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JOURNAL OF VIROLOGY, May 2011, p. 4974–4981 Vol. 85, No. 10
0022-538X/11/$12.00 doi:10.1128/JVI.00057-11
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Contact Transmission of Tobacco Mosaic Virus: a Quantitative Analysis
of Parameters Relevant for Virus Evolution
䌤
Soledad Sacrista´n,* Maira Díaz,† Aurora Fraile, and Fernando García-Arenal
Centro de Biotecnología y Geno´mica de Plantas (UPM-INIA) and E.T.S.I. Agro´nomos, Universidad Polite´cnica de Madrid,
Campus Montegancedo, 28223 Pozuelo de Alarco´n, Madrid, Spain
Received 10 January 2011/Accepted 21 February 2011
Transmission between hosts is required for the maintenance of parasites in the host population and
determines their ultimate evolutionary success. The transmission ability of parasites conditions their evolution
in two ways: on one side, it affects the genetic structure of founded populations in new hosts. On the other side,
parasite traits that increase transmission efficiency will be selected for. Therefore, knowledge of the factors and
parameters that determine transmission efficiency is critical to predict the evolution of parasites. For plant
viruses, little is known about the parameters of contact transmission, a major way of transmission of important
virus genera and species. Here, we analyze the factors determining the efficiency of contact transmission of
Tobacco mosaic virus (TMV) that may affect virus evolution. As it has been reported for other modes of
transmission, the rate of TMV transmission by contact depended on the contact opportunities between an
infected and a noninfected host. However, TMV contact transmission differed from other modes of transmis-
sion, in that a positive correlation between the virus titer in the source leaf and the rate of transmission was
not found within the range of our experimental conditions. Other factors associated with the nature of the
source leaf, such as leaf age and the way in which it was infected, had an effect on the rate of transmission.
Importantly, contact transmission resulted in severe bottlenecks, which did not depend on the host suscepti-
bility to infection. Interestingly, the effective number of founders initiating the infection of a new host was
highly similar to that reported for aphid-transmitted plant viruses, suggesting that this trait has evolved to an
optimum value.
During the last 30 years, important efforts have been made
to model the evolution of parasites, since understanding par-
asite evolution is necessary to develop sustainable strategies
for the control of infectious diseases and to anticipate, prevent,
and control new emergences. This is particularly so in the case
of RNA viruses, which make up the largest fraction of emerg-
ing pathogens of humans, animals, and plants (6, 24, 47). Ex-
perimental work has not followed the pace of theory, and there
is a paucity of information on the values of key parameters in
evolutionary models, on the relationship among various pa-
rameters, and on the applicability of model assumptions. Data
on parameters related to virus transmission are particularly
scant, despite the fact that horizontal transmission is a critical
step of the infective cycle of parasites. Indeed, between-host
transmission and within-host multiplication are the two main
components of parasite fitness, and thus, knowledge on the
rates of transmission and multiplication and on the relation-
ship between the two parameters is required to understand
parasite evolution. Most models of parasite evolution consider
that within-host multiplication and between-host transmission
are linked traits, so that parasites must multiply up to a certain
level for transmission to occur and that rates of transmission
are positively correlated with rates of multiplication, at least
within a range of values (14, 17, 32, 33). Although this is a
reasonable assumption, its experimental support for plant vi-
ruses derives from a limited number of systems that represent
few transmission mechanisms (see below). Also, since viruses
may multiply to very high levels within the infected hosts, virus
evolution has often been analyzed using purely deterministic
models in which selection is considered to be the primary
evolutionary factor acting on virus populations (9, 10). How-
ever, the relevant evolutionary parameter is not the total num-
ber of individuals in the population but the effective population
number (N
e
), which can be grossly assimilated to the number
of individuals that pass their genes to the next generation. At
small N
e
values, random genetic drift will predominate over
selection (7). The occurrence of population bottlenecks during
the virus life cycle will result in the reduction of N
e
, hence the
interest in identifying and quantifying these bottlenecks. Pop-
ulation bottlenecks may occur when a new virus population is
initiated by horizontal transmission, resulting in a type of ge-
netic drift known as the founder effect, given that the new virus
population is initiated from a small number of genotypes ran-
domly sampled from the mother population. It has been shown
that severe population bottlenecks may occur during horizon-
tal transmission of plant viruses, as infection of a new host may
be started by just a small number of individuals (12, 27). Once
again, however, this information derives from just a few sys-
tems that represent only one of the various possible mecha-
nisms of plant virus transmission.
In nature, plant viruses are horizontally transmitted either
* Corresponding author. Mailing address: Centro de Biotecnología y
Geno´mica de Plantas (UPM-INIA) and E.T.S.I. Agro´nomos, Univer-
sidad Polite´cnica de Madrid, Campus Montegancedo, 28223 Pozuelo
de Alarco´n, Madrid, Spain. Phone: 34 91 336 4563. Fax: 34 91 715
7721. E-mail: soledad.sacristan@upm.es.
† Present address: Departamento de Cristalografía y Biología Es-
tructural de Proteínas, Instituto de Química Física Rocasolano, Ser-
rano 115, 28006 Madrid, Spain.
䌤
Published ahead of print on 2 March 2011.
