1

Diagnosis of Invisible Photosynthetic Injury Caused by a Herbicide (Basta) with
Chlorophyll Fluorescence Imaging System
Kotaro Takayama, Atsumi Konishi, Kenji Omasa
Department of Biological and Environmental Engineering
Graduate School of Agricultural and Life Science
The University of Tokyo
Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japanaomasa@mail.ecc.u-tokyo.ac.jp
Abstract
Chlorophyll fluorescence imaging is a powerful tool in obtaining physiologicalinformation on plant leaf in non-destructive and non-invasive ways. Effects of Basta,
one of the most popular commercially available foliar application-type herbicides, on insitu kidney bean leaf was analyzed with a developed chlorophyll fluorescence imaging
system. Immediately after the Basta treatment, CO2 assimilation rate and ΦPSΙΙ, which
represents the photochemical yield of photosystem ΙΙ, decreased and NPQ, which
represents the heat dissipation of absorbed light energy, increased in the treated area.
A positive linear correlation was confirmed between assimilation rate and ΦPSΙΙ, while anegative linear correlation was confirmed between assimilation rate and NPQ. These
results demonstrated that the early diagnosis of invisible photosynthetic injury causedby Basta was feasible and the chlorophyll fluorescence imaging can be used as a systemfor early detection of photosynthetic inhibition of growing plants caused by herbicides.
[Keywords] chlorophyll fluorescence, CO2 assimilation rate, heat dissipation,
photochemical yield of photosystem ΙΙ, photosynthetic injury
Introduction
In the controlled greenhouse and precision agriculture, the information on plant
response and growth environments measured by sensors is used to achieve theoptimization of the controlled system, increase in plant production and sustainableagriculture (Hashimoto et al., 1993, Sigrimis, 2001, De Baerdemaeker et al., 2001).
Many studies have been done for the application of biotic information, obtained throughnon-destructive and non-invasive measuring methods, to diagnose invisible plant
responses in the research field. Such an approach is called “Speaking Plant Approach(SPA)” (Hashimoto, 1989).
In particular, imaging techniques, e.g. multi- or
hyper-spectral image sensing, thermal image sensing, and fluorescence image sensing,have been intensively investigated for use in diagnosis because image information is themost intuitive, easily comprehensible, and provides copious amount of information(Omasa, 1990; Omasa et al., in press). Multi- or hyper-spectral image sensing
measures the reflection spectrum from plants, which contains information on pigments
and water status in plants (Myers, 1983; Omasa and Aiga, 1987; Omasa, 1990; Omasa,
in press). Thermal image sensing measures surface temperature, which provides
information on stomatal response and gas exchange (Omasa et al., 1981ab; Hashimoto
et al., 1984; Omasa and Aiga, 1987; Omasa, 1990; Omasa and Croxdale, 1992; Jones,
1999; Omasa, 2002). Fluorescence image sensing is an active sensing technique, forexample laser-induced fluorescence (LIF) imaging and chlorophyll fluorescenceimaging (Omasa 1990; Lichtenthaler, 1996; Omasa, 1998, Govindjee and Nedbal, 2000;
Kim et al., 2002; Omasa and Takayama, 2002). These sensing techniques provide
K. Takayama, A. Konishi, and K. Omasa. “Diagnosis of Invisible Photosynthetic
Injury Caused by a Herbicide (Basta) with Chlorophyll Fluorescence Imaging System”.
Agricultural Engineering International: the CIGR Journal of Scientific Research andDevelopment. Manuscript IT 03 001. June, 2003.

