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New Phytol. (1999), 142, 483–494
Effects of geminivirus infection and
growth irradiance on the vegetative growth
and photosynthetic production of
Eupatorium makinoi
S. FUNAYAMA* I. TERASHIMA
Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305–8572,
Japan
Received 5 October 1998; accepted 8 March 1999
Eupatorium makinoi plants with or without geminivirus infection were grown in shading frames with 70, 15 and
5.5% sunlight. Growth characteristics of these plants in the early vegetative phase were compared by means of
growth analysis. We also measured leaf photosynthetic gas exchange rates and examined relationships between leaf
photosynthesis and whole-plant growth. Relative growth rate (RGR¯ (1}W)¬(dW}dt), where W is plant dry
mass) of virus-infected plants was lower than that of uninfected plants under all three light conditions. The
reduction of RGR by infection was increased with irradiance. The net assimilation rate (NAR¯ (1}A)¬(dW}dt),
where A is total leaf area of the plant) was also reduced both by infection and shading. NARs that were estimated
from light-response curves of leaf photosynthesis, in situ measurements of irradiance, and respiration rates of
leaves, stems and below-ground parts, agreed very well with the values obtained by conventional growth analysis
techniques. Decreases in the estimated NAR value from infection and shading were mostly explained by the
decreases in leaf photosynthesis. These results clearly showed that lowered RGR in virus-infected plants was
attributed mainly to impaired photosynthesis in virus-infected leaves.
Key words: Eupatorium makinoi, geminivirus, growth analysis, NAR, leaf photosynthesis.
Disease is one of the most important biotic factors
which affect plant fitness (Paul & Ayres, 1986a,b;
Jarosz & Burdon, 1992; for a review, see Jarosz &
Davelos, 1995). It is usually considered that foliar
pathogens will reduce fitness of their host plants by
impairing photosynthetic production. From the
ecological point of view, it is important to clarify,
quantitatively, the effects of plant pathogens on host
fitness.
Many studies have reported impaired photosyn-
thesis of diseased leaves (Balachandran et al., 1994)
and some reports show impaired growth of their host
plants (Paul & Ayres, 1986a; Navas et al., 1998).
However, little effort has been made to examine,
quantitatively, the relationship between whole-plant
performance and photosynthetic properties of dis-
eased leaves. Studies by Ben-Kalio & Clarke (1979)
*Author for correspondence (present address) : Department of
Biology, Graduate School of Science, Osaka University,
Machikaneyama-cho 1-16, Toyonaka, Osaka 560-0043, Japan (fax
81 6 6850 5808; e-mail funayama!chaos.bio.sci.osaka-u.ac.jp).
and Sims et al. (cited as an unpublished study in a
review by Balachandran et al., 1997) are exceptions.
Ben-Kalio & Clarke (1979) found that the growth
rate of Senecio vulgaris, infected by powdery mildew,
decreased, due not only to reduced leaf area but also
to a smaller net assimilation rate (NAR). Sims et al.
observed, in Plantago lanceolata grown under weak
light conditions, that the decrease in RGR by
infection with tobacco mosaic virus (TMV) was not
due to impaired leaf photosynthesis but accelerated
leaf turn-over. Both studies successfully applied
conventional growth analysis techniques. When
growth analysis is conducted concomitantly with the
measurement of leaf photosynthesis, as was done by
Sims et al., quantitative relationships between
whole-plant performance and leaf photosynthesis are
obtained. This would greatly improve our mech-
anistic understanding of growth impairment in
diseased plants. Based on such relationships, one can
even argue fitness of the infected plants under
various environmental conditions in their natural
habitats.
We have already reported that the photosynthetic
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484 S. Funayama and I. Terashima
rate of virus-infected leaves of E. makinoi was
depressed both in the initial slope and light-saturated
regions of the light-response curve (Funayama et al.,
1997b). The initial slope of the light-response curve
decreased with the reduction in chlorphyll (Chl)
content, but the light-saturated rate was not de-
pressed until leaf Chl content was !c. 0.1 mmol m−#
(Funayama et al., 1997b). The depression of the
initial slope therefore occurs even in leaves with
moderate disease symptoms, while depression of the
light-saturated rate of photosynthesis occurs only in
severely infected leaves. We have also reported that
the performance of virus-infected E. makinoi plants
differed depending on light environment: shading
accelerated the adverse effects of virus infection
(Funayama et al., 1997a). In these studies, however,
the relationships between leaf photosynthesis and
performance of the whole plants were not fully
examined.
In the present study, therefore, we conducted
growth analysis and measurements of leaf photo-
synthesis on the same E. makinoi plants to examine
to what extent the effects of virus infection on leaf
photosynthesis explained the difference in whole-
plant performance.
Study system
Eupatorium makinoi Kawahara et Yahara is a short-
lived perennial, and is frequently infected by a
geminivirus in natural habitats. Infected plants are
distinguished by visible symptoms: yellow vein. The
detailed characteristics of E. makinoi and geminivirus
have been described previously (Funayama et al.,
1997a).
Growth conditions
On 14 April 1997, 300 plants of E. makinoi (150
virus-infected and 150 uninfected) were collected
from a population on Mt. Futago, Kanagawa
Prefecture, Japan (35° 17« N, 139° 36« E, 207 m
above sea level). The infection status of the plants
was determined by visible symptoms. Until trans-
plantation, these plants were kept in cool dark boxes.