4974
by contact or by means of vectors. Transmission by aphids,
which vector the highest number of virus genera, has been best
analyzed. The relationship between virus accumulation in the
source leaf and transmissibility by aphids has been analyzed for
a number of systems, showing that both traits, as assumed in
evolutionary models, are positively correlated (3, 13, 16, 29). It
has also been shown that nonpersistent transmission by aphids
results in stochastic effects in the composition of the transmit-
ted population (1), thus indicating the existence of founder
effects due to the low number of particles transmitted. Accord-
ingly, the effective number of founders (N
ef
) after aphid trans-
mission has been estimated to be on the order of units (4, 37).
In contrast, little is known about the parameters of trans-
mission by contact, despite the fact that this is the major way of
transmission during field epidemics of important viruses in
genera such as Tobamovirus,Potexvirus, and Hordeivirus (15).
The little attention paid to contact transmission may be ex-
plained by the general assumption that it should not be essen-
tially different from experimental mechanical inoculation,
which is a routine technique in plant virology for transferring
virus populations to new hosts. Mechanical inoculation consists
of applying a suspension of viral particles or RNA (for viruses
with a single-stranded RNA genome of messenger sense) on
the leaf surface, in which infection occurs after the leaf is
gently rubbed. Early work by plant virologists extensively ana-
lyzed the process of mechanical inoculation (reviewed in ref-
erence 20). However, no attempts have been made to analyze
if these results could be applied to understand the nature of
contact transmission.
The goal of this work is to characterize the evolutionarily
relevant parameters of contact transmission of plant viruses.
We used experimental populations of Tobacco mosaic virus
(TMV) to address two major questions: (i) if the rate of con-
tact transmission is positively correlated with virus accumula-
tion in the source leaf and (ii) if contact transmission results in
population bottlenecks leading to founder effects. Our data
show that the rate of transmission does not depend primarily
on the virus titer in the source leaf. We also show that contact
transmission results in severe population bottlenecks.
MATERIALS AND METHODS
Biological material and virus inoculations. Two TMV genotypes were used:
wild-type TMV (wt TMV) and the coat protein mutant P20L-TMV, which has
the transition C5656U, resulting in the amino acid replacement P 3L at position
20 of the coat protein. These TMV genotypes were derived from biologically
active cDNA clones that have been described elsewhere (8) and were a gift of
W. O. Dawson and J. N. Culver. Infectious RNA was transcribed from these
clones with T7 (for wt) or SP6 (for P20L mutant) RNA polymerase as previously
described (8) and was inoculated into Nicotiana tabacum cv. Samsun plants
suspended in 0.1 M Na
2
HPO
4
. Virus particles were purified from plants infected
with RNA transcripts as described previously (5). Virus suspensions in 10 mM
sodium phosphate buffer (pH 7.2) were used for further inoculations. Two
tobacco genotypes were used: Samsun, which is fully susceptible to systemic
infection by TMV, and Xanthi-nc, which is hypersensitively resistant to TMV
infection, thus showing necrotic local lesions (nlls) around infection foci. All
plants were kept in a greenhouse at 20 to 25°C with 16 h of light.
Detection of TMV genotypes and quantification of their accumulation in
infected tobacco leaves. TMV genotypes present in individual nlls were detected
in lesion prints on nylon membranes that were hybridized with 5⬘
32
P-labeled
oligonucleotide probes specific for each TMV genotype. The genotype-specific
oligonucleotide probes used for these analyses and hybridization conditions have
been described previously (44). Virus accumulation was quantified as viral RNA
accumulation. Total RNA was extracted from 0.2 g (fresh weight) tobacco leaves
(36) and resuspended in 50 l of distilled water. Viral RNA in each sample was
quantified by dot blot hybridization with genotype-specific
32
P-labeled oligonu-
cleotide probes. In each blot, internal standards for each genotype were included
as a 2-fold dilution series of purified RNA (2 to 0.015 g) in nucleic acid extracts
from noninoculated tobacco plants, as described previously (44). Total RNA of
mock-inoculated tobacco plants was used as a negative control. Different
amounts of nucleic acid extracts of each sample were blotted to ensure that the
hybridization signal was in the linear portion of the RNA concentration-versus-
hybridization signal curve. The RNA hybridization signal was detected using a
Typhoon 9400 scanner (GE Healthcare, Chalfont St. Giles, United Kingdom)
after exposure of the labeled samples to Eu
2⫹
-containing phosphor screens and
was quantitated using Image-Quant (version 5.2) software (Molecular Dynamics,
GE Healthcare).
Statistical analyses. All the statistical analyses are described in references 40
and 45. Means were compared by the nonparametric Wilcoxon signed rank test.
Correlation between data was analyzed by the nonparametric Spearman rank
correlation test, and significance was obtained from 1,000 random pairings of
data. Analyses of variance (ANOVAs), general linear models (GLMs), and
Student-Newman-Keuls post hoc contrasts of means were calculated using the
software Statgraphics Centurion XV (StatPointTechnologies, Warenton, VA).
Data were transformed when necessary in order to meet the assumptions for
parametric analyses.
