Ann. appl. Biol. (197s), 80, W i t h 2 plates
181-184
181
Printed in Great Britain
A probable rhabdovirus infecting ryegrass (Lolium spp.) BY R. T. PLUMB AND MAR1 JAMES* Rothamsted Experimental Station, Harpenden, Hertfordshire
(Accepted I 9 December I 974) SUMMARY
A particle with morphology characteristic of the rhabdovirus group was found in leaf-dips and thin sections of symptomless Lolium spp. from several places in Britain. The particle was not transmitted mechanically nor by a number of groups of possible arthropod vectors. Despite the lack of conclusive evidence for the viral nature of the particle its morphology and intracellular localization strongly suggest that it is a member of the rhabdovirus group. This is the first report of such a particle in Lolium spp. and in Gramineae in Britain. The presumed virus is tentatively called ryegrass bacilliform virus (RBV). INTRODUCTION
The rhabdovirus group includes viruses with bullet-shaped or bacilliform morphology that infect animals and plants, twenty-four of the latter being noted by Knudson (1973). Seven of these infect members of the Gramineae but none has been reported from Lolium spp. Examination of leaf-dip preparations from Lolium spp. showed occasional particles with characteristic rhabdovirus morphology, tentatively called ryegrass bacilliform virus (RBV). We report some details of the morphology of the particles, their intracellular localization and the results of transmission experiments. METHODS
Infected plants were found initially at two sites: a pesticide experiment at Wye College, Ashford, Kent and a National Institute of Agricultural Botany demonstration at the National Agricultural Centre, Stoneleigh, Warwickshire. The presence of bacilliform particles was confirmed in leaf-dip preparations using 2 % neutral sodium phosphotungstate stain and carbon covered mounts, and parts of the same leaf were fixed in 2-5yo (v/v) glutaraldehyde in 0.05 M phosphate buffer, post fixed in I % (w/v) osmium tetroxide, dehydrated in acetone and embedded in Epon. Tissue was stained with uranyl acetate during dehydration and, after sectioning, with lead citrate. Aphids, Rhopalosiphum padi, and planthoppers, Javesella pellucida, used in transmission tests were from virus-free cultures. Insects collected by sweep nets from grass swards were also tested for transmission. The Italian ryegrass (Lolium multiforum) S. 22 and hybrid ryegrass (L. multiforum x L . perenne) Grasslands Manawa were used as test plants throughout.
*
Present address : School of Plant Biology, U.C.N.W., Bangor, N. Wales.
I82
R. T. PLUMBAND MARIJAMES RESULTS
Symptoms and hosts. The plants in which RBV was first found showed symptoms of ryegrass mosaic virus (RMV) and were examined to confirm the presence of RMV. Experience showed that the only symptom which distinguished plants infected with RBV as well as RMV was slightly more chlorosis than in simple RMV infections. Subsequent samples were correctly classified on this basis but no symptom reliably indicated infection by RBV alone. Both initial discoveries of RBV were in the hybrid ryegrass Grasslands Manawa, but RBV has since been found in the perennial variety S. 23 in North Wales, Somerset and Kent. Particles were not found in plants of these varieties grown from seed in the glasshouse. Transmission tests. The bacilliform particle was not transmitted mechanically. The aphid R. padi and the planthopper J. pellucida also failed to transmit after long acquisition and infection feeds. The following potential vectors were collected from grass swards known to contain plants infected with RBV; beetles, Oulema spp. (Coleoptera : Chrysomelidae) ;planthoppers, J . pellucida (Homoptera :Delphacidae) and five species of aphids, R. padi, Metopolophium dirhodum, M. festucae, Macrosiphum (Sitobion) avenae and M ( S ) .fragariae, but RBV particles were not found in test plants on which they fed. Negative stain. The morphology of all particles in negative stain was similar (PI. I, fig. I).The mean dimensions of twenty-four measured particles were 210& 42'9 x 67.4& 5.7nm, with some showing regular cross-banding at an interval of 3-5 nm (PI. I, fig. 2). They had one flat and one round end and were surrounded at a distance of 5 nm by a thin discontinuous layer (PI. I, figs I, 2) possibly formed by surface projections, similar to those occurring on other rhabdoviruses (Peters & Kitajima, 1970;Howatson, 1970;Knudson, 1973).The flat end may be caused by breakage (Peters & Kitajima, 