Scientia Horticulturae 209 (2016) 229–235

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Assessment of technological maturity parameters and anthocyanins in

berries of cv. Sangiovese (Vitis vinifera L.) by a portable vis/NIR device
Alejandra Ribera-Fonseca a,b,∗ , Massimo Noferini c , Emilio Jorquera-Fontena d ,
Adamo D. Rombolà e
a
Departamento de Producción Agropecuaria, Facultad de Ciencias Agropecuarias y Forestales, Universidad de La Frontera, Avenida Francisco Salazar
01145, P.O. Box 54-D, Temuco, Chile
b
Center of Plant-Soil Interaction and Natural Resources Biotechnology, Scientific and Technological Bioresources Nucleus (BIOREN-UFRO), Universidad de
La Frontera, Avenida Francisco Salazar 01145, P.O. Box 54-D, Temuco, Chile
c
FA.MO.SA S.r.l. (FArm MOnitoring Systems for Agriculture), Via Selice 84/a, 40026 Imola, Italy
d
Facultad de Ciencias Agropecuarias y Forestales, Universidad de La Frontera, Avenida Francisco Salazar 01145, P.O. Box 54-D, Temuco, Chile
e
Department of Agricultural Sciences, School of Agriculture and Veterinary Medicine, Alma Mater Studiorum, University of Bologna, Viale G. Fanin 44,
40127 Bologna, Italy

a r t i c l e i n f o a b s t r a c t

Article history: In grape berries the balance between technological parameters such as soluble solids and titratable
Received 11 March 2016 acidity and phenolic maturity such as anthocyanins concentration, is a key factor for obtaining qual-
Received in revised form 22 May 2016 ity wines. Grapevine berries are commonly harvested on the base of technological maturity parameters
Accepted 7 June 2016
determined by traditional analysis methods, often without considering properly phenolic maturity. We
investigated the potential use of a portable and non-invasive device based on visible and near infrared
Keywords:
(vis/NIR) spectroscopy (Cherry-Meter), which provides an Index of Absorbance Difference (IAD ) based
Grapevines
on two wavelengths peaks (560 and 640 nm), to measure soluble solids concentration (SSC), titratable
Cherry-Meter
Maturity
acidity (TA), firmness (DI) and anthocyanins (total and monomeric) in cv. Sangiovese grapes. Berries were
Technological parameters separated in ten IAD classes according to the Cherry meter data ranging from 0.4 to 1.8, and then analyzed
Principal component analysis for technological parameters and anthocyanins by using conventional methods. Linear and non-linear
Sangiovese regression analysis showed that IAD values were significantly correlated to SSC (R2 = 0.92), TA (R2 = 0.87),
DI (R2 = 0.89), and monomeric and total anthocyanin concentration (R2 ranging from 0.68 to 0.97). A
Principal Component Analysis (PCA) was applied to analyze relationship among IAD classes, obtaining
four different clusters based on increasing level of maturity defined by means of technological parame-
ters and anthocyanins concentration. This is the first approach demonstrating that the use of IAD values
obtained from Cherry-Meter could be useful for monitoring both technological maturity parameters and
anthocyanin concentration and composition of grape berries.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction (Ribéreau-Gayon et al., 2000) and this condition can be accentuated

Grapevines clusters are characterized by a high spatial and tem-
poral heterogeneity of berry maturation in the vineyard (Bramley,
2005; Tuccio et al., 2011). The balance between technological matu-
rity parameters and phenolic maturity plays a crucial role in wine
quality (Conde et al., 2007; Kontoudakis et al., 2011). Technological
maturity of grapevine berries normally precedes phenolic maturity
∗ Corresponding author at: Departamento de Producción Agropecuaria, Facul-
tad de Ciencias Agropecuarias y Forestales, Universidad de La Frontera, Avenida
Francisco Salazar 01145, P.O. Box 54-D, Temuco, Chile.
E-mail address: alejandra.ribera@ufrontera.cl (A.D. Rombolà).
as a consequence of climatic conditions (Sadras and Moran, 2012)
and agronomic practices such as heavy bunch thinning (Gatti et al.,
2012).
