Remote Sensing of Chlorophyll a Fluorescence of Vegetation Canopies: 2. Physiological Significance of Fluorescence Signal In Response to Environmental Stresses*

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REMOTE SENS. ENVIRON. 47:29-35 (1994)

Remote Sensing of Chlorophyll a Fluorescence of Vegetation Canopies: 2. Physiological

Significance of Fluorescence Signal In Response to Environmental Stresses*

**R. Valentini, t G. Cecchi,* P. Mazzinghi, § G. Scarascia Mugnozza, t G. Agati, § M. Bazzani, ~ P. De Angelis, t F. Fusi, § G. Matteucci, t and V. Raimondi ~**

M e a s u r e m e n t s of laser-induced chlorophyll fluores- cence of living leaves were compared to ecophysiological parameters, both in near and far field conditions. Near field measurements were carried out with a two-wave- length portable fluorometer, both in the laboratory and in the field. Results show significant changes of the 690 nm and 730 nm chlorophyll fluorescence bands in different environmental conditions. Water stress and carboxylation limitations also affect the fluorescence spectra. Far field measurements from a ground-operated fluorescence LI- DAR system confirm those results. The F 6 9 0 / F 7 3 0 fluo- rescence ratio is then demonstrated as a good index for vegetation remote sensing.

INTRODUCTION

Remote sensing of laser-induced fluorescence in green terrestrial plants shows good potentials for species identification, green biomass estimation, leaf area index, and canopy structure (Chapelle et al., 1984). Airborne systems for laser induced fluorescence measurements are today available (O'Neal et al., 1980, Hoge et al.,

* Research supported by National Research Council of Italy, Special Project RAISA, Subproject 2.4.6 UR 2.36, Paper n. 1129.

t University of Tuscia, Department of Forest Resources (DISA- FRI), Viterbo, Italy

*Research Institute on Electromagnetic Waves (IROE) of the National Research Council of Italy (CNR), Firenze, Italy

~Institute of Quantum Electronics (IEQ) of the National Re- search Council of Italy (CNR), Firenze, Italy

Address correspondence to Giovanna Cecchi, Research Inst. on Electromagnetic Waves (IROE) of the National Research Council of Italy (CNR), Via Panciatichi 64, 1-50127 Firenze, Italy.

Received 1 October 1992; revised 1 May 1993.

0034-4257 / 94 / $6.00

©Elsevier Science Inc., 1994

655 Avenue of the Americas, New York, NY 10010

1983; Diebel-Langohr et al., 1985; Pantani and Cecchi, 1992). In the future, according to the development of specific research projects, such as the European EUREKA Project LASFLEUR (EU380), a larger data- base of airborne fluorescence data will be collected on terrestrial ecosystems. Nevertheless, the feasibility of remote sensing techniques to the identification of physiological processes and to the assessment of the impact of environmental stresses on plant physiology is still under discussion.

Chlorophyll fluorescence has been used for many years as a powerful tool in plant physiology to understand the primary events of photosynthesis and the stress development affecting photochemistry. In particular, the quantum efficiency of the Photosystem II (PSII) and the electron transport to carbon metabolism are currently investigated by pulse-modulated fluorometers and saturating light pulses on intact leaves under natural conditions (Schreiber et al., 1986). The analysis of the fluorescence signal quenching components has provided useful information on the action of different environmental stresses (temperature, water, high light, etc.) (Snel and van Kooten, 1990). The developed indexes, such as the ratio of the variable fluorescence and the maximum fluorescence (F~ / Fm), have been used to moni- tor stresses on intact leaves, both in laboratory and in natural conditions (Demming and Winter, 1988).

These physiological indexes derived by pulse-modulated fluorometry are in practice undetectable by airborne remote sensing, especially when dark preadapta- tion of leaves is requested. However, since fluorescence spectral shape can be measured with remote sensing techniques, some spectral parameters have been developed for the detection of physiological stresses (Lichten-

2 9

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30 Valentini et al.

thaler and Rinderle, 1988). In particular, the ratio of the fluorescence bands at 690 nm and 730 nm (F690/

F730) was considered by Lichtenthaler and Rinderle (1988) as a possible index of plant stresses.

