Photoacoustics as a tool for the diagnosis of radicular stress: Measurements in
eucalyptus seedlings
P. R. Barja, A. M. Mansanares, E. C. da Silva, and P. L. C. A. Alves 
 
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REVIEW OF SCIENTIFIC INSTRUMENTS VOLUME 74, NUMBER 1 JANUARY 2003

 This art
Photoacoustics as a tool for the diagnosis of radicular stress:
Measurements in eucalyptus seedlings

P. R. Barja
Universidade do Vale do Paraı´ba-UNIVAP, Research and Development Institute-IP&D, Av. Shishima Hifumi
2911, 12244-000 Sa˜o Josédos Campos, SP, Brazil

A. M. Mansanaresa) and E. C. da Silva
Instituto de Fı´sica Gleb Wataghin, Universidade Estadual de Campinas, Unicamp, Caixa Postal 6165,
13083-970 Campinas, Sa˜o Paulo, Brazil

P. L. C. A. Alves
Departamento de Biologia Aplicada, FCAVJ-Unesp, 14870-000 Jaboticabal, SP, Brazil

~Presented on 26 June 2002!

In reforesting companies~cellulose industry!, eucalyptus is usually cultivated in small plastic
containers ~50 mL!. As seedlings remain for about 120 days in these containers—until
transplantation—their roots become space restricted, with consequent limitations in water and
nutrient absorption. These restrictions may lead to plant stress, decreasing productivity. In this work,
we used the photoacoustic technique to evaluate the photosynthetic activity ofEucalyptus grandis,
E. urophylla and E. urograndisseedlings subjected to this limited space availability, seeking a
correlation with morphological parameters and fluorescence measurements in these seedlings.
Photoacoustic, fluorescence, and morphological analysis were conducted every 15 days, from 45 to
120 days after sowing. Fluorescence and photosynthetic rate were evaluatedin vivo andin situ, the
latter one using the open photoacoustic technique. Data show that root dry matter diminished
markedly at 90 and 120 days after sowing; this behavior showed a high correlation with the gas
exchange component of the photoacoustic signal, as well as with the fluorescence ratioFv/Fm.
These results indicate that the soil volume of the container becomes insufficient for the roots after
90 days, probably leading to a nutritional deficiency in plants, which explains the decrease observed
in the photosynthetic rate of seedlings. ©2003 American Institute of Physics.
@DOI: 10.1063/1.1518567#
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I. INTRODUCTION

The utility of the photoacoustic technique in studyin
leaf photosynthesis is corroborated by the reviews publis
in this area.1–4 The photoacoustic technique is based on
absorption of modulated light by the sample with the succ
sive release of modulated heat and~in the case of vegeta
leaves! gas evolution.

The first photoacoustic measurements of leaf photos
thesis were performed with the leaf usually being cut a
enclosed in the cell.5 This has changed with the developme
of the open photoacoustic cell~OPC!6–8 composed by an
electret microphone that uses its own chamber as the ac
tic cell, with the sample acting as one of the walls. Since
sample itself closes the chamber, it is not necessary to c
leaf disk, or to detach the leaf from the plant for measu
ments, so the OPC allowsin vivo and in situ monitoring of
the photosynthetic activity in plants, avoiding dehydration
the sample. Part of the leaf remains exposed to the outs
capturing external CO2, which minimizes changes in th
photoacoustic chamber atmosphere. Several studies on
tosynthesis were carried out with the OPC technique, suc
the evidence of heterosis in maize hybrids through photos

a!Electronic mail: manoel@ifi.unicamp.br
7090034-6748/2003/74(1)/709/3/$20.00

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thesis induction measurements9 and the energy storag
determination.10

The two most cultivated species of eucalyptus in Bra
are the fast growingEucalyptus grandisand theE. urophylla
that grows lower, but is more tolerant of water-limite
conditions.11,12 The crossing of these two species genera
the hybridE. urograndis, which presumably grows faster an
is more resistant to dry regions.13 For these reasons, theE.
urograndishybrid has been the most planted by reforest
companies in Brazil in recent years.

In this work, in vivo andin situ photosynthesis induction
measurements were performed in leaves ofEucalyptus gran-
dis, Eucalyptus urophylla, andEucalyptus urograndisplants
for different ages of the seedlings. This was done to de
mine if the photosynthetic behavior of these plants was
fluenced by a root stress due to the limited volume of
receptacle utilized for each sample.

II. MATERIALS AND METHODS

A. Photoacoustic setup

The experimental setup is already depicted elsewher10

There are two light sources: a xenon arc lamp~Oriel,
mod.6128, 1000 W! and a halogen lamp~Ushio/ELC, 250
W!. A chopper ~PAR, mod. 192! and a monochromato
© 2003 American Institute of Physics

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710 Rev. Sci. Instrum., Vol. 74, No. 1, January 2003 Barja et al.

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~Oriel, mod. 77250! are used in front of the xenon lamp i
order to obtain modulated light of a determined wavelen
~680 nm!. Measurements were carried out at 17 Hz. Opti
filters limit the light of the halogen lamp to the visible part
the spectrum. Intensity of the modulated red light, used
photosynthesis induction, was 10 W/m2, while that of con-
tinuous white light, used for photosynthesis saturation, w
about 350 W/m2. A double-branched optical cable is used
guide each light beam up to the photoacoustic cell. T
chopper and the photoacoustic cell microphone are c
nected to a lock-in amplifier~PAR-EG&G, mod. 5210! that
measures the amplitude and phase of the microphone si
The lock in is connected through a general purpose inter
bus to a microcomputer for data acquisition. The typical ti
constant used is 1 s, which gives the time response of
setup.

