Journal of Berry Research 13 (2023) 195–210 195

DOI:10.3233/JBR-220067
IOS Press

Research Report

Influence of growing environment and

pruning techniques on storage and quality
traits of Vaccinium corymbosum L., cv
‘Duke’
Alice Varaldo∗ , Valentina Chiabrando and Giovanna Giacalone
Department of Agricultural, Food and Forest Sciences, University of Turin, Turin, Italy

Received 5 October 2022

Accepted 17 May 2023
Pre-press 9 June 2023
Published 14 September 2023

Abstract.
BACKGROUND: Due to the increasing market demand for blueberries, the amount of land dedicated to their production is
expanding. Therefore, cultivation techniques to ensure high quality and a longer shelf life have become key concerns for the
blueberry industry and consumers.
OBJECTIVE: This study investigated the impact of various growing environments and pruning techniques on the quality
and shelf life of highbush blueberry (Vaccinium corymbosum L. cv. ‘Duke’) during cold storage.
METHODS: In this study, six commercial orchards were selected based on the type of pruning (light or hard) and the
plot’s altitude (lowland, hill, or mountain). After harvest, the blueberries were stored at 1◦ C and 90% RH for 21 days. Quality
analyses were performed on days 0, 10, and 21 of storage. The data were processed with a factor-designed analysis of variance
(one-way ANOVA).
RESULTS: Blueberries from the lowland cultivation area with light pruning had a longer shelf life. At harvest, the lowland
area had indeed produced firmer blueberries that were sweeter and less acidic, and cold storage positively preserved these
characteristics compared to fruit grown at other altitudes.
CONCLUSIONS: In summation, the lowland area produced firmer berries that retained their characteristics after 10 days
of storage, especially those harvested from hard-pruned plants. Notably, the bioactive compounds were not affected by the
pruning method. However, when combined with 10 days of storage, light pruning was associated with better preservation of
the total phenol content.

Keywords: Highbush blueberry, growing habitat, pruning, quality, shelf life

∗ Corresponding author: Alice Varaldo, Department of Agricultural, Food and Forest Sciences, University of Turin, Turin, Italy, Largo
Paolo Braccini 2, Grugliasco (TO), E-mail: alice.varaldo@unito.it.

ISSN 1878-5093/$35.00 © 2023 – IOS Press. All rights reserved.

196 A. Varaldo et al. / Influence of growing environment and pruning techniques

Abbreviations:

a.s.l. above sea level

AA antioxidant activity
DDT digital durometer test
DT deformation test
PT penetration test
RH relative humidity
TA titratable acidity
TAC total anthocyanin content
TPC total phenol content
TSS total soluble solids

1. Introduction

Recently, blueberries have become more popular, no longer representing a moderate market segment, but
an emerging sector that is rapidly growing and recruiting new consumers [1]. This upward trend is driven by
the recognized health benefits of blueberry consumption. Studies have shown that blueberries have antioxidant
and anticancer properties and are an excellent source of vitamins B, C, and K, polyphenols [2, 3], and several
minerals, such as K, P, Ca, Mg, and Mn, of relevant health importance [4].
Due to the rapid increase in blueberry demand, new growing regions, innovative genetic materials, and
agronomic techniques have been developed [5–7].
As in the rest of the world, in Italy, mainly in the Piedmont and Trentino-Alto Adige regions, the land
dedicated to blueberry production is increasing. The main cultivar grown in these regions is the Duke variety, an
early ripening blueberry.
The main training system applied to blueberry plants is the free bush consisting of 5 or 6 productive branches.
Every year, a pair of the oldest branches is replaced with more vigorous shoots of 1 to 2 years of age. The
main purpose of pruning is to balance both the productive load and crop vegetative renewal. Cutting back too
drastically causes an unnecessary vegetative boost, while very light pruning causes early aging and a bush that
is no longer sufficiently covered with productive branches.
Many growers whose production comes mainly from mixed branches prefer harder pruning, and those who
favour production on brindils (light shoots) consequently prefer lighter pruning. In the Piedmont area, where
blueberry cultivation is increasing, a uniform pruning technique has not yet been applied due to the lack of
consolidated experience in the management of blueberry bushes. This can be attributed to the limited number
of studies highlighting the effects of pruning methods on the berries’ quality and shelf life. Some studies report
that hard pruning leads to better fruit production and size due to a better distribution of photosynthates but
has no effect on nutritional attributes [8–10]. Other studies, in contrast, show that light pruning, resulting in a
45–55◦ angled bush conformation, leads to very productive yields [11–13]. Finally, many trials conducted in
North America support the idea that growing blueberry bushes in a V-shape is easier for mechanical harvesting
[14]. Nevertheless, this method cannot be used in italian production because orchards are commonly on slopes.
Moreover, consumers’ desire for high product quality limits the mechanical harvesting of blueberries for fresh
markets [15].
Considering these issues, the objective of this study was to compare two pruning techniques applied to highbush
blueberry plants grown in three landscapes (lowland, hill, and mountain) and to identify which pruning method
and cultivation site provided the best balance of blueberry quality characteristics and shelf-life performance.
 A. Varaldo et al. / Influence of growing environment and pruning techniques 197

Quality traits were evaluated at harvest and monitored over a period of 21 days in cold storage to identify the
best blueberry growing conditions to optimize shelf life.

2. Materials and methods

The study was carried out in six commercial orchards of highbush blueberry (Vaccinium corymbosum) located
in the Saluzzo area (Piedmont, Italy). Five growing sites located at different altitudes were used: lowland < 300 m
a.s.l. (Revello, Envie); hill 300–600 m a.s.l. (Castellar, Pagno), and mountain > 600 m a.s.l. (Sanfront).

