horticulturae

Article
Controlled Atmosphere Storage Alleviates Hass Avocado Black
Spot Disorder
Claudia Fuentealba 1 , Juan Vidal 2 , Claudio Zulueta 3 , Excequel Ponce 2 , Virgilio Uarrota 2 ,
Bruno G. Defilippi 4 and Romina Pedreschi 2, *

1 Facultad de Ciencias Agronómicas y de los Alimentos, Escuela de Alimentos, Pontificia Universidad Católica
de Valparaíso, Waddington 716, Playa Ancha, Valparaíso 2360100, Chile; claudia.fuentealba@pucv.cl
2 Facultad de Ciencias Agronómicas y de los Alimentos, Escuela de Agronomía, Pontificia Universidad Católica
de Valparaíso, Calle San Francisco s/n, La Palma, Quillota 2260000, Chile; juan.vidal@pucv.cl (J.V.);
excequel.p.g@gmail.com (E.P.); virgiliouarrota@gmail.com (V.U.)
3 Sociedad Gardiazabal y Mena Ltda, Calle Blanco 512, Quillota 2260890, Chile; czulueta@gama.cl
4 Unidad de Poscosecha, Instituto de Investigaciones Agropecuarias, INIA-La Platina, Santiago 8831314, Chile;
bdefilip@inia.cl
* Correspondence: romina.pedreschi@pucv.cl; Tel.: +56-32-237-2912

Abstract: As it was previously reported, black spot development in the skin of Hass avocado has
been related to a decreased antioxidant defense system. The aim of this study was to investigate the
effect of different postharvest storage conditions on controlling black spot development targeting
their effect on the antioxidant system (non-enzymatic and enzymatic) of the skin. Four postharvest
treatments (T1: regular air storage (RA) at 5 ◦ C for 40 d; T2: controlled atmosphere storage (CA) of
4 kPa O2 and 6 kPa CO2 at 5 ◦ C for 40 d; T3: 10 d RA + 30 d CA and T4: 5 µM methyl jasmonate



(MeJA) for 30 s + 10 RA + 30 d CA) were tested on controlling black spot incidence in fruit from six


orchards from different agroclimatic zones and harvests. Then, on two selected orchards and harvests,
Citation: Fuentealba, C.; Vidal, J.;
the evolution of total phenolics (TPC), antioxidant capacity (AC) and antioxidant enzymes (catalase
Zulueta, C.; Ponce, E.; Uarrota, V.;
(CAT), polyphenol oxidase (PPO), superoxide dismutase (SOD), peroxidase (POD), phenylalanine
Defilippi, B.G.; Pedreschi, R.
Controlled Atmosphere Storage
ammonia lyase (PAL)) was monitored. Results revealed that incidence of black spot disorder was not
Alleviates Hass Avocado Black Spot associated to an agroclimatic zone and harvest stage. Immediate application of CA (T2) controlled
Disorder. Horticulturae 2022, 8, 369. black spot development during prolonged storage (40 d) and under these conditions TPC content
https://doi.org/10.3390/ remained higher compared to the other treatments. No clear role of CAT, PPO, SOD, POD and PAL
horticulturae8050369 on controlling black spot was observed. The results obtained are of value for the Hass avocado supply
Academic Editors: Isabel Lara and
chain since a clear performance of CA was evidenced that will result in reduction of postharvest
Brian Farneti losses associated to this problem.

Received: 27 March 2022 Keywords: epicarp; phenolics; antioxidant activity; quality attributes
Accepted: 16 April 2022
Published: 22 April 2022

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in 1. Introduction
published maps and institutional affil- The avocado mesocarp is recognized by its highly nutritional oil which is rich in
iations. monounsaturated fatty acids such as oleic acid. In addition, its composition includes
vitamin E, proteins, bioactive compounds and 10% of unsaponifiable compounds such as
sterols and volatile acids [1]. The main phenolic compounds found in the epicarp and seed
are epicatechin derivatives and flavonoids such as quercetin derivatives [2].
Copyright: © 2022 by the authors.
Originally from Central America, the cultivation of avocado (Persea americana Mill)
Licensee MDPI, Basel, Switzerland.
This article is an open access article
has spread to various regions of the world, mainly towards tropical, subtropical and
distributed under the terms and
Mediterranean climates. Mexico is the main producer of avocados in the world, contributing
conditions of the Creative Commons 34%, while Chile produces 2.5%. The predominant variety in Chile is Hass, which represents
Attribution (CC BY) license (https:// 90% of the national production concentrated between the Norte Chico and the Central
creativecommons.org/licenses/by/ Zone of the country [3,4].
4.0/).