These plants were planted in unglazed clay pots (25
cm diameter, 20 cm height, one plant per pot) filled
with vermiculite. The pots were placed in a frame
with 15% sunlight, in the Agricultural and Forestry
Centre of University of Tsukuba (36° 6« N, 140° 6«E). On 6 May, the plants were transferred to three
frames with different light conditions so that each
frame had 50 each of the infected and uninfected
plants. The high-light (HL) frame was covered with
a sheet of transparent polyethylene film. The
medium-light (ML) and low-light (LL) frames were
covered with three and five layers of black shading
screen. Light conditions of these frames were: 70%
of full sunlight for HL, 15% for ML and 5.5% for
LL. The plants were watered daily and also supplied
with 500 ml of the 1}1000 strength of Hyponex, a
commercial fertilizer (Murakami Bussan Co.,
Tokyo, Japan), twice weekly throughout the ex-
periment. Positions of the pots were rotated within
each experimental block twice a week to avoid
positional effects. Since geminivirus infecting E.
makinoi is transmitted by an insect vector, we applied
insecticide to prevent uninfected plants from being
infected during the experimental period (Admire,
Bayer Co., Kansas City, MO, USA). For con-
venience, the six blocks (virus-infected or
uninfected¬three PPFR levels) are abbreviated as
infected}HL and uninfected}LL etc.
Experimental design
Plants were allowed to acclimate to new light
environments for c. 1 month before plant measure-
ments commenced on 4 June 1997. For non-
destructive growth analysis, growth of 20 each of the
infected and uninfected plants was followed for each
of the three light conditions. The mean values of the
initial plant size (estimated g dry mass on 4 June) for
these plants were adjusted to be nearly equal between
the experimental blocks (one-way ANOVA, P¯0.982), and ranged from 1.05³0.22 to 1.12³0.26
(mean³SD, n¯20) for infected}HL to uninfected}LL blocks. Height of the main shoot and lamina
length of all the leaves were measured to the nearest
1 mm and stem diameter was measured at ground
level with calipers to 0.1 mm. For convenience, the
time periods from 4 to 14 June and from 14 to 25
June are termed T1 and T2, respectively.
On 4 and 25 June, 10 each of both infected and
uninfected plants were harvested from each light
condition. Height and basal diameter of the main
shoot, and lamina length and leaf area of all leaves
were determined for each plant. Three disks of 1-cm
diameter per leaf were obtained from fully expanded
youngest leaves of three or four plants in each block
and stored at ®80°C until Chl determination.
Thereafter, plants were divided into leaf, stem and
root parts. These parts were dried for at least 72 h at
70°C, and weighed. Leaf area of each plant was
determined with an automatic leaf area meter (model
AAM-7, Hayashi Denko, Tokyo, Japan). From these
destructive harvests, the relationships between dry
mass of the whole plants and the non-destructive
parameters, height (H in cm) and basal diameter (D
in mm), were determined using the equation:
Biomass¯ a(D#H)$b(D#H)#c(D#H)
(a, b, and c are constants (10) g m−$ ; see Table 1).
This equation was obtained for each block and used
to calculate the biomass throughout the experiment.
The relationships between leaf area and lamina
length of single leaves were also determined for each
of the blocks and expressed as a quadratic function
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Effects of virus infection and irradiance on growth of Eupatorium makinoi 485
Table 1. Constants (10) g m−$) used in the equations
for biomass estimation
a(¬10"!) b(¬10') c(¬10$) r#
High Light
Infected 1.39 ®1.28 5.25 0.97
Uninfected 0.744 ®0.702 4.64 0.97
Medium Light
Infected 3.20 ®2.07 5.00 0.95
Uninfected 1.81 ®1.45 4.31 0.94
Low Light
Infected 18.8 ®4.81 4.85 0.87
Uninfected 16.5 ®4.66 5.12 0.89
(all correlation coefficients exceeded 0.95, data not
shown). These relationships were used to estimate
leaf area from lamina length.
RGR, NAR and leaf area ratio (LAR) were
calculated using estimated data of biomass (W) and
leaf area (A). RGR and NAR were calculated
according to the method of Venus & Causton (1979).
LAR corresponding to RGR and NAR for each
period was calculated as the mean of leaf area:
biomass ratios at the beginning and the end of the
period under consideration. Leaf weight ratio (LWR
¯ leaf mass: total plant mass) and specific leaf area
(SLA¯ leaf area: leaf mass) were calculated using
the data from the two sample dates.
CO#
exchange rates
Light-response curves of photosynthesis were
obtained for three attached leaves at different
insertion levels for each plant during period T2. The
plants in pots were brought to the laboratory and
measurements were made with a portable gas-
exchange system (LI-6400, Li-Cor, Lincoln, NE,
USA). E. makinoi has decussate phyllotaxis and
plants with nine leaf pairs were used for measure-
ments. Photosynthetic rates were measured on three
leaves for each plant, and these leaves included the
youngest fully-expanded leaf from the third leaf pair
from the top (uppermost leaf, U-leaf), a leaf from the
fifth leaf pair (middle leaf, M-leaf), and a leaf from
the seventh leaf pair (lower leaf, L-leaf). Leaves were
pre-illuminated with white light at PPFR of 500–800
µmol m−# s−" for 15–20 min to allow photosynthetic
induction.
During the measurements, light was provided by
red light emitting diodes (LEDs, 6400–02, Li-Cor).