2.1. Plant material and climate characterization

The study was performed from April to August 2021 on even-aged (10 y) blueberry cv ‘Duke’ orchards
managed according to the organic agriculture protocol (Reg. CE n◦ 834/2007). Six orchards were selected based
on the type of pruning. Three of the six (Table 1) were pruned using hard techniques (production on vigorous
shoots), and the remaining three received light pruning (production on light shoot – brindile). Each of the three
selected locations (lowland, hill, and mountain) had one light-pruning orchard and one hard-pruning orchard.
Both pruning methods were applied to 8–10 productive branches, a procedure that is usually performed every
year in the winter (last 10 days of February) during the dormancy phase of the plant. The difference between the
techniques is that hard pruning is carried out to obtain production mainly on vigorous shoots, and light pruning
is done for production predominantly on light shoot - brindle. In other words, hard pruning removes more wood
from the bush than light pruning does.
Meteorological data were collected from the Arpa Piemonte [16] database to study the climate characteristics
of each selected location. The data from 2021 were collected until June, the month of blueberry harvest.
In April, three bushes located near the centre of the orchard were chosen. These bushes were similar in
vigour and number of flowering bunches. The blueberries were hand-harvested from June 14 to July 14
through weekly pickings. Samples of blueberries from the second week were stored at 1◦ C and 90% RH
(relative humidity) for 21 days at the Lagnasco Group’s warehouse in Saluzzo (Piedmont, CN, Italy). The
storage conditions were chosen based on those normally used by the company. Defect-free fruits were ran-
domly divided into 48 RPET baskets of 500 g each: 8 baskets were selected for each treatment (orchard/pruning
method).

Table 1
Properties of the six blueberry orchards selected for research purposes

ORCHARDS LOCALITY ALTITUDE RANGE PRUNING METHOD

Envie lowland Light
Revello lowland Hard
Castellar hill light
Pagno hill hard
Sanfront Mountain light
Sanfront Mountain hard
198 A. Varaldo et al. / Influence of growing environment and pruning techniques

2.2. Quality analysis

Blueberry quality was evaluated at harvest (day 0) and after 10 and 21 days of cold storage at the DISAFA
laboratory, University of Turin (Grugliasco, TO) after they were transported from the warehouse to the laboratory.

2.3. Firmness

The fruit firmness of each treatment group was evaluated by performing a digital durometer test (DDT) using
a digital durometer (53215 TP-Turoni, Forlı̀, Italy), with firmness measurements expressed on the Shore scale
(10–90). The analyses were performed at room temperature in the equatorial zone on 30 fruits per sample.
Mechanical properties were also determined using a Texture Analyzer TA-XT2i (Stable Micro System, United
Kingdom). Two tests were performed: a penetration test (PT) and a deformation/compression test (DT). The
samples were cooled to 6◦ C before testing to ensure the same fruit surface temperature, as the texture analyser
is a very accurate instrument.
The PT was performed at a speed of 1 mm/s with a 3-mm Ø (P/3) cylindrical probe and a 3-mm penetration
in the berries’ equatorial zone. The DT was carried out by applying 2% compression to the sample with 50%
deformation using a 75-mm Ø flat probe (P/75) and a speed of 1.2 mm/s.
Firmness was measured as the maximum force recorded (N) in a force-time curve obtained during the penetra-
tion and deformation cycles. The PT and DT were carried out in the berries’ equatorial zone at room temperature
on 15 fruits per sample.

2.4. Titratable acidity (TA)

Total acidity was measured on 10 ml of clear blueberry juice and titrated automatically with 0.1 N NaOH to
a pH of 8.1 (Titralab AT1000-HACH, France). The analysis was performed in triplicate, and the results were
expressed as meqNaOH /l.

2.5. Total soluble solids (TSS)

The total soluble solid content (TSS) of clear blueberry juice was determined using a digital refractometer
(ATAGO-PR-32, Italy). The juice was obtained with a juice extractor and then centrifuged at 2,500 rpm for 10
minutes using the AVANTIM J-25 centrifuge (Beckamn Instruments Inc.) to obtain clear juice. The analysis was
performed using the supernatant in triplicate, and the results were expressed in ◦ Brix.

2.6. Total anthocyanin content (TAC)

The total anthocyanin content (TAC) analysis followed the pH differential protocol [17]. From the blueberry
samples, clear juice was extracted by adding 12.5 ml of MeOH (100%) to 5 g of fruit and homogenising the result
at 16,128 g for 1 min using an Ultra-Turrax T18 basic (Janke and Kunkel, IKA® -Labortechnik, Germany). The
juice (20 ␮l) was diluted separately with 2 ml each of pH 1 (potassium chloride 0.025 mol/L) and pH 4.5 (sodium
acetate 0.4 mol/l) buffer solution. The absorbance values of the solution were determined spectrophotometrically
at both ␭ 520 nm and ␭ 700 nm (U-5100, Hitachi, Japan).
The TAC was calculated using the following formula:
103
TAC = A × MW × DF × × L [17]
ε
TAC: total anthocyanin content in mg pelargonidin-3-glucosidine/l.
A: difference in absorbance ((A520 nm − A700 nm)pH1 − (A520 nm − A700 nm)pH4.5 ))
 A. Varaldo et al. / Influence of growing environment and pruning techniques 199

MW: molecular weight of pelargonidin (433.2 g/mol)

DF: dilution coefficient (10)
L: optical path in cm
E: extinction coefficient (48,340 l/mol*cm)
Three replicates per treatment were used.