The physiological meaning of this spectral index is still under debate. The fluorescence spectrum of a chloroplast suspension shows a very low emission at 730 nm with respect to the 690 nm peak. The chlorophyll reabsorption in the 690 nm band, however, changes the ratio of the two peak intensities to values around 1 in mature leaves. The fluorescence spectral shape of leaves is therefore sensitive to the chlorophyll concentration (Dahn et al., 1992). This factor is very important by itself, since many kinds of damage lead to a reduction in leaf chlorophyll content. Anyway, chlorophyll concentration is expected to change slowly in response to most environmental changes. So the change in spectral shape due to this effect is not very suitable as an early indicator of stress conditions.

On the other hand, the variation of chlorophyll concentration can be taken into account (Agati et al., 1993) using the information provided by passive reflectance/transmittance spectra. Information concerning the physiological state must be extracted, in remote sensing, by fluorescence spectra. In this work, the effect of change in chlorophyll concentration was neglected, since rapid changes in the chlorophyll fluorescence spectra on selected targets were mainly measured, as daily cycles on natural grown plants or artificially induced short term stresses.

In a remote sensing arrangement, as an airborne fluorescence lidar, the fluorescence reabsorption must be taken into account. Anyway, this first step is neces- sary to evaluate which fluorescence spectral parameters are more sensitive to vegetation health state.

This article presents some results of laboratory and field measurements, obtained with the near field systems (LEAF and PAM fluorometers) and the far field system (FLIDAR-3), described in Part 1. The aim of the present work is a critical review of the relationships between physiological processes and some indexes, like the F690 / F730 ratio and the single fluorescence bands, to in- vestigate their usefulness in vegetation remote sensing.

This work is not exhaustive of the subject since it will require a big deal of common effort, as pointed out in Part 1. However, the most important factors affecting chlorophyll fluorescence were pointed out in some key laboratory experiments and, whenever possible, compared with results of field and remote sensing experiments.

MATERIAL AND METHODS

The fluorescence spectra show peaks at wavelengths that shift slightly in the range of 5 nm for the F685 band and even 20 nm for the /7730 band, depending

mainly on vegetation type and leaf structure. Thus, the term red Fluorescence Ratio (RFR) is accepted to replace the well-known F690/F730 ratio. LEAF mea- sures the two bands at 6 8 5 + 5 nm and 7 3 0 + 5 nm, while FLIDAR detects the complete spectrum from 500 nm to 800 nm (see Part 1).

Near Field Measurements

Near field measurements of fluorescence signal were obtained with LEAF portable fluorometer (described in Part 1). The first experiment was performed on leaves of

Populus alba

L. and

Quercus ilex

L. seedlings in good water conditions. Leaves were enclosed in a controlled chamber where air temperature was maintained at 25 + 0.5°C and vapor pressure deficit at 0.6 + 0.05 KPa.

Light was provided with two 400 W HQI OSRAM lamps and filtered with a water bath for thermal shielding.

Different light levels were obtained with plastic shields, placed over the cuvette.

Diurnal cycle measurements of fluorescence with the LEAF fluorometer clip-on probe (described on Part 1) were collected every minute, as an average of 15 measurements on an isolated 15-year-old tree

(]uglans regia

L.). Photosynthetic photon flux density (PPFD) was measured with a cosine corrected sensor (LICOR), placed very close to the leaf and with the same orienta- tion of the leaf. The sensor output was recorded by a separate channel of the fluorometer itself, simultaneously to fuorescence measurement.

LEAF measurements on

Fagus sylvatica

L. were collected in the forest on mature trees, in good water conditions, averaging about 20 fluorescence data points relative to different locations on each leaf surface. Photo- synthesis measurements were carried out with a gas exchange portable porometer (ADC).

Experiments on water stressed

Populus alba

L. seedlings were carried out inside greenhouse. Different water stress levels were induced on clones of the same plant. The LEAF data are the average of about 20 points on each leaf. The data were taken at two different times of day.

Far Field Experiments

Remote sensing measurements were carried out on

Fa- gus sylvatica

L. and on

Quercus pubescens

Wild. young trees with FLIDAR-3 (described in Part 1) at distances ranging between 15 m and 60 m. Using a folding mirror settled just over the tree to deflect the lidar beam, the trees were irradiated also from the top, simulating an airborne remote sensing experiment.