RESULTS
Efficiency of contact transmission under controlled condi-
tions. Contact transmission between plants was simulated by
brushing one leaf of a recipient plant with an infected source
leaf. Source leaves were collected from Samsun tobacco plants
systemically infected after inoculation with 600 ng of wt-TMV
particles per leaf. Three types of source leaves were used:
inoculated leaves (L0 leaves) collected either at 4 or at 7 days
postinoculation (dpi) and the systemically infected second leaf
above L0 (L2 leaves) collected at 10 dpi. Five leaves of each
type, each from a different infected tobacco plant, were used to
inoculate two sets of 20 5-week-old Xanthi-nc tobacco plants,
with the recipient leaf always being the youngest fully ex-
panded leaf of each plant. One set of plants was inoculated by
brushing the recipient leaf once with the source leaf; the other
set was inoculated by brushing the recipient leaf 10 times with
the source leaf. In this way, the transmission efficiency during
a single contact event (1 brush) and the transmission efficiency
during a more prolonged contact (10 brushes) were compared.
In each source leaf, TMV accumulation in the area that had
been in contact with the recipient leaf was quantified. At 4 days
postinoculation, the recipient Xanthi-nc tobacco leaves were
collected and local lesions were counted.
The number of plants with nlls is a measure of the number
of successful transmission events. Ten brushes always resulted
in more plants infected than a single brush (Table 1), and both
values were positively correlated for each source leaf (Spear-
man correlation coefficient r
S
⫽0.80, P⫽0.003). In addition,
the mean number of lesions was higher in recipient leaves
brushed 10 times than in those brushed once (Wilcoxon signed
rank test, Pⱕ0.004; Table 1). No correlation was found be-
tween virus accumulation in the source leaf and either the
number of plants with nlls or the mean number of nlls per
recipient leaf (P⫽1 in both cases). The number of plants
showing nlls was higher after they were brushed with the sys-
temically infected source L2 leaves than with L0 leaves (P⬍
0.01; Table 1), although the viral titer in L2 leaves was signif-
icantly lower than that in L0 leaves (P⫽0.03; Table 1). There-
fore, the number of successful transmission events depended
on the number of contact opportunities between the source
VOL. 85, 2011 CONTACT TRANSMISSION OF TOBACCO MOSAIC VIRUS 4975
and recipient leaves but not on the viral titer in the source leaf.
Since systemically infected leaves were a more efficient source
than inoculated leaves, factors other than contact opportunity,
related to the nature of the source leaf, must also determine
the efficiency of contact transmission: L2 leaves are younger
than L0 leaves, which suggests that leaf age might be one such
factor. Results also suggest that the mode in which the source
leaf had been infected (directly inoculated or systemically in-
fected) might be another factor.
Source leaf factors that determine efficiency of contact
transmission. In order to determine which factors associated
with the nature of the source leaf affect its infectivity, an
experiment was performed in which the effects of age and
mode of infection of the source leaf (directly inoculated or
systemically infected) were analyzed, together with the effect of
virus titer. Two sets of three and eight plants, respectively,
were inoculated (inoculation sets S1 and S2, respectively; Ta-
ble 2), in order to obtain source leaves of different ages that
had been infected either directly (L0 leaves) or systemically
(L2 and L4 leaves, second and fourth leaves above L0, respec-
tively). These source plants were inoculated with a lower viral
dose than in the experiment described in the previous section
(50 ng of virus particles of wt TMV per leaf), in order to
promote variation of viral accumulation at the source leaves
over a larger range of values. S2 plants were inoculated 6 days
later than S1 plants, but in both cases plants were inoculated at
the same age of 30 days. Since the tobacco leaf plastochron
under our growing conditions is 3 days, the difference in age
between two alternate leaves (L0 and L2 leaves or L2 and L4
leaves) was 6 days. This allowed us to use L0, L2, and L4
source leaves of the same and of different ages (Table 2) in a
single transmission experiment, with inoculations of recipient
leaves taking place at 16 and 10 dpi for S1 and S2 plants,
respectively. Thus, the type of source leaf factor could be
differentiated from the source leaf age factor. At the time of
inoculation of source plants, L2 leaves were 4 days old and L4
TABLE 1. Number of infected plants and mean number of nll in the recipient leaf per source leaf
Source leaf
and time
postinoculation
a
TMV
accumulation
in source leaf
b
No. of infected recipient plants
c
No. of nlls per infected recipient leaf
d
1 brush 10 brushes 1 brush 10 brushes
L0
4 dpi 5.9 5 14 1.0 ⫾0.0 1.4 ⫾0.2
5.4 1 13 1.0 ⫾0.0 2.3 ⫾0.3
5.0 3 14 1.0 ⫾0.0 1.6 ⫾0.3
6.0 2 13 1.0 ⫾0.0 1.6 ⫾0.2
Mean ⫾SE
e
5.6 ⫾0.2 2.8 ⫾0.9 13.5 ⫾0.3 1.0 ⫾0.0 1.7 ⫾0.1
7 dpi 6.4 3 10 1.7 ⫾0.5 3.6 ⫾1.0
6.9 2 17 1.0 ⫾0.0 5.2 ⫾1.1
4.7 14 19 3.6 ⫾1.2 38.6 ⫾6.7
4.7 15 20 8.6 ⫾3.6 33.9 ⫾8.3
Mean ⫾SE
e
5.7 ⫾0.6 8.5 ⫾3.5 16.5 ⫾2.3 5.5 ⫾1.7 23.1 ⫾3.7
L2 2.4 18 20 8.5 ⫾1.7 39.7 ⫾4.9
10 dpi 4.5 16 20 3.6 ⫾0.9 25.0 ⫾4.4
3.5 17 20 6.0 ⫾1.2 24.5 ⫾4.1
3.5 20 19 31.2 ⫾3.9 85.2 ⫾12.6
Mean ⫾SE
e
3.5 ⫾0.4 17.8 ⫾0.9 19.8 ⫾0.3 13.2 ⫾1.8 43.2 ⫾4.5
a
Time postinoculation at which the source leaf was used for inoculations. L0, inoculated leaf; L2, systematically infected second leaf above L0.
b
Viral accumulation in the source leaf expressed as micrograms of viral RNA per gram (fresh weight) of leaf.
c
Two sets of 20 Xanthi-nc tobacco plants were inoculated per source leaf by brushing the source leaf either once or 10 times.
d
Data are means and standard errors for the infected recipient leaves.
e
Data are means and standard errors for four source leaves.
