The Journal of Horticultural Science and Biotechnology

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/thsb20

Unravelling factors associated with ‘blackspot’

disorder in stored Hass avocado (Persea americana
Mill) fruit

Virgílio Gavicho Uarrota , Ignacia Hernandez , Excequel Ponce Guequen ,

Juan Vidal Cruz , Claudia Fuentealba , Bruno G. Defilippi , Vicente Lindh ,
Claudio Zulueta , Rosana Chirinos , David Campos & Romina Pedreschi

To cite this article: Virgílio Gavicho Uarrota , Ignacia Hernandez , Excequel Ponce Guequen ,
Juan Vidal Cruz , Claudia Fuentealba , Bruno G. Defilippi , Vicente Lindh , Claudio Zulueta ,
Rosana Chirinos , David Campos & Romina Pedreschi (2020): Unravelling factors associated
with ‘blackspot’ disorder in stored Hass avocado (Persea�americana Mill) fruit, The Journal of
Horticultural Science and Biotechnology, DOI: 10.1080/14620316.2020.1763860

To link to this article: https://doi.org/10.1080/14620316.2020.1763860

View supplementary material Published online: 30 May 2020.

Submit your article to this journal Article views: 133

View related articles View Crossmark data

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=thsb20
THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY
https://doi.org/10.1080/14620316.2020.1763860

Unravelling factors associated with ‘blackspot’ disorder in stored Hass avocado

(Persea americana Mill) fruit
Virgílio Gavicho Uarrota a, Ignacia Hernandeza, Excequel Ponce Guequena, Juan Vidal Cruzb,
Claudia Fuentealbab, Bruno G. Defilippic, Vicente Lindha, Claudio Zulueta a, Rosana Chirinos d
,
David Campos d and Romina Pedreschi a
a
Facultad de Ciencias Agronómicas y de los Alimentos, Escuela de Agronomía, Pontificia Universidad Católica de Valparaíso, Quillota,
Chile; bFacultad de Ciencias Agronómicas y de los Alimentos, Escuela de Alimentos, Pontificia Universidad Católica de Valparaíso,
Valparaiso, Chile; cInstituto de Investigaciones Agropecuarias, INIA-La Platina, Santiago, Chile; dInstituto de Biotecnología (IBT),
Universidad Nacional Agraria La Molina (UNALM), Lima, Perú

ABSTRACT ARTICLE HISTORY

This study aimed to understand the involvement of agro-climatic conditions, external quality Accepted 15 April 2020
and defence system of Hass avocado fruits on blackspot disorder development after prolonged
KEYWORDS
cold storage. The results prompt us to conclude that the weight of the fruits at harvest and External parameters; quality;
weight loss during the storage period is involved in blackspot disorder development. Large epidermis; enzymes;
incidence of lenticelosis in fruits and lower activity of SOD, CAT, POD, PAL, phenolics and defence system; blackspot
epicatechin levels may contribute to blackspot spot development during cold storage. Non-
supervised techniques pointed for epicatechin and derivatives, temperature, relative humidity
and enzymes as important specific variables that affect blackspot disorder while predictive
models showed positive correlation with minimum temperature (below 5°C) and maximum
temperature and relative humidity displayed negative association with blackspot.

