Bioimpedance Parameters as a Risk Factor to Assess Pine Decay

An Innovative Approach to the Diagnosis of Plant Diseases

E. Borges

, M. Sequeira

, André F. V. Cortez

, H. Catarina Pereira

, T. Pereira

, V. Almeida

T. M. Vasconcelos

, I. M. Duarte

, N. Nazaré

, J. Cardoso

and C. Correia

Instrumentation Center, Physics Department of the University of Coimbra, Rua Larga, Coimbra, Portugal

Centro de Estudos de Recursos Naturais Ambiente e Sociedade, Escola Superior Agrária de Coimbra

of the Instituto Politécnico de Coimbra, Bencanta, Coimbra, Portugal

Keywords: Electrical Impedance Spectroscopy, Bioimpedance, Early Detection, Physiological States, Pinewood

Disease, Pinewood Nematode, Plant Diseases, Hydric Stress, Pinus pinaster Aiton, Bursaphelenchus

xylophilus Nickle.

Abstract: Electrical impedance spectroscopy, EIS, has been proving efficacy and utility in a wide range of areas, from

the characterization of biological tissues to living organisms. Several commercial solutions, with high

precision and resolution, are available. Nonetheless, the typical equipments are expensive, unfeasible for in

vivo and in field applications and unspecific for concrete applications. These features, together with the

lately demands in the vegetal field, fundament this work. Actually, the fast spread of asymptomatic forest

diseases, with no cure available to date, such as the pinewood disease, PWD, constitute a problem of

economical and forestall huge proportions. Herein is proposed a portable EIS system, for biological

applications, able to perform AC current or voltage scans within a selectable frequency range. The

procedure and the results obtained for a population of 24 young pine trees (Pinus pinaster Aiton) are also

presented. Pine trees were kept in a controlled environment and were inoculated with the nematode

(Bursaphelenchus xylophilus Nickle), that causes the PWD, and also with bark beetles (Tomicus destruens

Wollaston). Some degree of discrimination between different physiological states was achieved. These

results may constitute a first innovative approach to the diagnosis of such types of diseases.

1 INTRODUCTION

Electrical impedance measurements performed in a

wide frequency range give rise to a great number of

techniques able to characterize solids, liquids and

suspensions (Callegaro, 2009). Lately, the method

has proved its value also in the characterization of

biological tissues and fluids, either in vitro or in vivo

(Callegaro, 2009), and also to living plants (Fukuma,

2001); (Repo et al., 2000); (Väinölä and Repo,

2000); (Bauchot et al., 2000), animals (Dean et al.,

2008); (Willis and Hobday, 2008) and humans (Kyle

et al., 2004); (Giouvanoudi and Spyrou, 2008).

Concerning the vegetal field, the applications of

electrical impedance spectroscopy, EIS, techniques

have been claiming significant and growing

acceptance, especially as a measure of the water

content in the quality control processes (Vozáry and

Mészáros, 2007); (Fukuma, 2001); (Pliquett, 2010)

of fruits (Fang et al., 2007) and vegetables (Hayashi

et al., 1992); (Dejmek and Miyawaki, 2002), as well

as in the monitoring of the maturation process of

fruits (Bauchot et al., 2000); (Harker and

Maindonald, 1994) and the physiological state of

living plants under adverse environmental conditions

(Repo et al., 2000); (Väinölä and Repo, 2000).

The electrical impedance of a biological

material, or simply bioimpedance, is a passive

electrical property that measures the opposition

relatively to an alternating current flow applied by

an external electric field. The current I, as it passes

across a section of a material of impedance Z, drops

the voltage V, established between two given points

of the same section, yielding the well-known

generalized Ohm’s law: V=IZ, where V and I are

complex scalars and Z is the complex impedance.

The law can be rewritten as V=I|Z|e

since, at a

given frequency, the current flow I lag the voltage V

by a phase of Ѳ (i.e. the current signal is shifted

(Ѳ/2π)T s to the right with respect to the voltage

Borges E., Sequeira M., F. V. Cortez A., Pereira H., Pereira T., Almeida V., M. Vasconcelos T., M. Duarte I., Nazaré N., Cardoso J. and Correia C..

Bioimpedance Parameter as a Risk Factor to Assess Pine Decay - An Innovative Approach to the Diagnosis of Plant Diseases .

DOI: 10.5220/0004230500350046

In Proceedings of the International Conference on Biomedical Electronics and Devices (BIODEVICES-2013), pages 35-46

ISBN: 978-989-8565-34-1

 2013 SCITEPRESS (Science and Technology Publications, Lda.)

signal, in the time domain). Hence, the result of the

EIS measurements is a set of complex (magnitude

and phase) of impedance versus frequency.