2.7. Total phenol compounds (TPC) and antioxidant activity (AA)

For the tests, fruit extracts were prepared according to the protocol in Šavikin et al. [18]. Extraction was
performed by adding 12.5 ml of MeOH (100%) to 5 g of fruit and homogenising the result for 1 min with
an Ultra-Turrax T18 basic (Janke and Kunkel, IKA® -Labortechnik, G). The samples were then placed in an
ultrasonic bath (VWR Ultrasonic cleaner, Germany) containing water at 50◦ C for 20 minutes. The solutions
were centrifuged at 2.5 g for 10 minutes using the AVANTIM J-25 centrifuge (Beckamn Instruments Inc.). The
clear juice (supernatant) was collected and stored at - 26◦ C until analysis.
Total phenol compounds (TPCs) were determined with the Folin–Ciocalteu reagent [19], using gallic acid as a
standard. Absorption was measured at 760 nm. The results were expressed as mg gallic acid equivalents (GAE)
per 100 g of fresh berries. Three replicates of each treatment were performed.
Antioxidant activity (AA) was determined according to the ferric-reducing antioxidant power (FRAP) method
[20] modified for fruits [21]. It is based on the reduction of the Fe3+ -TPTZ (2,4,6-tripidyl-s-triazine) complex
to an Fe2+ (ferrous) iron form at a low pH.
The reduction of iron in the TPTZ-ferric chloride solution (FRAP reagent) results in the formation of a blue-
coloured product (ferrous tripyridyltriazine complex), the absorbance of which was read spectrophotometrically at
595 nm 4 min after the addition of berry extract to the FRAP reagent. The results were expressed as mmolFe2 + /kg
of fresh berries. Three replicates per treatment were used.

2.8. Statistical analysis

A statistical analysis was conducted using R Studio software version 4.1.2 (Integrated Development for R., R
Studio, PBC, Boston, MA, USA). Factor-designed analysis of variance (one-way ANOVA) was applied to the
quality data. The factors considered were pruning (hard/light), growing area (lowland/hill/mountain), storage
(0/10/21 days) and their interactions. Least significant differences at a significance level of 0.05 (p ≤ 0.05) were
used to compare means with Tukey’s test.

3. Results and discussion

3.1. Meteorological data

The meteorological data obtained from the Arpa Piemonte database provided the thermo-hygrometric values
recorded by the automatic weather stations. Based on the locations of the case study areas, three weather huts
were selected: one in Revello (Table 2) as a representative of the lowland area, one in Saluzzo (Table 3) for the
hilly area and one in Paesana Erasca (Table 4) to represent the mountain area.
Concerning rainfall (Fig. 1), it emerged that February and March were characterised by low rainfall, compared
to January and May, when precipitation was most abundant. In addition, it should be noted that the hilly area,
represented by the weather hut in Saluzzo, was characterised by less rainfall than the lowland and mountain
areas.
Regarding the temperature trend (Figs. 2, 3), it appeared that January and June recorded the lowest and
highest average maximum temperatures, respectively. On the contrary, the highest and lowest average minimum
200 A. Varaldo et al. / Influence of growing environment and pruning techniques

Table 2
Blueberry firmness (determined by DDT: digital durometer test, PT: penetration test and DT:
deformation test) as affected by storage period (0, 10 and 21 days of storage). Results:
means, standard deviation (SD) and Tukey’s post hoc test (same letters indicates no
statistical difference among column; p ≤ 0.05)

STORAGE FIRMNESS
DDT (Sh) PT (N) DT (N)
Day 0 52.0 ± 0.479c 2.21 ± 0.050a 16.13 ± 0.346b
Day 10 58.6 ± 0.494a 2.33 ± 0.043a 19.56 ± 0.455a
Day 21 53.8 ± 0.374b 2.19 ± 0.036a 18.92 ± 0.513a

Table 3
Blueberry firmness (determined by DDT: digital durometer test, PT: penetration test and DT:
deformation test) as affected by the growing area (lowland, hill and mountain). Results:
means, standard deviation (SD) and Tukey’s post hoc test (same letters indicates no
statistical difference among column; p ≤ 0.05)

AREAL FIRMNESS
DDT (Sh) PT (N) DT (N)
Lowland 56.8 ± 0.400a 2.51 ± 0.040a 19.02 ± 0.385a
Hill 53.5 ± 0.539b 2.13 ± 0.038b 18.65 ± 0.466a
Mountain 54.1 ± 0.506b 2.09 ± 0.040b 17.08 ± 0.523b

Table 4
Blueberry firmness (determined by DDT: digital durometer test, PT: penetration test and DT:
deformation test) as affected by pruning technique (hard and light). Results:
means, standard deviation (SD) and Tukey’s post hoc test (same letters indicates no
statistical difference among column; p ≤ 0.05)

PRUNING FIRMNESS
DDT (Sh) PT (N) DT (N)
Hard 54.6 ± 0.374a 2.25 ± 0.039a 18.23 ± 0.351a
Light 55.0 ± 0.434a 2.24 ± 0.033a 18.21 ± 0.413a

Fig. 1. Cumulative monthly rainfall (mm) of the case study areas; source: Arpa Piemonte database.
 A. Varaldo et al. / Influence of growing environment and pruning techniques 201

Fig. 2. Average maximum temperatures (◦ C) of the case study areas; source: Arpa Piemonte database.

Fig. 3. Average minimum temperatures (◦ C) of the case study areas; source: Arpa Piemonte database.

temperatures were attributed to June and February, respectively. The mountain environment, represented by the
weather hut in Paesana Erasca, reported the coldest climate compared to the plains and hills. However, it appeared
that the climatic trends of the average maximum temperatures in the mountain and hilly cultivation areas were
almost coincident (Fig. 2). A similar trend was also recorded in the average minimum temperature values among
the lowland and hill environments (Fig. 3).