Horticulturae 2022, 8, 369. https://doi.org/10.3390/horticulturae8050369 https://www.mdpi.com/journal/horticulturae

Horticulturae 2022, 8, 369 2 of 12

In recent years, the exportation of avocados has been challenged because a defect has
been found in the fruit epicarp known as a black spot, manifested as a dark circular spot on
the skin during storage, and that mainly affects the perception of the quality of the fruit.
This disorder is confused with anthracnose, which is a disease caused by a fungus that
develops dark and irregular spots on the epicarp but even compromises the mesocarp of
the fruit. Moreover, other disorders mimic black spot symptoms such as chilling injury
during storage and lenticel breakdown. Recent research distinguishes black spot from
anthracnose, indicating that the possible factors associated with the incidence of black spots
on the epicarp of avocados are produced by cold storage for more than 10 days; in addition,
to a drastic decrease in the enzymatic defense system of the epicarp [5]. Pre-harvest factors
such as maximum temperatures and relative humidity during growth and development
have also been reported to be negatively associated with black spot development [5].
Similarly, Lindh et al. [6] reported that lenticel damage does not result in black spots, and
the harvest system did not correlate to the incidence of black spots. Controlled atmosphere
(CA) has also been studied showing a significant reduction in black spot incidence, even
though this disorder is orchard dependent [5,7].
In the need to meet fruit quality standards for exports, growers have taken measures to
prevent anthracnose with the application of fungicides both at preharvest and postharvest
stages. Some recent studies indicate that the application of methyl jasmonate (MeJA)
reduces cold damage during avocado storage and prevents anthracnose by altering its fatty
acid content and compounds related to stress responses [8–10]. However, no application
of MeJA has been studied to control black spot. Based on the previous antecedents that
indicate that black spot is correlated with a drastic decrease in the defense system of the
epicarp during the first 10 days, substances of the GRAS (generally recognized as safe) type
such as MeJA and others could help to control the appearance of this physiological disorder.
Regarding this, the central objectives of this research were to evaluate the effect of different
postharvest treatments on: (i) the black spot incidence in orchards of three agroclimatic
zones and two harvests; and (ii) the enzymatic and non-enzymatic defense system of the
epicarp and its evolution during storage.

2. Materials and Methods

2.1. Selection of Orchards and Plant Material
Five thousand avocado cv. Hass fruits were harvested in the 2019/2020 season from six
orchards located in three agroclimatic zones of the Region of Valparaíso. Two maturity stages
were considered: early harvest (23–27% dry matter) and middle harvest (>27–30% dry matter).
The orchards from the coastal zone (orchards A and B) were located at a distance of less than
10 km from the sea and between 100–250 m above sea level. Those in the intermediate zone
(orchards C and D) were located at a distance between 20 and 45 km from the sea and between
300–400 m above sea level, and those in the interior zone (orchards E and F) were located at a
distance greater than 45 km from the sea and between 300–900 m above the sea level.

2.2. Postharvest Treatments

Four hundred fruits were selected from each orchard per harvest and were split into
four treatments: (T1) 40 d of storage in regular air (21 kPa O2 and 0.04 kPa CO2 ) at 5 ◦ C,
(T2) 40 d of storage in a controlled atmosphere of 4 kPa O2 and 6 kPa CO2 at 5 ◦ C, (T3) 10 d
of storage in regular air at 5 ◦ C plus 30 d in a controlled atmosphere at 5 ◦ C and 4 kPa O2
and 6 kPa CO2 , (T4) application of 5 µM MeJA for 30 s, fruit drying at room temperature
and application of 10 d of storage in regular air at 5 ◦ C followed by 30 d in a controlled
atmosphere at 5 ◦ C and 4 kPa O2 and 6 kPa CO2 .
In addition, five fruits were sampled per treatment from the orchards that presented
the highest incidence of black spot (B and E), at times 0, 3, 6, 10 and 40 d, respectively. The
epicarp or skin of each fruit was removed and immediately frozen and stored at −80 ◦ C
for further analyses. The remaining fruits were subjected to a shelf-life period (20 ◦ C and
65–60% RH) until the fruit reached the ready-to-eat stage (RTE, 4–14 N firmness).
Horticulturae 2022, 8, 369 3 of 12