2

information on the bleaching of plant pigments and about photosynthetic functions.
Especially, chlorophyll fluorescence imaging, originally developed by Omasa et al.
(1987) and Daley et al. (1989), is a highly sophisticated technique for the evaluation of
the heterogeneous distribution of photosynthetic activities, e.g. photosynthetic electrontransport, assimilation rate, and stomatal opening over a plant leaf surface (Omasa and
Takayama, 2002).
Since chlorophyll fluorescence is emitted from chlorophyll a pigments and is
the reemission of light energy trapped by antenna chlorophyll not used inphotosynthesis, a chlorophyll fluorescence image represents the heterogeneous
distribution of light utilization and photosynthetic activity over a leaf surface (Krause
and Weis, 1991; Govindjee, 1995). Furthermore, several effective fluorescenceparameters indicating the activities of photosynthetic electron transport or heatdissipation of absorbed light energy calculated by “Saturation pulse method” had beendeveloped (Quick and Horton 1984; Genty et al., 1989; Bilger and Bjőrkman, 1990).
Taking advantage of the chlorophyll fluorescence imaging, it has been applied to thediagnosis of photosynthetic injury caused by biotic and abiotic stress factors(Balachandran et al., 1994; Mott 1995; Osmond et al., 1998; Omasa and Takayama,
2002). Especially, the diagnosis of invisible photosynthetic injury caused by herbicidewith chlorophyll fluorescence imaging has been widely investigated (Genty and Meyer,
1994; Rolfe and Scholes, 1995; Omasa et al., 2001; Takayama et al., 2000; Takayama
and Omasa, 2001ab).
In this study, we have demonstrated the early diagnosis of invisible
photosynthetic injury caused by Basta with a developed chlorophyll fluorescenceimaging system.
Basta is the most popular commercially available foliar
application-type herbicide, for many genetically engineered crops that have resistance tothe herbicide (Lea and Ridley, 1989). Furthermore, the weeding process of Basta onplant leaf was analyzed with continuous measurements of chlorophyll fluorescenceimaging.
Materials and Methods
Plant material
Plants of Phaseolus vulgaris L. cv. Shin-edogawa were grown in anenvironmentally controlled growth chamber for 5 weeks after sowing in pots (12 cm indiameter and 10 cm in height). The pots were filled with artificial soil (mixture ofvermiculite and perlite, 1:1 v/v). The plants were illuminated for 12 h each day withwhite fluorescent lights that have a light intensity of approximately PPFD 300 μmol
m-2s-1. Air temperature was 26.5 ºC during the day and 20.0 ºC at night. Relative
humidity was 45 % during the day and 60 % at night. Plants were watered daily with anutrient solution (1:1000 dilution of HYPONex).
Mature in situ Phaseolus vulgaris
L. leaves were used in the experiments.
Herbicide and the treatment
Basta (AgrEbo) is composed of 18.5 % Glufosinate-ammonium <Ammonium-DL-homoalanine-4-YL-(methyl)phosphinate> and 81.5 % mixture of water anddetergents. The active ingredient of Basta is glufosinate. It inhibits the activity ofglutamine synthetase, which is essential for the removal of toxic ammonia produced in
the metabolism of living systems in plants (Lea and Ridley, 1989). The application of
glufosinate leads to increased levels of ammonia in the plant tissues. This causes
photosynthesis to stop and the plant dies within few days (Lea and Ridley, 1989). In
this experiment, a 1:100 dilution of Basta with pure water was used according to label
directions for killing annual plants. After the steady-state photosynthesis had been
attained under light condition, the measurement of chlorophyll fluorescence and visual
K. Takayama, A. Konishi, and K. Omasa. “Diagnosis of Invisible Photosynthetic
Injury Caused by a Herbicide (Basta) with Chlorophyll Fluorescence Imaging System”.
Agricultural Engineering International: the CIGR Journal of Scientific Research andDevelopment. Manuscript IT 03 001. June, 2003.

3

images for the control was done. Thereafter, the herbicide treatment was performed
with the following procedure. The upper surface area on the left side of the leaf wascovered with a gauze soaked in the Basta dilute solution for 10 min. After thetreatment, the measurements of chlorophyll fluorescence and visual images were
performed at appropriate intervals for 24 h.
Chlorophyll fluorescence imaging system
Figure 1 shows a schematic diagram of the chlorophyll fluorescence imaging
system installed in a laboratory. Two different light intensities, actinic light andsaturation light pulse, were used in the chlorophyll fluorescence measurements. The
measurement method is called “Saturation pulse method” (Quick and Horton, 1984).
Optical fiber
Actinic lightsourceDigital video
recorderDigital stillcameraShort-passfilterComputer
Attached leaf