3.2. Effect of cold storage and environmental factors on fruit softening

Firmness is one of the most important quality traits for soft fruits such as blueberries and is often considered a
shelf-life-limiting factor. For this reason, it was decided to evaluate three different parameters of berry consistency.
The DDT evaluates surface hardness thorough the measurement of the maximum force to the compression
distance. The PT is linked to the thickness of the fruit epidermis, and the DT measures the compactness of the
berry itself. These tests conducted on textural properties showed how storage and growing area significantly
affected the blueberries’ texture.
Table 2 displays the significant differences resulting from days of storage as indicated by the DDT and DT. The
samples were found to be firmer after 10 or 21 days of storage than at day 0. In several works, it has been observed
that the firmness of blueberries increases in the early stages of storage and then decreases with prolonged storage
[22, 23]. Such behavior is due to moisture loss, which is the major cause of textural changes during cold storage
[24], mainly initially, when moisture loss is greater than intercellular cohesion loss [25].
202 A. Varaldo et al. / Influence of growing environment and pruning techniques

The lowland cultivation areas provided more consistent fruits than the hill and the mountain (Table 3). The
DDT and PT revealed that the softest berries were those grown on hills and mountains, while the DT only
indicated the mountainous zone, with no significant differences between the lowland and hill areas. The DT
generally gives information on the compactness of the fruit itself. The differences in PT (which is a much more
accurate measurement than DDT and is closely linked to the thickness of the fruit epidermis) can be attributed
to differences in the skin structure, as previously observed [26]. These results, however, are not in agreement
with those of Rolle et al. [27], who found a trend of increasing grape skin thickness with increasing altitude.
The discrepancies could be due to the diversity of the fruits, which belong to different species despite all being
berries, and especially to the agronomic techniques used.
Turning to the pruning techniques, the statistical analysis demonstrated how the blueberries’ textural properties
were not influenced by the choice of either hard or light pruning. The ANOVA, in fact, did not reveal any significant
differences between the samples (Table 4).
Higher DDT values were found after 10 days of storage with both hard and light pruning (Fig. 4). By contrast,
the PT and DT identified the hardest berries at 10 days of storage with hard pruning only. These findings are in
agreement with those of other researchers; e.g. some studies [28] have found that a low canopy density, which
results from intense pruning, creates more consistent berries, while other studies [29] discovered that a strong-cut
mango tree provides firmer fruits. The least consistent blueberries were recorded in low-cut orchards and not
cold-stored (day 0). These results, therefore, indicate that a short period of conservation and hard pruning produce
more consistent fruits.
The interaction between storage period and growing area (Fig. 5) indicated that the firmest fruits (according
to the DDT, PT and DT) were those collected on the hill zone without cooling (day 0). The ANOVA revealed
the softest samples to be from day 10 in fruits grown in the lowland. The interpretation of the data in relation
to the PT and DT was more complex; however, one trend that could be observed is that the lowest data for the
penetration test were found for the lowland plot as early as after 10 days, while it was after 21 days in the hilly
and mountain zones. For the compression test, instead, the results highlighted how the lowest values occurred
after 10 days (lowland) and 21 days (mountain) of cold storage.
Considering the interaction between pruning technique and growing area (Fig. 6), the DT findings did not
show statistically significant differences. By contrast, the fruits defined by the best firmness (DDT) were those
collected on lowland-grown plants with both hard and light pruning systems. The softest berries were found in
hill areas with both hard and light pruning and in mountain-cultivated berries grown with a hard pruning of the
bush. The results of the PT showed strong consistency in berries grown in the lowland with hard pruning and in
the hilly environment with light pruning.

Fig. 4. Blueberry firmness (determined by DDT: digital durometer test, PT: penetration test and DT: deformation test) as affected by storage
period (0, 10, and 21 days of storage) and pruning technique (hard and light). Results: means, standard deviation (SD) and Tukey’s post hoc
test (same letters indicates no statistical difference among column; p ≤ 0.05).
 A. Varaldo et al. / Influence of growing environment and pruning techniques 203

Fig. 5. Blueberry firmness (determined by DDT: digital durometer test, PT: penetration test and DT: deformation test) as affected by storage
period (0, 10 and 21 days of storage) and growing area (L: lowland, H: hill and M: mountain). Results: means, standard deviation (SD) and
Tukey’s post hoc test (same letters indicates no statistical difference among column; p ≤ 0.05).

Fig. 6. Blueberry firmness (determined by DDT: digital durometer test, PT: penetration test and DT: deformation test) as affected by pruning
techniques (hard and light) and growing area (L: lowland, H: hill and M: mountain). Results: means, standard deviation (SD) and Tukey’s
post hoc test (same letters indicates no statistical difference among column; p ≤ 0.05).

3.3. Effect of cold storage and environmental factors on chemical properties

The chemical traits assayed suggested that the storage period does not affect TA (Table 5) and the pruning
techniques do not influence the TSS (Table 6). On the other hand, TSS showed an increasing trend from day 0 to
day 21. This pattern may be related to weight loss. As the fruit loses moisture, the sugar concentration increases.
Since blueberry fruit does not have starch to support soluble sugar synthesis after harvest, the small increase in
TSS may be a consequence of cell wall degradation [30]. Furthermore, light pruning provides a lower blueberry
acidity than hard cutting.
Among the chemical traits evaluated, ANOVA indicated how the growing area is one of the most influential
factors for TA and TSS (Table 7). Specifically, the blueberries with the lowest acidity and highest sweetness were
those grown in lowland areas. While hill and mountain cultivation areas provided more acidic and less sugary
fruits than the plains area, this charateristic is therefore the most appropriate in terms of chemical properties.
These results are in agreement with the rainfall recorded in 2021. The hill, in fact, turned out to be the least rainy
area, a phenomenon that could be responsible for increased sugar concentration in the blueberries.
The storage area factor interaction (Fig. 7) showed significant TSS differences. These results demonstrate that
lowland-cultivated blueberries can maintain high TSS during cold storage. The results also revealed how TA is
204 A. Varaldo et al. / Influence of growing environment and pruning techniques

