Journal of General Virology (1991), 72, 2243-2246. Printed in Great Britain
2243
Effect of recombinant beet necrotic yellow vein virus with different RNA
compositions on mechanically inoculated sugarbeets
R. Koenig,* W. Jarausch, Y. Li, U. Commandeur, W. Burgermeister, M. Gehrke and P. Liiddecke
Biologische Bundesanstalt fftr Land- und Forstwirtschaft, Institut J~r Biochemie und Pflanzenvirologie, Messeweg 11,
D-W-3300 Braunschweig, Germany
Beet necrotic yellow vein virus (BNYVV) inocula with
different RNA compositions were prepared from
infectious transcripts of RNAs 3 and 4 and the Rg 1
isolate, which has a genome consisting only of RNAs 1
and 2. The recombinant viruses were inoculated on 6to 8-day-old sugarbeet seedlings by 'vortexing'. Inocula
containing RNAs 1 and 2 or 1, 2 and 4 produced some
growth reduction, but the most dramatic effects, with
yield reductions of about 95 % in a highly susceptible
variety, were seen when RNA 3 was also present in the
inoculum. Under these conditions the side roots were
brown and brittle and often deteriorated, but 'root
beardedness' was not observed. This might be due to
the fact that our experiments were done in the absence
o f P o l y m y x a betae. Alternatively, the heavy inoculation
at a very young age may either have weakened the
plants to such an extent that extensive root proliferation was impaired or it may have led to rapid
deterioration of the proliferating rootlets, which would
therefore be lost prior to or during removal of the tap
roots from the soil. In the presence of RNA 3 the virus
concentrations in tap roots were markedly increased
suggesting that this RNA facilitates the multiplication
and/or spread of the virus in root tissues.
The genome of beet necrotic yellow vein virus
(BNYVV), the causal agent of sugarbeet rhizomania,
consists of several RNA species. Four (Koenig et al.,
1986; Lemaire et al., 1988), or in Japan sometimes even
five (Tamada et al., 1989), different viral RNAs with
considerable homology at their 3' ends (Bouzoubaa et al.,
1987) have been found in naturally infected sugarbeets.
After mechanical transmission to Chenopodium quinoa or
Tetragonia expansa, RNAs 3, 4 and 5 are often partially
deleted or, especially after prolonged local lesion
passages, are lost entirely, thus yielding isolates with only
RNAs 1 and 2 (Kuszala et al., 1986; Burgermeister et al.,
1986; Koenig et al., 1986; Tamada et al., 1989). Such
isolates cause only mild symptoms in various hosts and
in Beta vulgaris move only poorly systemically (Koenig &
Burgermeister, 1989; Koenig & Ehlers, 1989; Tamada et
al., 1990). Tamada et al. (1989) and Tamada & Abe
(1989) have selected BNYVV isolates with different
RNA compositions on T. expansa. These were retransmitted mechanically to sugarbeets and further transmitted to young sugarbeet seedlings using Polymyxa betae.
All isolates with RNA 3 caused a massive proliferation
of fine rootlets, foliage symptoms and a severe reduction
of the sugar content in the tap roots. Isolates lacking
RNA 3 caused no conspicuous symptoms although there
was a slight reduction in the weight of the tap roots
(Tamada et al., 1990). This strongly suggested that RNA
3 has a major role in the production of rhizomania
symptoms although it could not be excluded that some of
the differences in symptoms might also have been due to
differences in RNAs 1 or 2 in the natural virus isolates.
We have recently been able to obtain full-length
cDNA clones of BNYVV RNAs 3 and 4 in the vector
pRT103 which contains the cauliflower mosaic virus 35S
promoter and polyadenylation signal (Jarausch et al.,
1990; Li et al., 1990; U. Commandeur et al., unpublished
results). These clones were infectious when they were coinoculated on test plants together with the Rg 1 isolate of
BNYVV which contains only RNAs 1 and 2 (Burgermeister et al., 1986). This enabled us to produce different
virus inocula which all had identical RNAs 1 and 2, but
which differed in either containing or not containing
RNAs 3 and/or 4. In order to avoid confusion of the
effects caused by P. betae and by the virus, we have
introduced the recombinant viruses directly into young
sugarbeet seedlings in the absence of P. betae by means of
a recently developed mechanical inoculation procedure
(Koenig & Burgermeister, 1989; Koenig & Stein, 1990).
