J. gen. Virol. (1983), 64, 2489-2492.
Printed in Great Britain
2489
Key words: ultrastructure/TMV/bean golden mosaic virus~mixed infection/phloem restriction
Evidence that Bean Golden Mosaic Virus Invades Non-phloem Tissue in
Double Infections with Tobacco Mosaic Virus
By R. J. C A R R AND K. S. K I M *
Department of Plant Pathology, University of Arkansas, Fayetteville, Arkansas 72701, U.S.A.
(Accepted 5 August 1983)
SUMMARY
Bean leaves that had been doubly infected systemically with the legume strain of
tobacco mosaic virus (CP-TMV) and bean golden mosaic virus (BGMV) were studied
ultrastructurally. Virus particles and the cytopathological changes associated with each
virus in single infections occurred within the same cell when plants were doubly
infected, indicating that individual systemically infected host cells can multiply viruses
of different nucleic acid composition. Although the cytopathic effects induced by
BGMV are limited to phloem-associated cells in singly infected plants, the same effects
were found in most leaf cell types in mixedly infected plants.
Mixed infections by plant viruses are common but have been relatively little studied
(Matthews, 1981). Thus, information concerning the ultrastructure of mixed virus infections is
sparse, although recent reports by Gill & Chong (1981) and Carr & Kim (1983), indicate that
ultrastructural features in mixedly infected cells may be useful in discriminating related viruses
or strains of the same virus.
This paper describes cytopathological effects occurring in leaf cells infected with the legume
strain of tobacco mosaic virus (CP-TMV), an R N A virus with rod-shaped particles, and bean
golden mosaic virus (BGMV), an isometric, DNA-containing geminivirus. The results suggest
that BGMV infects cell types in doubly infected tissue that are not infected in singly infected
leaves.
Bean plants (Phaseolus vulgaris L. cv. Cherokee Wax) were cultivated in a growth chamber in
subdued light for a 16 h day and at a constant 32 °C, and 5- to 7-day-old bean plants were
inoculated mechanically using sap from infected beans. Mixed inocula contained equal amounts
of sap from plants infected with each virus. Inoculating either virus as part of a mixture did not
appear to affect the efficiency of inducing mixed infections. Tissue samples were taken from
infected trifoliate leaves 15 to 20 days after inoculation and prepared for electron microscopy as
described previously (Carr & Kim, 1983).
Infection of bean with CP-TMV either singly or in mixed infection with BGMV resulted in
the production of massive arrays of virus particles that were aligned along cell walls (Fig. 1a) or
protruded into the vacuoles of all leaf parenchyma cells. Particles of CP-TMV were also present
in the chloroplasts and nuclei of infected cells (Fig. 1a, d). The occurrence of CP-TMV particles
in the nuclei of cells infected singly with CP-TMV, as well as in mixed infection with BGMV
(see below), distinguishes this strain from the common strain of TMV (Honda & Matsui, 1969).
Particles of the common strain of TMV occurred in the nuclei of tobacco leaf cells only if they
were also infected with cucumber mosaic virus (Honda & Matsui, 1969).
The most consistently observed ultrastructural effects of BGMV infection were nuclear
hypertrophy, segregation of the nucleolar fibrillar and granular regions, formation of
intranuclear fibrillar rings (Fig. 1 and 2), and the appearance of small (18 to 20 nm) isometric
virus-like particles (Fig. 1b and 2a) in the nuclei of phloem parenchyma cells. These effects were
the same as those previously described in P. vulgaris cv. Top Crop by K i m et al. (1978) except
that there was an accumulation of what appeared to be perichromatin and interchromatin
granules in the nuclei of infected P. vulgaris cv. Cherokee Wax leaf cells (Fig. 1 b, c). These
granules were similar in appearance to granules described by Daskal (1981), and to
Downloaded from www.microbiologyresearch.org by
0022-1317/83/0000-5765 $02.00© 1983 SGMIP: 88.99.165.207
On: Mon, 19 Jun 2017 00:32:29
2490
Short communication
Fig. 1. Cytopathic effects of mixed infection with CP-TMV and BGMV. (a) A portion of a leaf
mesophyll cell showing an intranuclear fibrillar ring (arrow) induced by B G M V , as well as clusters of
CP-TMV partictes in the cytoplasm and the chloroplasts (arrowheads). (b) Section through a mesophyll
cell nucleus (N) showing fibrillar rings (arrowheads), virus-like particles (circled area; see also Fig. 2a)
and a cluster of perichromatin granules (arrow) associated with B G M V infection, as well as CP-TMV
particles (squared area). (c) Enlargement of perichromatin granules (arrowheads) as indicated by an
arrow in (b). (d) Enlargement of the squared area in (b) showing CP-TMV particles (arrowheads). Bar
markers represent 1 ~m in (a, b) and 100 nm in (c, d).
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 19 Jun 2017 00:32:29
Short communication
2491
Fig. 2. (a) A higher magnification of the circled area of the nucleus in Fig. 1(b) showing BGMV-like
particles (arrowheads). Some particles are somewhat paired. Bar marker represents 100 nm. (b) An
epidermal cell infected with CP-TMV and BGMV showing a nucleus containing a multilobed fibrillar
ring (arrowhead) characteristic of BGMV infection. Bar marker represents 5 ~tm.
perichromatin granules found in Datura stramonium cells infected by another geminivirus, the
Florida isolate of Euphorbia mosaic virus (Kim & Martin, 1982).