Leaf temperature was 25³1°C (range) and the CO#
partial pressure was 35 Pa. The vapour pressure
deficit, the difference between the water vapour
pressure in the ambient air and the saturated water
vapour pressure at the leaf temperature, was main-
tained at !1 kPa throughout the measurements of
photosynthesis. The rates of photosynthesis were
first measured at 1500 µmol m−# s−" in HL-grown
plants and 800 µmol m−# s−" in ML- and LL-grown
plants. The irradiance was then decreased in a
stepwise manner. The rate of dark respiration was
determined at the end of the measurement sequence.
Gross photosynthetic rate was assumed to be equal
to net gas exchange rate plus the rate of dark
respiration.
Estimation of leaf and whole-plant assimilation
The net assimilation of leaves per unit leaf area per
day (PLn) was evaluated using measurements of the
PPFRs incident on the leaves and the photosynthetic
light-response curves.
Diurnal changes in PPFR at the top of the plants
were measured on 27 June 1997, a clear day, and 28
June 1997, a cloudy day, with Ga-AsP photodiodes
(G1118, Hamamatsu Photonics, Hamamatsu,
Japan). PPFR values were recorded every 5 min.
The photodiode was set at the plant surface level for
each frame. The outputs of the photodiodes were
calibrated against a quantum sensor (LI-190SA, Li-
Cor). All the outputs of photodiodes were recorded
on a data logger (Thermodac-E, Eto Denki, Tokyo,
Japan).
Photosynthetically active photon fluence rates
incident on U-, M- and L-leaves of a plant relative
to that of the top of the plant (R-PPFR) were
measured on 28 June using two quantum sensors
(LI-190SA, Li-Cor). Measurements of leaf photo-
synthesis and R-PPFR were conducted using the
same plants. Since these R-PPFRs were not different
between infected and uninfected plants in the same
light condition, the average values were used for
calculations. Average R-PPFRs for U-, M- and L-
leaves, respectively, were 0.98, 0.49 and 0.41 in HL,
0.98, 0.68 and 0.65 in ML, and 0.98, 0.90 and 0.72
in LL. We assumed that the leaves were horizontal.
The non-rectangular hyperbolic equation was
used to express a photosynthetic light-response
curve (Thornley, 1976; Terashima & Takenaka,
1986):
P¯φIP
max®o(φIP
max)#®4φIθP
max
2θ®r
dEqn 1
(P¯net photosynthetic rate, Pmax
¯gross photo-
synthetic rate at light saturation, I¯ incident photon
fluence rate, φ¯ initial slope of the light-response
curve, θ¯ convexity index, rd¯dark respiration
rate).
Instantaneous net assimilation per unit leaf area
was estimated by substituting calculated PPFR
incident on the leaves for I in Eqn 1. The summation
of the instantaneous net assimilation rate for every 5
min for 1 d was regarded as daily PLn.
To clarify which of the two parameters, φ or Pmax
,
was the more important determinant of PLn, we
substituted the photosynthetic parameters (φ, Pmax
,
or both) of the infected leaves for those of uninfected
leaves and calculated daily assimilation.
NAR of the whole plant was estimated in the
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486 S. Funayama and I. Terashima
Table 2. (a) ANOVA summary table for the effects of infection (infected and uninfected) and growth light
conditions (High light (HL), Medium light (ML) and Low light (LL)) on the growth parameters. (b) Growth
parameters (Relative growth rate (RGR), Net assimilation rate (NAR), Leaf area ratio (LAR)) of virus-infected
and uninfected Eupatorium makinoi plants under three light conditions (High light (HL), Medium light (ML),
Low light (LL)) in two growth periods (T1 and T2)
(a)
Parameter
Growth
period Source of variation df SS F P
RGR T1 Infection 1 757.07 3.436 0.0664 ns
Light 2 108554.88 246.33 !0.0001 ***
Infection¬Light 2 2053.61 4.66 0.0113 *
Residual 114 25119.63
T2 Infection 1 13350.39 59.89 !0.0001 ***
Light 2 66503.50 149.16 !0.0001 ***
Infection¬Light 2 6938.79 15.56 !0.0001 ***
Residual 114 25414.24
NAR T1 Infection 1 2.13 1.59 0.2103 ns
Light 2 640.09 239.01 !0.0001 ***
Infection¬Light 2 21.51 8.03 0.0005 ***
Residual 114 152.65
T2 Infection 1 104.68 124.99 !0.0001 ***
Light 2 521.89 311.57 !0.0001 ***
Infection¬Light 2 81.25 48.50 !0.0001 ***
Residual 114 95.48
LAR T1 Infection 1 0.98 0.11 0.7391 ns
Light 2 1191.20 67.43 !0.0001 ***
Infection¬Light 2 114.36 6.47 0.0022 **
Residual 114 1007.02
T2 Infection 1 181.03 25.12 !0.0001 ***
Light 2 1775.44 123.18 !0.0001 ***
Infection¬Light 2 11.13 0.77 0.4644 ns
Residual 114 821.56
ns, not significant; ***P!0.001; **P!0.01; *P!0.05.