CCD
cameraLong-passfilterFixingapparatus

Saturation lightpulse source

Figure 1. Schematic diagram of chlorophyll fluorescence imaging
system. An attached plant leaf was placed on the fixing apparatus. The
5 x 5 cm2 measurement area is illuminated by the actinic or saturation
light pulse sources. The downward solid blue arrow represents theactinic and the saturation pulse lights transmitted through the short-passfilter. The upward solid blue arrow represents the reflection of the blue
illuminations. The upward solid red arrow transmitted through thelong-pass filter represents chlorophyll a fluorescence emitted from the leaf
induced by the blue illuminations.
Actinic light (PPFD 350 μmol m-2s-1) for photosynthesis was provided with a 180 W
metal halide lamp (Sumita Optical Glass, Inc., LS-M180) equipped with a short-passfilter (Sumita Optical Glass, Inc., Blue-filter; λ < 570 nm) through an optical fiber.
Saturation light pulse (PPFD 2700 μmol m-2s-1 for 1 s) that caused a transient saturationof photochemistry was provided with three 180 W metal halide lamps (Sumita Optical
Glass, Inc., LS-M180) equipped with a short-pass filter (Sumita Optical Glass, Inc.,
Blue-filter; λ < 570 nm) through the optical fiber. The spectral property of theshort-pass filter was represented as the blue solid line shown in Figure 2. The optical
K. Takayama, A. Konishi, and K. Omasa. “Diagnosis of Invisible Photosynthetic
Injury Caused by a Herbicide (Basta) with Chlorophyll Fluorescence Imaging System”.
Agricultural Engineering International: the CIGR Journal of Scientific Research andDevelopment. Manuscript IT 03 001. June, 2003.

4

fiber has four small optical fiber branches, one of those branches was connected to theactinic light source and three of those branches were connected to the saturation lightpulse sources. The optical fiber collects the blue illuminations provided by the actinicand saturation light pulse sources and provides the blue illumination on an attachedplant leaf. The variation in the distribution of the blue illumination within themeasurement area (5 x 5 cm2) was kept within less than ±5 %.
Chlorophyll fluorescence induced by the blue illumination was captured by a
B/W chilled charge-coupled device (CCD) video camera (Hamamatsu Photonics,
C5985) equipped with a long-pass filter (Corning, 2-64; λ > 640 nm) in order to remove
the reflectance of the blue light illumination. Spectral property of the long-pass filter
was represented as the red solid line shown in Figure 2. The chlorophyll fluorescence
images were recorded on a digital video recorder (SONY, DSR-V10) as 480H × 720V
pixels/frame with an 8-bit resolution. Visual changes of plant leaves after the
herbicide treatment were observed by visual images obtained with a digital still camera(Sony, DSC-70). The capturing of visual image was performed after a series ofchlorophyll fluorescence measurements.
After the experiment, the recorded
chlorophyll fluorescence images were transferred to a computer and analyzed.
The fixing apparatus for a plant leaf attached to the plant was placed at a
distance of 0.12 m from the optical fiber and 0.55 m from the CCD camera. The sizeof illuminated area was 7 x 7 cm2 and the measurement area was 5 x 5 cm2 located at
the center of the illuminated area. The attached plant leaf was horizontally set on the
fixing apparatus normal to the axis of the blue illumination from the optical fiber.
Upper and lower surfaces of the plant leaf on the fixing apparatus were opened to the
air.

Transmittance (%)

100
80

Figure 2. Spectral properties of the
optical filters used in the chlorophyll
fluorescence imaging system. The blue
solid line represents the spectral propertyof the short-pass filter. The red solid line
represents the spectral property of thelong-pass filter.
Transmittances were
measured with MSR-7000.