2.3. Quality Parameters

The incidence of black spot was determined by visual inspection of the epicarp after
prolonged postharvest storage under the different treatments and furthermore at the RTE
stage, where 0 = without incidence and 1 = with incidence. The results were expressed as
a percentage of the fruits per treatment [5].
Fruit weight was measured individually at harvest, after postharvest treatments, and
at the RTE stage. The values were then used to calculate the percentage of weight loss.
Dry matter was determined in the epicarp and mesocarp at harvest using 20 fruits per
orchard. The epicarp was separated from the mesocarp and the seed was discarded, they
were chopped and placed separately in an oven at 100 ◦ C for 24 h. The dry matter content
was expressed as a percentage (g of dry matter per 100 g of epicarp or mesocarp).
Non-destructive firmness was evaluated at harvest and after postharvest treatments
in five fruits for each treatment as described by Ochoa-Ascencio et al. [11] with minor
modifications. A texture analyzer (Model TA.XT plusC, Stable Micro Systems Ltd., Surrey,
United Kingdom equipped with a 10 mm Ø cylindrical probe, with an initial force of 0.5 N
and a speed of 8 mm s−1 was used. The compressive force was recorded in Newton (N) at
2 mm deformation.

2.4. Analysis of Epicarp Non-Enzymatic Defense System

Total Phenolic Compounds and Antioxidant Capacity
For the extraction of total phenolic compounds and antioxidant capacity, the method
described by Saavedra et al. [12] was used. Briefly, 50 mg of lyophilized epicarp were
homogenized with 2 mL of acetone: water (70:30 v/v) and mixed in a shaker (VorTemp 56,
Labnet International, Inc., New York, NY, USA) at 1300 rpm and 3 mm shaker orbit for 1 h.
Then, it was centrifuged at 10,000× g and 4 ◦ C for 20 min (Labnet International, Inc., New
York, NY, USA). Two hundred µL of the supernatant was evaporated with nitrogen gas
and the remaining pellet was resuspended with 400 µL of methanol and stored (−20 ◦ C)
for subsequent analysis of total phenolics and antioxidant capacity.
Total phenolic compounds were quantified by mixing 240 µL of water (HPLC grade),
20 µL of 1 N Folin-Ciocalteu reagent, 20 µL of 5% (w/v) sodium carbonate, and 20 µL of
the extract in microplates. Samples were incubated for 2 h in darkness at room temperature
and the absorbance was measured at 765 nm in a UV-Vis microplate spectrophotometer
(Multiscan GO type, Thermo Fisher Scientific, Vantaa, Findland). Total phenolic compounds
were calculated using gallic acid as a standard curve (50–200 µg mL−1 , y = 0.0031x − 0.012,
r2 = 0.995) and the results were expressed as µg gallic acid equivalent (GAE) per mg of dry
weight (DW) sample.
The antioxidant capacity was obtained by mixing 20 µL of extract and 125 µL of
60 µM of 2,2-diphenyl-1-picrylhydrazyl (DPPH). The mixture was incubated for 30 min
in darkness and the absorbance was measured at 517 nm. The antioxidant capacity was
calculated using a trolox as a standard curve (20–140 µM mL−1 , y = 0.41x − 1.107, r2 = 0.998)
and expressed as µM of trolox equivalent (TE) per mg of sample (DW).

2.5. Analysis of Epicarp Enzymatic Defense System

2.5.1. Enzyme Extraction
The extraction and enzymatic activity methods were performed as described by Uar-
rota et al. [5] with minor modifications. One hundred mg powdered samples were mixed
with 500 µL of extraction buffer pH 7.0 (potassium phosphate buffer (50 mM, pH 7.0, with
25 µM ethylenediaminetetraacetic acid (EDTA) and 0.025% Triton X-100) and 20 µL of 125
mM of phenylmethylsulfonyl fluoride (PMSF). The sample was vortexed and centrifuged
at 10,000× g for 10 min at 4 ◦ C (Z216 MK refrigerated microcentrifuge, Ø = 11 mm). The
supernatant was kept at −20 ◦ C.
The content of total soluble proteins was determined by the Bradford method using
bovine serum albumin (BSA) as the standard curve [13]. Total soluble protein was expressed
µg g−1 of fresh avocado epicarp.
Horticulturae 2022, 8, 369 4 of 12