The effects of virus inocula with different RNA
compositions on the growth of shoots and roots of a
highly susceptible and a partially resistant sugarbeet
variety were compared.
0001-0345 © 1991 SGM
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2244
Short communication
A
Table 1. ELISA readings and total weights of the
B
C
D
E
sugarbeets depicted in Fig. 2
ELISA readings with sap*
from
Sugarbeet variety
Total weight (g)
and RNA composition Cotyledons Tap roots
of tap roots
of inoculum
2 weeks p.i.t 12 weeks p.i. 12 weeks p.i.
Highly susceptible
Buffer control
1 and 2
1, 2 and 4
1, 2 and 3
1, 2, 3 and 4
Partially resistant
Buffer control
1 and 2
1, 2 and 4
1, 2 and 3
1, 2, 3 and 4
0.00
1.19
1.09
1.06
1.37
0.00
0.07
0.16
1.10
1.13
184.1
99.0
86.7
6.6
9.1
0-00
1.14
0-97
1.29
1.33
0-00
0.06
0-09
0.55
0.42
139.4
68.3
68.2
23.5
31.4
* The dilution of the sap in ELISA sample buffer (Clark & Adams,
1977) was l :10 for the cotyledons and 1:20 for the tap roots.
t P.i., Post-inoculation.
B N Y V V with different R N A compositions was
obtained f r o m T. expansa in the second or at a later
passage after the original inoculation with B N Y V V
isolate Rg 1 and plasmids containing full-length c D N A
sequences o f R N A s 3 and/or 4 (Li et al., 1990; U.
C o m m a n d e u r et al., unpublished results). At least 30 6- to
8-day-old sugarbeet seedlings were inoculated with each
type o f B N Y V V by 'vortexing' in freshly expressed sap
from heavily infected T. expansa leaves w h i c h had been
diluted 1 : 4 in 0.05 M-phosphate buffer p H 7.2. Serological tests indicated that B N Y V V with different R N A
compositions reached similar concentrations in T.
expansa leaves. This was also suggested by the similarity
of the virus concentrations which were observed in the
sugarbeet cotyledons, which in our procedure (Koenig &
Stein, 1990) are directly exposed to the various inocula
(Table 1). Control plants were mock-inoculated with 0.05
M-phosphate buffer p H 7.2. The inoculated seedlings
were potted and kept in a greenhouse for up to 12 weeks.
T h e B N Y V V R N A composition in T. expansa and
sugarbeets was checked by N o r t h e r n blot analyses
(Burgermeister et al., 1986). E L I S A was done as
described by Clark & A d a m s (1977).
Plants which had received an inoculum containing
R N A 3 developed yellow spots on the cotyledons and
also on the first true leaves which had not yet developed
at the time o f inoculation. T h e plants grew poorly
c o m p a r e d to the control plants or those which had been
inoculated with virus containing only R N A s 1 and 2 or 1,
2 and 4 (Fig. 1). These effects were more p r o n o u n c e d
with the highly susceptible than with the partially
resistant variety (Fig. 1, upper and lower panels). T h e tap
Fig. 1. Sugarbeet plants of a highly susceptible (upper panel) and a
partially resistant (lower panel) variety 9 weeks after inoculation with
BNYVV isolates of different RNA composition: B, RNAs 1 and 2; C,
RNAs 1, 2 and 4; D, RNAs l, 2 and 3; E, RNAs 1, 2, 3 and 4. The
control plants (A) were mock-inoculated with 0.05 ~l-phosphate buffer
pH 7-2. Thirty plants were inoculated in each group; the photographs
show one typical plant from each group.
A
B
C
D
E
Fig. 2. Sugarbeets harvested from plantsofa highly susceptible (upper
panel) and a partially resistant (lower panel) variety 12 weeks after
inoculation of the seedlings with BNYVV isolates of different RNA
composition: B, RNAs 1 and 2; C, RNAs 1,2 and 4; D, RNAs 1, 2 and
3 ; E, RNAs 1, 2, 3 and 4. Control plants (A) were mock-inoculated with
0.05 M-phosphate buffer pH 7.2. Each group consisted of 30 plants. For
total weights of the beets harvested in each group and ELISA readings
see Table 1.
