horticulturae
Article
Calcium Nutrition in Fig Orchards Enhance Fruit Quality
at Harvest and Storage
Jackson Mirellys Azevêdo Souza 1 , Sarita Leonel 2, * , Magali Leonel 3 , Emerson Loli Garcia 3 ,
Luiza Rocha Ribeiro 2 , Rafael Bibiano Ferreira 2 , Rafaelly Calsavara Martins 2 , Marcelo de Souza Silva 4 ,
Laís Naiara Honorato Monteiro 2 and Anita Santos Duarte 2
1
2
3
4
*
Citation: Souza, J.M.A.; Leonel, S.;
Leonel, M.; Garcia, E.L.; Ribeiro, L.R.;
Ferreira, R.B.; Martins, R.C.; de Souza
Silva, M.; Monteiro, L.N.H.; Duarte,
A.S. Calcium Nutrition in Fig
Department of Agronomy, Federal University of Viçosa (UFV), Viçosa 36570-900, MG, Brazil
Department of Horticulture, School of Agriculture, São Paulo State University (UNESP),
Botucatu 18610-034, SP, Brazil
Center for Tropical Root and Starches, São Paulo State University (UNESP), Botucatu 18610-034, SP, Brazil
Faculty of Higher Education and Integral Training, FAEF, Garça 17400-000, SP, Brazil
Correspondence: sarita.leonel@unesp.br; Tel.: +55-14-3880-7503
Abstract: Due to the high perishability of figs, calcium stands out as an important nutrient for
orchard management. This study aims to study the pre-harvest applications of calcium chloride in
fig orchards. The fig trees were sprayed with 0, 0.5, 1.0, 1.5 and 2.0% CaCl2 , and fruits were thereafter
stored for 0, 7, 14 and 21 d. The variables analysed were the concentration of calcium in leaves and
fruits, weight loss, fruit decay, pectinolytic enzyme activity, and physical and chemical attributes
of the fruits; CaCl2 sprays enhanced fruit Ca2+ concentration in leaves (14.03%) and fruits (29.3%)
and were effective in reducing polygalacturonase and pectin methylesterase activity, weight loss and
fruit decay. Pre-treatment with 1% CaCl2 provided fruits with larger diameters, greater firmness, and
higher levels of total phenolic compounds in both fruit peel and pulp. The pre-harvest application at
1% CaCl2 showed to be a promising technique in producing high quality fruits and extended storage
by approximately 7 to 10 days. These findings may be useful in planning new cultural practices for
fig orchards that produce high-quality fruit with desirable characteristics for growers and consumers.
Keywords: Ficus carica L.; fruit decay; pectin methylesterase; polygalacturonase; weight loss
Orchards Enhance Fruit Quality at
Harvest and Storage. Horticulturae
2023, 9, 123. https://doi.org/
10.3390/horticulturae9010123
Academic Editors: Gustavo Brunetto,
Danilo Eduardo Rozane,
Arcângelo Loss, Corina Carranca
and Elena Baldi
Received: 22 December 2022
Revised: 7 January 2023
Accepted: 11 January 2023
Published: 16 January 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
1. Introduction
Historically, ancient cultures have exploited figs as a cultural food, and figs are fruits
full of health benefits [1]. The Mediterranean basin has long been a site of fig production,
especially Egypt, Turkey and Algeria. Brazil is the 11th largest producer in the world,
accounting for 19.6 thousand metric tonnes in 2020 [2]. The cultivar ‘Roxo de Valinhos’ is
the most used by Brazilian producers; the fruits are extremely perishable, though. This
perishability is due to the fig being a climacteric fruit with rapid physiological breakdown
and thin skin that makes it susceptible to pathogen attack [3].
Pre-harvest spraying with calcium in fig orchards is a promising cultural practice.
Calcium-pectin cross-links promote the stabilisation of cell wall structures and limit their
degradation by the action of enzymes [4], such as pectin methylesterase (PME) and polygalacturonase (PG). These pectin-degrading enzymes are the most implicated in fruit-tissue
softening, wherefore the exogenous application of calcium helps stabilise the plant cell wall
by maintaining tissue firmness and reducing weight loss in fruits [5].
Furthermore, some studies have shown that calcium applications have reduced the
incidence of microorganisms in various fruits [3,6,7]; others have reported the involvement
of calcium in phenolic compound synthesis and antioxidant enzyme activity [8–10]. The
concentration of phenolic compounds is important, as consumer trends drive demand for
functional foods. Thus, the likely increase in these substances makes fig a great diet for
4.0/).