The OPC is already described in literature.7,14 It is com-
posed by an electret microphone, with the photoacou
~PA! microphone chamber being closed by the leaf its
The sensitivity is of about 10 mV/Pa.

B. Plant materials

All eucalyptus plants utilized in this work were cult
vated in small pots~50 ml! under 50% shade conditions an
were irrigated and fertilized daily. Measurements were p
formed in eucalyptus leaves from seedlings between 45
120 days old, in fifteen days intervals. Fully expanded lea
of the second pair were selected for measurements. Seed
were dark adapted at ambient temperature for about 24
the laboratory before measurements. After this dark per
plants were moved to the experimental setup, and a sele
part of an undetached leaf was fixed to the OPC and expo
to the modulated light. Average values were taken over ab
four measurements for each sample and condition.

III. RESULTS AND DISCUSSION

Figure 1 shows a typical curve of the photobaric sig
amplitude in photosynthesis induction measurements in aE.
urograndisleaf. At time t50, modulated light was switche
on and the PA signal~amplitude and phase! was recorded as

FIG. 1. Typical photosynthesis induction curve~amplitude of the photobaric
component as a function of time! obtained forE. urograndisleaf ~45 days
old seedling! previously dark adapted for 24 h. Modulated light attribute
l5680 nm, modulation frequency of 17 Hz, and intensity of 10 W/m2.
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a function of time. For all species of eucalyptus studie
photobaric component presents a fast increase in the si
amplitude followed by a decrease to the initial level in t
first minute of illumination. After this, amplitude increase
up to a steady state, reached after about 4 min.

Figure 2 shows the amplitude of the steady-state pho
baric signal as a function of the age of the seedlings for
three species. These data indicate that all species stu
reach maximal photosynthesis rate between 60 and 75
old. Measurements in older samples reveal a sudden decr
in the photosynthetic rate for 90 days old seedlings.

The reduction of the photosynthetic activity observed
90 days old seedlings could be correlated with the stress
to the insufficiency of space in the pot. Figure 3 shows
dried root weight as a function of the age of the seedlings
E. urophylla. As one can see, there is a minimum around
days, thus correlating to the photoacoustic measuremen
the oxygen evolution. Similar results were obtained for t
other species. In fact, the deficit of nutrients and lack
space in the pot may inhibit both mineral absorption a
growing of the root system, reducing nutrient acquisition.
a result, activation/synthesis of enzymes involved in pho

FIG. 2. Amplitude of the steady-state photobaric signal as a function of
age of the eucalyptus plants~previously dark adapted for 24 h!. Each point
is the mean value of about four measurements. Solid line/triangles:E. uro-
grandis; dashed line/squares:E. urophylla, and dotted line/circles:E. gran-
dis leaves.

FIG. 3. Dried root weight and the ratio between the variable fluoresce
Fv and maximum fluorescenceFm as a function of the age of the seedlin
for E. urophylla.
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711Rev. Sci. Instrum., Vol. 74, No. 1, January 2003 Photoacoustic and photothermal phenomena

 This art
synthetic processes may be damaged, with immediate ef
in photosynthesis.

Figure 3 also shows the ratio between the variable fl
rescenceFv and the maximum fluorescenceFm plotted
against the age of the seedlings. The measurement o
fluorescence of the chlorophyll is made in function of tim
after the start of incidence of intense white light on t
sample~previously in the dark!. In the beginning, there wa
a low level of fluorescenceF0 , since the electron accepto
in the Photosystem II were available. As time goes by, th
acceptors become unavailable because of the large amou
electrons produced as a result of light absorption. The p
tochemical path is therefore saturated, and fluorescenc
chlorophyll increases, reaching finally a maximum valueFm.
The difference (Fm-F0) is named variable fluorescenceFv.
Hence, the ratioFv/Fm is a measure of the availability o
electron acceptors in the Photosystem II in the absenc
light. Figure 3 presents a reduction in the ratioFv/Fm after
90 days forE. urophylla. This result also indicates that th
photosynthetic system is being affected by the restriction
the volume of pot.

Comparison between species in measurements for
days old seedlings shows thatE. grandisseems to be the
most resistant to this stress, whileE. urograndisis the most
affected. However, subsequent measurements show that
month after the decay of photosynthesis activity attributed
radicular stress,E. urograndistotally recuperates the sam
photosynthetic capacity verified in 45 days old seedlin
Concluding, one can say that all eucalyptus species stu
presented a partial restoration of their photosynthetic cap
ties one month after the manifestation of the radicular str
This recuperation may never be complete because of the
ited supply of nutrients in the small pot used for ea
sample.

IV. CONCLUSIONS

The study presented here shows the application of
OPC in investigating how the photosynthetic behavior of
calyptus leaves is affected by the availability of soil a
icle is copyrighted as indicated in the article. Reuse of AIP content is subje

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nutrients for the seedlings. This kind of study can only
performed throughin vivo and in situ measurements, sinc
suspensions of chloroplasts can not reproduce the beha
of living samples and the response of detached leaves ca
distorted by dehydration.

Data presented here show that parameters as the
volume available to each sample have a strong influenc
the development of the eucalyptus plants. Results indic
that the photosynthetic behavior of seedlings has been
fected by a radicular stress, with the soil volume of the p
becoming insufficient for the roots, causing a nutritional d
ficiency in plants. Investigating this stress through the p
toacoustic technique, the present work can be helpful for
optimized planning of eucalyptus reforestation.

ACKNOWLEDGMENTS

The authors wish to thank the Brazilian agenc
CAPES, CNPq, and FAPESP, for financial support, and
torantim Celulose e Papel Ltda., for supplying the sampl

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