roots were harvested 12 weeks after inoculation and the
effects o f different virus inocula were assessed visually
(Fig. 2) and by weight determination (Table 1, last
column). W h e n R N A s 1 and 2 or R N A s 1, 2 and 4 had
been present in the inoculum, there was some reduction
in yield which varied s o m e w h a t from one experiment to
another. It was rather p r o n o u n c e d in the experiment
described in Fig. 2 and Table 1, but not quite as strong in
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Table 2. ELISA readings for the tap roots of sugarbeets* of
a highly susceptible variety 6 and 9 weeks after mechanical
inoculation with B N Y V V isolates of different RNA
compositions
ELISA reading
RNA composition
of inoculum
1 and 2
1, 2 and 4
I, 2 and 3
1, 2, 3 and 4
6 weeks p.i.
9 weeks p.i.
0.03
0.04
0-40
0.49
0.02
0.02
0-28
0-29
* Each group consisted of 30 plants. The sap from the tap roots was
diluted 1:10 in ELISA sample buffer (Clark & Adams, 1977).
some other experiments, e.g. the one depicted in Fig. 1.
In all our experiments however, a very drastic reduction
in yield, amounting to about 9 5 ~ in the highly
susceptible variety, was observed when R N A 3 had been
present in the inoculum (Fig. 1 and 2, Table 1). The effect
of R N A 4 in the presence of R N A 3 again varied
somewhat in different experiments. In the experiment
described in the upper panel of Fig. 1, R N A 4 further
increased the damage done by R N A 3, but in the
experiment described in Fig. 2 and Table 1 no such
enhancing effect was seen. The yield losses under the
influence of R N A 3 were much more pronounced in the
highly susceptible than in the partially resistant variety
(Fig. 2, Table 1).
Twelve weeks after inoculation the virus concentration in the tap roots was much higher in plants which had
received an inoculum containing R N A 3 than in those
which had been inoculated with virus lacking R N A 3,
although 2 weeks after inoculation similar virus concentrations were found in the cotyledons of all inoculated
plants (Table 1). An at least 10-fold increase in the virus
concentration in the presence of R N A 3 was also
observed in the tap roots of plants which were harvested
6 to 9 weeks after inoculation (Table 2). These
observations, like earlier ones by Tamada & Abe (1989)
and Tamada et al. (1990), suggest that R N A 3 in
sugarbeet roots facilitates the replication and/or the
translocation of the virus.
Our results also confirm, with well defined inocula
from recombination experiments rather than field
isolates, the recent conclusion of Tamada et al. (1990)
that it is mainly R N A 3 that is responsible for the
damaging effect of the virus on sugarbeets. A difference
between our observations and those of Tamada et al.
(1990) is that they also observed a massive proliferation
of fine rootlets, i.e. the typical symptom of root
'beardedness', when R N A 3 had been present in the
inoculum. In our experiments the side roots were brown
2245
and very brittle and often deteriorated but neither in the
highly susceptible nor in the partially resistant variety
did we encounter excessive root proliferation. Since
Tamada et al. (1990) used P. betae as a vector for the virus
and we employed mechanical inoculation, the differences in the observations might suggest a possible
involvement of the vector P. betae in the production of
root 'beardedness'. Alternatively, the exposure of the
sugarbeet seedlings at a very young age to a heavy
inoculum as under our experimental conditions may
weaken the plants to such an extent that massive root
proliferation is impaired. Alternatively, it may lead to an
early deterioration of proliferating rootlets which would
be lost prior to or while the plants are removed from the
soil. The tap roots from plants that received an inoculum
containing R N A 3 were very easily removed from the
soil. This was not the case with plants which had been
mock-inoculated or inoculated with BNYVV lacking
R N A 3, and had developed normal side roots.
The financial support given by the Deutsche Forschungsgemeinschaft (grant Ko 518/11-1) and the Gemeinschaft zur F6rderung der
privaten deutschen Pflanzenziichtung e.V. for different aspects of this
research is gratefully acknowledged.
References
BOUZOUBAA,S., QUILLET, L., GUILLEY, H., JONARD, G. & RICHARDS,
K. (1987). Nucleotide sequence of beet necrotic yellow vein virus
RNA-I. Journal of General Virology 68, 615-626.
BURGERMEISTER, W., KOENIG, R., WEICH, H., SEBALD, W. &
LESEMANN, D.-E. (1986). Diversity of the RNAs in thirteen isolates
of beet necrotic yellow vein virus in Chenopodium quinoa detected by
means of cloned cDNA. Journal of Phytopathology 115, 229 242.