60
40
20
0
400

500
600
700
Wavelength (nm)

Chlorophyll fluorescence analysis with “Saturation pulse method”
Chlorophyll fluorescence analysis with saturation light pulse is called“Saturation pulse method”(Quick and Horton, 1984; Genty et al., 1989; Bilger andBjőrkman, 1990). This method has been applied to the chlorophyll fluorescenceimaging analysis (Daley et al., 1989; Genty and Meyer, 1994). Figure 3 shows aschematic diagram of time course of chlorophyll fluorescence imaging and the state of
the blue illuminations in the saturation pulse method. First, an attached plant leaf
placed in the fixation apparatus was acclimated to a dark condition for at least 1 h.
After the acclimation, an Fm image was captured during the saturation light pulse (SL in
K. Takayama, A. Konishi, and K. Omasa. “Diagnosis of Invisible Photosynthetic
Injury Caused by a Herbicide (Basta) with Chlorophyll Fluorescence Imaging System”.
Agricultural Engineering International: the CIGR Journal of Scientific Research andDevelopment. Manuscript IT 03 001. June, 2003.

5

Fig.3) by a CCD camera with a shutter speed of 0.03 s. Next, the leaf was illuminatedwith an actinic light source (AL in Fig.3). After the photosynthesis reached a steadystate, an Fm' image was obtained during the saturation light pulse under actinic light by a
CCD camera with a shutter speed of 0.03 s. An F image was captured just before theFm' measurement under actinic light by a CCD camera with a shutter speed of 0.5 s.
Nomenclature of fluorescence parameters is according to van Kooten and Snel (1990).
The relationship between fluorescence intensity, which was emitted from a fluorescentsheet illuminated with blue actinic light through the optical fiber illustrated in Fig. 1,
and the digital output from the camera was calibrated. Figure 4 shows therelationships between fluorescence intensity and analog to digital (A/D) conversion
level obtained at the shutter speeds of 0.03 s (● in Fig.4) and 0.5 s (■ in Fig.4) and theregression lines for each shutter speed.

Light intensity Fluorescence intensity

Dark period

Light period

Fm
Fm'
F
1s
1s

Time

Dark
treatment

SL

AL

Figure 3. Schematic diagram of time courseof “Saturation pulse method”. Dashed arrow
represents the dark treatment before thechlorophyll fluorescence imaging.
SL
represents the irradiation time of the saturation
light pulse. AL represents the start time oflight period. Fm, F, and Fm' represent thechlorophyll fluorescence imaging time points.

SL

A/D conversion level

250
200

y = 7.2293x + 23.123
R2 = 0.9996

150
100
50

y = 0.4877x + 21.422
R2 = 0.9999

Relationships
betweenFigure4.
fluorescence intensity and analog to digital(A/D) conversion level obtained under two
different shutter speed settings, 0.03 s (●) and
0.5 s (■). The regression lines were used in
the conversion of the A/D conversion level tothe relative fluorescence intensity.

00
100 200 300 400
Fluorescence intensity (Relative unit)

In order to determine photosynthetic properties from chlorophyll fluorescencemeasurements, the fluorescence intensity images (Fm', F, and Fm) must be converted into
relative fluorescence yield images (ΦFm', ΦF, and ΦFm) with the following correction
formulae developed from formulae indicated in Fig.4 and the ratio (Ratio = 7.71) of
saturation light pulse intensity to actinic light intensity.
K. Takayama, A. Konishi, and K. Omasa. “Diagnosis of Invisible Photosynthetic
Injury Caused by a Herbicide (Basta) with Chlorophyll Fluorescence Imaging System”.
Agricultural Engineering International: the CIGR Journal of Scientific Research andDevelopment. Manuscript IT 03 001. June, 2003.

6

Fm − 21.422
0.4877F '− 21.422
ΦF m ' = m
0.4877F − 23.123
× RatioΦF =7.2293
ΦF m =

Using the relative fluorescence yield images, images of the photochemicalyield of photosystem ΙΙ (PSΙΙ) (ΦPSΙΙ) and nonphotochemical quenching (NPQ) werecomputed. Images of ΦPSΙΙ obtained under actinic light represent the activity of linear
electron transport in PSΙΙ and ranged from 0 to 1 (Genty et al., 1989). Images of ΦPSΙΙ
can be used as relative maps of CO2 fixation in non-photorespiratory condition and werecomputed for each corresponding pixel using the following formula (Genty and Meyer,
1994; Rolfe and Scholes, 1995):