Cell membranes, intracellular fluid (cytosol) and

extracellular fluid are the major contributors of the

impedance of biological tissues (Kyle et al., 2004);

(Pliquett, 2010).The cytosol and the extracellular

fluid are mostly constituted by water, consist in

electrolytes, and act like ohmic resistors, while the

insulating membranes behave like capacitors (Kyle

et al., 2004); (Pliquett, 2010). It is, therefore,

possible to depict the behaviour of a biological

tissue by the representation of capacitive and

resistive elements of a respective equivalent

electrical circuit. A commonly used circuit to

represent biological tissues consists of a parallel

arrangement between a resistor, simulating the

extracellular fluid, and a second serial arrangement

connecting a resistor, this one of the cytosol, and a

capacitor, of the membrane (Kyle et al., 2004) - see

figure 1. Since the time constant for loading cell

membranes is typically of the order of the

microsecond (Pliquett, 2010), tissue impedance can

be measured in a frequency range up to tens of MHz

(Callegaro, 2009). In this range of frequencies the

membrane performs like an almost perfect capacitor,

allowing an estimation of the combined ohmic value

of the cytosol and the extracellular fluid. On the

other hand, using direct current level, DC, (low

frequency), the current does not cross the membrane

due to its insulator behaviour. This short circuit-like

actuation forces the current to flow exclusively

through the extracellular fluid providing, thus, a

measure of its ohmic value. However, due to

technical limitations and multiple dispersions (α

dispersions at low frequencies – tissues’ electrolyte

behaviour – and γ dispersions at very high

frequencies – tissues’ aqueous behaviour Ivorra,

2003), the usage of DC and very high frequency AC

currents is restricted (Kyle et al., 2004). Therefore, it

becomes quite more convenient to determine the

ohmic values by prediction. The model commonly

used to predict such values is the Cole bioimpedance

model, in which the bioimpedance spectra is

represented by means of a Cole-Cole plot (see figure

1), that explores resistance versus reactance,

allowing the determination of the ohmic values of

the cytosol and the extracellular fluid. The

mathematical expression descriptive of the Cole-

Cole plots is the Cole equation (here expressed has

in Grimnes and Martinsen, 2008):







∆











, ∆







(1)

Where Z is the impedance value at frequency ω

(with ω=2πf), Z

∞

is the impedance at infinite

frequency (high frequencies) (note: this term is

misleading and is replaced by an ideal resistor R

∞

), j

is the complex number, R

is the impedance at DC

frequency, τ is the characteristic time constant and α

is a dimensionless parameter with a value between 0

and 1.

Figure 1: Bode and Cole-Cole diagrams obtained by

simulation with Matlab® for an electrical circuit

representing a hypothetical biological tissue (right top of

the figure).

The relationship between reactance and

resistance, perceived in a Cole-Cole plot, expresses

the electrical properties of tissues. Diseases and

nutritional or hydration levels may change their

physiological state. These changes have direct

influence in the impedance spectra. The phase angle

and other interrelated indices, such as Z

∞

(Kyle et

al., 2004) and Z

(Hayashi et al., 1992), have

been used to extract information about the

physiological condition of biological materials. The

index Z

gains some significance since it is at the

50 kHz that the current starts passing through both

cytosol/membranes and extracellular ﬂuid, although

-2

x 10

Frequency (Hz)

Module (Ohms)

-2

Frequency (Hz)

Phase Shift (º)

1.5 2 2.5 3 3.5 4 4.5

x 10

-5000

5000

10000

15000

20000

Resistance

Reactance

Ω

BIODEVICES2013-InternationalConferenceonBiomedicalElectronicsandDevices

the proportion varies from tissue to tissue (Kyle et

al., 2004).

The nature of the impedance excitation signal

varies depending on the application. It is possible to

excite the sample with a current and measure a

voltage or to do the exact opposite. The discussion

on what source, voltage or current, is the most

convenient remains. Current sources, CS, provide

suitably controlled means of current injection

(Rafiei-Naeini et al., 2007) and present reduced

noise due to spatial variation when compared with

voltage sources, VS (Ross et al., 2003). However,

CS accuracy decreases with high frequency (Yoo et

al., 2010), especially due to their output impedance

degradation (Ross et al., 2003). Since the impedance

measurements are limited to field strength where the

current is linear with respect to the voltage applied

(Pliquett, 2010), or vice-versa, CS need high-

precision components (Saulnier et al., 2006) and a

limited bandwidth operation range (Yoo et al., 2010,

Saulnier et al., 2006) to overcome the stated

limitation. On the other hand, VS, although

producing less optimal EIS systems (Saulnier et al.,

2006), can operate over a sufficient broad frequency

range (Yoo et al., 2010); (Saulnier et al., 2006) and

are built with less expensive components (Saulnier

et al., 2006).