(b)
Growth Light
RGR (mg g−" d−") NAR (g m−# d−") LAR (m# kg−")
period conditions Infected Uninfected Infected Uninfected Infected Uninfected
T1 HL 92.8³20.0 108³15 6.37³1.74 7.64³1.43 15.6³3.0 15.1³2.5
ML 87.4³12.8 82.4³16.3 5.04³1.04 4.24³1.12 18.0³2.7 20.3³2.4
LL 27.9³11.2 32.7³10.9 1.21³0.53 1.53³0.59 24.3³4.2 21.8³2.7
T2 HL 61.4³16.9 102³14 3.91³1.20 8.04³1.31 16.0³2.8 12.8³1.5
ML 35.0³16.1 54.2³17.8 1.69³0.76 2.90³0.93 20.7³2.2 19.0³2.5
LL 23.1³12.7 26.6³10.8 0.93³0.44 1.19³0.47 25.0³4.0 22.5³2.6
Mean³SD (n¯20).
following way. First, we integrated PLn for a whole
plant taking account of cumulated leaf areas at three
leaf insertion levels. With respect to both photo-
synthetic characteristics and incident PPFR, we
assumed that the first to third pairs of leaves from the
top of the plants were identical to U-leaves, the
fourth to sixth pairs to M-leaves, and the seventh to
ninth pairs to L-leaves, respectively. Next, we
calculated the average of daily net production of all
the leaves in a plant for 1 wk taking account whether
each day in period T2 was fine or cloudy. We
calculated net production rate of the whole plant
expressed on leaf area basis, NAR, by subtracting
the respiration rates of below-ground parts
(rhizomes and roots) and aerial stems reported for
Solidago altissima (stem¯0.4 mg CO#g−" DW h−",
below-ground parts¯0.2 mg CO#
g−" DW h−" in
Iwaki et al., 1966). We assumed that 45% of dry
mass is carbon (i.e. 1 mol CO#corresponds to 26.7 g
of dry mass).
Determination of chlorophyll
Chlorophyll was extracted from leaf discs with N,
N-dimethylformamide and Chl contents were de-
termined according to Porra et al. (1989).
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Effects of virus infection and irradiance on growth of Eupatorium makinoi 487
160
120
80
40
00 1 2 3 4 5
Biomass (g)
RG
R (
mg
g–1
d–1
)
Fig. 1. Relationships between relative growth rate (RGR)
and total dry mass in virus-infected and uninfected
Eupatorium makinoi under three light conditions in two
growth periods (T1 and T2). Means³SD (n¯20) are
shown. Virus-infected plants in High light (HL) (closed
circles) ; uninfected plants in HL (open circles) ; virus-
infected plants in Medium light (ML) (closed squares) ;
uninfected plants in ML (open squares) ; virus-infected
plants in Low light (LL) (closed triangles) ; uninfected
plants in LL (open triangles).
Table 3. (a) ANOVA summary table for the effects of infection (infected and uninfected) and growth light
conditions (High light (HL), Medium light (ML) and Low light (LL) on Leaf weight ratio (LWR) and Specific
leaf area (SLA). (b) LWR and SLA in virus-infected and uninfected Eupatorium makinoi plants
(a)
Parameter
Sampling
date Source of variation df SS F P
LWR 4 Jun Infection 1 0.00626 1.941 0.1695 ns
Light 2 0.00008 0.013 0.9872 ns
Infection¬Light 2 0.01291 2.002 0.1454 ns
Residual 52 0.16764
25 Jun Infection 1 0.01228 6.185 0.0159 *
Light 2 0.00701 1.765 0.1808 ns
Infection¬Light 2 0.00113 0.284 0.7536 ns
Residual 55 0.10917
SLA 4 Jun Infection 1 0.33075 0.877 0.3532 ns
Light 2 190.26230 252.215 !0.0001 ***
Infection¬Light 2 0.24232 0.321 0.7266 ns
Residual 54 20.36784
25 Jun Infection 1 3.62224 24.770 !0.0001 ***
Light 2 105.65045 361.242 !0.0001 ***
Infection¬Light 2 1.37874 4.714 0.0129 *
Residual 55 8.04277
Data were obtained from two samplings made on 4 June (at the beginning of experiment), and 25 June (at the end of
experiment). ns, not significant; ***P!0.001; **P!0.01; *P!0.05.
(b)
Sampling Light
LWR (g g−") SLA (cm# g−")
date conditions Infected Uninfected Infected Uninfected
4 Jun HL 0.348³0.026 0.346³0.045 3.11³0.46 2.82³0.21
ML 0.345³0.070 0.348³0.037 5.55³0.35 5.38³0.46
LL 0.381³0.062 0.317³0.087 7.30³0.76 7.32³1.04
25 Jun HL 0.451³0.062† 0.411³0.035 4.04³0.43† 3.16³0.42
ML 0.423³0.038 0.398³0.031 5.72³0.24 5.30³0.27
LL 0.417³0.045 0.397³0.047†† 6.87³0.40 6.71³0.47
Mean³SD (no mark n¯10, † n¯11, †† n¯9).
Statistical analysis
Analysis of variance (ANOVA) was used to assess
differences between infected and uninfected plants
and among growth light conditions and}or among
leaf insertion levels for each growth analytical
parameter, Chl content, Chl a:b ratio and dark
respiration rate. Data were analysed with StatView
version 4.5 (Abacus Concepts, Berkeley, CA, USA).