Introduction
Belonging to the family Lauraceae, avocado (Persea
americana Mill) fruit is rich in monounsaturated (71%
- MUFAs) and polyunsaturated (13% - PUFAs) fatty
acids and has high economic and health importance
globally (Dreher & Davenport, 2013; Pedreschi et al.,
2019). It is quite unique not only from a compositional
point of view but also shows a very distinct and com-
plex physiology compared to other fruit. For example,
flowering period can last up to 3 months; thus, a broad
range of fruit physiological ages can be found on the
same tree, which will be evident during postharvest
storage and management (Lewis, 1978).
A total of 74% of the world production comes from
Americas, followed by Africa (11.7%), Asia (11.2%),
Europe (1.6%) and Oceania (1.4%). Chile is currently
the ninth top producer (245 000 t in 2018 year) in the
world and the fifth exporter of avocado fruit in 2018,
being preceded by Mexico, the Netherlands, Peru and
Spain (FAOSTAT, 2018; FreshPlaza, 2019) respec-
tively. Therefore, an important issue to be considered
is arrival at market of high-quality fruit with good
external appearance (i.e. without black spots on the
epidermis) even after prolonged days of shipping, cold
and controlled atmosphere conditions of storage.
Blackspot disorder was reported for the first time in
Peruvian Hass avocado fruits (Everett et al., 2015)
stored in cold storage and relatively recently in
Chilean exported avocado with fruit losses of up to
20%. Fruit with blackspot disorder are not marketable
as fresh fruit due to unsightly lesions. Blackspot is
characterized by brown to black spots of 2–3 cm in
diameter on the epidermis but not on the mesocarp
tissue. The symptoms are only visible after long sto-
rage (cold and/or controlled atmosphere storage) and
are usually attributed or confused with mechanical
damage, pathogen attack (anthracnose caused by
Colletotrichum gloeosporioides) and/or mechanical
damage combined with detrimental environmental
conditions that favour severe lenticelosis (Everett,
Hallett, Rees-George, Chynoweth, & Pak, 2008;
Guetsky et al., 2005). It is highly possible, that the
problem is physiological in origin but as the fruit
becomes more susceptible, and given the optimal
environmental conditions and inoculum load, it can
further be attacked by pathogens (Genga et al., 2011).
Blackspot as a physiological disorder can have its
origin in a disturbed fruit metabolism. Plant metabo-
lism generates reactive oxygen species (ROS), which
are key regulators that mediate signalling pathways