Nowadays, instruments with high precision, high

resolution and frequency ranges extending from

some Hz to tens of MHz are commercially available

(Callegaro, 2009). However, in what concerns to the

range of low or high frequencies (already above 100

kHz), the degradation of the excitation signal affects

the accuracy of the measurements (Callegaro, 2009).

Besides, the typical solutions consist in impedance

analyzers and LCR meters which are desktop

instruments (Callegaro, 2009), unfeasible for in vivo

(Callegaro, 2009) and in field applications.

Those EIS features, together with the lately

demand in the vegetal applications, fundament this

work. In fact, there are several plant pest and

diseases affecting different cultures of huge

economic and forestall importance, not only in our

country but also around the world. This is the case of

esca disease in vineyard, ink disease in chestnuts or

pinewood disease, PWD, and bark beetles in pinus

stands, among others. It is known that PWD, the

case study presented in this paper, is caused by the

nematode Bursaphelenchus xylophilus Nickle, that is

housed in the tracheas of pine sawyer Monochamus

galloprovincialis Olivier. Bark beetles in general

and pine shoot beetle (Tomicus destruens

Wollaston), in particular, play an important role in

nematode establishment since they are responsible

of pine decay, condition required for M.

galloprovincialis oviposition. The PWD disease

leads to a rotting process from within the plant

(therefore, inside the stem) so that symptoms are

difficult to see from the outside. Furthermore, there

is still no cure available and the only solution to

discontinue the progress of the disease throughout

the culture is to identify and isolate the specimens

that seem to have contracted the disease.

The authors propose a portable EIS system able

to perform AC scans within a selectable frequency

range. The system implements the phase sensitive

detection, PSD, method and can drive either a

current or a voltage signal to excite a biological

sample in field or in vivo. The instrumentation was

designed to be cost-effective and usable in several

applications.

The design specifications are listed in Table I.

Table 1: Summary of speciﬁcations of the EIS system.

Range

Parameter Current Mode Voltage Mode

Measuring method 2 electrodes

Frequency 1 kHz to 1 MHz

Signal amplitude 25 uA 4.6 V

Impedance magnitude 100 Ω to

100 kΩ

1.5k Ω to

2.2 MΩ

Impedance phase -π rad to π rad -π rad to π rad

Mean absolute magnitude error 1675.45 Ω 709.37 Ω

Mean absolute phase error 2.45 % 2.06 %

Mean distortion 0.29 % 0.48 %

Mean SNR 117.0 dB 118.8 dB

Calibration Automatically calibrated by

software

A first interesting case study is presented for a

population of 24 young pine trees (Pinus pinaster

Aiton), from a controlled environment. Pine trees

were inoculated with the nematode that causes the

PWD and also with bark beetles (T. destruens

Wollaston).

2 SYSTEM DESIGN

2.1 General Description

The developed EIS system employs two electrodes

and consists of three main modules: signal

conditioning unit, acquisition system (PicoScope®

3205A) and a laptop for data processing (Matlab®

based software), as figure 2 depicts. There were built

two different versions: one OEM for lab studies and

another miniaturized version for field acquisitions.

The electrodes being used are beryllium cooper

gold platted needles with around 1.02 mm in

BioimpedanceParameterasaRiskFactortoAssessPineDecay-AnInnovativeApproachtotheDiagnosisofPlant

Diseases

diameter. The bioimpedance measurement requires

the most superficial possible penetration of the

electrodes in order to reduce the dispersion of the

needles surface current density (Grimnes and

Martinsen, 2008), and also to reduce damage on the

biologic sample.

Figure 2: Schematics of the EIS OEM system – 1)

Biologic sample; 2) electrodes; 3) short coaxial cables; 4)

EIS system conditioning unit and acquisition system, with

the Picoscope®3205A incorporated; 5) laptop/PC.

The digital oscilloscope PicoScope® 3205A has

dual functionality: 1) synthesizes and provides the

excitation AC signal to the conditioning unit (ADC

function); 2) digitizes both excitation and induction

signals at high sampling rates (12.5 MSps) and

transfers data to the computer via USB where it is

stored. The signal conditioning unit receives the

exciting AC signal, coming from the PicoScope®,

and amplifies it to be applied, through an electrode,

to the specimen under study. The induced AC signal

is collected by a second electrode and is redirected

to the conditioning unit where it is also amplified.

Both excitation and induced signals are conduced to

the PicoScope® to be digitized.

It is also important to remark that the

conditioning unit has an external switch that allows

the user to select the mode type of excitation: by AC

current or AC voltage. As previously mentioned, it

is more advantageous to choose a mode of excitation

over another, depending on the type of application.

The features of both excitation modes are

described below.

2.2 Design Specifications

The current mode circuit employs the current-

feedback amplifier AD844 in a non-inverting ac-

coupled CS configuration (see figure 3), already

studied by Seoane et al., 2006.