This experimental setup is suitable for young plants grown at the border of the forest. Near field and physiological measurements were made simultaneously, di- rectly on the same target leaf (or leaves), or on leaves belonging to the same branch and close to the target

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Fluorescence Response to Environmental Stresses 31

ones. The monitored leaves (see Part 1) were enclosed in an environmental controlled cuvette (CMS, Walz) for measurement of physiological parameters. The cuvette temperature was maintained equal to the external air.

In addition, a stress was induced on the controlled tree

(Ouercus pubescens

Wild., isolated tree). The tree behavior from normal to stress conditions was monitored with the whole equipment. In particular, the stress was produced simply by cutting the tree branch, inducing mainly a water stress at first.

RESULTS AND DISCUSSION

For the sake of clarity, the data presented here are split into two parts, concerning the results carried out on vegetation in the near and far field, respectively.

Near Field Detection of Fluorescence Signal with LEAF Fluorometer

Figure 1 presents the RFR behaviour in comparison with different steady-state light conditions. The data show a linear decrease of RFR, increasing light, both for

Quercus ilex

and

Populus alba.

The decrease of RFR values in the range from 0 to 2 0 0 0 / t m o l m -2 s-~ is

Figure 1.

Variation of the RFRindex and of the total fluorescence

Ft

(F690 + F735) as a function of light

(Quercus ilex

and

eopulus alba).

Quercus flex

1.8 700

I - B - R F R

1.8i r ~ Ft

1,4 ~ []

0.8 ~

O.(t = i i

0 5OO 1000 1600

PPFD (umol m's')

Populus alba 1.2

8OO

"11 500 4oo 8 ~

S O 0 ~

2 0 0

1 0 0 2 0 0 0

700

1.1

O:

O.8

0.7

O.e 0

R F R []

• Ft

[]

~ • ~ [ ] -

400 800 1200 1000

PPFD (~nol m=s ~)

80O "n

500

4 0 0 ~

3 0 0

200

1 0 0 2O00

2 0 0 0

Juglans regia

1.3

1500

"E ,~ 1000

5 O 0

• RFR o PPFD ]

~0

⁰ "" •

• ~ , : ~ ' ~ , ~ . ~ : .

o o

• ~ o . . *. • . 0 ". . " O;

o .'. o'~,~~

© •

d~

, o o

p --t ~ I I I I I I

2 4 6 8 10 12 14 16 18 20 22

time of day

1.2

1.1

1 :0

0.9 018

0.7 0.6 24

Figure 2.

Diurnal behavior of the RFR index and of the photosynthetic photon flux density (PPFD). The PPFD was measured close to the fluorescence detection point.

25% for

Quercus ilex

and 18% for

Populus alba.

The total fluorescence

(Ft)

shows a similar behavior and decreases increasing light levels. The decrease is more pronounced than that of RFR and the variations are about 50% for both

Quercus ilex

and

Populus alba.

The same behavior was confirmed by a daily cycle of RFR taken in the field on a leaf of

Juglans regia L.

(Fig. 2). The fluorescence ratio decreases during the day and reaches its minimum value at the highest value of PPFD. RFR ranges from about 1.1 during night to about 0.75 during day, in full sunlight conditions (PPFD = 1800/tmol m -2 s-t).

The decrease of RFR with increasing light level was found in all the experiments to be determined by a relative higher decrease of the 685 nm band with respect to the 720 nm one. Figure 3 presents an example of this behavior, where the two bands, normalized to total fluorescence

(Ft),

are plotted as a function of PPFD.

RFR behavior was also investigated as a function of photosynthetic rates, at constant PPFD levels. This was analyzed exposing leaves of

Quercus ilex

and

Populus alba

to different levels of CO2 concentration.

Figure 3.

Changes of the relative contribution of the F690 and F730 bands to total fluorescence

(Ft

= F690 + F730)

( Quercus ilex).

70

I 60©

*~ 50 u."

40

Quercus flex

F6851Ft F 7 2 0 I F t

N

0

30 0

i i i

500 1000 1500

PPFD ( ~ o I m's')

2000

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32 Valentini et al.