1970);stain often penetrated a central canal c. 20 nm in diameter, from this end (PI. I, fig. I). Thin sections. In section the mean length of thirty particles was 287.9116.2 nm, the diameter 68.2 6.0nm. They occurred most frequently in mesophyll cells (PI. I , fig. 3), although cells containing virus were rare in infected plants. Most particles occurred within membrane-bound bags (Pl. I, fig. 3), some containing many particles (Pl. I , fig. 4). Occasionally aggregates (Pl. I, fig. 5 ) were not confined within a membrane. Large aggregates often had a palisade of particles just within and at right angles to the bounding membrane (PI. I, fig. 4),and particles in the centre with no regular orientation. Particles were frequently cut transversely in aggregates (Pl. I, fig. 6),and showed two concentric electron-dense layers each c. 5-8 nm wide with a small, dense axial core. The inner ring had a diameter of 20-25 nm and the outer a diameter of c. 50 nm. Tubular structures 40 nm in diameter were also present in the cytoplasm of some cells (PI. 2, fig. 7); cross-sections (PI. 2, fig. 8) suggest they could be RBV particles lacking their outer layer. It has not been possible to follow the sequence of infection and particle development because we have not transmitted the virus and all our evidence comes from thin sections of natural infections that are probably well established. However, small
Rhubdovirus of ryegrass
183
aggregates of both complete particles and ‘shells’, apparently consisting only of the outer layer of particles, do occur in the perinuclear space (Pl. 2, fig. 9). No evidence was seen of continuity between host membranes and virus or virus ‘shells’. T h e ‘shells’ were also a common feature of some cytoplasmic aggregates of virus (Pl. I, fig. 3)DISCUSSION
There is as yet no proof that the bacilliform particles are those of a rhabdovirus, as their morphology suggests. There are no records of rhabdovirus in ryegrass and we have not been able to compare RBV with other rhabdoviruses infecting Gramineae nor, as yet, to transmit it. Th e lack of symptoms is not unusual because other hosts of rhabdoviruses often show ephemeral symptoms (Knudson, 1973) or none, as in Melilotus latent virus (Kitajima, Lauritis & Swift, 1969). Several membranes have been implicated as the sites of maturation of rhabdoviruses (Kitajima et al. 1969; Howatson, 1970; Francki, 1973; Knudson, 1973). We have not been able to follow the development of RBV but particles are associated with the nuclear membrane and membrane-bound vesicles in the cytoplasm. T h e regular array of particles at the periphery of the vesicles suggests that mature particles may be formed by budding through the membrane. Th e regular arrangement is presumably lost, as in the centre of the aggregates, when particles are released from the membrane. Hull (1970) grouped rhabdoviruses according to their apparent sites of maturation, perinuclear or cytoplasmic, although this is not definitely known for any rhabdovirus (Francki, 1973). I n RBV-infected cells the ‘shells’ in the perinuclear space were often as numerous as complete particles, and may be abortive enclosures of nucleocapsids, as their diameter is similar to that of mature particles. No virus nucleocapsids were seen in the nucleus but they were occasionally present in the cytoplasm, although their occurrence was usually associated with cellular disorganization and they may represent a stage in degradation rather than formation. Most rhabdoviruses that occur in the perinuclear space are also found within cytoplasmic, membrane-bound vesicles, and these are thought to be extensions of the endoplasmic reticulum, which is continuous with the outer layer of the nuclear membrane (Francki, 1973). Th e greater number of mature RBV particles in the cytoplasmic vesicles than in the perinuclear space may relate more to stage of infection that site of maturation. All present comparison with other rhabdoviruses suggests perinuclear maturation but transmission of RBV will need to precede further studies. Despite doubts about sites of synthesis, RBV agrees in all its known characters with other confirmed rhabdoviruses except in the periodicity (3.5 nm) of cross banding on negatively stained particles as compared with 4.5 nm for other rhabdoviruses (Howatson, 1970). We are grateful for the help of the officers of the National Institute of Agricultural Botany and the Agricultural Development and Advisory Service in providing some of the initial samples.