Effects of virus infection on growth analysis
parameters
The basic parameters of growth analysis (RGR,
NAR, LAR) at T1 and T2 during the growth
experiment are shown in Table 2. At T1, no
significant differences were found between infected
and uninfected plants for either RGR or NAR
(Table 2a), whereas both RGR and NAR signifi-
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488 S. Funayama and I. Terashima
10
5
0
0 400 800 1200
PPFR (µmol photons m–2 s–1)
Net
ph
oto
syn
thet
ic r
ate
(µm
ol C
O2
m–2
s–1
)
1600 0 400 800 1000200 600 0 400 800 1000200 600
15
20
10
5
0
15
20
10
5
0
15
20HL ML LL
Upper
Middle
Lower
Fig. 2. Light-response curves of net photosynthesis on a leaf area basis in virus-infected and uninfected leaves
in growth period T2. Horizontal axes are incident PPFR values. The data obtained with the uppermost, middle
and lower leaves of the plants grown under three light conditions (HL, High light; ML, Medium light; LL,
Low light) are shown. The symbol and bar denote mean³SD (n¯3). Virus-infected leaves (closed circles) ;
uninfected leaves (open circles). Upper, middle and lower indicate the position of the leaves. See Table 4 for
parameter values.
Table 4. Gross photosynthetic rate at light saturation (Pmax
), initial slope
of the light response curve (φ) and the convexity index (θ), obtained from
Fig. 2 light-response curves of net photosynthesis on leaf area in virus-
infected and uninfected leaves of Eupatorium makinoi
Light
conditions
Leaf insertion
level
Infected}uninfected φ θ P
max
HL U Infected 0.019 0.87 5.94
Uninfected 0.061 0.067 17.5
M Infected 0.049 ®0.048 9.91
Uninfected 0.062 0.43 16.5
L Infected 0.052 0.69 8.06
Uninfected 0.062 0.21 10.3
ML U Infected 0.046 0.81 9.09
Uninfected 0.067 0.75 11.5
M Infected 0.050 0.85 7.26
Uninfected 0.049 0.96 9.68
L Infected 0.041 0.73 7.24
Uninfected 0.060 0.83 6.94
LL U Infected 0.060 0.83 7.42
Uninfected 0.067 0.080 6.87
M Infected 0.051 0.92 4.47
Uninfected 0.057 0.85 4.67
L Infected 0.055 0.82 3.69
Uninfected 0.054 0.88 3.43
Light conditions: HL, High light; ML, Medium light; LL, low light.
Leaf insertion level : U, uppermost leaves; M, middle leaves; L, lower leaves.
cantly increased with increasing growth PPFR
(Table 2a). At T2, significant differences in RGR
and NAR were found not only among growth light
conditions, but also between infected and uninfected
plants (Table 2a).
RGR and NAR in infected plants were signifi-
cantly smaller than those in uninfected plants under
all light conditions except for those in ML at T1
(Table 2b), and the effects of infection significantly
increased with increasing growth PPFR (Table 2b).
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Effects of virus infection and irradiance on growth of Eupatorium makinoi 489
Table 5. (a) ANOVA summary table for the effects of infection (infected and uninfected) and growth light
conditions (High light (HL), Medium light (ML), Low light (LL)) on Chl contents and Chl a:b ratios. (b)
Chlorophyll contents and Chl a:b ratios in virus-infected and uninfected Eupatorium makinoi leaves
(a)
Parameter
Sampling
date Source of variation df SS F P
Chls ab 4 Jun Infection 1 0.10028 26.539 !0.0001 ***
Light 2 0.00352 0.466 0.6348 ns
Infection¬Light 2 0.07264 9.612 0.0014 **
Residual 18 0.06802
25 Jun Infection 1 0.35604 182.412 !0.0001 ***
Light 2 0.01552 3.975 0.0473 *
Infection¬Light 2 0.04215 10.796 0.0021 **
Residual 12 0.02343
Chl a:b 4 Jun Infection 1 2.14175 31.666 !0.0001 ***
Light 2 4.92721 36.425 !0.0001 ***
Infection¬Light 2 0.72420 5.354 0.0150 *
Residual 18 1.21743
25 Jun Infection 1 2.64207 20.608 0.0007 ***
Light 2 7.81142 30.464 !0.0001 ***
Infection¬Light 2 2.30921 9.006 0.0041 **
Residual 12 1.53847
Data were obtained from two samplings made on 4 June (at the beginning of experiment), and 25 June (at the end of
experiment). ns, not significant; ***P!0.001; **P!0.01; *P!0.05.
(b)
Sampling Light
Chlorophyll ab (mmol m−#) Chlorophyll a:b
date conditions Infected Uninfected Infected Uninfected
4 Jun HL 0.16³0.06 0.42³0.05 4.50³0.62 3.48³0.06
ML 0.31³0.06 0.31³0.08 3.11³0.03 2.94³0.05
LL 0.22³0.05 0.34³0.06 3.33³0.13 2.73³0.04
25 Jun HL 0.082³0.017 0.45³0.10 5.13³0.70 3.41³0.18
ML 0.18³0.02 0.50³0.03 3.27³0.17 2.70³0.13
LL 0.22³0.01 0.37³0.02 2.79³0.38 2.79³0.25
Mean³SD (4 June, n¯4; 25 June, n¯3).
The light-dependencies of RGR and NAR changed
between T1 and T2 (Table 2b), partly because
symptoms in infected leaves had not fully developed
by T1, especially for ML-frame (see Table 5a). As a
consequence, the growth rates of infected plants in
ML were as fast as in HL at T1.
RGR and biomass were negatively correlated
except for uninfected}HL (Fig. 1). At T1, no
significant differences were found between the LAR
of infected and uninfected plants (Table 2a), whereas
LAR significantly decreased with increasing growth
PPFR (Table 2a). At T2, significant differences in
LAR were found not only among growth light
conditions, but also between infected and uninfected
plants (Table 2a). The LAR of infected plants was
significantly greater than uninfected plants (Table
2a).