Horticulturae 2023, 9, 123. https://doi.org/10.3390/horticulturae9010123
https://www.mdpi.com/journal/horticulturae
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humans, since phenolic compounds can help preventing and/or treating several diseases
when vegetables and fruits are consumed [11].
However, calcium is poorly phloem-mobile; therefore, the accumulation of calcium in
fruits depends on the water that is pulled up through the xylem by the force of transpiration,
but the fruits have low rates of transpiration when compared to leaves [12]. This results in
low calcium accumulation in the fruits, making it necessary to adopt techniques that will
meet the need for this mineral, especially in trees with vigorous vegetative growth, such as
fig trees.
Alternatively, calcium can be supplied to the figs by immersing the fruits in CaCl2
solution up to 4% after harvest. Moreover, considering that figs are highly perishable
fruits, alternative methods to immersion, such as pre-harvest spraying, avoid handling
and possible damage to fruits. There are no reports of pre-harvest spraying of calcium
on figs, but studies have shown promising results in other fruits, such as blueberries [10],
guavas [13], blackberries [14] and pears [4]. In Californian-style black and green olives,
the effect of calcium strategies by immersion in the fruit is well known [15]. However,
soaking olives in calcium may provide some negative bitterness attributes provided by this
mineral [16]. These reports [15,16] indicated that CaCl2 is a firming agent, which is often
added to the package table olive top brine to improve olive firmness due to calcium ions
that help maintain structural firmness and cell wall stability and the cell turgor of fruits that
form cross-links between pectin molecules. This strengthens the plant cells and prevents
this breakdown.
Importantly high Ca2+ concentrations in fruits result in cellular toxicity and abnormalities in plant development. Large amounts of calcium can move from vacuoles and intercellular spaces to the cell cytoplasm, triggering greater activity of the ascorbate-glutathione
cycle and consequently promoting greater synthesis of ascorbic acid. This process is a
reflection of the plant’s protection system against oxidative stress. Ascorbic acid acts as
an antioxidant in the plant, regulating the amounts of reactive oxygen species (ROS) that
contribute to injuries resulting from stress, causing cell damage, which are visible on leaves
in the form of lesions, especially when excess calcium is applied [12].
The effects of pre-harvest applications of calcium chloride on fruit quality still have
not been well investigated. There is a shortage of information regarding the source, the
concentration, the number, and the period of calcium application onto the trees to get an
effective response to fruit quality [8,9]. Therefore, studies that come to enable the adoption
of safety concentrations by fig growers seeking the production of higher quality fruit.
Consequently, this study aimed to find an appropriate calcium concentration for fig based
on fruit quality and storage.
2. Materials and Methods
2.1. Characteristics of the Experimental Area
The experimental field was installed at the farm for the School of Agriculture, São
Paulo State University (UNESP), located in the city of São Manuel, São Paulo (22◦ 44′ 28” S,
48◦ 34′ 37” W at an altitude of 740 m). Based on the Köppen classification, the climate in
this area is of the Cwa type (temperate hot—mesothermal). Rainfall is concentrated from
November to April (summer season), and the average value of precipitation is 1376.70 mm.
The temperature of January, the hottest month, is 22 ◦ C. The soil is classified as a sandytextured Latossolo Vermelho distroférrico according to the Brazilian system of soil classification [17], that is, a dystrophic Typic Hapludox [18].
The fig orchard was established in June 2013 by planting the fig tree (Ficus carica L. cv
Roxo de Valinhos) at 3 m spacing between rows and 2 m between trees. The correction of
the soil and fertilisation of the entire orchard were based on previous soil analyses and crop
recommendations. One hundred and twenty trees were conducted in a rain-fed system,
receiving all the standard cultural practices recommended for the crop. Twenty guard trees
were used to preserve some treatments from others.
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2.2. Pre-Harvest Applications of Calcium
Calcium chloride (CaCl2 ) with 27% of Ca2+ was used as a source of calcium in this
study. CaCl2 was applied with a backpack sprayer at 0; 0.5; 1.0; 1.5 and 2.0%. The product
was diluted with water and adhesive spreader (2 mL L−1 —Assist® , São Paulo, Brazil). The
first CaCl2 application was performed as soon as fruit emission began, that is, October 2019.
Then, 10 applications were made at 15 d intervals during the 2019 and 2020 harvest seasons.
Calcium chloride was applied when the fruit setting began and finished in a ripening stage
less unripe, with firmer fruits [19]. Five hundred millilitres of solution per tree were used
per application at ambient temperature. The sprayings were directed towards the entire
aerial part of the trees (i.e., leaves and mainly fruits).
2.3. Harvest and Storage of Figs
The fruits were harvested when at least 50% of their surfaces had a red-purple colour.