A common problem inherent to bioimpedance

measurements is the charging of the dc-blocking

capacitor between the source and the electrode due

to residual DC currents (Seoane et al., 2006). This

effect lead to saturation of the transimpedance

output of the AD844. The DC feedback of the

implemented configuration maintains dc voltage at

the output close to 0V without reducing the output

impedance of the source. Subsequently, the output

current, is maintained almost constant over a wide

range of frequencies.

Figure 3: Schematic of the EIS system conditioning unit -

1) AC current source; 2) AC voltage source; 3)

current/voltage sense.

The high speed voltage-feedback amplifier

LM7171 is employed in the voltage mode circuit

(see figure 3). This behaves like a current-feedback

amplifier due to its high slew rate, wide unit-gain

bandwidth and low current consumption.

Nevertheless it can be applied in all traditional

voltage-feedback amplifier configurations, as the

one used. These characteristics allow the

maintenance of an almost constant voltage output

over a wide range of frequencies.

Current or voltage signals resulting from voltage

or current excitation modes, respectively, are sensed

by a high speed operational amplifier, LT1220 (see

figure 3), which performs reduced input offset

voltage and is able of driving large capacitive loads.

Gain values of both current excitation source and

voltage excitation source can be changed in order to

extend the range of impedance magnitude. The

transductance gain of the LT1220 is currently set to

5.1 kΩ and defines the gain of the system. Since the

gain values are known and also the amplitude of the

AC excitation signal, V

sin

, from the PicoScope®, the

EIS system is calibrated automatically by software.

2.3 Cables Capacitance

The characteristics of the cables that connect

between the conditioning unit and the sample under

study are also crucial. For an optimized signal-to-

noise ratio, coaxial cable must be used.

LT1220

BIODEVICES2013-InternationalConferenceonBiomedicalElectronicsandDevices

Nevertheless, this type of cable is prone to introduce

high equivalent parasitic capacitances, which

translate in errors in the bioimpedance

measurements, especially at high frequencies. To

overcome this effect, the employed RG174 RF

coaxial cables (capacitance of 100 pF.m-1) are as

short as possible (around 15cm). It was also

implemented a driven shield technique to the coaxial

cables, which permits to partially cancel the

capacitive effect, that otherwise is generated

between the internal and the external conductors, by

putting both at the same voltage (Yamamoto et al,

1985). Reductions in the capacitive effect of 20.4%,

in the current mode, and around 35.8%, in the

voltage mode, at the highest frequencies are verified.

Figure 4 depicts the capacitive effect reduction by

the usage of the driven shield technique.

Figure 4: Bode and Cole-Cole diagram showing the

reduction of cables capacitive effect by the application of

the driven shield technique. The voltage mode excitation

was used to analyze the circuit at the right top. The

reduction is more noticeable at high frequencies where the

capacitive effects have more influence.

When assessing bioimpedance, the capacitive

effects from cables are not the only exerting

influence. In fact, phase shift effects, perceptible

especially in the high frequencies range, are

introduced mainly by the amplifiers. The influence

of phase shift errors has a cumulative effect that is

translated, in the impedance spectra, as an inflexion

that occurs at high frequencies (see figure 4).

This behaviour can be simulated by an

equivalent circuit as it is like the system analyzes

any load always in parallel with a capacitor.

The impedance magnitude, at high frequencies,

is also affected. It presents a characteristic decline as

the bode diagrams of the figure 4 show. In the

developed EIS system, the slight decline of the

impedance magnitude is due to the loss of the

product gain-bandwidth of the LT1220 for high

frequencies.

Since stray capacitances are considered

systematic errors of the system, thus affecting all the

measurements, theirs influence doesn’t directly

affect the results. Although, it is convenient to have

an approached sense of the real equivalent circuit

(see figure 5), in such a way that the effect of all the

parasitic elements can be considered and/or

discounted where justified.

Figure 5: Equivalent electric circuit of all parasitic

elements affecting impedance measurements of a load,

LOAD

. The effect of the stray capacitances from cables,

CABLE

, is minimized by the driven shield. Other stray

capacitance effect, C

STRAY

, due primarily to the phase shift

of amplifiers, can be minimized by software.