LWR was not affected by growth PPFR at both
sample times (Table 3a). However, at the later
period, the LWR of infected plants was significantly
greater than that of uninfected plants (Table 3a).
SLA decreased with increasing growth PPFR in
both infected and uninfected leaves at both times
(Table 3a). The difference in SLA between infected
and uninfected plants was significant only at the later
stage of the experiment (Table 3a), when a syn-
ergistic interaction between light and infection was
also found (Table 3a).
Photosynthetic properties of virus-infected and
uninfected leaves
The stomatal conductance of infected leaves was not
appreciably different from uninfected leaves (data
not shown). However, photosynthetic rate decreased
in infected leaves under all light conditions and at all
insertion levels (Fig. 2, Table 4). In HL, decreases in
both initial slope (φ) and Pmax
in infected leaves were
most severe in the U-leaves. Pmax
of M-leaves was
highest in infected plants, while, in uninfected
plants, Pmax
decreased with the decrease in the leaf
insertion level. In ML, the depression of the
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490 S. Funayama and I. Terashima
Table 6. (a) ANOVA summary table for the effects of infection (infected
and uninfected), growth light conditions (High light (HL), Medium light
(ML) and Low light (LL)) and leaf insertion level (upper, middle and lower
leaves) on dark respiration rates. (b) Dark respiration rates of virus-infected
and uninfected Eupatorium makinoi leaves
(a)
Source of variation df SS F P
Infection 1 0.0001 0.003 0.9588 ns
Light 2 2.4572 24.355 !0.0001 ***
Insertion level 2 0.3824 3.790 0.0324 *
Infection¬Light 2 0.1829 1.813 0.1782 ns
Infection¬Insertion level 2 0.0335 0.332 0.7198 ns
Light¬Insertion level 4 0.1028 0.510 0.7289 ns
Infection¬Light¬Insertion level 4 0.1391 0.690 0.6041 ns
Residual 35 1.7656
ns, not significant; ***P!0.001; **P!0.01; *P!0.05.
(b)
Light
Leaf
insertion
Dark respiration rate
(µmol CO#
m−# s−")
conditions level Infected Uninfected
HL Upper leaves 0.80³0.02 1.1³0.6
Middle leaves 0.71³0.33 0.87³0.24
Lower leaves 0.62³0.24 0.52³0.12
ML Upper leaves 0.52³0.12 0.33³0.10
Middle leaves 0.29³0.17 0.41³0.15
Lower leaves 0.34³0.32 0.38³0.12
LL Upper leaves 0.43³0.13 0.31³0.06
Middle leaves 0.47³0.13 0.20³0.01
Lower leaves 0.28³0.01 0.18³0.05
Mean³SD (n¯3).
photosynthetic rate was more striking in the U- and
M-leaves than in L-leaves, but unlike plants at HL,
a vertical gradient of Pmax
within the plants was
found in both infected and uninfected plants (Fig. 2,
Table 4). In LL, photosynthetic rates were not
markedly different between infected and uninfected
leaves for any of the leaf insertion levels. On the
other hand, the dependency of the gradient of Pmax
on insertion level was obvious in both infected and
uninfected plants (Fig. 2, Table 4).
The differences in the Chl content between
infected and uninfected leaves were significant in
both measurements (Table 5a). A significant in-
teraction between the effects of light and infection
was found (Table 5a) and the differences in Chl
content between infected and uninfected leaves
increased with growth PPFR (Table 5b). Chl a:b
ratio increased with decreasing leaf Chl content in
infected leaves (Table 5b). For uninfected leaves, the
Chl a:b ratio was greater in HL than in ML and LL
(Table 5b).
There was no significant difference in dark
respiration rate between infected and uninfected
leaves (Table 6a), while the differences among
growth PPFRs and among leaf insertion levels were
significant (Table 6a). The dark respiration rate
increased with growth PPFR and increased with
height of the insertion level (Table 6b).
Estimation of leaf and whole-plant assimilation
PLn was estimated for each leaf from the light-
response curve of photosynthesis (Fig. 2, Table 4),
and the measured PPFR data on a clear day (27
June), on a cloudy day (28 June), and the average R-
PPFR. PLn was reduced by virus infection under all
light conditions and on both clear and cloudy days
(Fig. 3). The relative decrease in PLn caused by
infection was greatest in HL, and moderated with
the decrease in growth PPFR. The relative decrease
in PLn by infection was greatest at the highest leaf
insertion levels. This trend was found on both clear
and cloudy days. Under the same light conditions,
the relative decrease in PLn by virus infection was
greater on a cloudy day than that on a clear day. In
LL on a cloudy day, PLn in uninfected leaves was
positive, whereas that in infected leaves was negative.
Fig. 4 shows PLn calculated for uninfected leaves
and for model leaves with φ, Pmax
, or both of them
from the infected leaves. In all leaves in HL and U-
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Effects of virus infection and irradiance on growth of Eupatorium makinoi 491
200
100
0
Upper Middle
Leaf insertion level within a plant
Net
ph
oto
syn
thet
ic p
rod
uct
ion
(m
mo
l CO
2 m
–2 d
–1)
Clear day Cloudy day
HL
Lower Upper Middle Lower
300
400
200
100
0
300
400
200
100
0
300
400
Upper Middle Lower Upper Middle Lower
Upper Middle Lower Upper Middle Lower
ML
LL
Fig. 3. Daily net photosynthetic production in infected and uninfected Eupatorium makinoi leaves at different
insertion levels of the plants. Values were calculated using the data in Fig. 2, measured PPFR at the top of the
plants and the average relative PPFR on each leaf to the top of the plants. Virus-infected leaves (shaded
columns); uninfected leaves (open columns).
leaves in ML, both the decreases in φ and in Pmax
contributed to the decrease in PLn. However, for a
clear day, the decrease in Pmax
exerted a greater effect
on the decrease in daily photosynthetic production
than φ, while on a cloudy day, φ exerted a greater
effect. In ML, except for U-leaves, and LL, the PLn
of the model leaf with φ of the infected leaves and
Pmax
of the uninfected leaves was virtually equal to
that calculated for the model leaf with both Pmax
and
φ of the infected leaves.