After harvesting, they were cleaned in running water and placed on benches for natural
drying. Subsequently, the fruits were placed in polystyrene trays covered with polyvinyl
chloride (PVC) film and were therefore placed in a cold chamber (5 ± 1 ◦ C; 90–95% R.H.)
for 0, 7, 14 and 21 storage days.
2.4. Calcium Concentration in Leaves and Fruits
The levels of calcium in the leaves and fruits were measured soon after harvest.
Samples were washed with deionised water to avoid contamination and were dried in an
oven under forced-air ventilation at 60 ◦ C until the weight remained constant, then ground
in a Willey mill. Calcium concentration was determined according to Malavolta et al. [20].
After sample digestion, the calcium had the absorbance measured in an atomic absorption
spectrophotometer at 422.2 nm, expressed in g kg−1 .
2.5. Weight Loss and Fruits Rot Decay
At fruit packing time, trays were randomly selected and identified for the evaluation
of weight loss and fruit rot decay (conidia of Aspergillus fungi). For weight loss, the same
fruits were used for the initial weight and the final weight. The trays were weighed on
an analytical balance during storage (0, 7, 14 and 21 days) and the difference between the
initial and final weight of each interval was calculated. The following equation was used:
Weight loss (%) = [(WI − WF/WI)] × 100, where WI is the initial weight (g) and WF is the
weight on the evaluation day (g).
The percentage of fruit rot decay was assessed by means of the presence or absence
of pathogenic rot decays or peel spots (lesion diameters greater than 3 mm). Results were
expressed as the percentage of decayed fig fruits. Evaluations were performed at 2-day
intervals from day 0 (harvest) to the 22nd day of storage. The number of fruits per replicate
was therefore taken to calculate the percentage of decayed fruits by using the following
equation: % decayed fruits = [(number of decayed fruits/total number of fruits)] × 100,
according to the methodology described by Shiri et al. [5].
2.6. Degrading Enzyme Activity
Samples from each treatment corresponding to the CaCl2 concentrations (plot) at
each storage time (subplot) were sliced and ground in a pestle and mortar with liquid
nitrogen. The action of the enzyme’s polygalacturonase (PG) and pectin methylesterase
(PME) was measured using the protocol introduced by D’Innocenzo and Lajolo [21]. The
extraction was performed by diluting 5 g of the sample in 5 mL of buffer with pH 5.0 (12%
polyethylene glycol + 0.2% sodium bisulfide). Then, the extract was homogenised in turrax
(IKA T10—90 s, Staufen, Baden-Würetmberg, Germany) and centrifuged (Helttich Mikro
220 R—24,510 g, 30 min, 4 ◦ C, St. Louis, MO, USA). The recovered pellet was homogenised
with sodium acetate (0.1 M) buffer (pH 4.5) with NaCl (0.1 M) in an ice bath under constant
agitation. The extract was again centrifuged (12,255 g, 20 min, 4 ◦ C) and the supernatant
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was dialysed in 0.1 M sodium acetate buffer (pH 4.5) with 1.0 M NaCl. This dialysate was
used as a source of enzymes.
2.7. Fruit Firmness, Size and Colour
The firmness, size and colour analyses of the figs were performed at 0, 7, 14 and 21 d
of storage. For storage time evaluation, 32 figs (4 trays of 8 fruits) were evaluated for
each CaCl2 concentration. For firmness, the penetration resistance evaluated the texture
of fig fruit, and the measurements were performed using a TA.XT Plus Texture Analyzer
equipped with a cylindrical probe (SMS P/2N). Tests were performed in two equatorial
regions of the fruits. The penetration depth was set at 5 mm (50 mm s−1 ), results were
expressed in Newton (N). The size of the fruits was measured by their diameter and
length, measured with a digital vernier calliper (Starrett 799A-6/150, Athol, MA, USA)
and expressed in millimetres (mm). For colour parameters, fruit colour was evaluated
with a CR-400 Chroma Meter (Konica Minolta, Osaka, Japan). The colour changes were
evaluated using the CIELAB colour space. The peel colour was measured on opposite sides
of the mid-equatorial region of each fruit surface. Chromaticity (C) and hue angle (◦ h)
were evaluated.
2.8. Chemical Evaluation of Fruits
The same fruits used for physical properties were used for chemical analyses, also
performed on the day after harvest, 0 d and at 7, 14 and 21 d. The samples were crushed
with the aid of mixed fruit (Philips Walita—RI1364, Varginha, Minas Gerais, Brazil) to form
a homogeneous extract of the figs. However, for the analysis of total phenolic compounds
and antioxidant activity, the preparation of the samples differed. The figs were sliced and
frozen in liquid nitrogen and then ground with the aid of a mortar and pestle.
