLP. The origin of the bodies and vesicles and the purification of the VLP were not considered at this stage in our studies, neither was their relation to the etiology of the yellows disease. Thus, the question whether these structures are causative of the aster yellows disease or are part of a secondary infection, remains to be answered. There is a possibility that the bodies may be degenerated organelles such as mitochondria or plastids. Careful examination of our infected preparations show few intact mitochondria. Intact plastids which were in good number in the phloem of healthy explants, appeared to be rare in the phloem of infected explants. Several plant hosts susceptible and not susceptible to aster yellows were mechanically inoculated with our preparations, but they failed to show any recognizable symptom. We were unable to transmit the aster yellows disease from our cultures with leafhopper vectors. It is possible that the conditions used in our cultures might have induced the need for an abnormally long acquisition time for the vector or an abnormally long incubation period to establish infectivity as suggested by Mitsuhashi and Maramorosch (25). Another explanation for the lack of infectivity might be that some components of the cultural medium inhibited the causative agent of the disease. We think this is unlikely, although it could explain why the structures discussed here were not~found in peripheral cells of our infected preparations. @ork is in progress to isolate and identify the viruslike particles described above. To our knowledge, this is the first time that these structures have been reported in plant tissue cultures.
ASTER YELLOWS IN PLANT TISSUE CULTURES
41
Fro. 8. LongitudinaI section of the phloem of infected carrot callus showing intra- and extranuclear inclusions (Ii and le) and P-protein (P). × 13 800. FIo. 9. Enlargement of the inclusion (Ii) of Fig. 8. The nonfilamentous structure is very apparent at this magnification, x 42 000.
42
JACOLI AND RONALD
We thank Miss Esther Lo for electron microscopy, Mr. F. E. Skelton for infected materials and infectivity tests, and Mr. Howard Severson for photography.
REFERENCES 1. ALLEN, T. C., Virology 47, 467 (1972). 2. - - - - ibid. 47, 491 (1972). 3. ATANASOFr,D., Biol. Zentralbl. 88, 571 (1969). 4. BANTTARI,E. E. and ZEYEN, R. J., Phytopathology 60, 1282 (1970). 5. Bov~, J. M., Bull. Soc. Fr. Physiol. VOg. 12, 307 (1966). 6. CARROLL,T. W., Virology 42, 1015 (1970). 7. CHALCROFT,J. and MATTHEWS,R. E. F., Virology 28, 55 (1966). 8. CRONSHAW,J. and ESAU, K., J. Cell Biol. 34, 801 (1967). 9. - - - - ibid. 38, 292 (1968). 10. DAVIDSON,E. M., Virology 37, 694 (1969). 11. DAVIS, R. E. and WHITCOMB,R. F., Annu. Rev. Phytopathol. 9, 119 (1971). 12. DE ZOETEN, G. A., GAARD, G. and DIEZ, F. B., Virology 48, 638 (1972). 13. ESAU, K. and HOEFERT,L. L., Virology 48, 724 (1972). 14. EVERT, R. F. and DESHPANDE,B. P., J. Cell Biol. 44, 462 (1970). 15. GAUTt~ERET,R. J. (Ed.), La Culture des Tissus V6g6taux, p. 92, Masson, Paris, 1959. 16. GEROLA,F. M., BASSI, M. and G1USSANI,G., Caryologia 19, 457 (1966). 17. GIANNOTTI,J., DEVAtJCHELL~:,G. and VAGO, C., Proe. Int. Congr. Mierobiol. lOth p. 222, Mexico City, 1970. 18. HAMPTON,R. O., Annu. Rev. Plant Physiol. 23, 389 (1972). 19. HARRISON,B. D., STEFANAC,Z. and ROBERTS,I. M., J. Gen. Virol. 6,127 (1970). 20. HATTA,R., NAKAMOTO,T., TAKAGI,Y. and USHIYAMA,R., Virology 45, 292 (197l). 21. HmUMI, H. and MARAMOROSCH,K., Phytopathol. Z. 75, 9 (1972). 22. HITCHBORN,J. H. and HILLS, G. J., Virology 35, 50 (1968). 23. HULL, R., Rev. Plant Pathol. 50, 121 (1971). 24. LUI~T,J. H., J. Biophys. Bioehem. Cytol. 9, 409 (1961). 25. MITSUHASHI,J. and MARAMOROSCtt,K., Virology 23, 277 (1964). 26. MURASHIGE,T. and SKOOG,F., Physiol. Plantarum 15, 473 (1962). 27. RUBIo-HUERTOS,M., VELA, A. and LOeEz-ABELLA,D., Virology 32, 438 (1967). 28. USmYAMA,R. and MATrHEWS, R. E. F., Virology 42, 293 (1970). 29. WALK,Y, D. G. A. and WEBB, M. J. W., J. Gen. Virol. 7, 159 (1970).