NAR was calculated by incorporating PLn of the
leaves, the respiration rates of stems and below-
ground parts and biomass allocation to the leaves,
stems and below-ground parts. The calculated NAR
was similar to the NAR obtained by the conventional
destructive procedure (Fig. 5a). The contribution of
respiration in the leaf, stem and below-ground part
to the gross production of the whole plant is shown
in Fig. 5b. As mentioned in the Materials and
Methods section, respiration rates of stem and
below-ground part were estimated using data
obtained for S. altissima (Iwaki et al., 1966).
However, since the carbon losses due to respiration
rates of stem and below-ground parts were at most
3–8% of daily gross production of plant, the error
due to any difference in the respiration rate between
E. makinoi and S. altissima, relative to the NAR
values, will be small.
Effects of virus infection on whole-plant performance
The small effect of virus infection on RGR, NAR
and LAR at T1 was probably due to the slow
development of symptoms (Tables 2, 4). However,
in period T2, the RGR of infected plants showed
significant decreases (Table 2). The NAR of infected
plants also decreased, whereas LAR, SLA and LWR
increased in infected plants (Tables 2,3). These
results suggested the close relationships between
RGR and NAR. An increase in LAR is often
reported for plants whose leaves are damaged by
herbivory or fungal pathogens (for a review, see
Prins & Verkaar, 1992). The increase in LAR is
interpreted as a ‘compensation’ for the impaired
function of leaves and}or decreased amounts of
leaves. The growth rates of plants with infected
NAR but uninfected LAR at T2 were calculated,
and the ratios of the growth rates of infected and
modelled plants to that of uninfected plants were
0.61 and 0.49 in HL, 0.64 and 0.58 in ML, and 0.87
and 0.78 in LL, respectively. These results con-
firmed the compensatory function of increased LAR
in infected plants. However, compensation by in-
creased LAR was much smaller than the decrease in
NAR by infection. For the present case, therefore,
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492 S. Funayama and I. Terashima
40
20
0Upper Middle
Leaf insertion level within a plant
Pro
po
rtio
n o
f p
ho
tosy
nth
etic
pro
du
ctio
nin
infe
cted
/mo
del
leav
es t
o u
nin
fect
ed le
aves
(%
)
Clear day Cloudy day
HL
Lower Upper Middle Lower
100
120
Upper Middle Lower Upper Middle Lower
Upper Middle Lower Upper Middle Lower
ML
LL60
80
40
20
0
100
120
60
80
40
20
0
100
120
60
80
Fig. 4. Daily net photosynthetic production of uninfected leaves and the model leaves with Pmax
, initial slope
(φ) or both of infected leaves. Proportions of the photosynthetic production of the uninfected leaves are shown.
Photosynthetic parameters used in the calculations are obtained from data in Fig. 2. Dark respiration rates used
in the calculations are those in uninfected leaves (Table 6). Parameter combinations used in the calculations are
as follows: control (open columns), leaf with uninfected φ and uninfected Pmax
(¯uninfected leaf) ; low φ
(hatched columns), model leaf with infected φ and uninfected Pmax
; low Pmax
(light shaded columns), model leaf
with uninfected φ and infected Pmax
; and, low φ and Pmax
(dark shaded columns), model leaf with infected φ and
infected Pmax
. Low φ and Pmax
leaves are almost identical to the infected leaves, although the convexity terms
of the curves and dark respiration rate may be different.
we can conclude that the decreased NAR was the
predominant factor explaining the decrease in RGR
in virus-infected plants.
Changes in RGR are generally explained by
variations in LAR, especially when plants having the
same growth forms are compared under optimal
conditions (Poorter & Remkes, 1990). However,
several studies report a strong correlation between
RGR and NAR, as in the present study. These
findings generally apply to comparisons of the same
species under different conditions, such as different
CO#concentrations (Poorter et al., 1988), continuous
light and fluctuating light (Watling et al., 1997) or
with or without VA-mycorrhizal infection (Lovelock
et al., 1996).Therefore, the generalization that
variation of RGR is explained by LAR is valid only
for the comparison between species. On the other
hand, as revealed by the present study, variation of
RGR within the same species under different growth
conditions may be mainly explained by NAR.
The relationship between leaf photosynthesis and
NAR
The calculated NAR values agreed well with those
obtained by the conventional method (Fig. 5a). NAR
is often termed as a ‘physiological ’ component, but,
strictly speaking, it is a complex intermingling of
photosynthetic characteristics of the leaves with dark
respiration, biomass allocation, chemical compo-
sition of leaves and with leaf area formation (for a
review, see Lambers & Poorter, 1992). In the present
case, virus infection affected leaf photosynthetic rate
and the daily net photosynthetic production of leaves
(PLn) under all light conditions (Figs 2, 3). The
relative decreases in photosynthetic rate and PLn in
virus-infected leaves were enhanced by the increas-
ing PPFR like the decrease in NAR at T2 (Figs 2, 3,
Table 2). These results suggested that the decrease
in leaf photosynthetic rate would lead to smaller
NAR. However, it is frequently reported that
Printed from the CJO service for personal use only by...