3 SOFTWARE AND ANALYSIS

PROCESSING

3.1 General Specifications

The software interface, developed with Matlab®

tools, allows the operator to choose the parameters

of the bioimpedance analysis and to monitor the data

acquisition. The operator can perform an analysis for

one specific frequency or alternatively can carry out

a true bioimpedance spectroscopy. These two

software functioning modes can be programmed for

continuous monitoring, where the number of

acquisitions and the intervals between them are

specified by the user. The interface includes a basic

function that allows a preview of the Bode and Cole-

x 10

Module (Ohms)

Frequency (kHz)

Phase Shift (º)

Frequency (kHz)

2 2.5 3 3.5

x 10

-2000

2000

4000

6000

8000

10000

12000

14000

Reactance

Resistance

CABLE

LOAD

CABLE

STRAY

CABLE



CABLE



CABLE



Ω

180

BioimpedanceParameterasaRiskFactortoAssessPineDecay-AnInnovativeApproachtotheDiagnosisofPlant

Diseases

Cole diagrams of the acquired data. Bioimpedance

*txt or *mat files are saved in a pre-determined

directory with a filename, previously chose by the

user, to which date and time are associated. Each file

contains information about magnitude, phase shift

and real and imaginary parts of the measured

impedance, for each frequency.

The type of bioimpedance spectroscopy

implemented consists in a frequency AC sweep,

whose limit values are 1 kHz and 1 MHz.

Notwithstanding, the software allows the operator to

choose other frequency limits, as well as the number

of intervals between them. In addition, it can be

choose a linear or logarithmic analysis. Therefore,

the frequencies, f(i), over which the impedance of a

sample is analysed, are determined by the following

equations:

For a linear analysis:





f_starti∗

f_stopf_start

n1

,

∀i⊂



0,n1



∧n∈N

(2)

For a logarithmic analysis:





f_start∗10







_

_













,

∀i⊂



0,n1



∧n∈N

(3)

Where f_star and f_stop are, respectively, the first

and final frequencies of the AC sweep, and n the

number of intervals between them.

3.2 PSD Method

To assess the impedance phase shift it is

implemented a digital Phase Sensitive Detection,

PSD, method with a novel implication. As stated in

the literature, the PSD method is a quadrature

demodulation technique that implements a coherent

phase demodulation of two reference (matched in

phase and quadrature) signals (He et al, 2008);

(Dobrev et al., 2008). It is also known that this

method is preferable over others especially when

signals are affected by noise (Dobrev et al., 2008).

The signal from the Picoscope® that corresponds

to the current, V

=Bsin(ωt+φ

), is set as the

reference signal. Since the phase of the signal V

not controlled, it is easily understandable that it

doesn’t necessarily contain a null phase. This

statement remains valid whether V

is used to excite

the sample, in the current mode, or whether it

corresponds to the current passing through the

sample, in the voltage mode. The signal from the

Picoscope® that corresponds to the voltage, V

sin(ωt+φ

), also contains a non-null phase. Both

amplitudes, A and B, are also different from each

other and none equals to 1.

The developed PSD algorithm was tested with

Matlab® for several phases and amplitudes without

the theoretical requirements (i.e., ensure that the

reference signal has null phase at the origin and that

its amplitude equals to 1 (He et al., 2008)). For all of

them it was showed an always corrected phase shift

assessment, when compared to the results obtained

for a reference signal with the theoretical

characteristics.

In addition, the mathematical resolution for the

demodulation of two signals with non-null phases

and amplitudes not equal to 1, corresponds to the

phase difference between both signals. The

following mathematical demonstration and the

schematic block diagram (shown in figure 6) support

the results obtained with the simulation.

Assuming that the analog input signals V

(t) and

(t) are sine waves of frequency f, amplitude A and

B, respectively, and initial phase φ

and φ

respectively:







Asin2π

φ





(4)







Bsin2π

φ





(5)

The digitized input signals V

(n) and V

(n) are

obtained from V

(t) and V

(t), respectively, by

sampling at a frequency f

, where f

is a multiple of

the f:







Asin

2πfn



φ



,n∈0,N1

(6)







Bsin

2πfn



φ



,n∈0,N1

(7)

Where N is the number of samples. N/f

is the

measurement time and must be an exact multiple of

1/f, so that there is whole number of cycles of the

sine wave.

Figure 6: Schematic of Phase-sensitive Demodulator

implemented in the EIS system.



∑



∑









2/







PicoscopeCh.B











2/







PicoscopeCh.A











2/







PicoscopeChannelAshifted90º









cos













sin





BIODEVICES2013-InternationalConferenceonBiomedicalElectronicsandDevices

The signal V

(n) is set as reference. The

quadrature reference signal, V

(n), results from the

reference signal shifted by a phase of 90º.

Consequently, V

(n) is cosine with the same

frequency, amplitude and initial phase as V

(n):







Bsin

2πfn



φ



,n∈0,N1

(8)







Bcos

2πfn



φ



,n∈0,N1

(9)

The output voltages of the system shown at figure 6

are:





V







V



n





(10)





V







V



n





(11)

The multiplication between two sine signals, with

the same frequency, results in a sum of a DC signal

and a sine signal with a frequency that is the double

of the original. The double frequency component

can be suppressed since the time is a multiple of the

period of the input sine signal. Therefore, it remains

only the DC component which amplitude is

dependent on the amplitude of the individual sine

signals and their relative phase:





cosφ



φ





(12)





sinφ



φ





(13)

From the expressions above, the resulting amplitude

and phase can be determined:



φ



arctan







(14)

A



V







V







(15)

The determined phase is actually a phase difference

between the demodulated signal, V

and the

reference signal, V

, i.e., it corresponds to the phase

difference between voltage and current signals.