R. T. PLUMBAND MARIJAMES REFERENCES
FRANCKI, R. I. B. (1973). Plant rhabdoviruses. Advances in Virus Research 19,257-345. HOWATSON, A. F. (1970). Vesicular stomatitis and related viruses. Advances in Virus Research 16, 195-256. HULL,R. (1970). Large RNA plant-infecting viruses. In The Biology of Large RNA Viruses (ed. R. D. Barry and B. W. J. Mahy). London: Academic Press. KITAJIMA, E. W., LAURITIS, J. A. & SWIFT, H. (1969). Morphology and intracellular localization of a bacilliform latent virus in sweet clover. Journal of Ultrastructure Research 29, 141-150. Virology 20, 105-130. PETERS, D. & KITAJIMA,E.W. (1970). Purification and electron microscopy of sowthistle yellow vein virus. Virology 41, 135-150. KNUDSON,D.L. (1973). Rhabdoviruses. Journal of General
EXPLANATION OF PLATE PLATE I
Fig. I. Rhabdovirus-like particle in negative stain. Fig. 2. Rhabdovirus-like particle in negative stain showing cross-banding. Fig. 3. Cytoplasm of infected cell showing membrane bound aggregates. Fig. 4. Large aggregate of particles showing the palisade layer at right angles to the bounding membrane. Fig. 5. Unenclosed aggregates of particles. Fig. 6. Transverse section of particles showing the double ring structure. PLATE 2 Fig. 7. Enveloped nucleoprotein ' cores '. Fig. 8. Single ring structure of nucleoprotein ' cores '. Fig. 9. Complete particles and shells in the perinuclear space.
Annals of Applied Biology, Lrol. 80, No.
R. T. PLUMB
ASD
MAR1 J A l l E S
2
Plate
I
( F a c i q p . I 84)
Annals of Applied Biology, J’ol. 80, *Yo. 2
Plate
2
181-184
181
Printed in Great Britain
A probable rhabdovirus infecting ryegrass (Lolium spp.) BY R. T. PLUMB AND MAR1 JAMES* Rothamsted Experimental Station, Harpenden, Hertfordshire
(Accepted I 9 December I 974) SUMMARY
A particle with morphology characteristic of the rhabdovirus group was found in leaf-dips and thin sections of symptomless Lolium spp. from several places in Britain. The particle was not transmitted mechanically nor by a number of groups of possible arthropod vectors. Despite the lack of conclusive evidence for the viral nature of the particle its morphology and intracellular localization strongly suggest that it is a member of the rhabdovirus group. This is the first report of such a particle in Lolium spp. and in Gramineae in Britain. The presumed virus is tentatively called ryegrass bacilliform virus (RBV). INTRODUCTION
The rhabdovirus group includes viruses with bullet-shaped or bacilliform morphology that infect animals and plants, twenty-four of the latter being noted by Knudson (1973). Seven of these infect members of the Gramineae but none has been reported from Lolium spp. Examination of leaf-dip preparations from Lolium spp. showed occasional particles with characteristic rhabdovirus morphology, tentatively called ryegrass bacilliform virus (RBV). We report some details of the morphology of the particles, their intracellular localization and the results of transmission experiments. METHODS
Infected plants were found initially at two sites: a pesticide experiment at Wye College, Ashford, Kent and a National Institute of Agricultural Botany demonstration at the National Agricultural Centre, Stoneleigh, Warwickshire. The presence of bacilliform particles was confirmed in leaf-dip preparations using 2 % neutral sodium phosphotungstate stain and carbon covered mounts, and parts of the same leaf were fixed in 2-5yo (v/v) glutaraldehyde in 0.05 M phosphate buffer, post fixed in I % (w/v) osmium tetroxide, dehydrated in acetone and embedded in Epon. Tissue was stained with uranyl acetate during dehydration and, after sectioning, with lead citrate. Aphids, Rhopalosiphum padi, and planthoppers, Javesella pellucida, used in transmission tests were from virus-free cultures. Insects collected by sweep nets from grass swards were also tested for transmission. The Italian ryegrass (Lolium multiforum) S. 22 and hybrid ryegrass (L. multiforum x L . perenne) Grasslands Manawa were used as test plants throughout.