TABLE 2. Relationship between transmission efficiency and age and mode of infection of the source leaf
Type of
source leaf
S1
a
S2
Age
b,c
Surface
c,d
TMV
accumulation
c,e
No. of infected
recipient
plants
c,f
No. of nlls
per infected
recipient leaf
g
Age Surface TMV
accumulation
No. of infected
recipient plants
No. of nlls per
infected
recipient leaf
L0 26 269 ⫾26* 2.9 ⫾0.3* 6.7 ⫾0.7* 4.7 ⫾0.9* 20 446 ⫾20* 8.8 ⫾2.6* 8.1 ⫾0.8* 9.3 ⫾0.9***
L2 20 457 ⫾38** 7.9 ⫾1.9** 10.0 ⫾0.0** 24.9 ⫾3.2** 14 592 ⫾28** 27.7 ⫾6.1** 8.9 ⫾1.1* 14.6 ⫾1.3*
L4 14 410 ⫾23** 19.5 ⫾0.2† 10.0 ⫾0.0** 15.7 ⫾2.9** 8 236 ⫾19† 40.3 ⫾4.9** 9.1 ⫾0.4* 5.6 ⫾0.5**
a
Two sets of three (S1) and eight (S2) Samsun tobacco plants were inoculated with a lag period of 6 days. Different symbols in each column indicate significant
differences in Student-Newman-Keuls contrasts of means at 95% confidence.
b
Age (days) of the source leaf L0, inoculated leaf; L2 and L4, 2nd and 4th leaves above the inoculated leaf, systematically infected at the time of the transmission
experiment.
c
Data are means ⫾standard errors of three (S1) or eight (S2) source leaves.
d
Surface (in cm
2
) of the source leaf at the time of the transmission experiment.
e
Viral accumulation in the source leaf expressed as micrograms of viral RNA per gram (fresh weight) of leaf.
f
Number of plants with lesions out of 10 Xanthi-nc tobacco plants inoculated with each source leaf.
g
Data are means ⫾standard errors for the infected recipient leaves.
4976 SACRISTA
´NETAL. J. VIROL.
leaves would appear 2 days later. Systemic infection of those
leaves may have happened at any time after 3 dpi (44), when
both leaves had already started expansion. Therefore, we can
assume that L2 and L4 leaves have been infected for the same
period of time (a maximum of 13 days for inoculation set S1
and 7 days for inoculation set S2). Each source leaf was used to
inoculate a set of 10 5-week-old Xanthi-nc tobacco plants by
brushing 10 times their youngest fully expanded leaf. In each
source leaf, virus accumulation in the area that had been in
contact with the recipient leaf was quantitated. At 4 days post-
inoculation, the recipient Xanthi-nc tobacco leaves were col-
lected and local lesions were counted.
Viral accumulation in the source leaves was compared in a
two-way ANOVA using type of leaf and inoculation set as
factors. Viral accumulation differed significantly according to
type of leaf (F
2,27
⫽9.77; P⫽6⫻10
⫺6
), and it was lower in
the inoculated L0 leaves than in the systemically infected L2
and L4 leaves (Table 2 and Fig. 1A). Also, viral accumulation
was lower in L2 leaves than in L4 leaves. Viral accumulation
was also significantly different according to inoculation set, and
it was higher in S2 than in S1 (F
1,27
⫽7.78; P⫽0.01). There
was no interaction between the two factors (P⫽0.66).
To analyze the effects of the different factors on the effi-
ciency of transmission, the number of inoculated plants with
nlls and the mean number of nlls per recipient leaf were ana-
lyzed using GLMs, in which the factors type of leaf (L0, L2, or
L4 leaves), inoculation set (S1 or S2), and viral accumulation
were included. Only the factor type of leaf had a significant
effect on the number of infected plants (F
2,30
⫽3.84; P⫽0.03),
and transmission was significantly less effective from inocu-
lated leaves (L0 leaves) than from systemically infected L2 or
L4 leaves (Student-Newman-Keuls contrast of means, P⬍
0.05). Hence, the number of infected plants was not dependent
on viral accumulation or the inoculation set. The mean number
of nlls in recipient leaves depended both on the type of source
leaf and on viral accumulation in the source leaves, and the
effect of these factors differed according to the inoculation set
(F
7,25
⫽4.92; P⫽0.001, data not shown). For S1, L0 leaves
(L0S1) were the least effective source leaves (P⬍0.05; Table
2 and Fig. 1B), while for S2, L4 leaves (L4S2) were the least
effective ones and L2 leaves (L2S2) were the most effective
ones (P⬍0.05; Table 2 and Fig. 1B). When S1 and S2 data are
compared, significant differences were found only with L4
leaves (P⬍0.05). L4S2 leaves were the youngest and least
effective source ones, despite having the highest viral content
(Table 2). They were 8 days old when they were used as source
leaves and not yet completely expanded, as shown by their
smaller surface (Table 2). However, source leaf surface was not
a significant factor on transmission efficiency when it was in-
troduced in GLMs (data not shown).Therefore, efficiency of
transmission depended on the age of the source leaf and on the
way it was infected.
