effects of geminivirus infection and growth irradiance on the vegetative growth and photosynthetic...

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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


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


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



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).



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).


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


Residual 52 0.16764

25 Jun Infection 1 0.01228 6.185 0.0159 *

Light 2 0.00701 1.765 0.1808 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 ***


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-



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).




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



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¬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-



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


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,



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



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



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



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

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