Table 5
Blueberry titratable acidity (TA) and total soluble solids (TSS) as affected by storage
period (0, 10 and 21 days of storage). Results: means, standard deviation (SD) and
Tukey’s post hoc test (same letters indicates no statistical difference
among column; p ≤ 0.05)

STORAGE TA TSS
(meq/l) Brix
Day 0 93.5 ± 6.603a 9.00 ± 0.070b
Day 10 88.1 ± 4.694a 9.16 ± 0.108ab
Day 21 89.7 ± 4.719a 9.32 ± 0.089a

Table 6
Blueberry titratable acidity (TA) and total soluble solids (TSS) as affected by pruning
technique (hard and light). Results: means, standard deviation (SD) and Tukey’s
post hoc test (same letters indicates no statistical difference
among column; p ≤ 0.05)

PRUNING TA TSS
(meq/l) Brix
Hard 97.1 ± 5.116a 9.09 ± 0.073a
Light 83.7 ± 3.010b 9.23 ± 0.079a

Table 7
Blueberry titratable acidity (TA) and total soluble solids (TSS) as affected by growing
area (lowland, hill and mountain). Results: means, standard deviation (SD) and
Tukey’s post hoc test (same letters indicates no statistical difference
among column; p ≤ 0.05)

AREAL TA TSS
(meq/l) ◦ Brix

Lowland 77.9 ± 6.013b 9.49 ± 0.084a

Hill 100.8 ± 4.254a 9.03 ± 0.082b
Mountain 84.8 ± 4.910ab 9.12 ± 0.081b

not impacted by the storage area factor interaction. Accordingly, the pruning area interaction (Fig. 8) suggested
that the sweetest and least sour fruits came from plains-grown orchards managed with light pruning. Furthermore,
it is interesting to observe how the samples were able to preserve TA and TSS during storage with both hard and
light pruning (Fig. 9).

3.4. Effect of cold storage and environmental factors on bioactive compounds

The TAC and AA suffered significant losses during storage, while the TPC remained unchanged until the
21st day of storing (Table 8). During the 21 days of storage, TAC and AA decreased significantly (p < 0.05).
This TAC reduction may be related to greater use as a respiratory substrate of anthocyanins compared to the
non-anthocyanin component of polyphenols [31]. These values are in accordance with other studies that reported
 A. Varaldo et al. / Influence of growing environment and pruning techniques 205

Fig. 7. Blueberry titratable acidity (TA) and total soluble solids (TSS) as affected by storage period (0, 10 and 21 days of storage) and growing
area (L: lowland, H: hill and M: mountain). Results: means, standard deviation (SD) and Tukey’s post hoc test (same letters indicates no
statistical difference among column; p ≤ 0.05).

Fig. 8. Blueberry titratable acidity (TA) and total soluble solids (TSS) as affected by pruning techniques (hard and light) and growing area
(L: lowland, H: hill and M: mountain). Results: means, standard deviation (SD) and Tukey’s post hoc test (same letters indicates no statistical
difference among column; p ≤ 0.05).

Fig. 9. Blueberry titratable acidity (TA) and total soluble solids (TSS) as affected by storage period (0, 10 and 21 days of storage) and
pruning techniques (hard and light). Results: means, standard deviation (SD) and Tukey’s post hoc test (same letters indicates no statistical
difference among column; p ≤ 0.05).

an enzymatic degradation of anthocyanin during storage in the presence of oxygen [32]. This is compatible with
the current study since the fruits were stored was in cold cells without atmospheric control. Thus, in the presence
of high O2 concentrations, there is greater degradation of anthocyanin fraction and total antioxidant capacity
than of total polyphenols.
The pruning methods did not affect the nutraceutical properties. The synthesis of these compounds is connected
to several factors, e.g. soil, agronomic practices and climatic trends [33, 34]; in this situation, based on the results
206 A. Varaldo et al. / Influence of growing environment and pruning techniques

Table 8
Blueberry total anthocyanin content (TAC), total phenol content (TPC) and antioxidant
activity (AA) as affected by storage period (0, 10 and 21 days of storage).
Results: means, standard deviation (SD) and Tukey’s post hoc test
(same letters indicates no statistical difference among
column; p ≤ 0.05)

STORAGE TAC TPC AA

mg/ml mg(GAE) /100 g mmol/kg
Day 0 21.4 ± 0.334a 175.6 ± 6.273a 30.1 ± 0.320a
Day 10 13.1 ± 1.579b 197.4 ± 8.232a 29.3 ± 0.480ab
Day 21 11.0 ± 0.510b 194.0 ± 5.548a 28.5 ± 0.451b

Table 9
Blueberry total anthocyanin content (TAC), total phenol content (TPC) and antioxidant
activity (AA) as affected by pruning technique (hard and light). Results: means,
standard deviation (SD) and Tukey’s post hoc test (same letters indicates
no statistical difference among column; p ≤ 0.05)

PRUNING TAC TPC AA

mg/ml mg(GAE) /100 g mmol/kg
Hard 14.6 ± 1.431a 188.3 ± 5.435a 29.2 ± 0.341a
Light 15.8 ± 1.180a 190.7 ± 5.675a 29.4 ± 0.381a