Estimation of effective number of founders starting an in-
fection by contact transmission. The estimation of the number
of founders starting an infection by contact transmission was
based on the probability of segregation of two alleles in demes
originated from a mother deme (44). Given a source leaf that
is infected by genotypes A and B with frequencies p
sA
and p
sB
,
respectively, assuming that the probability of transmission of
each genotype is given by its frequency in the source leaf and
that both probabilities are independent (p
sA
⫹p
sB
⫽1), the
genetic composition of the transmitted viral population will be
given by the binomial distribution 共psA ⫹psB兲Nef. Hence, the
probability P
0B
that a plant that has been in contact with that
source leaf is infected only by genotype A is given by the
equation
P0B⫽psANef (1)
N
ef
can be estimated from this expression by estimating p
sA
and
P
0B
experimentally. Conversely, the probability P
0A
that a plant
that has been in contact with that source leaf is infected only by
genotype B is given by the expression
P0A⫽psBNef ⫽共1⫺psA兲Nef (2)
If genotypes A and B are not equally fit and selection occurs in
coinfected leaves, the least-fit genotype can be lost before
observation, and hence, the probability of no infection by that
genotype can be overestimated. Therefore, the actual effective
number of founders should lie between those obtained from
equations 1 and 2. The transmission probability of each TMV
genotype will depend on both its frequency in the source leaf
and its transmissibility. In case both genotypes had different
transmissibilities, it would be necessary to correct p
sA
and p
sB
accordingly (4).
FIG. 1. TMV accumulation (A) and efficiency of contact transmis-
sion (B) from different types of source leaves. (A) TMV accumulation
in the source leaf. (B) Mean number of nlls produced by the source
leaf in 20 recipient leaves. Source leaves L0S1, L2S1, and L4S1 cor-
respond to inoculation set S1, and source leaves L0S2, L2S2, and L4S2
correspond to inoculation set S2. Data are means and standard errors
(error bars) from 3 (S1) and 8 (S2) plants.
VOL. 85, 2011 CONTACT TRANSMISSION OF TOBACCO MOSAIC VIRUS 4977
N
ef
can also be maximum likelihood estimated by comparing
the observed frequencies of plants infected with either geno-
type A, genotype B, or both genotypes with those expected
from the binomial distribution 共psA ⫹psB兲Nef.
To estimate the transmissibility of each TMV genotype, two
sets of five Samsun tobacco plants were inoculated with 600
ng/leaf of either wt-TMV or P20L-TMV particles. At 4 dpi, the
inoculated leaf of each plant was collected and used as the
source leaf to inoculate the youngest fully expanded leaf of 20
Xanthi-nc tobacco plants by brushing them 10 times. In each
source leaf, the wt-TMV or P20L-TMV accumulation in the
area that had been in contact with the recipient leaf was quan-
tified (Table 3). In the recipient leaves, nlls were counted at 4
dpi. The transmissibility for each TMV genotype was calcu-
lated as the ratio between the number of plants with lesions
produced by each source leaf and the virus accumulation in
that leaf and was not significantly different for the two different
viral genotypes (P⫽0.46; Table 3). Therefore, N
ef
can be
calculated from equations 1 and 2 with no need for correction
for transmissibility.
To estimate P
0B
and P
0A
, eight Samsun tobacco plants were
inoculated with equal amounts (600 ng/leaf) of wt-TMV and
P20L-TMV particles. This inoculum dose was chosen on the
basis of previous single-lesion infectivity experiments in which
it was shown that the number of nlls induced in Xanthi-nc
tobacco did not differ significantly between the two TMV ge-
notypes at 300 ng/half leaf. At 4 dpi, the inoculated leaf of each
plant was collected and used as the source to inoculate the
youngest fully expanded leaf of 20 Xanthi-nc tobacco plants, as
described above. In each source leaf, the wt-TMV and P20L-
TMV accumulation in the area that had been in contact with
the recipient leaf was quantified (Table 4). In the recipient
leaves, nlls were counted at 4 dpi and the TMV genotype(s)
present in each nll was determined. The number of plants
TABLE 3. Transmissibility by contact of wt and P20L TMV genotypes
TMV genotype
inoculated in
source leaf
Recipient Xanthi-nc tobacco plants Recipient Samsun tobacco plants
TMV
accumulation
in source leaf
a
No. of infected
recipient plants
b
Transmissibility
c
TMV
accumulation
in source leaf
No. of infected
recipient plants Transmissibility
wt 1.1 12 10.7 2.8 8 2.9
1.1 4 3.7 0.4 7 17.5
0.7 8 11.0 1.2 12 10.0
1.0 10 10.1 0.8 16 20.0
2.4 9 3.7 0.9 20 22.2
Mean ⫾SE
d
1.3 ⫾0.3 8.6 ⫾1.3 7.8 ⫾1.7 1.2 ⫾0.4 12.6 ⫾2.4 14.5 ⫾3.6
P20L 0.3 9 29.0 0.9 6 6.7
1.1 13 12.1 0.3 5 16.7
1.1 5 4.6 0.4 1 2.5
0.2 2 9.1 0.3 2 6.7
0.3 2 5.9 0.3 10 33.3
Mean ⫾SE
d
0.6 ⫾0.2 6.2 ⫾2.1 12.1 ⫾4.4 0.4 ⫾0.1 4.8 ⫾1.6 13.2 ⫾5.6
a
Viral accumulation in the source leaf expressed as g of viral RNA per gram (fresh weight) of leaf.