Figure 4 presents the result; RFR is plotted as a function of intercellular CO2 concentration (Ci) at two different light levels (80 and 800/~mol m -2 s -1) for

Quercus ilex

and three light levels (100/~mol m -2 s -;, 500/~mol m -2 s -1, and 1400 pmol m -2 s -;) for

Populus alba.

Increasing Ci, the RFR index increases for both the species. Since the photosynthetic rate increases with increasing CO2 concentrations, this behavior is opposite to that of Figure 1, where RFR decreases with increasing PPFD. Also,

Populus alba

shows higher RFR variations (30%) with respect to

Quercus ilex

(11%), opposite to the variation with PPFD.

On the contrary, the total fluorescence decreases increasing Ci, showing a similar behavior to that of Figure 1. Nevertheless, the two bands behave differently with respect to Figure 3: The 690 nm band decreases slower than the 730 nm one (data not shown).

RFR measurements as function of the maximum photosynthetic rates (Amax) in

Fagus sylvatica

leaves are shown in Figure 5. Although leaves were exposed to a constant light level, RFR decreases when Am~x increases.

Figure 4.

Variation of the RFR values with intercellular CO2 concentration under different light conditions

(Quercus ilex

and

Populus alba).

O u e r c u s i l e x 1.8

1.76

rr 1.7 [J. rr

1 . 6 5

1.6

1.55 0

1.4

1.2

n- i,

rr 1

0 . 8

0.6

I -b PPFD = 800 lamol m ' g ' i

I I I I

1 0 0 200 3 0 0 4 0 0 6 0 0

CI ( p p m ) P o p u l u s a l b a

. . . + . . . ~ . . . - ~ . . . ~. . . .

, + . + • +

........

• P P F D = 1400 I~mol re's"

i i i i

1 0 0 2 0 0 3 0 0 4 0 0 6 0 0 0 0 0

CI ( p p m )

1.1

li

0.9 n-

0.8

F a g u s s y l v a t i c a

. * i i i

i[] i

0.7 . . . . : ~

i i i i I

0 . 6 - - ~

0.5 1 1.5 2 2.5 3 3.5 4 4.5

A.,,,(0mol CO 2 m's') []

i i

2 5 0 0

2 0 0 0

1500 0

1 0 0 0 3~

500

Figure 5.

Relationship between net photosynthesis (Amax) and the RFR values for

Fagus sylvatica

leaves in natural conditions.

In this experiment, the chlorophyll content of the sam- pled leaves was not found to be correlated with the RFR values, probably due to the small variations of chlorophyll concentrations among beech leaves.

The effect of water stress on RFR was monitored on clones of

Populus alba

L. grown in greenhouse. In Figure 6, RFR values, taken at two different times of day, are plotted as a function of water potential. The data reveal an appreciable difference between the stressed and unstressed conditions. Actually, the stressed plants show higher values of RFR than the unstressed ones.

This RFR variation is more pronounced for the data taken in the afternoon, when the lack of water enhances the difference between stressed and unstressed plants.

Far Field D e t e c t i o n o f F l u o r e s c e n c e Signal with the FLIDAR System

Far field measurements on different plant species were carried out with the FLIDAR system. The plants were monitored simultaneously with the near field equipment, including physiological measurements. The data

Figure 6. Variation of the RFR ratio for water-stressed Popu- lus alba seedlings. The water stress condition is expressed by the levels of predawn water potential.

Populus alba

O . 6 5

N o t s t r e s s e d W a t e r s t r e s s e d

0.6

[]

r~

~: 0.55

0.5 ~ _[] D a t a s e t 1 (H 12:30) ] [] []

s [] ~ D a t a s e t 2 (H 16:20)/ I

0.45 i i

0 0.6 1 1.5 2

P r e d a w n W a t e r Potential (Mpa)

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Fluorescence Response to Environmental Stresses

33

presented here refer to Fagus sylvatica L. and Ouercus pubescens Wild. species.

Figure 7a shows RFR values, obtained in vivo on a leaf of Fagus sylvatica. The measurements were taken in a beech wood, on the Appenines mountains in the central part of Italy. The beech tree under control was a healthy young plant, at the border of the forest. The fluorescence signal was monitored for several hours, observing the night-to-day transition. The relative PPFD values together with net photosynthesis measurements are shown in Figure 7b.