Effects of virus infection and irradiance on growth of Eupatorium makinoi 493
HL
Infected
Pro
po
rtio
n t
o g
ross
pro
du
ctio
n (
%)
ML LL LL ML LL
Uninfected
(b)
0Conventional NAR (g m–2 d–1)
(a)
2 4 6 8 10
2
4
6
8
10
Cal
cula
ted
NA
R (
g m
–2 d
–1)
Fig. 5. (a) The relationship between calculated NAR and
NAR obtained by growth analysis (conventional NAR) in
growth period T2. Lines indicate that the calculated NAR
is conventional. Virus-infected plants in High light (HL)
(closed circles) ; uninfected plants in HL (open circles) ;
virus-infected plants in Medium light (ML), (closed
squares) ; uninfected plants in ML (open squares) ; virus-
infected plants in Low light (LL) (closed triangles) ;
uninfected plants in LL (open triangles). (b) Proportions
of leaf, stem and below-ground parts respiration and net
photosynthetic production to daily gross photosynthetic
production in infected and uninfected plants under three
light conditions in T2. Net photosynthetic production
(open columns); leaf respiration (hatched columns); stem
respiration (shaded columns); below-ground parts res-
piration (closed columns).
respiration rate increases with infection (for reviews,
see Matthews, 1991; Farrar, 1992). In the present
study, consistent increases were not observed (Table
6). However, in infected}LL, the respiration rates
tended to be enhanced by infection. This caused the
underestimation of NAR in infected}LL (Fig. 5a).
Since the estimated NAR closely agreed with the
conventional NAR at T2, except for infected}LL,
and since there were no significant differences in leaf
respiration rate, it is concluded that the impaired leaf
photosynthesis in the infected plants is the pre-
dominant factor lowering NAR and thereby RGR of
the infected plants.
The relationship between leaf photosynthesis, RGR
and growth irradiance
Leaf Chl content in virus-infected leaves decreased
with increasing PPFR (Table 5). As shown by our
previous study (Funayama et al., 1997b), there was a
clear relationship between leaf Chl content and leaf
photosynthetic capacity in virus-infected plants.
Therefore, the effects of virus infection on leaf
photosynthesis in E. makinoi increased with in-
creasing growth PPFR (Fig. 2). The difference in
RGR between infected and uninfected plants was,
therefore, enhanced by the increase in growth PPFR
(Table 2). In infected leaves, both φ and Pmax
decreased, but the contributions of φ and Pmax
to the
decrease in RGR were different depending on
growth irradiance (Fig. 4). In ML and LL, the
maximum incident PPFRs were lower than those
needed for light saturation of photosynthesis, which
explains that the depression of Pmax
by virus infection
did not contribute to the depression of photo-
synthetic production in ML and LL (Fig. 4). In HL
the effects of virus infection on leaf photosynthesis
were greater than in ML and LL, and moreover, the
maximum incident PPFR was much greater than
that needed for light saturation.
The results of the present study were apparently
inconsistent with the results of our previous study
(Funayama et al., 1997a) in which we studied the
effects of virus infection and growth irradiance on
integrated yearly growth. The main result of the
previous study was that shading enhanced the
adverse effects of virus infection on yearly growth.
Integrated yearly growth rates are generally de-
pendent not only on the assimilatory capacity of the
plant, but also on various factors such as the length
of the growth period and other environmental
factors. Growth rates in the early vegetative phase
are nearly equal to maximal values realized in the
phase of intensive growth but plant growth charac-
teristics generally change with time (Hunt, 1978).
For example, as shown in this study (Fig. 1) RGR,
the product of NAR and LAR, generally decreases
with increasing plant size, especially in plants of
erect growth form. This is because large plants
require more non-photosynthetic supporting organs
at the expense of leaves (lower LAR) and an increase
in self-shading (lower NAR).Therefore, the yearly
growth of below-ground parts, which was measured
in the previous study, may not be directly affected by
the growth characteristics in the early vegetative
phase. However, it should be stressed here that the
differences in RGR between infected and uninfected
plants became greater with increasing plant size
(Fig. 1) and the difference in the final size would be
significant even in LL-plants. Also the extremely
low PPFR level used in the previous study just
enabled the uninfected plants to grow, but not the
infected plants. This possibility is clearly indicated
by PLn in LL on a cloudy day (Fig. 3).
Concluding remarks
Virus infection decreased the RGR of E. makinoi in
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494 S. Funayama and I. Terashima
an early vegetative growth phase under all light
conditions. Infected plants allocated more dry matter
to the leaves than uninfected plants and an increased
LAR in infected plants partially compensated for the
lowered photosynthetic rate but this response was
much less than that needed to compensate for the
decreased NAR. It was concluded, therefore, that a
reduction of NAR, mainly caused by reduced leaf
photosynthesis, was the predominant factor
explaining the decrease in RGR in virus-infected E.
makinoi.
We thank Dr A. Takenaka for many helpful discussions,
Dr S.-I. Ishikawa for suggestions on statistical analysis
and two anonymous reviewers for constructive comments.
This research was supported by the Research Fellowships
of the Japan Society for the Promotion of Science for
Young Scientists.
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