b
Number of infected plants out of 20 plants inoculated with each source leaf.
c
Ratio between the number of infected plants from each source leaf and the virus accumulation in that leaf.
d
Data are means and standard errors for five source leaves.
TABLE 4. Frequency of Xanthi-nc tobacco plants and of nlls infected with only one TMV genotype after contact transmission
from double-infected source leaves
TMV
accumulation
in source leaf
a
Proportion of each genotype in
the source leaf
b
No. of infected
recipient plants
c
Frequency of single infected
recipient plants
d
No. of nlls per
infected
recipient leaf
e
Frequency of nlls with just one
TMV genotype
f
p
sA
p
sB
P
0B
P
0A
P
L0B
P
L0A
0.9 0.90 0.10 12 0.67 0.00 4.4 ⫾1.4 0.74 0.26
0.6 0.94 0.06 3 0.67 0.00 4.0 ⫾2.0 0.92 0.08
1.5 0.93 0.07 4 1.00 0.00 2.0 ⫾1.0 1.00 0.00
1.3 0.90 0.10 9 0.56 0.00 4.7 ⫾1.8 0.81 0.17
0.8 0.75 0.25 10 0.70 0.00 4.2 ⫾1.1 0.86 0.12
0.9 0.87 0.13 9 0.44 0.11 2.3 ⫾0.9 0.67 0.33
2.0 0.87 0.13 12 0.17 0.33 4.1 ⫾1.0 0.69 0.31
1.5 0.86 0.14 5 0.80 0.00 4.2 ⫾1.2 0.95 0.05
1.2 ⫾0.2
g
0.88 ⫾0.02
g
0.12 ⫾0.02
g
8⫾1
g
0.63 ⫾0.09
g
0.06 ⫾0.02
g
3.7 ⫾0.4
g
0.83 ⫾0.04
g
0.17 ⫾0.04
g
a
Viral accumulation in the source leaf expressed as g of viral RNA per gram (fresh weight) of leaf.
b
Fraction of wt-TMV (p
sA
) or P20L-TMV (p
sB
) RNA in the total viral RNA in each leaf.
c
Number of plants with lesions out of 20 plants inoculated with each source leaf.
d
Fraction of plants infected by only wt-TMV (P
0B
) or P20L-TMV (P
0A
).
e
Data are means and standard errors for the infected recipient leaves.
f
Fraction of nlls infected by only wt-TMV (P
L0B
) or P20L-TMV (P
L0A
).
g
Means ⫾standard error values for 8 source leaves.
4978 SACRISTA
´NETAL. J. VIROL.
showing nlls ranged from 3 to 13 per source leaf and was not
correlated with virus accumulation in the source leaf (P⫽0.09;
Table 4). The number of nlls per recipient leaf ranged from 1
to 15, with a mean of 3.7 (mean numbers per source leaf are
shown in Table 4). P
0B
and P
0A
were calculated as the number
of plants with lesions caused only by wt-TMV and only by
P20L-TMV, respectively (Table 4). The frequency of each
TMV genotype in the source leaf (p
sA
and p
sB
) was estimated
from the fraction of wt-TMV or P20L-TMV RNA in the total
viral RNA in each leaf (Table 4). The effective number of
founders transmitted by contact was estimated from the data in
Table 4 using equations 1 and 2 and was between 1.4 and 3.6,
with a maximum-likelihood estimate of 2 (P⫽0.70). More
than one virus particle may be involved in initiating an infec-
tion focus, as shown by the occurrence of nlls in which both
TMV genotypes were present (Table 4). Applying the same
model used to estimate the effective number of founders that
infect a leaf to the data on frequency of lesions with only one
TMV genotype, the mean number of founders initiating an
infection focus was estimated to be between 0.9 and 1.4, with
a maximum-likelihood estimate of 1 (P⫽0.95).
In nature, virus outbreaks occur only between susceptible
plants and transmissibility of a virus could be different for
susceptible (e.g., Samsun tobacco for TMV) and resistant (e.g.,
Xanthi-nc tobacco for TMV) plants. Since there is no infor-
mation on a possible relationship between host susceptibility
and effective number of founders, N
ef
was estimated in an
experiment simultaneously and in a manner similar to that
described above, using Samsun tobacco as recipient plants. It
was shown that transmissibility to Samsun tobacco plants of
wt-TMV and of P20L-TMV did not differ (P⫽0.35) and that
for neither of them was it different from transmissibility to
Xanthi-nc tobacco plants (P⫽0.50; Table 3). The genetic
composition of the virus population in the recipient leaves of
Samsun tobacco plants is shown in Table 5. Once again, the
number of infected plants was not correlated with the virus
concentration in the source leaf (P⫽0.08; Table 5). P
0B
and
P
0A
were calculated as the number of plants infected only with
wt-TMV and only with P20L-TMV, respectively (Table 5).