CLARK, i . e. & ADAMS,A. N. (1977). Characteristics of the microplate
method of enzyme-linked immunosorbent assay for the detection of
plant viruses. Journal of General Virology 34, 475-483.
JARAUSCH, W., COMMANDEUR, U., L1, Y., KOENIG, R., BURGERMEISTER, W. • LESEMANN,D.-E. (1990). Infectious in vi~o transcripts of
beet necrotic yellow vein virus cDNA clones containing the 35S
promoter. First Symposium of the International Working Group on
Plant ViruseswithFungal Vectors,Braunschweig,Germany,August 2124, 1990, Abstracts of Papers, p. 4.
KOENIG, R. & BURGERMEISTER, W. (1989). Mechanical inoculation of
sugarbeet roots with isolates of beet necrotic yellow vein virus having
different RNA compositions. Journal of Phytopathology 124, 249255.
KOENIG, R. & EHLERS, U. (1989). Influence of the RNA composition of
beet necrotic yellow vein furovirus isolates and of the sugarbeet
cultivar on the translocation of the virus in mechanically inoculated
sugarbeet roots. EPPO Bulletin 19, 527-530.
KOENIG, R. & STEIN, B. (1990). Distribution of beet necrotic yellow
vein virus in mechanically inoculated sugarbeet plantlets of cultivars
with different degrees of rhizomania resistance. Schriftenreihe der
Deutschen Phytomedizinischen Gesellsehaft, vol. 1. Proceedings of the
First Symposium of the International Working Group on Plant Viruses
with Fungal Vectors, Braunschweig, Germany, August 21-24, 1990, pp.
87-90.
KOENIG, R., BURGERME1STER,W., WEICH, H., SEBALD, W. & KOTHE,
C. (1986). Uniform RNA patterns of beet necrotic yellow vein virus
in sugarbeet roots, but not in leaves from several plant species.
Journal of General Virology 67, 2043-2046.
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On: Sun, 18 Jun 2017 06:29:19
2246
Short communication
KUSZALA, M., ZIEGLER, V., BOUZOUBAA,S., RICHARDS, K., PUTZ, C.,
GUILLEY, H. & JONARD,G. (1986). Beet necrotic yellow vein virus:
different isolates are serologically similar but differ in RNA
composition. Annals of Applied Biology 109, 155 162.
LEMAIRE, O., MERDINOGLU, D., VALENTIN, P., PUTZ, C., ZIEGLERGRAFF, V., GUILLE¥, H., JONARD, G. & RICHARDS, K. (1988). Effect
of beet necrotic yellow vein virus RNA composition on transmission
by Polymyxa betae. Virology 162, 232-235.
LI, Y., JARAUSCH, W., COMMANDEUR, U., KOENIG, R., BURGERMEISTER, W. & LESEMANN, D.-E. (1990). Infectious in vivo transcripts of
beet necrotic yellow vein virus cDNA clones containing the 35S
promoter. Schriftenreihe der Deutschen Phytomedizinischen Gesellschaft, vol. 1. Proceedings of the First Symposium of the International
Working Group on Plant Viruses with Fungal Vectors. Braunschweig,
Germany, August 21-24, 1990, pp. 49-52.
TAMADA, T. & ABE, H. (1989). Evidence that beet necrotic yellow vein
virus RNA-4 is essential for efficient transmission by the fungus
Polymyxa betae. Journal of General Virology 70, 3391 3398.
TAMADA,T., SIqlRAKO,Y., ABE, H., SAITO, M., KIGUCHI, T. & HARADA,
T. (1989). Production and pathogenicity of isolates of beet necrotic
yellow vein virus with different numbers of RNA components.
Journal of General Virology 70, 3399-3409.
TAMADA,T., SAITO,M., KIGUCHI, T. & KUSUME, T. (1990). Effect of
isolates of beet necrotic yellow vein virus with different RNA
components on the development of rhizomania symptoms. Schriftenreihe der Deutschen Phytomedizinischen Gesellschaft, vol. I. Proceedings of the First Symposium of the International Working Group on Plant
Viruses with Fungal Vectors, Braunschweig, Germany, August 21-24,
1990, pp. 41-44.
(Received 23 April 1991 ; Accepted 5 June 1991)
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