RFR shows an opposite behavior compared to that of PPFD. Actually, high RFR values correspond to low PPFD, and PPFD variations, mainly due to meteorologi- cal conditions (wind and clouds) induce variations on RFR. RFR decreases from 0.95 to 0.6 during the night- to-day transition and at high light condition PPFD reaches a value of 1200/~mol m -z s -]. The near field measurements, obtained with LEAF fluorometer, show the same RFR behavior confirming FLIDAR results.

Photosynthetic rates range from a minimum of - 0.5 /~mol m- 2 s-~ during the dark respiration to a maximum of 8/~mol m-2 s-1. Figure 8 presents two fluorescence spectra detected during dark and light conditions, respectively. RFR and total fluorescence decrease in sunlight, confirming near field measurements. The total

Figure 7. Diurnal behavior of far field RFR measurements on Fagus sylvatica: a) RFR values during the night-day transition; b) net photosynthesis and PPFD values of the target.

a) Fagus sylvatica

1.1

1

0.9 r,- i, r,- 0.8

0.7

0.6

0.5

2000

1600

"E ..~ 1OO0

6OO

0 4

b)

~ ~ ~ ~ 1;) 1~ 12

time of day

Fagus sylvatica

I ~ A --~--PPFD 1 ~ ~ 6

2

0

- 2

6 6 7 8 9 10 11 12

time of day

o o

6 0 0

Fagus sylvatica

5 0 0 ... d a r k ,, ', . . . .... -. '...

- - s u n l i g h t / '. . ..

:. 4 0 0 " .. . . .

300

200 100 ..~

0

₆₀₀ _6~o _~oo _T~o

wavelength ( n m )

Figure 8. Laser-induced fluorescence spectra of Fagus syl- vatica in dark and light conditions.

fluorescence decrease is related to quenching processes involved in photosynthesis, but for far field measurements this decrease is much lower than that expected and also measured by LEAF and PAM fluorometers.

This is probably due to other mechanisms, concerning the different excitation (wavelength and pulse duration).

However, from a remote sensing point of view, the total fluorescence intensity has a lower interest.

Actually, these absolute measurements require the knowledge of all those parameters, such as geometrical factors, and atmospheric transmission, which do not play a role in differential measurements.

Fluorescence spectra were also teledetected with FLIDAR-3 on unstressed and water stressed Quercus pubescens tree. As shown in Figure 9 for the unstressed target, the red fluorescence ratio changes from 0.98 at 6:00 a.m. (dark conditions) to 0.76 at 1:00 p.m. (full sunlight).

Figure 9. Diurnal behavior of the RFR values on Quercus pubescens tree under unstressed and water stressed conditions.

1,1

1

0,9 I, n-

0,8

0,7

0,6 4

Quercus pubescens

" ~ 3 /

^". ~ ... •

... - ... u n s t r e s s e d [ " ~

• stressed ]

6 8 10 12 14 16 18

time of day

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34

Valentini et al.

350

Quercus pubescens

300

, ~ . 250

-.i

200

f-

o 1so

... u n s t r e s s e d ... - ~

/.

- - stressed ...

..' ". :.

. . ... "-..

100

/

°oo 650

wavelength (nm)

Figure 10. Fluorescence spectra of Quercus pubescens under unstressed and water stressed conditions, showing the peak at 713 nm.

A similar behavior, but with lower RFR index values, is shown for the water-stressed target: The values vary from 0.94 at 8:00 a.m. to 0.67 at 3:00 p.m. The spectral analysis reveals (Fig. 10) that under stress conditions the peak at 690 nm is strongly decreased. More- over, a small peak, placed by a gaussian fit at 713 nm, appears and remains in all the spectra obtained during the afternoon. This band is probably related to changes in pigment composition. Also in this experiment LEAF measurements confirmed the RFR behavior detected by FLIDAR-3.

CONCLUSIONS

This work shows that chlorophyll fluorescence spectra are a sensitive tool for vegetation remote sensing. The connection between plant physiology parameters and RFR was demonstrated by several laboratory and field key experiments, However, since many parameters affect RFR, the extraction of a vegetation stress index from remote sensing spectra is not easy.