Estimates of p
sA
and p
sB
were calculated as described above
(Table 5). The effective number of founders transmitted by
contact to a susceptible host estimated from the data in Table
5 according to equations 1 and 2 was between 1.3 and 3.3, with
a maximum-likelihood estimate of 2 (P⫽0.72). Hence, the
effective number of founders did not differ for a resistant and
a susceptible host, although the efficiency of transmission to
the susceptible host was higher (P⫽0.007; Tables 4 and 5).
DISCUSSION
In this work, factors involved in the efficiency of contact
transmission of TMV were analyzed, as these factors may de-
termine key parameters in virus evolution models. Direct con-
tact between infected and noninfected hosts is an important
way of transmission of animal and human viruses, and its
efficiency may result in severe epidemics (11, 31, 35, 41).
Therefore, parameters related to contact transmission are in-
cluded in epidemiological models aimed at predicting epi-
demic outbreaks or at assessing the effectiveness of control
strategies (30). Many plant viruses of economic importance for
crops, such as Tomato mosaic virus,Pepper mild mottle virus,
Potato virus X, and Pepino mosaic virus, are also transmitted by
contact during the course of epidemic outbreaks, regardless of
other mechanisms of vertical or horizontal transmission that
may be relevant in outbreak initiation (15, 25). However, the
epidemics of contact-transmitted plant viruses have been
scarcely studied (27), and no attempt has been carried out to
identify the relevant epidemiological and evolutionary param-
eters. Similarly, the analysis of the mechanisms involved in
contact transmission of plant viruses has been neglected, as it
has been considered a simple and passive phenomenon (20). In
addition, it has been assumed that contact transmission is es-
sentially similar to mechanical inoculation, an experimental
procedure that was extensively studied in the past (20). In
mechanical inoculation, the inoculum is rubbed on the leaf
surface and the use of abrasives increases its efficiency. This is
thought to be due to the generation of wounds that facilitate
the direct contact of infectious particles with an infectible site,
i.e., with a leaf cell whose transiently exposed protoplast is
susceptible to infection (19, 25). Contact transmission might
TABLE 5. Frequency of Samsun tobacco plant infection with only one TMV genotype after contact transmission
from double-infected source leaves
TMV
accumulation
in source leaf
a
Proportion of each genotype in source leaf
b
No. of infected
recipient plants
c
Frequency of single infected
recipient plants
d
p
sA
p
sB
P
0B
P
0A
0.7 0.88 0.12 14 0.50 0.00
0.8 0.88 0.12 17 0.24 0.12
1.1 0.91 0.09 16 0.50 0.19
0.9 0.88 0.12 9 0.89 0.00
0.8 0.90 0.10 10 0.80 0.10
1.9 0.87 0.13 16 0.94 0.06
0.6 0.90 0.10 20 0.70 0.00
2.2 0.88 0.12 18 0.83 0.00
1.1 ⫾0.2
e
0.89 ⫾0.004
e
0.11 ⫾0.004
e
15 ⫾1
e
0.67 ⫾0.09
e
0.06 ⫾0.03
e
a
Viral accumulation in the source leaf expressed as g of viral RNA per gram (fresh weight) of leaf.
b
Fraction of wt-TMV (p
sA
) or P20L-TMV (p
sB
) RNA in the total viral RNA in each leaf.
c
Number of plants with lesions out of 20 plants inoculated with each source leaf.
d
Fraction of plants infected by only wt-TMV (P
0B
) or P20L-TMV (P
0A
).
e
Means ⫾standard error values for 8 source leaves.
VOL. 85, 2011 CONTACT TRANSMISSION OF TOBACCO MOSAIC VIRUS 4979
differ from mechanical inoculation because the recipient leaf
does not enter into contact with a suspension of virus particles
but enters into contact with an infected source leaf from which
infectious particles must be released, and the factors that favor
inoculum release are unknown. Also unknown is whether con-
tact between the source and recipient leaves results in the
generation of transiently susceptible cells, similarly to leaf rub-
bing in mechanical inoculation. Because of these two uncer-
tainties, the probability that an infectious viral particle encoun-
ters an infectible site in the recipient leaf during contact
transmission might be largely different from that in mechanical
inoculation. In our experiments, the efficiency of contact trans-
mission, estimated to be the number of successful transmission
events from a source leaf, was positively correlated with the
number of contacts between the source and recipient leaves.
This result suggests that during leaf contact, microwounds are
produced in the surfaces of both leaves, which result in the
liberation of infectious particles from the source leaf and in the
induction of infectible sites in the recipient leaf, as it has been
hypothesized (19, 25). Although this result is not unexpected,
it is highly significant: all epidemiological models of parasite
evolution and dynamics assume that transmission rates are
positively correlated with the number of contacts (direct or
indirect) between infected and susceptible noninfected hosts
(2, 26, 42), and our result shows that this is a realistic assump-
tion for TMV and, possibly, other contact-transmitted plant
viruses.