Titratable acidity (TA) was achieved by titrating with sodium hydroxide (NaOH—
0.1 N) in a solution of 1 g of the homogenised fig extract, 50 mL of distilled water and
0.3 mL of phenolphthalein [22], expressed as % of citric acid. Soluble solid content (SSC)
was measured with a digital refractometer (Atago 3405 PR-32a, Minato-ku, Tokyo, Japan)
using 1 g of the homogenised fig extract and expressed as %. The SS/TA ratio was also
measured, which indicates the fruit ripeness index (expression of sweet taste).
Reducing, non-reducing and total sugars were evaluated using the phenol-sulphuric
method [18] and the DNS method (3.5-dinitrosalicylic acid) [23,24]. One gram of the
homogenised fruit extract diluted in 100 mL of distilled water was used. The evaluations
were performed using a UV-visible spectrophotometer (BEL photonics® , Piracicaba, São
Paulo, Brazil), with readings at a wavelength of 490 and 540 nm and results expressed as
percentage (%).
The same extract was used for total phenolic compounds and antioxidant activity.
Samples (100 mg) were diluted in 3 mL of pure methanol and kept in an ultrasonic bath for
30 min. Then, the samples were centrifuged (Helttich Mikro 220 R—24,510 g, 15 min, 4 ◦ C)
and the supernatant was collected for analysis.
The total phenolic concentration in the peel and pulp of the fruits was determined
using the Folin–Ciocalteau colorimetric method [25]. The analyses were performed on
a UV-visible spectrophotometer (BEL photonics® , Brazil) at 765 nm and the absorbance
was compared with a calibration curve for gallic acid, expressed as mg 100−1 gallic acid
equivalents (GAE).
Antioxidant activity was evaluated in the peel and pulp of the fruits by a method
based on the use of the DPPH reagent (2,2-Diphenyl-1-picrylhydrazyl) [26]. Readings were
taken at 517 nm on a UV-visible spectrophotometer (BEL photonics® , Brazil). From the
absorbance, the scavenging activity was calculated and hence they were compared on a
calibration curve obtained for Trolox. The results were expressed in mg 100 g−1 in Trolox
equivalent antioxidant capacity (TEAC).
−
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2.9. Experimental Design and Statistical Analysis
Randomised complete block design was adopted with subdivided plots, that is, plots
corresponding to the CaCl2 concentrations (0, 0.5, 1.0, 1.5 and 2.0%) and the subplots to the
storage time (0, 7, 14 and 21 d). In the field, each plot was composed of four replicates, with
five trees each, totalling 100 useful trees. In storage, each subplot was composed of four
replicates of 10 fruits each, totalling 40 fruits per storage time, 160 by CaCl2 concentration
and 800 total fruits.
The analysis was based on average data from the 2019 and 2020 seasons. The normality hypothesis was tested by the Shapiro–Wilk test, and the F test (1 and 5% levels
of significance) was used in the analysis of variance to detect the difference between the
factors. When a significant difference was found for the sources of variations, regression
analysis (linear and quadratic) was used to compare the treatment means for the data of
the plots (CaCl2 concentration) and subplots (storage time). To ensure the homogeneity
of variances in the regression analysis, Grubb’s test was applied to verify the presence of
outliers for each concentration level.
3. Results
3.1. Calcium Concentration in Leaves and Fruits
Pre-harvest CaCl2 spray significantly increased in calcium concentration in treated
leaves (p < 0.01) and fruits (p < 0.01). The calcium concentration in the leaves of Roxo de
Valinhos−figs increased linearly up to the concentration of 2.0% CaCl2 , when they reached
18.3 g kg−1 of calcium, corresponding to an increase of 14.03% in relation to the control
(Figure 1a). Similarly, the Ca concentration in fruits
increased linearly with the application
−
of 2.0% CaCl2 , reaching a value of 7.15 g kg−1 , that is, and an increase of 29.3% in relation
to untreated trees (Figure 1b). However, the leaves presented toxicity symptoms at 1.5 and
2.0% CaCl2 (Figure 2).
Figure 1. Calcium concentration in leaves (a) and fruits (b) of fig tree cv. ‘Roxo de Valinhos’ as
a function of different concentrations of pre-harvest CaCl2 spray. Bars in each column symbolise
standard errors (n = 4).
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a
b
Figure 2. Symptoms of Ca2+ toxicity in fig leaves cv. ‘Roxo de Valinhos’ as a function of pre-harvest
CaCl2 spray at 1.5% (a) and 2.0% (b).