Red grapevines (Vitis vinifera L.) berries are commonly harvested
based on soluble solids concentrations (Brix) and titratable acid-
ity. Nevertheless, these destructive methods can be carried out
merely on a restricted amount of fruits, being sometimes poorly
representatives of the vineyard and do not consider the grape phe-
nolic maturity (Cozzolino et al., 2006; Kontoudakis et al., 2011;
Urraca et al., 2015). Moreover, qualitative and quantitative anal-
ysis of anthocyanins, the main molecules providing the red/blue
color of mature red grapevine fruits (Boulton, 2001), is frequently

http://dx.doi.org/10.1016/j.scienta.2016.06.004
0304-4238/© 2016 Elsevier B.V. All rights reserved.
230 A. Ribera-Fonseca et al. / Scientia Horticulturae 209 (2016) 229–235
Fig. 1. IAD measurements in grape berries using the Cherry-Meter device (A) and visual aspect of berries from the different IAD classes, where C is the IAD class (B).
performed by using time consuming, invasive and expensive tech-
niques (Costa et al., 2009; Lorrain et al., 2013).
The use of non-destructive technologies to evaluate fruit qual-
ity and maturity parameters can represent a rapid and precise tool
to estimate the harvest date (Costa et al., 2003, 2009; Tuccio et al.,
2011; Urraca et al., 2015; Giovenzana et al., 2014). Hand-led visi-
ble/near infrared (vis/NIR) technologies have been assessed for the
estimation of ripening level in fresh blueberries (Guidetti et al.,
2009), fresh apricot (Camps and Christen, 2009), apple varieties
(Beghi et al., 2013), and red grapes (Cerovic et al., 2008; Ben Ghozlen
et al., 2010; Guidetti et al., 2010; Tuccio et al., 2011; Giovenzana
et al., 2014). One of these technologies are the Cherry-Meter, a
portable and friendly-use vis/NIR developed in 2005 at Bologna
University (Italy). This device provides an Index of Absorbance Dif-
ference (IAD ), calculated on the basis of two wavelengths (560 and
640 nm), which correlates with several fruit chemical and biochem-
ical parameters (Nagpala et al., 2013; Ribera et al., 2016). The, IAD
values have been used as suitable fruit harvest index in some cul-
tivars of peaches (Ziosi et al., 2008; Bonora et al., 2013; Spadoni
et al., 2016), plums (Infante et al., 2011; Infante, 2012), and apples
(Nyasordzi et al., 2013). Lately, Cherry-Meter has been also used
to estimate cherry maturity through the prediction of fruit skin
total anthocyanin of cherry and blueberry fruits with encouraging
results (Nagpala et al., 2013; Ribera et al., 2016).
The aim of this work was to perform a preliminary approach
about the use of IAD values as potential non-destructive predictor
of soluble solids, titratable acidity, firmness and anthocyanins in
cv. Sangiovese grapes.
2. Material and methods
2.1. Experimental site
The experiment was performed during 2014 growing season,
in a mature vineyard, planted in 2003 with cv. Sangiovese (clone
FEDIT 30 ESAVE; Vitis vinifera L.) grafted onto Kober 5BB and trained
to spur pruned cordon (VSP). The vineyard was located in Tebano
(Faenza, Emilia Romagna, Italy, 44◦ 17 7 N, 11◦ 52 59 E, 117 m
a.s.l.), on a medium slope, with southeast-northwest and downhill-
oriented rows. Vines were spaced 2.8 m × 1.0 m (3571 plants ha−1 ).
Starting in 2007, the vineyard is managed (irrigation, fertilizers
supply and phytosanitary managements) and certified as organic
in accordance with European Commission Regulation No. 834 (EC,
2007).
Table 1
Index of Absorbance Difference (IAD ) range of grapevine fruits from the ten different
IAD classes.
IAD classes IAD range
1 0.4–0.5
2 >0.5–0.6
3 >0.6–0.7
4 >0.7–0.8
5 >0.8–0.9
6 >0.9–1.0
7 >1.0–1.2
8 >1.2–1.4
9 >1.4–1.6
10 >1.6–1.8
2.2. Berry sampling
At August 28th (beginning of blooming period started on May
22th), about 1.5 kg berry sample was collected from ten randomly
selected mature (full-production) plants. Due to berries from clus-
ters normally ripen in a heterogeneous form, it was possible obtain
fruits with different coverage level of skin red-blue color at date in
which fruit was collected. It is important to stress that; overall, the
2014 vegetative season was marked by average temperatures well
below seasonal normal and high rainfall during summer. Such cli-
matic conditions hampered fully ripening of berries, consequently
the monitored vines displayed high heterogeneity in cluster com-
position, which enabled sampling of different berry ripening levels
from one cluster on one sample date. Thus, grapes from fully-green
to fully-blue were harvested. Berries were stored at 4 ◦ C for IAD data
acquisition and technological maturity parameters determination,
or at −80 ◦ C for quali-quantitative anthocyanins analyses.