Table 10
Blueberry total anthocyanin content (TAC), total phenol content (TPC) and antioxidant
activity (AA) as affected by growing area (lowland, hill and mountain). Results:
means, standard deviation (SD) and Tukey’s post hoc test (same letters
indicates no statistical difference among column; p ≤ 0.05)

AREAL TAC TPC AA

mg/ml mg(GAE) /100 g mmol/kg
Lowland 18.4 ± 1.67a 197.7 ± 8.46a 29.1 ± 0.547a
Hill 15.6 ± 1.18ab 193.4 ± 5.98a 29.5 ± 0.387a
Mountain 12.5 ± 1.36b 178.1 ± 6.91a 29.2 ± 0.447a

in Table 9, it is clear that the pruning method alone is not a source of variation in blueberries’ nutraceutical
profiles.
As for the cultivation region, the results highlighted that the blueberries’ best performance was in the lowland
area, where the samples showed the statistically highest TAC (Table 10). Similar studies on blueberries [35] and
other fruits, such as strawberries and pomegranates [36], found a lower amount of anthocyanins in fruits collected
at higher-altitude regions. These studies indicated a clear negative correlation between altitude and anthocyanin
synthesis, as was also indicated by the present research [37]. Otherwise, the cultivation region did not affect TPC
and AA, which were constant regardless of the growing area.
Significant P-values were estimated for interactions between storage and environmental conditions. The two-
way ANOVA factors of storage and pruning were significant for TAC and PC (Fig. 10). The TAC was considerable
 A. Varaldo et al. / Influence of growing environment and pruning techniques 207

Fig. 10. Blueberry total anthocyanin content (TAC), total phenol content (TPC) as affected by storage period (0, 10 and 21 days of storage)
and pruning techniques (hard and light). Results: means, standard deviation (SD) and Tukey’s post hoc test (same letters indicates no statistical
difference among column; p ≤ 0.05).

Fig. 11. Blueberry total anthocyanin content (TAC), total phenol content (TPC) as affected by storage period (0, 10 and 21 days of storage)
and growing area (L: lowland, H: hill and M: mountain). Results: means, standard deviation (SD) and Tukey’s post hoc test (same letters
indicates no statistical difference among column; p ≤ 0.05).

Fig. 12. Blueberry total anthocyanin content (TAC), total phenol content (TPC), as affected by pruning techniques (hard and light) and
growing area (L: lowland, H: hill and M: mountain). Results: means, standard deviation (SD) and Tukey’s post hoc test (same letters
indicates no statistical difference among column; p ≤ 0.05).

in non-stored (day 0) blueberries pruned with both techniques, but the highest TPC was linked to light-pruned
fruits cold-stored for 10 days. There were no statistically significant differences in AA between treatments (data
not reported).
208 A. Varaldo et al. / Influence of growing environment and pruning techniques

The interaction between storage and growing area indicated the highest TAC quantity at day 0, without
significant differences among the growing areas (Fig. 11). Blueberries with a higher TPC were associable with
hill-grown fruits stored for 10 days and lowland-grown fruits stored for 21 days. As in the previous case, AA
showed no statistically significant differences between treatments (data not reported).
The influence of the two factors – pruning and area – did not yield statistically significant differences (Fig. 12).
Accordingly, no strong positive influence can be attributed to a specific growing region or pruning technique in
terms of obtaining better nutraceutical properties (AA data not reported).
The constant values of AA were in accordance with the TPC, which remained unchanged in the one-way
ANOVA results for the three factors considered in the present study (Tables 8, 9 and 10).

4. Conclusions

The present study was designed to identify the best pruning method and cultivation area for V. corymbosum
production to preserve the best quality characteristics during post-harvest storage. As a result of the data pro-
cessing, it was possible to determine that a lowland cultivation area and light pruning technique allows a longer
blueberry shelf-life. These results are interesting since other studies on consumer preferences have outlined how
sweet, intense blueberry flavours yield the most positive purchase interest, whereas poor texture attributes are
the most detrimental to interest [38].
In terms of the fruits’ consistency, it was found that lowland areas produce firmer fruits that retain their
characteristics after 10 days of storage, especially if harvested from plants cultivated with hard pruning.
As for the blueberries’ chemical properties, it emerged that, at the harvest, the plains area produced more sugary
and less acidic fruits and that, because of cold storage, they better preserved these characteristics compared to
fruits grown in other environments. Moreover, by associating the lowland cultivation area with light pruning, we
could observe improved performance from the point of view of the maintenance of chemical properties.
Bioactive compounds, on the other hand, are not affected by the pruning method. However, if combined with 10
days of storage, it was found that light pruning better preserves TPC. Finally, the lowland area yields blueberries
(at day 0 of storage) that are richer in TAC and retain their nutraceutical properties better than those in hilly and
mountainous growing areas, even after 21 days of storage.
In conclusion, although the research identified the environmental characteristics that most positively affect
blueberry quality traits, future studies should focus on the main agricultural techniques involved, as well as
other cultivars. This could help overcome the limits of this research, which did use surveys or focus on other
agronomic practices, such as irrigation and fertilisation. It should also be stressed that conducting the research
over a single year did not allow definitive answers to be provided as to what the best pruning method is in relation
to the cultivation area. It would therefore be useful to continue this research in subsequent years by enlarging
the investigation to a wider number of cultivars, as each cultivar could exhibit different behaviours with respect
to the area in which it is grown.

Author contributions

G.G. conceived and designed the research; A.V. and V.C. performed the experiments. A.V. wrote the paper
and performed data analysis. G.G. reviewed and edited the paper. All the authors drafted and approved the final
manuscript.

Funding

The authors report no funding.

 A. Varaldo et al. / Influence of growing environment and pruning techniques 209

Conflict of interest

The authors have no conflict of interest to report.