*
Present address : School of Plant Biology, U.C.N.W., Bangor, N. Wales.
I82
R. T. PLUMBAND MARIJAMES RESULTS
Symptoms and hosts. The plants in which RBV was first found showed symptoms of ryegrass mosaic virus (RMV) and were examined to confirm the presence of RMV. Experience showed that the only symptom which distinguished plants infected with RBV as well as RMV was slightly more chlorosis than in simple RMV infections. Subsequent samples were correctly classified on this basis but no symptom reliably indicated infection by RBV alone. Both initial discoveries of RBV were in the hybrid ryegrass Grasslands Manawa, but RBV has since been found in the perennial variety S. 23 in North Wales, Somerset and Kent. Particles were not found in plants of these varieties grown from seed in the glasshouse. Transmission tests. The bacilliform particle was not transmitted mechanically. The aphid R. padi and the planthopper J. pellucida also failed to transmit after long acquisition and infection feeds. The following potential vectors were collected from grass swards known to contain plants infected with RBV; beetles, Oulema spp. (Coleoptera : Chrysomelidae) ;planthoppers, J . pellucida (Homoptera :Delphacidae) and five species of aphids, R. padi, Metopolophium dirhodum, M. festucae, Macrosiphum (Sitobion) avenae and M ( S ) .fragariae, but RBV particles were not found in test plants on which they fed. Negative stain. The morphology of all particles in negative stain was similar (PI. I, fig. I).The mean dimensions of twenty-four measured particles were 210& 42'9 x 67.4& 5.7nm, with some showing regular cross-banding at an interval of 3-5 nm (PI. I, fig. 2). They had one flat and one round end and were surrounded at a distance of 5 nm by a thin discontinuous layer (PI. I, figs I, 2) possibly formed by surface projections, similar to those occurring on other rhabdoviruses (Peters & Kitajima, 1970;Howatson, 1970;Knudson, 1973).The flat end may be caused by breakage (Peters & Kitajima, 1970);stain often penetrated a central canal c. 20 nm in diameter, from this end (PI. I, fig. I). Thin sections. In section the mean length of thirty particles was 287.9116.2 nm, the diameter 68.2 6.0nm. They occurred most frequently in mesophyll cells (PI. I , fig. 3), although cells containing virus were rare in infected plants. Most particles occurred within membrane-bound bags (Pl. I, fig. 3), some containing many particles (Pl. I , fig. 4). Occasionally aggregates (Pl. I, fig. 5 ) were not confined within a membrane. Large aggregates often had a palisade of particles just within and at right angles to the bounding membrane (PI. I, fig. 4),and particles in the centre with no regular orientation. Particles were frequently cut transversely in aggregates (Pl. I, fig. 6),and showed two concentric electron-dense layers each c. 5-8 nm wide with a small, dense axial core. The inner ring had a diameter of 20-25 nm and the outer a diameter of c. 50 nm. Tubular structures 40 nm in diameter were also present in the cytoplasm of some cells (PI. 2, fig. 7); cross-sections (PI. 2, fig. 8) suggest they could be RBV particles lacking their outer layer. It has not been possible to follow the sequence of infection and particle development because we have not transmitted the virus and all our evidence comes from thin sections of natural infections that are probably well established. However, small
Rhubdovirus of ryegrass
183
aggregates of both complete particles and ‘shells’, apparently consisting only of the outer layer of particles, do occur in the perinuclear space (Pl. 2, fig. 9). No evidence was seen of continuity between host membranes and virus or virus ‘shells’. T h e ‘shells’ were also a common feature of some cytoplasmic aggregates of virus (Pl. I, fig. 3)DISCUSSION