3.2. Weight Loss and Fruit Decay
Significant differences were found for the single effect of CaCl2 concentrations
(p < 0.01) and storage (p < 0.01) on the figs weight loss. When weight loss was evaluated upon CaCl2 concentrations, there was a quadratic decrease of up to 0.9% CaCl2 (value
obtained by the fit curve), reaching an average of 1.8%, equivalent to a reduction of 20.7%
(Figure 3a). Regarding the storage days, weight loss evolved linearly, reaching the highest
value at 21 d (Figure 3b).
Figure 3. Weight loss (a,b) and fruit decay (c,d) in ‘Roxo de Valinhos’ fig fruits as a function of CaCl2
concentrations and storage days at 5 ± 1 ◦ C and 90–95% R.H. Bars symbolise standard errors (n = 4).
Moreover, a significant difference was also found for the effect of the concentrations
(p < 0.05) on the decay percentage of fig fruits, whose averages decreased linearly with an
increase in CaCl2 percentage, resulting in a 66.8% reduction in the decay percentage of fig
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fruits (Figure 3c). However, fruit decay increased linearly with an increase in storage days
(p < 0.01). The first decay symptoms appeared only after 14 d of storage, regardless of the
CaCl2 concentration (Figure 3d).
3.3. Degrading Enzyme Activity
There was a significant effect of the interaction between CaCl2 concentrations and
storage for the PME (p < 0.01) and PG (p < 0.01) activities. Soon after harvest (day 0), high
PME activity was observed at 2.0% CaCl2 . However, there was a quadratic increase in
the averages of untreated and treated trees with 0.5% CaCl2 throughout storage time. In
general, PME activity remained low at 1.0 and 1.5% CaCl2 (Figure 4a). Similarly, high
PG activity was obtained in the untreated and treated trees with 2.0% CaCl2 . Therefore,
PG activity was fit to a quadratic regression model throughout storage, and the lowest
averages were mainly obtained at 1.5% CaCl2 (Figure 4b).
Figure 4. Pectin methylesterase (a) and polygalacturonase (b) activities of ‘Roxo de Valinhos’ fig
fruits as a function of CaCl2 concentrations and storage days at 5 ± 1 ◦ C and 90–95% R.H. Bars
symbolise standard errors (n = 4).
3.4. Fruit Firmness, Size and Colour
Significant differences were also found for the single effects of CaCl2 concentrations
(p < 0.01) and storage (p < 0.01) on fruit firmness. The applications promoted a quadratic
increase in fruit firmness up to 1.0% CaCl2 (Figure 5a). The firmness of the untreated
fruits was 0.28 N, while those that received about 1.0% CaCl2 was 0.45 N, that is, an
increase of 59.8%.
However, there was a linear reduction in fruit firmness from 0.48 N to 0.28 N throughout storage, that is, a decrease of 42.4% (Figure 5b). Fruit length was not affected by CaCl2
concentrations and storage, or even by the interaction of these factors, so the average fruit
length was 61.9 mm. On the contrary, the fruit diameter was significantly affected by CaCl2
concentrations (p < 0.05) and storage (p < 0.01). There was a quadratic increase in fruit
diameter up to 1.0% CaCl2 (0.8% by regression test) (Figure 5c). Regarding storage days,
there was a linear reduction in the diameters of the fruits (Figure 5d).
For chromaticity (p < 0.01) and hue angle (p < 0.01), there was a significant effect of the
interaction between CaCl2 concentrations and storage. During storage, the only increase
in the chromaticity of the fruit surface was observed at 1.0% CaCl2 concentration, while
the other concentrations (0.5, 1.5 and 2.0% CaCl2 ) presented a linear reduction. There was
a small increase in chromaticity up to 6 days of storage in the untreated trees, but also a
reduction after this period (Figure 5e).
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Figure 5. Firmness (a,b), diameter (c,d), chroma value (e) and hue angle (f) of ‘Roxo de Valinhos’
fig fruits as a function of CaCl2 concentrations and storage days at 5 ± 1 ◦ C and 90–95% R.H. Bars
symbolise standard errors (n = 4).
The untreated trees had a linear decrease in the hue angle values, while there was a
reduction until 10 days of storage, with a subsequent increase until 21 days in the trees
treated with 2.0% CaCl2 . Regarding the other concentrations, there was no adjustment of
the evaluated equations (linear and quadratic). This means that for this variable, there was
no regular pattern of response. However, it appears that the hue angle obtained with the
intermediate concentrations (0.5, 1.0 and 1.5% CaCl2 ) was higher than the others (Figure 5f).