Φ PSII =ΦF m '− ΦFΦF m '

NPQ, ranging from 0 to infinity, represents the extent of intrathylakoid pH gradient andthe ability of chloroplasts to dissipate excess excitation energy as heat (Bilger andBjőrkman, 1990; Osmond et al., 1998). Thereby, NPQ has been used as a sensitive
indicator of regulation and protection of photosynthesis in environments when lightenergy absorption exceeds the capacity for light utilization. Images of NPQ were
calculated for each corresponding pixel using the following formula (Osmond et al.,1998):
Images of ΦPSΙΙ and NPQ were stored as 480H×720V pixels/frame with an 8-bit
resolution.

NPQ =ΦFm − ΦFm 'ΦFm 'Measurement of CO2 assimilation rate with porometer
In order to evaluate the relationships between CO2 assimilation rate andchlorophyll fluorescence parameters (ΦPSΙΙ and NPQ) of a Phaseolus vulgaris L. leaf
after the Basta treatment, CO2 assimilation rate and chlorophyll fluorescence parameters
were simultaneously measured with a porometer (PPSYSTEMS, CIRAS-1) and thechlorophyll fluorescence imaging system, respectively. The leaf cuvette equipped withthe porometer measured CO2 assimilation rate within a selected circular leaf area (18mm diameter) before and after the Basta treatment. The leaf cuvette has a transparent
window at the upper side. Hence, chlorophyll fluorescence imaging was concurrently
performed within the circular leaf area selected by the cuvette through the transparent
window. The measurement was performed at appropriate time intervals in daytime for
2 consecutive days after the Basta treatment. Environmental conditions andexperimental protocols were the same as those of chlorophyll fluorescence imaging
described above.

K. Takayama, A. Konishi, and K. Omasa. “Diagnosis of Invisible Photosynthetic
Injury Caused by a Herbicide (Basta) with Chlorophyll Fluorescence Imaging System”.
Agricultural Engineering International: the CIGR Journal of Scientific Research andDevelopment. Manuscript IT 03 001. June, 2003.

7

Results and Discussion
Figure 5 shows changes in the distribution of chlorophyll fluorescenceintensity (F and Fm'), ΦPSΙΙ, and NPQ over the leaf surface and visual appearance during
24 h after the Basta treatment (Control, 30 min, 3 h, and 24 h). Image of Fm was
captured before the treatment and was used in the calculation for the NPQ image. The
treated area was covered with a gauze soaked in 1:100 dilution of Basta with pure water.

Fm

0

Fm

255

Control

Treated area
30 min

3h

24 h

0

F

F

255

Fm'

0

Fm'

255

ΦPSΙΙ

0.0

ΦPSΙΙ

1.0

NPQ

0.0

NPQ

5.5

VisualFigure 5. Effects of the Basta treatment on a Phaseolus valgaris L. leaf. The
fluorescence image titled “Treated area” represents the Basta treatment. Chlorophyll
fluorescence images (F and Fm') were captured before (Control), 30 min, 3 h, and 24 hafter the treatment and ΦPSΙΙ and NPQ images were calculated at each time.
Before the treatment, chlorophyll fluorescence intensity (F and Fm') was uniformly
distributed over the leaf surface except for the major veins and the neighboring areas.
ΦPSΙΙ and NPQ were also uniformly distributed in those areas and the mean values wereK. Takayama, A. Konishi, and K. Omasa. “Diagnosis of Invisible Photosynthetic
Injury Caused by a Herbicide (Basta) with Chlorophyll Fluorescence Imaging System”.
Agricultural Engineering International: the CIGR Journal of Scientific Research andDevelopment. Manuscript IT 03 001. June, 2003.