There is as yet no proof that the bacilliform particles are those of a rhabdovirus, as their morphology suggests. There are no records of rhabdovirus in ryegrass and we have not been able to compare RBV with other rhabdoviruses infecting Gramineae nor, as yet, to transmit it. Th e lack of symptoms is not unusual because other hosts of rhabdoviruses often show ephemeral symptoms (Knudson, 1973) or none, as in Melilotus latent virus (Kitajima, Lauritis & Swift, 1969). Several membranes have been implicated as the sites of maturation of rhabdoviruses (Kitajima et al. 1969; Howatson, 1970; Francki, 1973; Knudson, 1973). We have not been able to follow the development of RBV but particles are associated with the nuclear membrane and membrane-bound vesicles in the cytoplasm. T h e regular array of particles at the periphery of the vesicles suggests that mature particles may be formed by budding through the membrane. Th e regular arrangement is presumably lost, as in the centre of the aggregates, when particles are released from the membrane. Hull (1970) grouped rhabdoviruses according to their apparent sites of maturation, perinuclear or cytoplasmic, although this is not definitely known for any rhabdovirus (Francki, 1973). I n RBV-infected cells the ‘shells’ in the perinuclear space were often as numerous as complete particles, and may be abortive enclosures of nucleocapsids, as their diameter is similar to that of mature particles. No virus nucleocapsids were seen in the nucleus but they were occasionally present in the cytoplasm, although their occurrence was usually associated with cellular disorganization and they may represent a stage in degradation rather than formation. Most rhabdoviruses that occur in the perinuclear space are also found within cytoplasmic, membrane-bound vesicles, and these are thought to be extensions of the endoplasmic reticulum, which is continuous with the outer layer of the nuclear membrane (Francki, 1973). Th e greater number of mature RBV particles in the cytoplasmic vesicles than in the perinuclear space may relate more to stage of infection that site of maturation. All present comparison with other rhabdoviruses suggests perinuclear maturation but transmission of RBV will need to precede further studies. Despite doubts about sites of synthesis, RBV agrees in all its known characters with other confirmed rhabdoviruses except in the periodicity (3.5 nm) of cross banding on negatively stained particles as compared with 4.5 nm for other rhabdoviruses (Howatson, 1970). We are grateful for the help of the officers of the National Institute of Agricultural Botany and the Agricultural Development and Advisory Service in providing some of the initial samples.
R. T. PLUMBAND MARIJAMES REFERENCES
FRANCKI, R. I. B. (1973). Plant rhabdoviruses. Advances in Virus Research 19,257-345. HOWATSON, A. F. (1970). Vesicular stomatitis and related viruses. Advances in Virus Research 16, 195-256. HULL,R. (1970). Large RNA plant-infecting viruses. In The Biology of Large RNA Viruses (ed. R. D. Barry and B. W. J. Mahy). London: Academic Press. KITAJIMA, E. W., LAURITIS, J. A. & SWIFT, H. (1969). Morphology and intracellular localization of a bacilliform latent virus in sweet clover. Journal of Ultrastructure Research 29, 141-150. Virology 20, 105-130. PETERS, D. & KITAJIMA,E.W. (1970). Purification and electron microscopy of sowthistle yellow vein virus. Virology 41, 135-150. KNUDSON,D.L. (1973). Rhabdoviruses. Journal of General
EXPLANATION OF PLATE PLATE I
Fig. I. Rhabdovirus-like particle in negative stain. Fig. 2. Rhabdovirus-like particle in negative stain showing cross-banding. Fig. 3. Cytoplasm of infected cell showing membrane bound aggregates. Fig. 4. Large aggregate of particles showing the palisade layer at right angles to the bounding membrane. Fig. 5. Unenclosed aggregates of particles. Fig. 6. Transverse section of particles showing the double ring structure. PLATE 2 Fig. 7. Enveloped nucleoprotein ' cores '. Fig. 8. Single ring structure of nucleoprotein ' cores '. Fig. 9. Complete particles and shells in the perinuclear space.
Annals of Applied Biology, Lrol. 80, No.
R. T. PLUMB
ASD
MAR1 J A l l E S
2
Plate
I
( F a c i q p . I 84)
Annals of Applied Biology, J’ol. 80, *Yo. 2
Plate
2