2.5.2. Catalase (CAT) Activity

The protein extract (2.5 µL) was mixed with 250 µL of potassium phosphate buffer
(50 mM, pH 7.0) and 22.5 µL of 50 mM hydrogen peroxide. The absorbance was measured
at 240 nm every 10 s for 5 min. The catalase activity was calculated using Equation (1).
Where, “k” is the decay constant calculated as the natural logarithm (ln) of the ratio of
the absorbance measured at each moment and the absorbance at the starting point of the
measurement, “df ” is the dilution factor, t is the reaction time, “ε” the molar extinction co-
efficient and, “c” the protein concentration in the samples. The CAT activity was expressed
as mmol of hydrogen peroxide min−1 g−1 of protein.

k·df
CAT = (1)
t·ε·c

2.5.3. Polyphenol Oxidase (PPO) Activity

To 100 mg of pulverized sample, 500 µL of extraction buffer (0.1 M sodium phosphate
buffer, pH 6.5, and 2% polyvinylpyrrolidone (PVP)) was added. The mixture was vortexed
and then centrifuged (10,000× g at 4 ◦ C per 10 min). The enzymatic assay was performed by
adding to a microplate 240 µL of buffer, 20 µL of the extract, and 40 µL of 0.1 M catechol. The
formation of oxidized catechol polymer was monitored for 30 min by measuring the change
in absorbance at 410 nm every 2 min. The PPO activity was calculated based on the slope of
the linear portion of the curve and it was expressed as mmol of catechol min−1 g−1 of protein.

2.5.4. Superoxide Dismutase (SOD) Activity

The extract was prepared with 100 mg of sample and 0.5 mL of 0.1 M potassium
phosphate buffer (pH 7.8 and containing 0.1 mM EDTA), then homogenized and centrifuged
(13,000× g for 10 min at 0–5 ◦ C). Fifty µL of the extract were mixed with 250 µL of a working
solution prepared as follows: 194 mg of 13 mM methionine, 6 mg of 75 µM nitro blue
tetrazolium (NBT), and 3.73 mL of 1.3 µM riboflavin. The samples were illuminated with
a fluorescent lamp (Sylvania FC 12 T10 CW RS, JUC, Spain) for 15 min. Solvents without
sample extract were used as control and non-illuminated solutions were used as blank. The
absorbance was measured at 560 nm after illumination. One unit of SOD (U) was defined
as the amount of enzyme necessary to inhibit 50% of NBT and it was calculated according
to Equations (2) and (3).

ABSControl − ABSSample
% Inhibition = × 100 (2)
ABSControl

% Inhibition × Vol Sample (µL)

SOD (U/µg protein) =   (3)
(50%) × protein µg µL−1

2.5.5. Peroxidase (POD) Activity

The peroxidase activity was carried out according to Bi et al. [14] with modifications. To
100 mg of pulverized sample, 1.5 mL of extraction buffer (0.1 mM sodium phosphate buffer,
pH 7.0 containing 0.1 mM EDTA and 0.1% Triton X-100), 0.5% (w/v) PVP and 15 µL of 0.1 M
PMSF were added. The mixture was vortexed, sonicated at low temperature for 10 min and
centrifuged (13,000× g at 4 ◦ C per 20 min). The POD assay was performed by mixing 160 µL
of 20 mM Guaiacol in microplates with 40 µL of extract followed by incubation for 5 min at
30 ◦ C. Then, 80 µL of 50 mM hydrogen peroxide was added. The formation of the oxidized
tetra guaiacol polymer was monitored by measuring the change in absorbance at 460 nm every
30 s for 5 min. POD activity was calculated based on the slope of the linear portion of the curve
and it was expressed as mmol of oxidized tetraguaiacol min−1 g−1 of protein.
Horticulturae 2022, 8, 369 5 of 12

2.5.6. Phenylalanine Ammonia Lyase (PAL) Activity

The extract was prepared by homogenizing 100 mg of the powdered sample with 1 mL
of 0.1 M sodium borate buffer, pH 8.8, containing 5 mM β-mercaptoethanol, 2 mM EDTA,
and 2% (w/v) PVP in an ice bath. The mixture was centrifuged (10,000× g for 15 min at
4 ◦ C) and the supernatant recovered. Ten µL of extract, 230 µL of sodium borate buffer pH
8.8, and 50 µL of the substrate (60 mM L-phenylalanine) were mixed and incubated at 37 ◦ C
for 30 min. The reaction was stopped by adding 10 µL of 1 M trichloroacetic acid (TCA)
and the absorbance was measured at 290 nm. PAL activity was determined by cinnamate
production and calculated as in Equation (4). The sample and control OD absorbances
along with the dilution factor (df ), reaction time (t), molar extinction coefficient (ε), and
protein concentration (c) in the sample.
 