The ratio F690/F730 has been already considered by Lichtenthaler and Rinderle (1988) as a useful indicator of stress conditions in plants. Its usefulness for chlorophyll change determination was shown for several species and different growth conditions (Rinderle et al., 1991). The application of this spectral index of fluorescence showed significant changes varying environmental and physiological conditions. Also temperature showed a significant effect on the F690 / F'/30 ratio (Lipucci di Paola et al., 1992), with a variation of about 35% for a temperature change of 16°C.

The first factor to be considered using the F690/

F730 ratio is its relative dependence on the optical properties of leaves. In particular, the F690 band is strongly affected by reabsorption of chlorophyll. The relative concentration of chlorophyll was shown as a

primary factor determining the RFR value (D'Ambrosio et al., 1992; Dahn et al., 1992). This effect can be taken into account with a simple model using the combined information delivered by refectance and transmission spectra (Agati et al., 1993). So remote fluorescence spectra must be measured in close connection with passive reflectance spectra. The combination of the two measurements can deliver reliable information about chlorophyll concentration of the target, which is useful by itself.

Beyond this nearly static factor, RFR was found dependent on environmental conditions, especially PPFD, showing a daily, cyclic variation that cannot be attributed to variations in chlorophyll concentration.

This was also confirmed in laboratory experiments, with a direct relationship between RFR and light level (in addition, carboxylation rates and water stress modify RFR value).

RFR variations, due to increasing light levels, are essentially determined by the relative decrease of the 690 nm band with respect to the 730 nm band. The fluorescence intensity is anyway decreasing for both bands in these conditions. Reasons for this behavior could be attributed to changes of the optical properties of leaves, due to chloroplast movements or structural modifications of the leaf. This could explain the observed changes in RFR in water stress conditions. However, this is not likely to be the only factor affecting the RFR value. Actually, a variation of RFR without any change in leaf absorption during induction kinetics was recently demonstrated (Agati et al., 1992). The energy transfer processes, taking place at the onset of photosynthesis, can differently quench the two fluorescence peaks, showing a direct connection between RFR and the photosynthetic process.

This was also demonstrated by experiments with increasing carboxylation rates at a constant light level.

RFR variations were produced by a decrease of the 730 nm band, while the 690 nm one remains almost constant. Intercellular CO2 concentration (see Fig. 4) and photosynthetic rates (see Fig. 5) induce changes in RFR, showing that the F730 fluorescence band is quenched by photochemistry.

Remote RFR measurements carried out in the far field with fluorescence lidar techniques were successful on different trees and different environmental conditions. The RFR variations showed the same behavior of the laboratory experiments. The variations were also similar to the field measurements carried out on single leaves with LEAF fluorometer.

The main difference between near field and far field measurements was found in the total fluorescence intensity variation with PPFD, during daily cycles. The FLIDAR measured a nearly constant fluorescence intensity, while the LEAF (and PAM) fluorometer showed the usual fluorescence quenching induced by the actinic

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light. The fluorescence signal is generally quenched under light conditions by photochemical and nonphotochemical quenching (Shreiber et al., 1986). There- fore, lower fluorescence quantum yield is expected at increasing light levels.

A possible explanation of the observed contrasting behavior is that the FLIDAR fluorescence signal is induced by an excitation pulse, whose duration is about 10 ns. This is much shorter than the pulse used by the LEAF fluorometer that is longer than 1 ms. Under dark conditions, when reaction centres are open, a short pulse is equivalent to a measurement of Fo since most of excitation is trapped in the antenna chlorophylls.

During high light conditions most of the reaction centres are closed and the excitation gives a higher fluorescence signal, since the photochemical quenching has a longer time scale. It is not clear why the nonphotochemical quenching seems not to affect laser-induced fluorescence signal under full sunlight conditions. Further research work is needed to understand the physiological processes that are at the basis of the observed discrepan- cies. In particular the different duration of the excitation pulses should be taken into account to analyse photochemical processes.

The authors wish to thank Professor Luca Pantani for the useful discussions and Dr. llaria Ambrosini, Mrs. Maria Grazia Baldecchi, Mr. Faust0 Meiners, Mr. Bruno Radicati, Mr. Daniele Tire& Mr. Massimo Trambusti, and Mr. Giancarlo Valmori for their support during the laboratory and field experiments.

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