A second relevant result is that the efficiency of TMV con-
tact transmission, estimated to be the fraction of infected
plants, was not a function of virus titer in the source leaf for the
broad range of virus titers analyzed, which covered a 14-fold
increase in virus accumulation (between 2.9 and 40.3 g TMV
RNA/g fresh leaf tissue; Table 2). This is an unexpected result,
and if it is general, it would set contact transmission widely
apart from vector transmission. Indeed, it has been shown
repeatedly that the rate of plant virus transmission by ho-
mopterous insect vectors is positively correlated with virus titer
in the source leaf for virus titer ranges similar to those reported
here (3, 13, 16, 29). The consequences of this result for the
analysis of virus evolution are also important: if rates of trans-
mission are independent of within-host virus multiplication,
the applicability of most models of virulence evolution to con-
tact-transmitted plant viruses could be questioned. Indeed,
most of the theory on evolution of virulence rests on the
trade-off hypothesis, which assumes that within-host multipli-
cation, between-host transmission, and virulence of parasites
are positively correlated traits, among which trade-offs exist
(17). The underlying concept is that the parasite needs to
multiply within the host to a level that allows its transmission to
new hosts and that virulence would be an unavoidable conse-
quence of the within-host multiplication of the parasite. In our
case, such a correlation has not been found, although a mar-
ginal correlation (in one experiment out of four) was found
between the number of infection foci in the recipient leaf and
the virus titer in the source. Also, we obtained no evidence for
a lower threshold of TMV accumulation below which contact
transmission would not occur: efficient transmission was ob-
served from source leaves in which the level of TMV accumu-
lation was as low as 0.2 g RNA/g fresh tissue (data not
shown). Thus, if there is a threshold of TMV accumulation
below which contact transmission does not occur, it must be
very low.
Interestingly, the efficiency of TMV contact transmission
seems to be dependent on unsuspected factors associated with
the nature of the source leaf. One such factor was the devel-
opmental stage of the source leaf, with younger leaves being
less efficient source leaves than older ones. Age-associated
changes in thickness, structure, and chemical composition of
the cuticle (28, 39) might render older leaves more abrasive,
thus increasing the opportunities for contact transmission. Our
results also show that systemically infected leaves are better
sources for TMV transmission than directly inoculated leaves,
which could be related to the much more even virus invasion of
the mesophyll in systemically infected leaves than in directly
inoculated ones (21, 43, 46, 48).
As it is the case for all other modes of viral transmission
analyzed so far, contact transmission of TMV results in severe
population bottlenecks. The N
ef
initiating a TMV infection
after a contact transmission event is in the order of units
(between 1 and 4), with 2 being the most likely estimate. This
number imposes a severe bottleneck, since the number of
TMV particles in a tobacco leaf may reach 10
7
to 10
9
(23, 34,
38). The N
ef
after a contact transmission is similar to that
reported for nonpersistent aphid transmission (N
ef
of about 1
for Potato virus Y [37] and between 1 and 2 for Cucumber
mosaic virus [4]) or for mite transmission (3, as estimated from
reference 22). Mechanical inoculation also constitutes a severe
bottleneck, with values of N
ef
being between 1.6 and 7, highly
similar to those reported here (44). All these estimates are
based on similar probabilistic considerations, and future work
should reexamine N
ef
estimates on the basis of deep sequenc-
ing analyses of the virus population in source and recipient
leaves. However, current analyses are consistent in detecting
severe population bottlenecks associated with horizontal trans-
mission, which indicate that purely deterministic models of
virus and virulence evolution should be modified to integrate
the effects of random genetic drift. The effective number of
founders that initiate an nll was estimated to be 1. This value
agrees with older results from mechanical inoculation experi-
ments that lead to the one particle-one site hypothesis of the
initiation of infection (18, 19). If an nll is initiated by a single
genotype, comparison of the effective number of founders per
leaf with the number of observed nlls per leaf indicates that
most infection foci initiated during contact do not contribute to
the genetic diversity of the TMV population within a leaf. In
agreement with this hypothesis, the value of N
ef
did not depend
on the susceptibility to transmission of the recipient host,
which was much higher for Samsun than for Xanthi-nc tobacco
plants, which suggests that N
ef
has evolved to an optimum
value.
Thus, our results show unsuspected traits of contact trans-
mission that differ widely from vector transmission: one is that
the efficiency of contact transmission is not positively corre-
lated with virus titer in the source leaf, and the other is the
dependency of transmission efficiency on specific properties of
the source leaf. The higher rates of transmission from mature,
systemically infected leaves prompt us to speculate on a pos-
sible relationship between the rate of systemic host invasion
and the rate of transmission for a contact-transmitted virus.
Within the conceptual framework of the trade-off hypothesis of
4980 SACRISTA
´NETAL. J. VIROL.
the evolution of virulence, which assumes that within-host mul-
tiplication and between-host transmissions are linked traits, it
would thus be the rate of systemic host colonization rather
than the within-leaf multiplication rate that is the within-host
component of the viral fitness subject to selection. Future
experiments will be required to verify this hypothesis.
ACKNOWLEDGMENTS
This work has been supported by grant AGL2008-02458/AGR from
the Ministerio de Ciencia e Innovacio´n, Spain, to F.G.-A. M.D. was
supported by fellowship SENACYT-IFARHU of Panama-Programa
de Investigadores.
We thank Miguel Angel Iba´n˜ez, Department of Statistics, E.T.S.I.
Agro´nomos, Universidad Polite´cnica de Madrid, for advice with the
GLM analysis.
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