ODSample − ODControl × df
PAL (U/mg) = (4)
0.1 × t × ε × c

2.6. Statistical Analysis

Analysis of variance (ANOVA) followed by Tukey’s test was performed using Stat-
graphics Centurion XVI (StatPoint, Rockville, MD, USA) at 95% confidence.

3. Results and Discussion

3.1. Incidence of Black Spot
The incidence of the disorder on avocados from an early and middle harvest after
40 d of cold storage subjected to different treatments (T1-T4) is shown in Figure 1. Only
three orchards presented fruit with black spots (A, B, and E) in both harvests. No fruit
with the presence of anthracnose was found. Fruit stored in regular air (T1) showed a
higher incidence of black spot and it was the only treatment affecting fruit from the middle
harvest. On the other hand, fruit immediately stored under a controlled atmosphere after
the harvest (T2) showed the lowest black spot incidence. These results are in concordance
with the incidence observed on avocados from the 2017/2018 and 2018/2019 seasons,
where the orchards A, B, and E showed higher black spot incidence [5,7]. For the early
harvest fruit, all the treatments with CA (T2, T3, and T4) almost totally controlled the
incidence of black spot when compared to T1, except for one of the orchards in the interior
zone (orchard E). For orchards with a recurring history of black spot, the 10 d in regular air
can be critical to promote the disorder development and time should be reduced to fully
control the incidence of black spot as T2. Commercially, Chilean Hass avocados destined
for export remain over 7 d in regular air before the containers are consolidated and the
controlled atmosphere is applied. The effect of CA (reduced oxygen and increased carbon
dioxide concentrations) beyond delaying ripening [15], avoids the oxidation process of
the fruits preventing the appearance of black spot. In addition, reduced oxygen favors the
maintenance of pigments and antioxidant compounds [16].

3.2. Quality Parameters

Dry matter content of epicarp and mesocarp and the non-destructive firmness are
shown in Table 1. The dry matter of the epicarp varied between 21% and 26%, the highest
values were found in the intermediate zone (orchards C and D) and the lowest in the
coastal zone (orchards A and B). Similar dry matter contents in mesocarp were found and
ranged between 22% and 28% in both harvests. No statistical difference was observed in
the dry matter between early and middle harvests for both tissues although the expected
differences should be 3–4% between the harvest seasons. For orchards C and D, similar
contents of dry matter were attributed to a close harvest time. In general, the orchards that
presented black spot incidence after 40 d of cold storage in regular air, showed significantly
lower epicarp dry matter contents (A, B, and E with ranges from 21.1% to 22.4%) than
those that did not present the disorder (C, D, F with ranges from 23.4% to 26.1%). Besides
showing lower dry matter, the epicarp was thinner and less rough (observed data; not
Horticulturae 2022, 8, 369 6 of 12

shown) in addition to presenting a higher incidence of black spot. Therefore, probably

a higher concentration of some compounds in avocado epicarp could contribute to the
control of black spot development. A previous study on Hass avocado fruit produced in
regions of Mexico with a semi-warm and temperate climate revealed higher production of
lignin and total phenolics and a lower concentration of chlorophylls [17].

Figure 1. Evolution of the incidence of black spots for the different postharvest treatments evaluated
in early (a) and middle (b) harvests in different orchards after prolonged cold storage. T1: 40 d in
regular air (RA) at 5 ◦ C; T2: 40 d in a controlled atmosphere (CA) of 4 kPa O2 and 6 kPa CO2 at 5 ◦ C;
T3: 10 d in RA at 5 ◦ C plus 30 d in CA 4 kPa O2 and 6 kPa CO2 at 5 ◦ C; and T4: 5 µM MeJA plus 10 d
in RA at 5 ◦ C plus 30 d in CA 4 kPa O2 and 6 kPa CO2 at 5 ◦ C.

Table 1. Quality parameters (dry matter and firmness) of avocado fruit at harvest (before application
of postharvest treatments) in orchards from the coast (A and B), intermediate (C and D), and interior
(E and F) zones.