2.3. Cherry-Meter analysis
The Index of Absorbance Difference (IAD ) of all fruits (fresh
whole berries) was measured by using the Cherry-Meter (T.R. Tur-
oni S.r.l., Forlí, Italy; Fig. 1). Based on the Lambert-Beer’s law, the IAD
is calculated through differences between two wavelengths peaks
(560 and 640 nm) and the reference value at 750 nm (Noferini et al.,
2009). The instrument consists of a light source of six LEDs located
around a photodiode. For the IAD calculation, the light remitted
from the fruits is measured by the central photodiode, then con-
verted by an “Adc converter” (“analog to digital converter”) and
elaborated by a micro-controller (Costa et al., 2010). Because the
IAD obtained with Cherry-Meter has been correlated significantly
to ripening stages and levels of anthocyanins (Nagpala et al., 2013),
this devise was selected.
 A. Ribera-Fonseca et al. / Scientia Horticulturae 209 (2016) 229–235
Fig. 2. Spectrometric data (IAD values) against soluble solids (A), titratable acidity
(B) and Durofel Index (C) in ‘Sangiovese’ grapevines fruits, across the different IAD
classes. Regressions, standard deviation and coefficient of determination (R2 ) are
shown.
The time lapse between harvest and Cherry-Meter measure-
ments (at room temperature) was not higher than two hours by
taking two measurements on the center of each fruit and reading
the average value on the instrument screen. After Cherry-Meter
measurements, fruits were classified into ten different IAD classes
based on increasing values of IAD (Table 1). Each IAD class was com-
posed by an average of forty berries. From this initial sample, thirty
fruits were used to determine berry fresh weight, soluble solid con-
centrations, firmness and titratable acidity, whereas the remaining
ten fruits were used to anthocyanin extraction as described below.
2.4. Technological maturity parameters
Berry fresh weight (FW) was measured using a technical balance
(Gibertini Elettronica S.r.l., Milan, Italy) and expressed as grams
per berry (g berry−1 ). Then, berry firmness was measured accord-
ing to the method previously described Chiabrando et al. (2009)
231
for blueberry fruits, using the non-destructive instrument Duro-
fel (DFT 100, Forli, Italy) which has a dynamometer with a bolt of
3 mm diameter (0.10 cm2 ). This device operates on a scale of 1–60
(from soft to firm). The results were expressed as Durofel Index
(DI; N cm−2 ). According to this procedure, prior to berry firmness
analysis, fruit were exposed to room temperature (20 ◦ C) for 2 h,
due to commonly fruits decreasing firmness at increasing temper-
ature. Total soluble solids concentration (SSC) of berry juice was
determined using a manual thermo-compensated refractometer
(Maselli Misure S.P.A., Parma, Italy), standardized with distilled
water and expressed as Brix (◦ Bx). All these parameters were evalu-
ated on a total of thirty fruits, previously subjected to Cherry-Meter
measurements, from each IAD class. Finally, titratable acidity (TA)
was determined on diluted grape juice (5 mL, diluted on 25 mL
of distilled water) from fruits of each IAD class. This parameter
was analyzed through potentiometric titration (Crison Instrument
SA, Barcelona, Spain) on three repetitions for each IAD class and
expressed as g L−1 of tartaric acid.
2.5. Anthocyanins extraction and analysis
The anthocyanins analysis was performed on fruits previously
subjected to Cherry-Meter measurements and stored at −80 ◦ C. To
obtain the anthocyanin extracts, 0.3 g of fruit skin (obtained from
three whole berries approx.) were extracted with 3 mL of methanol
according to Mattivi et al. (2006). Three independent extractions
were performed for each IAD class. The anthocyanin profile was
evaluated using an HPLC apparatus (Jasco, Tokyo, Japan) equipped
with two binary pumps (model PU-1580), an autosampler (model
AS-2055 Plus) and a diode array UV–vis detector (model MD-
1510, quartz flow cell, 10 mm optical path). The HPLC analysis was
performed using a reversed-phase column RP18 250 × 4.6 mm (5-
␮m particle size) (Phenomenex, Torrance, CA, USA), according to
Mattivi et al. (2006), with minor modifications as described below.