8

0.44 and 0.96, respectively. The values of ΦPSΙΙ and NPQ indicated that thephotosynthetic activity in the mesophyll areas was at a moderate level over a leaf
surface (Genty and Meyer, 1994; Osmond et al., 1998). At 30 min after the treatment,
the patchy decrease in chlorophyll fluorescence intensity (F and Fm') within the treatedarea was confirmed. In those areas, ΦPSΙΙ decreased to 0.25 and NPQ increased to 2.26.
The time after the treatment suggested that Basta rapidly moved into mesophyll cells
from the leaf surface. Then, it began to inhibit the photosynthetic electron transport
activity and activated the heat dissipation pathway of absorbed light energy (Genty andMeyer, 1994; Osmond et al., 1998). The patchiness may be caused by heterogeneousabsorption of herbicide during the herbicide treatment through stomata. Uptake ofBasta in the areas with low ΦPSΙΙ and high NPQ seemed to be larger than in non-injured
areas. Thereafter, injured areas were connected with each other and expanded to theadjacent untreated area at 3 h after the treatment. This showed that Basta absorbed inleaf diffused in the leaf apoplast at first and then it was transported along veins andvienlets. The mean value of ΦPSΙΙ and NPQ in the injured areas was 0.11 and 3.47,
respectively. The values represented that photosynthetic electron transport activity
was severely inhibited and the heat dissipation pathway of absorbed light energy wassignificantly activated in those areas (Krause and Weis, 1991; Genty and Meyer, 1994;
Osmond et al. 1998). At that time, visual change was not yet observed. At 24 h after
the treatment, the injured area further expanded and occupied almost all of themeasurement area. The expansion rate between 3 h and 24 h after the treatment, along
the major vein was faster than that in mesophyll area. This indicated that the Basta
was transferred from the treated area to the untreated area located in the margin side ofthe leaf predominantly through the major vein and inhibited photosynthetic functions in
those areas. The browning areas (visible injury) were recognized in the untreated area
located adjacent to the treated area at that time. In those areas, re-increased chlorophyll
fluorescence intensity (F and Fm') was confirmed. Then, ΦPSΙΙ decreased to 0.01 and
NPQ re-decreased to 2.25. This suggested that the pigments emitting the chlorophyll
fluorescence still existed, however, both photochemical and nonphotochemical
processes were dramatically abolished (Krause and Weis, 1991; Govindjee, 1995).
The reason why the visible injury initially occurred in the untreated area and not in thetreated area could not be explained in this study. In the affected areas except for theareas with visible injuries, the mean value of ΦPSΙΙ and NPQ were 0.07 and 4.03,
respectively. In spite of no visible injury in those areas, photosynthetic electrontransport activity was severely inhibited, and the heat dissipation pathway was activated(Krause and Weis, 1991; Govindjee, 1995).
Figure 6 shows relationships between CO2 assimilation rate and ΦPSΙΙ (●) andbetween assimilation rate and NPQ (□) during 24 h after the Basta treatment. Whenthe assimilation rate decreased from 12.2 μmol m-2s-1 to 0.3 μmol m-2s-1, ΦPSΙΙ decreased
from 0.34 to 0.01, and NPQ increased from 0.45 to 2.64. A positive linear correlation
was confirmed between assimilation rate and ΦPSΙΙ, while a negative linear correlation
was confirmed between assimilation rate and NPQ. It showed that a low ΦPSΙΙ and ahigh NPQ indicate a low assimilation rate, and a high ΦPSΙΙ and a low NPQ indicate ahigh assimilation rate. These results prove that the assimilation rate in the areas withlow ΦPSΙΙ and high NPQ in Fig. 5 was low and vice versa.
Figure 7 shows a relationship between ΦPSΙΙ and NPQ during 24 h after the
Basta treatment. A close inverse correlation was confirmed between ΦPSΙΙ and NPQ.
This result implies that the decrease in photosynthetic electron transport activity was
mainly caused by the activation of heat dissipation of absorbed light energy. These
results could be interpreted as follows. First, Glufosinate, the active ingredient ofBasta, initially induced the accumulation of ammonia in mesophyll tissues (Lea andRidley, 1989). Second, it altered the extent of intrathylakoid pH gradient and theK. Takayama, A. Konishi, and K. Omasa. “Diagnosis of Invisible Photosynthetic
Injury Caused by a Herbicide (Basta) with Chlorophyll Fluorescence Imaging System”.
Agricultural Engineering International: the CIGR Journal of Scientific Research andDevelopment. Manuscript IT 03 001. June, 2003.