Dry Matter (%) Dry Matter (%)

Harvest Agroclimatic Zone Orchard Firmness (N)
Epicarp Mesocarp
A 22.1 ± 2.1 ab 23.3 ± 3.1 a,A 241 ± 14 b,A
Coast
B 21.1 ± 1.2 a 23.9 ± 2.4 a 241 ± 28 b
Early C 26.1 ± 1.2 c 27.9 ± 2.2 b 286 ± 9.3 c,B
Intermediate
D 25.9 ± 2.8 c 26.6 ± 2.7 b 249 ± 11 b
E 22.4 ± 2.1 a 22.3 ± 2.8 a,A 241 ± 14 a,B
Interior
F 23.4 ± 1.2 b 23.5 ± 1.7 a 239 ± 12 b
A 21.9 ± 1.0 a 26.5 ± 1.8 B 221 ± 9.4 a,B
Coast
B 21.8 ± 1.5 a 25.6 ± 2.8 248 ± 21 cd
C 24.9 ± 1.5 b 26.0 ± 1.9 229 ± 8.1 ab,A
Middle Intermediate
D 25.6 ± 1.7 b 24.9 ± 1.4 251 ± 11 d
E 21.9 ± 1.2 a 24.7 ± 2.4 B 238 ± 9.4 bc,A
Interior
F 24.3 ± 1.1 b 25.6 ± 2.5 249 ± 12 cd
Data expressed as mean ± standard deviation. Different lower-case letters in a column show statistical differences
among orchards at each harvest time and different upper-case letters in a column show statistical differences
between harvests (95% confidence).

Regarding mesocarp dry matter, no correlation was observed with the incidence of
black spot. Similar results were reported by Uarrota et al. ([5] and unpublished data) who
evaluated two seasons (2017/2018 and 2018/2019) and two harvests (early and middle).
The non-destructive firmness was evaluated at harvest and after application of evalu-
ated postharvest treatments. No significant differences or trends were observed between
harvest time and orchards. On the other hand, no significant changes were observed in
firmness after cold storage for all treatments. However, a desirable decrease in firmness at
20 ◦ C was observed, since the softening of the mesocarp is one of the main effects of the
ripening process explained by modifications in the composition and structure of the fruit
cell wall due to the enzymatic activity associated with the loss of turgor [18–20].
Horticulturae 2022, 8, 369 7 of 12

Regarding weight loss (Figure S1), fruit stored in regular air showed a greater loss
for both harvests (5–7%), and slightly higher values were observed in orchards from early
harvest. Similar results were reported by Escobar et al. [21], where weight loss ranged
5 to 6% under the same storage conditions. On the other hand, a lower weight loss was
observed in all fruit stored in CA, which did not exceed 2.2%, mainly due to the higher
water vapor pressure within CA containers. A significant increase in fruit weight loss
occurred during ripening related to the increase in temperature which generate an increase
in the transpiration rate of the fruit. Therefore, the CA treatment, in addition, presented
favorable conditions for preventing fruit weight loss.

3.3. Total Phenolic Compounds (TPC) and Antioxidant Capacity (AC)

TPC and AC were evaluated at harvest (Figure 2). No association was found between
TPC and AC with the incidence of black spot. However, fruit from early harvest showed
higher mean contents of phenolics and AC than those of middle harvest.

Figure 2. (a) Total phenolic compounds (TPC) expressed as gallic acid equivalents (GAE) and
(b) antioxidant capacity (CA) expressed as trolox equivalents (TE) in avocado fruit from orchards of
coast (A and B), intermediate (C and D), and interior (E and F) zones of early and middle harvests.
Different lower-case letters in bars show statistical differences among orchards and each harvest time
(95% confidence).