Figure 7 shows the consistence of the algorithm

when the impedance phase of a real data is

compared with a spice simulation in Cadence®.

The determination of impedance magnitude

cannot be achieved by the PSD method, since the

amplitude equation (equation 15) shows a

dependence on the amplitude of the reference signal,

which, in this case, is not equal to 1. Hence, to

assess amplitude, the EIS system algorithm

processes the root mean square, RMS, of both

signals V

(t)and V

(t) from de channel B and A,

respectively, of the Picoscope®. In this manner, the

impedance magnitude is given by the ratio between

the RMS value of the signal V

(t) and the rms value

of the signal V

(t):







∑















∑















, ∀∈0,1

(16)

Where Gain is the EIS system gain defined by the

transconductance gain of the LT1220 (see section

2.2).

Figure 7: Comparison between impedance phase of a real

data and Cadence® simulated data for a RC circuit. The

deviation that occurs between the graphics, at high

frequencies, is due to the influence of stray capacitances

(see section 2).

4 BIOLOGIC APPLICATION

STUDY

4.1 Materials and Methods

Twenty four young healthy pine trees (Pinus

pinaster Aiton), with about 2,5 meters tall and 2 to 3

centimetres in diameter, constituted the population

for the conducted study. The pine trees were placed

in vases in a controlled water environment at a

greenhouse. Half of the tree population was watered

during 5 minutes per day (~ 133,37 mL/day), while

the other half were watered during only 2 minutes

per day (~ 66,67 mL/day). This second half was less

watered to maintain a relevant level of hydric stress.

After one month elapsed since the pine trees

were placed in the greenhouse, the inoculations with

pinewood nematode, PWN, (Bursaphelenchus

xylophilus Nickle) and with the bark beetle (T.

destruens Wollaston) were performed. Six pines

were inoculated with PWN, other 6 pines were

BioimpedanceParameterasaRiskFactortoAssessPineDecay-AnInnovativeApproachtotheDiagnosisofPlant

Diseases

inoculated with bark beetles, other 6 pines were

inoculated simultaneously with PWN and bark

beetles, while the remaining 6 were kept under

normal conditions, i.e., healthy. The position of the

pines in the greenhouse was made so that each sub-

group had the same number of pines with normal

watering (5 min/day) and with reduced watering (2

min/day).

To perform the inoculations with bark beetles,

callow adults were collected immediately after

emergence. In each tree, a box containing 15 beetles

were placed in the middle and the device was

covered using Lutrasil tissue to avoid beetles escape.

The inoculation with the PWN followed an

innovative approach. Firstly, three 2 x 2 cm

rectangle of cork were removed from the first tiers

of the trunk (about 1,80 m above the soil) and

exposed phloem was erased with a scalpel in order

to increase the adhesion of the PWN. Afterward,

0,05 mL of of a PWN suspension was placed on in

each incision. In the total, 6000 nematodes were

inoculated per tree. To finalize the task, the removed

rectangle of cork was fixed in the respective place

and wrapped with plastic tape.

Seventy days after the inoculations, the EIS

measurements were performed in all the tree

population. At this time, the pine trees inoculated

with PWN presented some visually symptoms of the

PWD. The decay of those trees, rounded 40 %. Two

of the healthy pines died (decay of 100 %) due to

hydric stress. All remaining individual appeared

healthy.

To perform the EIS measurements, the electrodes

were placed in the trunk of each tree, in a diametric

position, and about 30 cm above the soil. It was used

the portable EIS system version in the voltage mode

of excitation and a frequency range between 1 kHz

and 1 MHz. There were taken two measurements for

each tree. The acquisitions took place between 11

a.m. and 13 p.m. since it was already verified in

previous studies that at this time period the trees

impedance is higher and presents few variation (see

figure 8 – section 4.2).

In order to relate the EIS data with the PWD and

the stage of the disease, the trunk of the pine trees

inoculated with PWN were cut in three distinct

regions to perform a count of nematodes. The cuts

were executed: a) immediately below the inoculation

incision (180 cm above the soil); b) 30 cm above the

soil (where EIS measurements took place); and c) in

the middle of the previous two cuts (approximately

80 cm above the soil).

After the EIS measurements, two healthy pines

were monitored by two independent portable EIS

systems. After a week of monitoring, the same pines

were inoculated with PWN, and the measurements

continued during 7 more weeks. The main purpose

of this last experiment was to study the variation of

the pine EIS profiles during the decay due to the

PWD.