3.5. Chemical Quality
The interaction between CaCl2 concentrations and storage time was significant for
soluble solids content (p < 0.05). Fruits treated with 1% CaCl2 showed a higher content
of soluble solids on the day of harvest (day 0). However, over storage time, there was a
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quadratic reduction of soluble solids for concentrations of 0 to 1.5% of CaCl2 and linear for
the concentration of 2.0% (Figure 6a). The highest means of soluble solids at 21 days were
found in the untreated fruits.
Figure 6. Soluble solids (a), reducing sugars (b), titratable acidity (c) and maturation index (d) of
‘Roxo de Valinhos’ fig fruits as a function of CaCl2 concentrations and storage days at 5 ± 1 ◦ C and
90–95% R.H. Bars symbolise standard errors (n = 4).
The contents of reducing sugars were significantly affected by the interaction between
concentrations and storage (p < 0.01). While there was a linear increase in reducing sugars
during storage in the untreated trees, a linear reduction was observed in the trees treated
with 1.0% CaCl2 . Furthermore, the means of reducing sugars of the other concentrations
did not adjust to the applied equations (Figure 6b). The contents of non-reducing and total
sugars were not affected by CaCl2 applications or even by storage, with averages of 0.8 and
6.7%, respectively.
There was a significant effect of the interaction between CaCl2 concentrations and
storage (p < 0.01) for titratable acidity, since there was an initial reduction in the averages
with a subsequent sharp increase in all treatments, reaching the highest values at the end
of the 21d storage period. Moreover, untreated and treated trees with 0.5% CaCl2 showed
the highest titratable acidity throughout storage (Figure 6c).
A high maturation index was found in fruit treated with 2.0% CaCl2 at the beginning
of the storage period due to the reduced titratable acidity of these fruits. The maturation
index decreased linearly throughout storage when concentrations were applied between 1.0
and 2.0%. Similarly, data was fit in a quadratic regression model in untreated and treated
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trees with 1.0% CaCl2 , as the maturation index increases followed by a reduction until
21 d (Figure 6d).
There was a significant effect of the interaction between CaCl2 concentrations and
storage for the total phenolics concentration of the peel (p < 0.05) and pulp (p < 0.01). In the
peel, the highest concentration of total phenols was obtained, with 1.0 and 2.0% CaCl2 at
harvest (day 0) (Figure 7).
Figure 7. Total phenolic content of peel (a) and pulp (b) and antioxidant activity of peel (c) and pulp
(d) of ‘Roxo de Valinhos’ fig fruits as a function of CaCl2 concentrations and storage days at 5 ± 1 ◦ C
and 90–95% R.H. Bars symbolise standard errors (n = 4).
Throughout storage, there was a reduction in these values, with the lowest in the
untreated fruits at the end of the 21d storage period (Figure 7a). Similarly, the pulp
had a lower concentration of total phenols in the untreated fruits throughout storage
(Figure 7b). For antioxidant activity, there was a significant effect of the interaction between
CaCl2 concentrations and storage (p < 0.05 for peel and pulp). In addition, there was a
certain relationship between phenol concentration and antioxidant activity of the peel and
pulp of the figs since the means during storage were similar between them. There was
less antioxidant activity in both the pulp and peel of the untreated fruits (Figure 7c,d).
Throughout the whole storage time, there was great antioxidant activity in the pulp of
fruits treated with 1.0% CaCl2 .
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4. Discussion
Pre-harvest CaCl2 sprays increased the calcium concentration in fig fruits; thus, a
promising and strategic method that improves human nutrition through biofortification of
figs, as it can supply dietary calcium deficiency. Previous studies have shown an increase
in Ca2+ in fruits with the exogenous application of calcium in grapes [27], papayas [28]
and kiwifruits [5]. Calcium-fortified fruits have been important not only from a nutritional
point of view but also because they involve several physiological processes. However, an
excessive or deficient amount of calcium can lead to major disturbances in plants [29].
Calcium deficiencies can lead to membrane breakdown and/or cell wall failure in
fruits [30], reinforcing the importance of an adequate supply for trees. On the contrary, if
tissue calcium concentration is high, this can result in cellular toxicity and developmental
abnormalities [12], explaining the necrosis observed in fig leaves treated with the highest
concentrations of CaCl2 . Calcium is moved in the plant metabolism by the transpiration
flow and part of the calcium reallocated in the fruits ends up being translocated to meristematic regions where there are higher transpiration rates, such as growing leaves. This
fact highlights the need for a constant supply of this nutrient during fruit growth [13]. This
report reinforced the need for the ten CaCl2 sprays needed in this study to improve final
fig fruit quality.