Based on our results and those reported by Uarrota et al. [5], the orchards with higher
black spot incidence (B and E) were chosen to evaluate the effect of postharvest treatments
on the total phenolic content and antioxidant capacity (Figure 3). At the beginning of cold
storage, TPC was higher in avocado epicarp of early harvest, which ranged from 40 to
50 µg GAE mg−1 epicarp (DW), whereas for middle harvest TPC ranged from 30 to 43 µg
GAE mg−1 . Similar results were reported by Uarrota et al. [5]. The controlled atmosphere
applied immediately after harvest (T2), besides fully controlling the incidence of black
spot, maintained higher levels of TPC after 40 d of cold storage (Figure 3a,b). Similar
behavior, but less marked, was observed for middle harvest, where the fruit from orchard
B with T2 showed the highest levels of total phenolics, mainly in the first days of storage
(Figure 3c). For the interior zone orchard (E) in early harvest, it was observed that the only
treatment that totally controlled the black spot during prolonged storage was T2, i.e., the
controlled atmosphere applied immediately after harvest. The treatments that included
10 d in regular air or MeJA plus 10 d in regular air (T3 and T4) showed a higher incidence
of black spot at 40 d. The control of black spot by T2 was related to a higher content of
phenolic compounds in this treatment compared to the other treatments. A similar trend
was observed for the middle harvest, the incidence of black spot was much lower, but only
T2 fully controlled the incidence of black spot and showed higher content of total phenolics
than the other treatments after 40 d of storage (Figure 3c,d). Saxena et al. [22] evaluated the
effect of a controlled atmosphere at different concentrations of O2 and CO2 on the content
Horticulturae 2022, 8, 369 8 of 12

of total phenolics in jackfruit. They reported that at high CO2 and low O2 concentrations
the phenolic compounds were maintained to a greater extent than under other conditions.

Figure 3. Evolution of the total phenolic content (TPC) expressed as gallic acid equivalents (GAE) and
the antioxidant capacity (AC) expressed as trolox equivalents (TE) of avocado fruit epicarp during
different postharvest treatments. (a) Early harvest orchard B; (b) middle harvest orchard B; (c) early
harvest orchard E; and, (d) middle harvest orchard E. T1: 40 d in regular air (RA) at 5 ◦ C; T2: 40 d in
controlled atmosphere (CA) 4 kPa O2 and 6 kPa CO2 at 5 ◦ C; T3: 10 d in RA at 5 ◦ C plus 30 d in CA
4 kPa O2 and 6 kPa CO2 at 5 ◦ C; and, T4: 5 µM MeJA plus 10 d in RA at 5 ◦ C plus 30 d in CA 4 kPa
O2 and 6 kPa CO2 at 5 ◦ C. TPC is represented by bars and AC by lines. The asterisk showed statistical
differences in each treatment (95% confidence).

In the case of the evolution of the antioxidant activity (line graphs in Figure 3), the
results were not as clear as for the phenolic compounds. For orchard B, the AC remained
slightly higher for T2 than the other treatments for both harvests. However, the differences
were not significant (Figure 3a). For orchard E, no differences were observed between
treatments (Figure 3b). The AC decreased in middle harvest when compared to early, which
ranged between 70–90 and 26–45 µM TE mg−1 epicarp (DW) in early and middle harvests,
respectively. Similar results were reported by Uarrota et al. [5]. The AC of avocados of
orchards of middle harvest was relatively stable during cold storage. However, the fruit of
early harvest showed a greater decrease in AC after 40 d. Although phenolic compounds
are substances that are involved in the stability of AC, there are other compounds such as
flavonoids, amino acids, tocopherols, and pigments that can be associated with AC [23].
On the other hand, López et al. [16] reported that the effect of controlled atmospheres
(4 kPa O2 + 96 kPa N2 ) in ripe Imperial tomato was more effective than the storage at
low temperature. The main antioxidant compound in tomatoes is ascorbic acid, which
is higher when stored at CA rather than RA. The ascorbic acid can be degraded by the
enzyme ascorbic acid oxidase, which acts in the presence of oxygen. The higher levels of
ascorbic acid in tomatoes stored in controlled atmospheres are due to the fact that they
degraded to a lesser extent due to the absence of oxygen.
Horticulturae 2022, 8, 369 9 of 12

3.4. Analysis of Epicarp Enzymatic Defense System

The evolution of enzymatic activities of SOD, PAL, CAT, POD, and PPO in the epicarp
of fruit from early and middle harvests during different postharvest treatments is shown in
Figure 4.

Figure 4. Evolution of enzymatic activity of superoxide dismutase (SOD), phenylalanine ammonia-

lyase (PAL), catalase (CAT), peroxidase (POD) and polyphenol oxidase (PPO) of avocado fruit epicarp
during different postharvest treatments. The (first) column shows fruit from the early harvest of
orchards B; the (second), from the early harvest of orchards E; the (third), from the middle harvest of
orchard B; and, the (last) column from the middle harvest of orchard E. T1: 40 d in regular air (RA) at
5 ◦ C; T2: 40 d in controlled atmosphere (CA) 4 kPa O2 and 6 kPa CO2 at 5 ◦ C; T3: 10 d in RA at 5 ◦ C
plus 30 d in CA 4 kPa O2 and 6 kPa CO2 at 5 ◦ C; and, T4: 5 µM MeJA plus 10 d in RA at 5 ◦ C plus
30 d in CA 4 kPa O2 and 6 kPa CO2 at 5 ◦ C.