4.2 Results

For each obtained impedance spectra there were

assessed several impedance parameters. Due to

paper space limitation and also because it is a well-

known impedance parameter, it will only be

presented the results obtained for the ratio Z

Note that it is used the index 1, that corresponds to

the lowest analyzed frequency (1 kHz), instead of

the index 0, as explained in section1.

4.2.1 Impedance Daily Oscillation

The EIS measurements revealed that EIS Cole

profiles have a daily oscillation. To analyze this

behavior it was calculated the R

ratio (R

represents module) for a period of 4 days.

Figure 8: Variation of the R

ratio during the

monitoring of a healthy pine tree. The impedance values

show a daily oscillation that is characteristic of the studied

trees.

To confirm the daily oscillation it was calculated

the fast fourier transform of this ratio. A frequency

of 11,57 µHz was clearly founded, which

corresponds to a frequency of 24 h.

The lower values of the ratio R

correspond

to the night period, while the higher values

correspond to the day period where the temperature

and luminance are higher (between 11 a.m. and 15

p.m.). Previous studies on plants also shown that,

during the day period, the variation of impedance

values is lower than the one observed at the night

Night Period

Day Period

BIODEVICES2013-InternationalConferenceonBiomedicalElectronicsandDevices

period. This was the main reason that lead to

performing the EIS measurements between the 11

a.m. and 14 p.m.

4.2.2 Discrimination between Physiological

States

In order to compare results between the different

physiological states of the trees, there were assessed

several impedance parameters. The impedance

parameter that showed better results was the Z

ratio – see figure 9.

Figure 9: Values of the impedance parameter Z

for

each of the 24 pine trees. Note that there are represented

two values for each pine.

The analysis of the obtained results shown that

the healthy pines and the pines inoculated with bark

beetles have similar Z

values. In fact, the bark

beetles doesn’t damage the inner structure of the

trees, therefore it was expected that the impedance

profiles were similar between healthy pines and

pines inoculated with bark beetles.

On the other hand, Z

values for the pines

inoculated with nematodes and also, for those

inoculated simultaneously with nematodes and bark

beetles, locates in the same region, different from

the previous one, of the graph of figure 9. Those

values present a relatively high dispersion in terms

of reactance. It was later confirmed that higher

reactance Z

values correspond to higher number

of nematodes in the tree (see figure 10 from section

4.2.3).

The pines that died due to hydric stress (decay of

100%) were also studied and the Z

parameter

present high resistance values in relation to all the

other pines.

4.2.3 Relation between the Number of

Nematodes and Impedance Parameters

The counting of nematodes in the several cut

sections revealed that the concentration of

nematodes was higher in the cut sections b) and c)

for the pines less watered (pines 1, 2 and 3) – see

table 2. It is known that the nematodes move toward

watered regions along the trunk. For this reason, the

concentration of nematodes in the lower parts of the

trunks was much higher for the pines with less

watering than for those with regular watering (pines

4, 5 and 6).

Table 2: Number of nematodes in the trunks of pine trees

per cut sections.

Tree Cut Section Number of nematodes in 0,05 mL

a 1

b 0

c 133

a 0

b 43

c 1

a 0

b 0

c 112

a 4

b 20

c 0

a 0

b 17

c 0

a 0

b 0

c 14

Figure 10: Values of the impedance parameter Z

for

the pines inoculated with nematodes and with low

watering (pines 1, 2 and 3 from the table 2). Note that

there are represented two values for each pine.

These results for the nematodes counting support

the already referred results obtained for the Z

impedance parameter. In fact, it is observed a clear

0 1 2 3 4 5 6 7 8 9 10

Resistance

Reactance

1.03 1.04 1.05 1.06 1.07 1.08 1.09 1.1 1.11

Resistance

Reactance

Legend:

■ Healthy Pines;

■ Pines inoculated with bark beetles;

■ Pines inoculated with nematodes;

■ Pines inoculated simultaneously

with nematodes and bark beetles;

■ Pines with a decay of 100 % due to

hydric stress (dead pines)

Legend:

■ Pine 1;

■ Pine 2;

■ Pine 3.

BioimpedanceParameterasaRiskFactortoAssessPineDecay-AnInnovativeApproachtotheDiagnosisofPlant

Diseases

relation between the number of nematodes and the

reactance dispersion for the Z

parameter, as

figure 10 shows. The higher the number of

nematodes is, the higher is the reactance value of

. It is considered that the dispersion in terms of

resistance is not significant when compared with

values from pines in other physiological condition –

see figure 9 from section 4.2.2.