Based on the results obtained, it can be suggested that leaf calcium concentrations
above 16 g kg−1 were toxic for fig tree cv. Roxo de Valinhos due to symptoms of leaf
necrosis. This record must be considered since excessive necrosis leads to a decrease in
photosynthesis areas and negatively affects the development and production of trees.
Calcium plays an important role in changes in cell turgor pressure and cell elongation
in fruits, in addition to being involved in auxin signalling and acidification of the cell
wall, allowing its expansion, which can lead to increased fruit weight [13]. However, the
reduction in averages during storage, which was observed in all treatments, is related to
the increase in weight loss in this period.
The increase in the calcium concentration in the figs was crucial to reduce weight loss,
especially at the estimated concentration of 1.0% CaCl2 (0.9% by fit curve–regression test).
This reduction is because Ca2+ provides structural integrity to cellular membranes. Previous research reported this efficient technique in reducing weight loss in red guavas [13],
blackberries [14], blueberries [31], apricots [32], apples [33], peaches [34] and kiwifruits [5].
Besides maintaining the cell wall structure, calcium reduced the hydraulic conductivity of
the fruits, which caused the lowest weight loss and is mainly due to water loss throughout
storage [29]. However, the same authors reported that when excess in the cytoplasm, Ca2+
causes damage to the membrane and increases respiratory rates. This explains the greater
weight loss of figs when concentrations greater than 1.0% CaCl2 are used.
Furthermore, the reduction in the percentage of decayed fig fruits can also be attributed
to the maintenance of the cell wall structure promoted by CaCl2 applications. In response
to pathogen attack, there is a cytosolic influx of Ca2+ followed by an apoplastic explosion
of reactive oxygen species (ROS), promoting the activation of the antioxidant defence
complex [29]. Irfan et al. [3] reported a lower incidence of aerobic bacteria, yeasts and
fungi in figs cv. Poona treated with 4% CaCl2 during post-harvest and, consequently, less
fruit deterioration.
The efficiency of calcium in reducing polygalacturonase and pectin methylesterase
activity has already been reported by Liu et al. [32] in apricot and by Ortiz, Graell and
Lara [33] in apple cv. Golden Reinders. Pectin methylesterase promotes the de-esterification
of pectin structure elements, forming a pectin with a lower degree of methylation; therefore,
it is more susceptible to the adverse effects of polygalacturonase, which promotes the
hydrolysis of (1–4) β-bonds between galacturonic acid residues present within the pectin
chains [12]. However, the interaction between Ca2+ and pectin forms a calcium-pectin gel
called calcium pectate, which results in greater resistance and less degradation of cell wall
components [4,29]. It is noteworthy that when in excess, the apoplastic Ca2+ leakage causes
damage to the cells, which explains the greater action of enzymes in fruits treated with 2.0%
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of CaCl2 . Thus, it can be said that pre-harvest applications of CaCl2 in safe concentrations
promote lower pectin methylesterase and polygalacturonase activity, as well as greater
fig firmness.
The great firmness of the fruits treated with 1.0% CaCl2 occurred due to the calciumpectin gel. Ca2+ ions induce a chain-chain association, thus forming junction zones responsible for the formation of the gel. These zones are popularly known as the “egg box
model.” In this mechanism, initially two antiparallel polyuronate chains form the egg-box
with Ca2+ and further aggregate to form multimers [24]. Similarly, Lobos et al. (2020) [10]
reported that early pre-harvest spraying of CaCl2 increased blueberry firmness compared
to untreated fruits.
High cytoplasmic concentrations of calcium cause damage to the membrane and
increase the respiratory rate [29,33]; consequently, firmness decreases when concentrations are above 1.0% CaCl2 . On the other hand, the reduction of firmness is the result of
cell wall hydrolases throughout storage, such as the enzymes pectin methylesterase and
polygalacturonase, which alter the cell wall compositions [12].
Extracellular auxin promotes Ca2+ transport in the plasma membrane, causing acidification of the cell wall solution that induces cell wall expansion. On the other hand, when
in excess, the Ca2+ ions compete with H+ for the same proton-binding sites, inhibiting cell
wall acidification and growth [12]. This explains the larger diameter of fruit treated with an
intermediate concentration of CaCl2 , as well as the smaller diameter of fruit treated with a
high concentration (2%).
The maintenance of higher chroma and hue angle values obtained in fruits from trees
treated with intermediate concentrations (i.e., 1.0% CaCl2 ) may be due to chlorophyll
retention, as calcium application reduces the respiratory rate and ethylene production in
the fruits, with a consequent slowdown in the ripening phase [7,29]. The retention of green
pigments is related to the accumulation of calcium in the cell wall and middle lamella,
which promotes thickening of the fruit’s cell wall due to the binding of Ca2+ with protein
molecules [3].