The mobile phase was prepared using acidified water [perchlo-
ric acid (HClO4 ) 0.3% v/v] (A) and 100% methanol (B) and a flow
rate of 1.1 mL min−1 was use. The binary gradient was applied as
follows: 0–39.9 min of 90%A–10%B, 40–41.9 min of 60%A–40%B,
42–48.9 min of 20%A–80%B, and 49–62 min of 90%A–10%B. All
individual glucosylated anthocyanins were quantified (mg L−1 ) by
means of a calibration curve obtained using a malvidin-3-glucoside
standard (Sigma-Aldrich, Milan, Italy). Total anthocyanin (ANTH)
concentration was calculated through the sum of all monomeric
anthocyanins detected. Three repetitions for each IAD class were
carried out.
2.6. Statistical analysis
Pearson correlations were used to test the relationships
between two response variables. Linear regressions between IAD
values and the different fruit technological parameters and total
anthocyanins were carried out. Additionally, a Principal Compo-
nent Analysis (PCA) was carried out with PAST version 1.75 to
discriminate the classes at a multivariate level (Hammer et al.,
2001). Methods used for fitting models were performed using
the software R version 2.15.0 (R Development Core Team, 2012)
through ‘lm’ and ‘nls’ functions. All statistical analyses were per-
formed on averaged data obtained in each IAD class.
3. Results
In the present work we evaluated the use of Cherry-Meter for
non-destructive estimation of technological maturity parameters
and anthocyanins, as phenolic maturity parameter, in cv. San-
giovese berries.
232 A. Ribera-Fonseca et al. / Scientia Horticulturae 209 (2016) 229–235

Fig. 3. Cherry-Meter measurements (IAD values) against total anthocyanins [ANTH] (A), delphinidin (B), petunidin (C), peonidin (D), cyanidin (E) and malvidin (F) concentration
in ‘Sangiovese’ grapevines fruits, across the different IAD classes. Anthocyanin concentrations are expressed in mg g−1 fresh berry skin. Regressions, standard deviation and
coefficient of determination (R2 ) are shown. At the lowest maturity stage (IAD class 1, green fruits), anthocyanins were undetected.

3.1. IAD as predictor of technological maturity in grape berries
In order to verify whether IAD had a good relation with the fruit
technological maturity variables under study (soluble solids, titrat-
able acidity, and firmness), linear and non-linear model were used.
The relation between IAD and SSC was better described by an expo-
nential model, with IAD accounting for 92% of the variance of SS
(Fig. 2A). On the other hand, our results showed that TA and DI were
negatively related to IAD values through potential models (Fig. 2B
and C) and IAD accounted for 87% and 89% of the variance of TA and
DI, respectively. At last, grapevine berry FW did not vary among the
different IAD classes (data not shown).
 A. Ribera-Fonseca et al. / Scientia Horticulturae 209 (2016) 229–235
(A) 2.5
1.5
10
9
8 0.5
PC 2 (4.73%)
6 0.0
-2.5 -1.5 -0.5 0.5
-0.5 2 SS
7
5 3
-1.5 4
-2.5
PC 1 (93.42%)
Fig. 4. (A) Scoring (PCA) and (B) loading (PC1) plot representing the technological parameters measured of grapevine fruits from the different IAD classes (A). Abbreviations:
SS = soluble solids; DI = Durofel Index (berry firmness); TA = titratable acidity; ANTH = total anthocyanins. In the PCA plot (A) numbers (1–10) represent IAD classes.
3.2. IAD as predictor of anthocyanins
The ANTH and IAD values showed to be positively and lin-
early related, with IAD accounting for 96% of the variance of ANTH
(Fig. 3A). In the same way, each monomeric anthocyanin positively
correlated to IAD values (Fig. 3), with R2 ranging from 68% (for mal-
vidin) to 97% (for cyanidin). Therefore, our data showed that the
IAD values measured by Cherry-Meter can be a suitable index for
predicting total anthocyanin concentrations in grape berry skins
(Fig. 3).