9activity of heat dissipation pathway, which resulted in an increase in NPQ (Krause andWeis, 1991; Govindjee, 1995; Osmond et al., 1998). Third, the photosynthetic
electron transport activity was reduced and which resulted in a decrease in ΦPSΙΙ (Genty
et al., 1989; Genty and Meyer, 1994). Under normal growing conditions, ΦPSII does
not necessarily relative to CO2 assimilation because the electrons transported thorough
PSII are used for photorespiration and other electron sinks (e.g. Mehler reaction) as well
as CO2 assimilation. In this experiment, however, the decrease in ΦPSII favorably
coincided with the decrease in CO2 assimilation rate after the Basta treatment. This
result showed that the activities of photorespiration and other electron sinks wereconcurrently inhibited by Basta as well as CO2 assimilation.

0.4

3.0

0.3

NPQ

Φ PSΙΙ

2.0
0.2
1.0
0.1

0.0
0.0

3.0

6.0

9.0

12.0

0.0
15.0

Relationships
Figure 6.
between CO2 assimilation ratemeasured with the porometermeasurement, ΦPSΙΙ (●) and NPQ(□)
calculatedfromthechlorophyll fluorescence imaging
during 2 days after the Basta
treatment.
The herbicide
treatment and environmental
conditions were the same as inFig. 5.

Assimilation rate (μmol CO2 m-2s-1)
3.0

Figure 7. Relationship between
ΦPSΙΙ and NPQ after the Basta
treatment. ΦPSΙΙ and NPQ were
calculated from the chlorophyll
fluorescence images.

NPQ

2.0

1.0

0.0
0

0.1

0.2

0.3

0.4

ΦPSΙΙ
Moreover, NPQ also quantitatively evaluated the decrease in CO2 assimilation rate after
the Basta treatment. The NPQ can be calculated only with the saturation light pulse,
so this result suggested that the simplified diagnostic procedure using NPQ could be
easily applied to the field experiment.
K. Takayama, A. Konishi, and K. Omasa. “Diagnosis of Invisible Photosynthetic
Injury Caused by a Herbicide (Basta) with Chlorophyll Fluorescence Imaging System”.
Agricultural Engineering International: the CIGR Journal of Scientific Research andDevelopment. Manuscript IT 03 001. June, 2003.

10

Conclusion

The experiments described here showed that the invisible photosynthetic
injuries caused by Basta could be detected at the early stage of the inhibitions by ΦPSII
and NPQ images obtained with chlorophyll fluorescence imaging. Furthermore, it also
provided topographical diffusion process of Basta in apoplast, veins, and veinlets. The
detected injury was identified as a decrease in CO2 assimilation rate by the porometermeasurement performed concurrently with chlorophyll fluorescence imaging.
The ΦPSII and NPQ do not necessarily relative to CO2 assimilation undernormal growing condition. In this experiment, however, the decrease in ΦPSII
favorably coincided with the decrease in CO2 assimilation rate after the Basta treatment.
This result showed that the activities of photorespiration and other electron sinks wereconcurrently inhibited by Basta as well as CO2 assimilation. Moreover, NPQ also
quantitatively evaluated the decrease in CO2 assimilation rate after the Basta treatment.
Consequently, this study provides evidence that the chlorophyll fluorescence imaging
proves to be a powerful tool in obtaining biotic information on plant leaf in
non-destructive and non-invasive ways.

Acknowledgement

We thank Takateru M. Uenishi and Ryosuke Endo for helpful discussion andcomments on the manuscript. We are grateful to Japan Society for the Promotion of
Science for funding this study.

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K. Takayama, A. Konishi, and K. Omasa. “Diagnosis of Invisible Photosynthetic
Injury Caused by a Herbicide (Basta) with Chlorophyll Fluorescence Imaging System”.
Agricultural Engineering International: the CIGR Journal of Scientific Research andDevelopment. Manuscript IT 03 001. June, 2003.