CONTACT Romina Pedreschi romina.pedreschi@pucv.cl Facultad de Ciencias Agronómicas y de los Alimentos, Escuela de Agronomía, Pontificia
Universidad Católica de Valparaíso, Calle San Francisco S/N, La Palma, Quillota, Chile
Supplementary data cited in the text is also made available together with the manuscript. An HTML report and script for analysis of variance performed
is made available as supplementary data.
Supplemental data for this article can be accessed here.
© 2020 The Journal of Horticultural Science & Biotechnology Trust
2 V. G. UARROTA ET AL.
involved in developmental processes and plant
responses to environmental fluctuations (Decros
et al., 2019). If there is an imbalance between ROS
with the antioxidant defence system, results in oxida-
tive stress which may be a potential causal factor of
blackspot in epidermis (Mittler, 2017, 2002).
This disorder represents a huge threat to Chilean
and other distant from export market countries.
Progress in understanding the factors involved in
blackspot development will help to define novel stra-
tegies for optimizing fruit quality and storage. In this
research it was hypothesized that agro-climatic condi-
tions of the production region, pre-and post-harvest
factors, biochemical imbalance of the fruit metabolism
due to stressors are potential drivers of blackspot dis-
order in avocado fruit during storage. Therefore, this
study was conducted (i) to evaluate the effect of agro-
climatic conditions on external fruit quality para-
meters; (ii) to evaluate the role of enzymatic and non-
enzymatic antioxidant defence system; (iii) to evaluate
blackspot disorder development after cold storage; (iv)
to correlate the external and defence system para-
meters with blackspot development in Hass avocado
fruits and (v) to predict blackspot development at
harvest using machine learning techniques.
Materials and methods
Orchard selection, geographical location and
agro-climatic data collection
The orchards were selected to be representative of
different Chilean agro-climatic zones of avocado pro-
duction. Three orchards of interior zone (A, B, and C),
three of intermediate (D, E, and F) and four of coastal
zone (G, H, I, and J) were selected. Orchards of inter-
ior zone were defined as those located at a distance
superior to 45 km from the sea and at 300–900 meters
above sea level (m.a.s.l). Those located at distance
between 20 km to 45 km and between 300 m.a.s.l
and 400 m.a.s.l were defined as intermediate zone
and orchards located at a distance inferior to 10 km
from the sea and between 100 m.a.s.l and 250 m.a.s.l
corresponded to coastal zone. Agro-climatic data col-
lected from each orchard were temperature (absolute
minimum, absolute maximum and average), relative
humidity (absolute minimum, absolute maximum and
average) and total rainfall.
Fruit sampling and storage conditions
Two harvests of the 2017/2018 season were collected:
early season (dry matter content of 23–26%) and late
season (27–30% of dry matter). Independently of the
harvest season, 150 fruits from each orchard were
collected and immediately transported to the labora-
tory for subsequent assessment of external quality
attributes before storage. Fruit were then separated
into 3 batches composed of 50 (batch 1), 50 (batch 2)
and 25 (batch 3) fruits, respectively. The first batch
was stored for 25 d in regular air at 5°C and the second
and third batches were stored for 40 d in the same
storage conditions. After cold storage, the batches (1
and 2) were left at room temperature (± 20 ° C and
65–70% RH) until ready-to-eat (RTE) stage. In total,
3000 fruits were analyzed.
External quality parameters
The presence of russet (type of skin-blemish variously
attributed to mechanical abrasion of young fruits by
the foliage, twigs, branches and other injuries when
the fruits are small and considered as the effect of wind
on avocado trees -California Avocado Commission,
2020; Carpenter, 1931), skin wounds (superficial skin
wound due to mechanical, impact, friction injury on
the fruit during growth, cultural management, harvest
or transport -Everett et al., 2008; Ramírez-Gil, López,
& Henao-Rojas, 2020) and lenticelosis (damage on
lenticel that covers the surface of the fruit that is
apparent immediately after harvest and packing as
small brown to dark spots about 1–5 mm diameter -
Everett et al., 2008) were assessed at harvest and after
cold storage and classified using a hedonic scale from 0
to 3, where 0 = absence, 1 = less than 25% of fruit area
is affected, 2 = more than 25% but less than 50% of the
fruit area is affected and 3 = more than 50% of the fruit
area is affected. The severity index was then calculated
based on the equation below as previously reported by
Baumgartner, Keller, and Schwendimann (1983) and
Gancarz (2018).