Regarding early harvest, only SOD showed differences between orchards, where B
showed higher activity than E. This difference was greater in the first 6 d of storage and it
is in agreement with those observed for TPC and AC. Superoxide dismutase is efficient at
scavenging reactive oxygen species (ROS), which may contribute to the antioxidant defense
Horticulturae 2022, 8, 369 10 of 12

system [5,24]. PAL is an entry-point enzyme in the phenylpropanoid pathway, which is one
of the most important pathways for the synthesis of phenolics and flavonoids [25]. Cold
storage stimulates the activity of PAL in fruits such as cucumber, grapes, and walnuts [25–27].
In our study, increasing activity of PAL was observed in the first days of cold storage (T1,
T2, and T3). However, PAL was significantly lower with the application of 5 µM MeJA (T4).
Glowacz et al. [8] reported that PAL activity increased in avocados treated with 100 µM MeJA,
but no changes were observed at 10 µM. The low activity of PAL with the application of MeJA
is consistent with the low contents of phenolics found in avocados with the same treatment.
On the other hand, all four postharvest treatments showed non-significant differences in the
activities of SOD, CAT, POD, and PPO. Nevertheless, enzymes such as SOD and PPO showed
slightly increasing activity during storage, whereas PAL and CAT decreased.
The avocado fruit from the middle harvest showed more marked differences in en-
zymatic activity between orchards. Orchard B showed higher enzymatic activity than E,
except for POD. POD enzymes are important ROS scavengers [25]. Most enzymes showed
increasing activities during storage, except for CAT which was not significant. CAT, as well
as SOD, are sensitive to oxidation, thus, antioxidant molecules (i.e., phenolics) are impor-
tant to counteract the effect of ROS [28]. All treatments applied did not show differences
among the enzyme’s activities studied. The previous study by Uarrota et al. [5] showed
higher black spot incidence in the orchards presenting decreases in SOD, CAT, POD, PAL
activities, and phenolics. As air conditions (regular air storage) favor oxidation of certain
antioxidant enzymes, thus, the non-enzymatic defense system (i.e., phenolics) becomes
crucial against black spot development.

4. Conclusions
Results of this study revealed that regular air storage cold conditions favor black spot
development in fruit from orchards displaying a history of this disorder. The disorder
could not be associated with a specific agroclimatic zone or harvest. Immediate application
of controlled atmosphere conditions of 4 kPa O2 and 6 kPa CO2 at 5 ◦ C controlled black
spot disorder up to the evaluated 40 d. These conditions resulted in fruit skin with a
higher content of total phenolics compared to the other evaluated treatments during the
prolonged storage period. The activity of antioxidant enzymes (CAT, PPO, SOD, POD,
PAL) did not show a clear trend in relation to controlling black spot disorder in the skin of
Hass avocados.
The results obtained are of practical application to the Hass avocado supply chain
and contribute to decreasing avocado food losses. Further studies will focus on deeply
understanding how CA controls black spot development at different omics levels.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10

.3390/horticulturae8050369/s1, Figure S1: Weight loss (%) of avocado fruit during different posthar-
vest treatments at the end of cold storage (a,b) and at ready-to-eat (RTE) (c,d) from early and middle
harvest.
Author Contributions: Conceptualization, R.P.; methodology, J.V., V.U., E.P. and C.F.; software, C.F.;
formal analysis, J.V., C.F., B.G.D. and R.P.; investigation, J.V., C.Z. and E.P.; resources, R.P. and B.G.D.;
writing—original draft preparation, C.F. and J.V.; writing—review and editing, R.P., V.U. and B.G.D.;
visualization, C.F.; supervision, R.P.; project administration, R.P.; funding acquisition, R.P. and B.G.D.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Comité de Paltas Chile and associated producers and
exporters (Santa Cruz, El Parque, Jorge Schmidt, Baika, Subsole).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All relevant data is shown in Figures.
Acknowledgments: We would like to thank Vicente Lindh for his technical assistance.
Horticulturae 2022, 8, 369 11 of 12

Conflicts of Interest: The authors declare no conflict of interest.

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