3.3. IAD class clustering analysis
The PCA analysis allowed to highlight similarities and differ-
ences among the 10 IAD classes within the bidimentional space
delimited by the first two PCAs (Brereton, 2003) and to identify the
ripening parameters that better define IAD classes. The PCA results
(Fig. 4A) indicated that the ten IAD classes were sorted in four differ-
ent clusters based on increasing level of maturity defined by means
of technological parameters and anthocyanins content. In partic-
ular, cluster I was composed only by the IAD class 1 (IAD values
ranging from 0.4 to 0.5), represented by the fruits with the lowest
maturity level whereas cluster II included the IAD classes 2–5 which
occupied the bottom right side of the bidimentional PCA space, the
cluster III was composed by the IAD classes 6 and 7, and Cluster
IV comprising IAD classes 8–10, the last ones represented by those
fruits with the highest maturity level with IAD values ranging from
1.4 to 1.8 (Fig. 4A). The results derived from the PCA analysis indi-
cated that the first component (PC1) accounted for 93.42% of the
variance, while the second one (PC2) for 4.72%. The loading of PC1
(Fig. 4B), revealed high r values for all evaluated ripening param-
eters. The PC1 was influenced by the different fruit variables as
follow SS > ANTH > DI > TA.
4. Discussion
Our results indicated that the relation between the IAD mea-
sured by using the Cherry-Meter device and fruit SSC, TA and
fruit firmness in cv. Sangiovese berries where highly significant,
with R2 values ranging from 0.87 to 0.92. Guidetti et al. (2010),
found good prediction models for SSC and TA, with root mean
square error of prediction (RMSEP) equal to 1.48 Brix and 1.48 g
tartaric acid dm−3 in fresh Nebbiolo grapes, respectively, using a
portable vis/NIR system (450–980 nm). Several studies showed that
233
1.0
(B)
1
0.5
Correlation to PC1
DI TA
1.5 2.5
DI TA ANTH
-0.5
-1.0
vis/NIR wavelength absorbance differences can represent a useful
tool to predict fruit maturity attributes through the achievement of
regression and calibration models (Giovenzana et al., 2014). More
recently, models based on vis/NIR spectroscopy ranging from 189.5
to 851.5 nm, showing to be accurate for predicting SSC, pH, TA in
cv. Cabernet Sauvignon, Cabernet Franc, and Syrah grapes were suc-
cessfully development by Fadock et al. (2016). In agreement with
our findings, Infante et al. (2011), Infante (2012) found that IAD data
were significantly correlated with fruit firmness (R = −0.95) and
SSC (R = 0.84) in plums. In contrast, Ziosi et al. (2008) showed that
the IAD correlated poorly to SSC and firmness in apples, whereas
Bonora et al. (2013) and Hale et al. (2013) observed a slight relation
between IAD and SSC in nectarine fruits.
Devices based either on vis/NIR or NIR spectrometry have also
been used for estimating fruit total anthocyanins concentration in
plant species, including blueberries (Guidetti et al., 2009; Ribera
et al., 2016), cherry (Nagpala et al., 2013) and red grapevines berries
(Cozzolino et al., 2006; Larraín et al., 2008; Cerovic et al., 2008;
Guidetti et al., 2010; Tuccio et al., 2011; Fadock et al., 2016).
It is accepted that the main conventional fruit anthocyanin anal-
ysis needs a previous step of anthocyanin extraction, which its
efficiency is influenced by the temperature, soluble solids con-
tents and the ability of this molecules to form resonance structures
through pH variation (Lapidot et al., 1999; Laleh et al., 2006; Dougall
and Baker, 2008; Hernández-Hierro et al., 2014). In addition, total
insoluble material in plant cell walls exhibits a big opposition
to anthocyanin extraction from grapes (Hernández-Hierro et al.,
2014). All these limiting factors could be counteracted through
the availability of non-invasive devices which would allow rapid,
reliable and less expensive acquisition of data concerning techno-
logical and phenolic maturity in grape. Nevertheless, to evaluate
the accuracy of a non-destructive device to estimate any plant
molecule, the comparison with wet chemistry is always needed.