References

[1] Romo-Muñoz R, Dote-Pardo J, Garrido-Henrı́quez H, Araneda-Flores J, Gil JM. Blueberry consumption and healthy lifestyles in an
emerging market. Span J Agric Res. 2020;17:e0111. https://doi.org/10.5424/sjar/2019174-14195.
[2] Jaglan P, Buttar HS, Al-bawareed OA, Chibisov S. Potential health benefits of selected fruits: apples, blueberries, grapes,
guavas, mangos, pomegranates, and tomatoes. Funct. Foods Nutraceuticals Metab. Non-Commun. Dis., Elsevier; 2022, p. 359-70.
https://doi.org/10.1016/B978-0-12-819815-5.00026-4.
[3] Norberto S, Silva S, Meireles M, Faria A, Pintado M, Calhau C. Blueberry anthocyanins in health promotion: A metabolic overview.
J Funct Foods. 2013;5:1518-28. https://doi.org/10.1016/j.jff.2013.08.015.
[4] Kuang L, Wang Z, Zhang J, Li H, Xu G, Li J. Factor analysis and cluster analysis of mineral elements contents in different blueberry
cultivars. J Food Compos Anal. 2022;109:104507. https://doi.org/10.1016/j.jfca.2022.104507.
[5] Hera O, Teodorescu R, Sturzeanu M. BLUEBERRY(VACCINIUM CORYMBOSUM) BREEDING PROGRAMME IN THE MAIN
CULTIVATING COUNTRIES 2021.
[6] Mazzoni L, Balducci F, Di Vittori L, Scalzo J, Capocasa F, Zhong C-F, et al. Yield and nutritional quality of highbush blueberry
genotypes trialled in a Mediterranean hot summer climate. J Sci Food Agric. 2020;100:3675-86. https://doi.org/10.1002/jsfa.10403.
[7] Bauer N, Visuals N. World Blueberry Acreage & Production 2008.
[8] Albert T, Kadri K, Marge S, Paàl T. The effect of mulching and pruning on the vegetative growth and yield of the half-high blueberry.
Agron Res. 2010;8:759-68.
[9] Jorquera-Fontena E, Alberdi M, Franck N. Pruning severity affects yield, fruit load and fruit and leaf traits of “Brigitta” blueberry. J
Soil Sci Plant Nutr. 2014:0-0. https://doi.org/10.4067/S0718-95162014005000068.
[10] Moura GCD, Vizzotto M, Picolotto L, Antunes LEC. PRODUCTION, PHYSICAL-CHEMICAL QUALITY AND BIOATIVE
COMPOUNDS OF MISTY BLUEBERRY FRUIT UNDER DIFFERENT PRUNING INTENSITIES. Rev Bras Frutic. 2017;39.
https://doi.org/10.1590/0100-29452017158.
[11] Douglas AP, Jeffrey GW. Pruning southern highbush blueberry in Florida. Hortic Sci Dep UFIFAS Ext. 2020.
[12] Lee SG, Cho JG, Shin MH, Oh SB, Kim HL, Kim JG. Effects of summer pruning combined with winter pruning on bush growth,
yields, and fruit quality of ‘Misty’ southern highbush blueberry for two years after planting. Hortic Environ Biotechnol. 2015;56:740-8.
https://doi.org/10.1007/s13580-015-0101-6.
[13] Strik B, Buller G, Hellman E. Pruning Severity Affects Yield, Berry Weight, and Hand Harvest Efficiency of Highbush Blueberry.
HortScience. 2003;38:196-9. https://doi.org/10.21273/HORTSCI.38.2.196.
[14] Takeda F, Krewer G, Andrews EL, Mullinix B, Peterson DL. Assessment of the V45 Blueberry Harvester on
Rabbiteye Blueberry and Southern Highbush Blueberry Pruned to V-Shaped Canopy. HortTechnology. 2008;18:130-8.
https://doi.org/10.21273/HORTTECH.18.1.130.
[15] Gallardo RK, Stafne ET, DeVetter LW, Zhang Q, Li C, Takeda F, et al. Blueberry Producers’ Attitudes toward Harvest Mechanization
for Fresh Market. HortTechnology. 2018;28:10-6. https://doi.org/10.21273/HORTTECH03872-17.
[16] Geoportale Arpa Piemonte n.d. https://geoportale.arpa.piemonte.it/app/public/ (accessed November 16, 2022).
[17] Roidoung S, Dolan KD, Siddiq M. Estimation of kinetic parameters of anthocyanins and color degradation in vitamin C fortified
cranberry juice during storage. Food Res Int. 2017;94:29-35. https://doi.org/10.1016/j.foodres.2017.01.013.
[18] Šavikin K, Zdunić G, Janković T, Tasić S, Menković N, Stević T, et al. Phenolic Content and Radical Scavenging Capacity of Berries
and Related Jams from Certificated Area in Serbia. Plant Foods Hum Nutr. 2009;64:212-7. https://doi.org/10.1007/s11130-009-0123-2.
[19] Slinkard K, Singleton VL. Total Phenol Analysis: Automation and Comparison with Manual Methods. Am J Enol Vitic. 1977:49-55.
[20] Benzie IFF, Strain JJ. The Ferric Reducing Ability of Plasma (FRAP) as a Measure of “Antioxidant Power”: The FRAP Assay. Anal
Biochem 1996;239:70-6. https://doi.org/10.1006/abio.1996.0292.
[21] Pellegrini N, Serafini M, Colombi B, Del Rio D, Salvatore S, Bianchi M, et al. Total Antioxidant Capacity of Plant Foods, Beverages and
Oils Consumed in Italy Assessed by Three Different In Vitro Assays. J Nutr. 2003;133:2812-9. https://doi.org/10.1093/jn/133.9.2812.
[22] Chen Y, Hung Y-C, Chen M, Lin H. Effects of acidic electrolyzed oxidizing water on retarding cell wall degradation and delaying
softening of blueberries during postharvest storage. LWT. 2017;84:650-7. https://doi.org/10.1016/j.lwt.2017.06.011.
[23] Chen H, Cao S, Fang X, Mu H, Yang H, Wang X, et al. Changes in fruit firmness, cell wall composition and cell wall degrading
enzymes in postharvest blueberries during storage. Sci Hortic. 2015;188:44-8. https://doi.org/10.1016/j.scienta.2015.03.018.
210 A. Varaldo et al. / Influence of growing environment and pruning techniques