Cells infected with both CP-TMV and BGMV contained particles and/or inclusions of both
viruses (Fig. 1 and 2). Generally, young leaves contained a larger proportion of mixedly infected
cells than did older leaves. Virus-like particles and inclusions associated with BGMV were
always confined to the nucleus, whereas particles of CP-TMV were observed in the cytoplasm,
nuclei and chloroplasts (Fig. 1 a, b, d).
Previously, BGMV (Kim et al., 1978) and several other geminiviruses (Esau & Hoefert, 1973;
Goodman, 1981) have been found to infect only phloem tissue cells. However, intranuclear
effects induced by BGMV, such as fibrillar bodies, and virus-like particles were found in most
cell types in plants infected with both BGMV and CP-TMV (Fig. 1 and 2). In some cells,
characteristic inclusions and/or particles of both viruses were located within the same nucleus
(Fig. 1b, d and 2a). The occurrence of these structures within the same cell shows that one cell
can harbour and probably assemble viruses having genomes composed of different types of
nucleic acid. Kamei et al. (1969) located particles of turnip mosaic virus (an R N A virus) and
cauliflower mosaic virus (a D N A virus) within the same cell in inoculated Brassica perviridis
leaves. The present study confirms the ability of single cells to harbour R N A and D N A viruses
in systemic as well as local infections.
One of the major problems in studying phloem-limited viruses is the difficulty of purifying
them (Takanami & Kubo, 1979), and low virus titre contributes to this difficulty. Because most
cell types contain BGMV in mixedly infected plants, double infection with CP-TMV may be a
way of increasing the concentration of BGMV in a propagation host. If a similar loss of host
tissue specificity occurs with other phloem-limited viruses in mixed infections, it may aid in the
study of these viruses.
Loss of virus host-tissue specificity as a result of mixed infection has been previously reported
for two isolates of barley yellow dwarf virus (BYDV) (Gill & Chong, 1981); virus that was
restricted to phloem in single infections was found in xylem tissue when plants were mixedly
infected with the two isolates. However, we found BGMV-induced ultrastructural effects to be
much more widespread in leaf cells than were BYDV particles in mixed infections. Perhaps the
large degree of unrelatedness between CP-TMV and BGMV led to the total loss of the tissue
specificity of BGMV observed here, whereas the relatively limited loss of tissue specificity in
mixed infection by BYDV isolates was due to the similarity of isolates of a single virus.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 19 Jun 2017 00:32:29
2492
Short communication
R e c e n t l y , it h a s b e e n s h o w n t h a t t o m a t o e s r e s i s t a n t to T M V b e c a m e s u s c e p t i b l e if t h e p l a n t s
h a d b e e n p r e - i n f e c t e d w i t h t h e u n r e l a t e d p o t a t o v i r u s X ( P V X ) as h e l p e r v i r u s ( T a l i a n s k y et al.,
1982). It w a s c o n c l u d e d t h a t P V X w a s f a c i l i t a t i n g t h e t r a n s p o r t o f T M V b e t w e e n t o m a t o cells,
a n d it is p o s s i b l e t h a t in o u r s t u d y C P - T M V a c t e d s i m i l a r l y as a h e l p e r v i r u s to allow B G M V to
m o v e f r o m p h l o e m to o t h e r cell t y p e s s u c h as m e s o p h y l l a n d e p i d e r m a l cells.
Published with the approval of the Director of the Arkansas Agricultural Experiment Station.
REFERENCES
CARR,R. J. & KIM,K. S. (1983). Ultrastructure of mixed plant virus infection: bean yellow mosaic virus with cowpea
severe mosaic or cowpea mosaic virus in bean. Virology 124, 338-348.
DASKAL, Y. (1981). Perichromatin granules. In The Cell Nucleus, vol. 8, pp. 117-137. Edited by H. Busch. New
York: Academic Press.
ESAU, K. & HOEFERT,L. L. (1973). Particles and associated inclusions in sugarbeet infected with the curly top virus.
Virology 56, 454-464.
GILL, C. C. & CHONG,J. ( 1981). Vascular cell alterations and predisposed xylem infection in oats by inoculation with
paired barley yellow dwarf viruses. Virology 114, 405-413.
GOODMAN, R. M. (1981). Geminiviruses. Journal of General Virology 54, 9-21.
HONDA, Y. & MATSUI, C. (1969). Occurrence of tobacco mosaic virus within the nucleus. Virology 39, 593-595.
KAMEI, T., GOTTO,T. & MATSUI,C. (1969). Turnip mosaic virus multiplication in leaves infected with cauliflower
mosaic virus. Phytopathology 59, 1795 1797.
KIM, K. S. & MARTIN, E. M. (1982). Nucleolar and extranucleolar perichromatin granules induced by Euphorbia
mosaic virus. Phytopathology 72, 938.
KIM, K. S., SHOCK, T. L. & GOODMAN,-R. M. (1978). Infection of Phaseolus vulgar& by bean golden mosaic virus:
ultrastructural aspects. Virology 89, 22-33.
MATTHEWS,R. E. F. (1981). Factors influencing the course of infection and disease. In Plant Virology, 2rid edn., pp.
427-453. New York: Academic Press.
TAKANAMI, Y. & KUBO, S. (1979). Enzyme-assisted purification of two phloem-limited plant viruses: tobacco
necrotic dwarf and potato leafroll. Journal of General Virology 44, 153-159.
TALIANSKY,M. E., MALYSHENKO,S. I., PSHENNIKOVA,E. S. & ATABEKOV,J. G. (1982). Plant virus-specific transport
function. II. A factor controlling virus host range. Virology 122, 327-331.
(Received 20 May 1983)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 19 Jun 2017 00:32:29