In our study we evaluated the relationship between Cherry-
Meter measurements and total and monomeric anthocyanin
concentration in cv. Sangiovese grape skins. The single antho-
cyanins detected in the present study by the HPLC analysis
(Fig. 3) are partially in accordance with those previously reported
by Mattivi et al. (2006) and Rombolà et al. (2011) for San-
giovese grapes, with malvidin-3-glucoside as the main anthocyanin
molecule. However, here we found a lower ratios between malvidin
and the other anthocyanins compared to those detected Mattivi
et al. (2006), which can be explained due to here berry sampling
was performed also before harvest.
234 A. Ribera-Fonseca et al. / Scientia Horticulturae 209 (2016) 229–235
Our outcomes concerning the relation between IAD and total and
monomeric anthocyanins are in agreement with Ben Ghozlen et al.
(2010) and Tuccio et al. (2011), who worked with fluorescence-
based optical sensors. Guidetti et al. (2010), Tuccio et al. (2011) and
Agati et al. (2013) that found very accurate calibrations to predict
anthocyanins in grapes using another non-invasive and portable
vis/NIR spectroscopy instruments. Suitable relationships among
Cherry-Meter measurements and total anthocyanin concentration
were beforehand reported only by Nagpala et al. (2013) and Ribera
et al. (2016) for intact cherry and blueberry fruits, respectively, but
not information was given regarding berry anthocyanins composi-
tion. Here we found significantly positive correlations among IAD
values and all single anthocyanins, which suggest that IAD could
also be useful as a predictor of anthocyanins composition and then,
health properties in grapevines fruits.
Otherwise, redness and blueness of anthocyanin molecules are
directly related to hydroxylation and methylation pattern of the
B ring (He et al., 2010). Therefore, whereas the redness intensifies
with the raising of the methylation of hydroxyl groups, the blue-
ness is enhanced with the increasing of free hydroxyl groups (He
et al., 2010). Most red grape cultivars, including Sangiovese, have
a predominance of tri-hydroxylated anthocyanins in their fruits,
with the reddest malvidin (di-methoxylated) as the prevalent form
(Castellarin et al., 2011). This is the first approach showing the
potential use of these non-invasive technologies to monitor not
only total anthocyanin concentration in red grapevine fruits.
The PCA has been previously used to test the variability of fruit
quality traits (Rajkumar et al., 2012). Our PCA results showed a
clear separation and clustering between grape fruits from the dif-
ferent IAD classes (from IAD class 1 to IAD class 10), suggesting that
each distinctive cluster has significantly different maturity levels.
Furthermore, PCA loading revealed that almost all grape maturity
parameters evaluated here (SSC, DI, TA and ANTH), as well as IAD
values, were highly correlated to the PC1. Finally, the PCA load-
ing confirm that using Cherry-Meter, grape berries with different
maturity levels could be adequately sorted in a non-invasive way.
Many researches focused on prediction of technological matu-
rity parameter of fruits have been developed via using wide
spectral ranges (Guidetti et al., 2010; Urraca et al., 2015), in which
data processing requires complex mathematical techniques to
explain chemical information encoded in spectral data (Cogdill
and Anderson, 2005; Cen and He, 2007). In contrast to other
vis/NIR-based portable devices, Cherry-Meter is based on only two
wavelengths. The selection of optimal wavelengths reduces the
size of the required measurement data while preserving the most
important information contained in the data (Sun, 2010).
5. Conclusions
Here we confirm that IAD can represent a reliable index to
the non-destructive estimation and monitoring of technological
maturity parameters and total and single anthocyanins in cv. San-
giovese grapes, without sample preparation. These findings can
represent an important tool for viticulture, which could allow a
quick and more precise assortment of grapes for obtaining high-
quality wines. Future applications of Cherry-Meter device could
also include a non-destructive qualitative analysis of wines compo-
sition. Further studies in-planta under different climatic conditions
and over different years, are necessary in order to validate our out-
comes.
Acknowledgments
The authors gratefully acknowledge to the ERASMUNS MUNDUS
LINDO Program for the post-doc scholarship to Dr. Alejandra Rib-
era F. and for the partial support of this work. This study was also
funded by Dr. Adamo Domenico Rombolà Research Group from
the Department of Agricultural Sciences(DIPSA), School of Agricul-
ture and Veterinary Medicine, Alma Mater Studiorum, University
of Bologna (Bologna, Italia).
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