[24] Paniagua AC, East AR, Hindmarsh JP, Heyes JA. Moisture loss is the major cause of firmness change during postharvest storage of
blueberry. Postharvest Biol Technol. 2013;79:13-9. https://doi.org/10.1016/j.postharvbio.2012.12.016.
[25] Li M, Rivera S, Franklin D, Nowak E, Hallett I, Kolenderska S, et al. Use of optical coherence tomography and light microscopy
for characterisation of mechanical properties and cellular level responses of ‘Centurion’ blueberries during weight loss. J Food Eng.
2021;303:110596. https://doi.org/10.1016/j.jfoodeng.2021.110596.
[26] Blaker KM, Plotto A, Baldwin EA, Olmstead JW. Correlation between sensory and instrumental measurements of standard and
crisp-texture southern highbush blueberries (Vaccinium corymbosum L. interspecific hybrids). J Sci Food Agric. 2014;94:2785-93.
https://doi.org/10.1002/jsfa.6626.
[27] Rolle L, Letaief H, Cagnasso E, Zeppa G, Gerbi V. STUDY OF MECHANICAL PROPERTIES OF “NEBBIOLO” GRAPES GROWN
IN DIFFERENT ENVIRONMENTS. Quad Vitic Ed Enol UNIV. 2006.
[28] Schöneberg T, Arsenault-Benoit A, Taylor CM, Butler BR, Dalton DT, Walton VM, et al. Pruning of small fruit crops can affect
habitat suitability for Drosophila suzukii. Agric Ecosyst Environ. 2020;294:106860. https://doi.org/10.1016/j.agee.2020.106860.
[29] Asrey R, Patel VB, Barman K, Pal RK. Pruning affects fruit yield and postharvest quality in mango (Mangifera indica L.) cv. Amrapali.
Fruits. 2013;68:367-80. https://doi.org/10.1051/fruits/2013082.
[30] Liu B, Wang K, Shu X, Liang J, Fan X, Sun L. Changes in fruit firmness, quality traits and cell wall constituents
of two highbush blueberries (Vaccinium corymbosum L.) during postharvest cold storage. Sci Hortic. 2019;246:557-62.
https://doi.org/10.1016/j.scienta.2018.11.042.
[31] Gergoff Grozeff GE, Alegre ML, Senn ME, Chaves AR, Simontacchi M, Bartoli CG. Combination of nitric oxide
and 1-MCP on postharvest life of the blueberry (Vaccinium spp.) fruit. Postharvest Biol Technol. 2017;133:72-80.
https://doi.org/10.1016/j.postharvbio.2017.06.012.
[32] Reque PM, Steffens RS, Jablonski A, Flôres SH, Rios A de O, de Jong EV. Cold storage of blueberry (Vaccinium spp.) fruits and juice:
Anthocyanin stability and antioxidant activity. J Food Compos Anal. 2014;33:111-6. https://doi.org/10.1016/j.jfca.2013.11.007.
[33] Lòpez MD, Illanes M, Jara P, Figueroa I, Fischer S, Wilckens R, et al. Phenolic compounds, pectin and antioxidant activity in
blueberries (Vaccinium corymbosum L.) infl uenced by boron and mulch cover. Bioagro. 2020;32:169-78.
[34] Yang B, Zheng J, Laaksonen O, Tahvonen R, Kallio H. Effects of Latitude and Weather Conditions on Phenolic Compounds in Currant
(Ribes spp.) Cultivars. J Agric Food Chem. 2013;61:3517-32. https://doi.org/10.1021/jf4000456.
[35] Mphahlele RR, Stander MA, Fawole OA, Opara UL. Effect of fruit maturity and growing location on the postharvest contents of
flavonoids, phenolic acids, vitamin C and antioxidant activity of pomegranate juice (cv. Wonderful). Sci Hortic. 2014;179:36-45.
https://doi.org/10.1016/j.scienta.2014.09.007.
[36] Rieger G, Müller M, Guttenberger H, Bucar F. Influence of Altitudinal Variation on the Content of Phenolic Compounds
in Wild Populations of Calluna vulgaris, Sambucus nigra, and Vaccinium myrtillus. J Agric Food Chem. 2008;56:9080-6.
https://doi.org/10.1021/jf801104e.
[37] Guerrero-Chavez G, Scampicchio M, Andreotti C. Influence of the site altitude on strawberry phenolic composition and quality. Sci
Hortic. 2015;192:21-8. https://doi.org/10.1016/j.scienta.2015.05.017.
[38] Gilbert JL, Olmstead JW, Colquhoun TA, Levin LA, Clark DG, Moskowitz HR. Consumer-assisted Selection of Blueberry Fruit
Quality Traits. HortScience. 2014;49:864-73. https://doi.org/10.21273/HORTSCI.49.7.864.
Copyright of Journal of Berry Research is the property of IOS Press and its content may not
be copied or emailed to multiple sites or posted to a listserv without the copyright holder's
express written permission. However, users may print, download, or email articles for
individual use.