4.2.4 EIS Monitoring during Pine Decay

There were monitored two healthy pines, one with

low watering (2 min/day) and another with regular

watering (5 min/day). After one week from the

beginning of the monitoring, both pines were

inoculated with nematodes. It was shown again a

dispersion of the reactance values of the Z

parameter, as figure 11 shows. As time passed the

reactance values became higher. The higher values

of reactance were achieved for the pine with less

watering. According to the previous presented

results, it was expected that the number of

nematodes increase in the below part of the trunk for

the pines with less watering; and consequently, to

observe a higher rising of the reactance of the Z

parameter. After the 6th week, pines start to decay

strongly and it was observed a relevant decrease of

the reactance and a significant increase of the

resistance for the same parameter – see figure 11.

The higher values of resistance were achieved for

the pine with less watering, and also in a shorter

period of time. At the end of the monitoring, the

decay of the pines, evaluated by an expertise, was

about 80 % for the pine with regular watering and

100 % for the pine with less watering.

From the figure 11 b), that represents a closer

view of the Z

values for the monitoring, it is

possible to observe that the path followed during the

period of nematodes population increasing is

different from the path followed during the period of

decay, i.e., it is observed an hysteresis-like behavior.

5 CONCLUSIONS

The EIS system was developed in order to ensure a

robust, efficient and fast bioimpedance analysis. The

adaptability to different biological applications, the

portability and the usage of easily accessible and

affordable components, were preferred aspects taken

into account. In this manner, the system allows the

user to choose the settings of the analysis that best

fit to a specific application. Furthermore, there were

built two versions of the equipment: one OEM

version for lab tests and a miniaturized version for

field applications.

Figure 11: a) Evolution of the Z1/Z50 during the

monitoring time (8 weeks). b) Closer view from the Z

evolution, showing a hysteresis-like behaviour.

The system is able to perform AC scans within a

frequency range from 1 kHz to 1 MHz. The

frequency limits and the number of intervals of the

scan can be selected at the user interface (developed

with Matlab® tools). The type of signal used to

excite de sample, voltage or current, can be

preselected by an external switch. This allows the

usage of the source with the best behaviour in a

concrete application.

The implemented PSD algorithm allows a very

good phase shift assessment without the need to use

a reference signal of amplitude equal to 1 and null-

phase at the origin. In fact, the signal set as reference

has undetermined phase and amplitude. All the

algorithm tests revealed results analogous to the

theoretical.

To overcome problems inherent to stray

capacitive effects from cables, a driven shield

technique is applied. The maximum phase shift

reduction is estimated at 20.4 % for the current

0 20 40 60 80 100

0.2

0.4

0.6

0.8

1.2

1.4

1.6

1.8

Resistance

Reactance

80 % of decay

BIODEVICES2013-InternationalConferenceonBiomedicalElectronicsandDevices

excitation mode and at 35.8% for the voltage mode.

The biological application study aimed at

discriminating between different pine tree

physiological states.

The obtained results suggest that the

implemented method may constitute a first

innovative approach to the early diagnosis of plant

diseases. In fact, the achieved impedance parameters

allow discriminating three different physiological

states: healthy trees, trees with PWD and trees in

hydric stress.

The trees with PWD present Z

ratio with

high values of reactance, suggesting that the current

flows preferably trough the cytosol. In fact, the

action of the nematodes inside the tree may destroy

cell membranes. This means that membranes

capacitor effect becomes less significant in the

impedance measurement.

It was also shown that the number of nematodes

and Z

impedance parameter are related. The

higher the number of nematodes is, the higher the

reactance of the ratio is.

The action of bark beetles seems not to interfere,

at least in measurable terms, in the level of hydric

stress of pine trees.

Healthy trees, with high values of hydric stress

(decays above 80 %), and also trees with PWD at

advanced stages, revealed low reactance and high

resistance for the same studied parameter. The high

values of resistance are justified due to the water

loss in the tree. Consequently, it means that for this

specific case, the method cannot distinguish between

trees with PWD or trees with high level of hydric

stress but with no disease. However, it is known that

advanced stages of PWD promote high levels of

hydric stress. This means that both cases represent,

in practical terms, the same situation, i.e., the tree

presents high probability to die. In addition, in the

stages where the method is able to distinguish

between healthy trees and trees with PWD, the

decay was determined to round the 40 %. Therefore,

if a cure is available, this diagnosis could help to

administrate a treatment and reverse the disease

evolution.

Hence, the main conclusion of the developed

study is that the studied method could be used to

assess physiological states of living pine trees, and

that the Z

impedance parameter could be

applied as a risk factor.

ACKNOWLEDGEMENTS

We acknowledge support from Fundação para a

Ciência e Tecnologia, FCT (scholarship

SFRH/BD/61522/2009).

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BIODEVICES2013-InternationalConferenceonBiomedicalElectronicsandDevices