The large decrease in the concentrations of soluble solids and reducing sugar contents
in fig fruits treated with CaCl2 can be attributed to the major stabilisation of pectin bonds
promoted by calcium [5]. The same authors reported that the soluble solid content in
papaya decreased with increasing CaCl2 concentrations. The effect of CaCl2 treatments on
titratable acidity presented variations between the evaluated concentrations and storage
times. This variable also presented different responses in other fruit trees. Ribeiro et al. [13]
reported that the pre-harvest application of CaCl2 (0.4%) decreased the titratable acidity
concentration in ‘Paluma’ red guava and Ali et al. [29] obtained results of high titratable
acidity in peaches treated with 1.0% CaCl2 .
The high concentration of total phenols and antioxidant activity observed in figs
treated with CaCl2 are probably related to the action of Ca2+ . Calcium stimulates the
activation of the NADPH oxidase to produce reactive oxygen species (O2 and H2 O2 ); thus,
as a defence measure, there is a synthesis of phenolic compounds and great antioxidant
enzyme activities formed by superoxide dismutase, peroxidase and catalase [8,29,34].
These findings were corroborated by Xu et al. [9], who reported that calcium treatment
in strawberries increased anthocyanin accumulation and total phenolic concentrations.
Similarly, Lobos et al. [10] reported the positive effects of CaCl2 sprays on antioxidant
activity and total phenol concentration in blueberries compared to untreated fruits.
There was a higher concentration of total phenols and, consequently, greater antioxidant activity of the peel in relation to the fruit pulp, regardless of CaCl2 concentration
or storage time. Similarly, Palmeira et al. [1] reported a higher concentration of phenolic
compounds in the peel (152.0 mg 100 g−1 ) compared to the pulp (54.2 mg 100 g−1 ) in a
study with Pingo de Mel figs. Therefore, consuming the peel with the pulp can boost the
total intake of these nutrients, since some consumers only end up eating the pulp, especially
in figs. Human diets composed of fruits and vegetables with great antioxidant activity act
as barriers against cancer [11]. Therefore, the pre-harvest CaCl2 application can be used
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as an important technique for producing high-quality figs, since provided calcium-rich
fig fruits, reduced weight loss and fruit rot decay, thereby enhancing the total phenolic
concentrations and antioxidant activity.
In summary, the concentrations of Ca2+ in the leaves and fruits of the fig tree increased
linearly as a function of CaCl2 applications. There was an increase of 14.03% in leaves and
29.3% in fruits, with a consequent reduction in the activity of pectinolytic enzymes, weight
loss and fruit rot decay percentage. The fig tree Roxo de Valinhos sprayed with 1.0% CaCl2
presented fruits with larger diameters and greater firmness, as well as higher total phenolic
concentrations and greater antioxidant activity for fruit peel and pulp.
CaCl2 pre-harvest sprays are easy management to implement by fig growers at low
cost. The main limitations were the number of applications needed to obtain significant
changes in the fruit characteristics, and at the same time, without causing symptoms of
calcium phytotoxicity on the leaves. Another research plan aims to perform a sensory and
volatile compound evaluation of the fruits, in addition to evaluating a smaller number of
sprays with CaCl2 .
5. Conclusions
Pre-harvest sprayings of CaCl2 1.0% is a promising cultural practice for the production
of high-quality fig fruit with greater nutritional value, reducing the activity of pectinolytic
enzymes and promoting less weight loss and incidence of rot, i.e., extending shelf-life by
approximately 7 to 10 days.
Author Contributions: Conceptualisation, S.L. and J.M.A.S.; methodology, S.L., J.M.A.S., M.L. and
E.L.G.; validation, J.M.A.S.; formal analysis, J.M.A.S.; investigation, J.M.A.S., E.L.G., L.R.R., R.B.F.,
R.C.M., M.d.S.S., L.N.H.M. and A.S.D.; resources, S.L. and M.L.; data curation, J.M.A.S.; writing—
original draft preparation, J.M.A.S. and S.L.; writing—review and editing, S.L.; supervision, S.L.;
project administration, S.L.; funding acquisition, S.L. All authors have read and agreed to the
published version of the manuscript.
Funding: This work was partially supported by the Brazilian National Council for Scientific and Technological Development (CNPq), grant numbers 302611/2021-5 and 302848/2021-5 and Coordination
for the Improvement of Higher Education Personnel (CAPES—PNPD scholarship).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article.
Acknowledgments: We thank Guilherme Augusto Rago Ferraz for providing language assistance.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
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