ðf1 :c1 þ f2 :c2 þ f3 :c3 þ fn :cn Þ
SIð%Þ ¼ 100 (1)
N:t
Where SI is the severity index of a particular disorder
(i.e. russet, skin wound, lenticelosis or external brown-
ing); c1, c2, c3 an cn represent specific class or level of
the disorder; f1, f2, f3, fn represent the number of fruits
of particular class or level of disorder; N is number of
classes of a specific disorder and t the total fruits
evaluated.
Fruit weight was evaluated individually at harvest,
after cold storage and at RTE stage. The values were
then used for weight loss calculation. Dry matter was
measured using 10 independent fruits. Briefly, the
epidermis, seed coat and seed were removed and the
remaining part sliced in small pieces and left to dry
using an oven at 100°C for 24 h. The dry matter was
expressed as mean percentage of 10 fruits. The firm-
ness of each fruit was also measured as described by
Ochoa-Ascencio, Hertog, and Nicolaï (2009) with
small modifications using a non-destructive texture
analyzer (Model TA.XT plusC, Stable Micro Systems
 THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY
Ltda, England) fitted with a cylinder probe of 10 mm
diameter, trigger threshold of 0.50 N and measuring
speed of 8 mm s−1. The compression force was
recorded in Newton (N) at 2 mm deformation and
was determined at two equidistant points on the equa-
torial region of each whole fruit. Results were
expressed in N.mm. Non-destructive firmness was
evaluated at harvest and after 25 and 40 days of cold
storage. Blackspot disorder was evaluated by visual
inspection, fruits were scored as 0 – no blackspot
presence and 1– the presence of blackspot, respectively
and expressed as percentage (%) of fruits affected per
batch. The number of days necessary to reach the
ready-to-eat (RTE) stage (4–8 N) was also assessed.
Antioxidant defence system of the epidermis
Enzymatic extraction
Part of milled epidermis samples (stored at −80°C)
was used for enzymatic analysis and the remaining
part was lyophilized and used for non-enzymatic
metabolite determinations. Five hundred micro litre
of extraction buffer (0.2 M potassium phosphate buf-
fer, pH 7.0 with 0.1 mM EDTA and 0.1% triton X-100)
and 20 μL of PMSF were added to 100 mg powdered
sample. The mixture was vortexed and centrifuged at
10 000 g and 4°C for 10 min. The supernatant was
collected and stored at −20°C for subsequent analysis.
Total soluble proteins
Total soluble proteins were determined using the
Bradford assay protocol (Bradford, 1976). A standard
curve of BSA (bovine serum albumin) from 5 μg.mL−1
to 100 μg.mL−1 (Y = 0.0023x + 0.00097, r2 = 0.999) was
built from the stock solution of 2000 μg .mL−1. Total
protein values were represented as mean ± standard
deviation and expressed in μg .mL−1.
Catalase activity (CAT)
Catalase activity was performed based on Aebi (1984)
with small modifications. Briefly, 250 μL of potassium
phosphate buffer (50 mM, pH 7.0) were mixed with
2.5 μL of the extract and 22.5 μL of hydrogen peroxide
and absorbance was read at 240 nm during 5 min and
expressed in mM .min−1 mg−1 of protein as means and
standard deviations (Equation (2)).
½CAT ¼ k df  ε c (2)
Where: (df) is dilution factor, (ε) extinction coefficient
and (c) protein content in the samples.
Superoxide dismutase activity (SOD)
SOD activity was determined as reported by
Giannopolitis and Ries (1977a, 1977b) and Bailly and
Kranner (2011). Samples in microplates were illumi-
nated with a fluorescent lamp (Sylvania FC 12 T10
CW RS) during 15 min in an incubator or box lined
3
with aluminium foil. The reaction was initiated by
turning on and off the lamp. The absorbance was
read at 560 nm after the illumination. One unit of
SOD (U) was defined as the quantity of enzyme
required to inhibit 50% of NBT and calculated accord-
ing to the Equations (3) and (4).


ðabsControl  absSampleÞ
%Inhibition ¼ 100
absControl
(3)
" #
U %Inhibition VOLsampleðμlÞ
SOD ¼ ¼  
proteinðμgÞ ð50%Þ proteins μg:μl1
(4)
Polyphenol oxidase activity (PPO) and Peroxidase
activity (POD)
PPO and POD were carried out based on the meth-
odologies reported by Woolf et al. (2013) and Bi,
Hemar, Balaban, and Liao (2015) with small modifica-
tions. The formation of oxidized catechol polymer was
monitored over 5 min by changes in absorbance at
410 nm. For POD, the formation of the oxidized
tetraguaiacol polymer was monitored by measuring
the change in absorbance at 460 nm during 5 min.
Enzyme activity was calculated based on the slope of
the linear portion of the curve and expressed as change
in absorbance.
Phenylalanine-ammonia-lyase (PAL)
Enzyme extraction and assay were carried out based
on the combined procedures of Popović et al. (2016),
Gerasimova, Pridvorova, and Ozeretskovskaya (2005),
Assis, Maldonado, Muñoz, Escribano, and Merodio
(2001) and Cheng and Breen (1991) with minor mod-
ifications. PAL activity was determined by the produc-
tion of cinnamate and calculated as in the Equation (5)
according to the protein concentration of the sample.
2  3
ODsample ODcontrol
  Vsample Cprotein Vtotal
6 7
6PAL U ¼ 0:01 7 (5)
4 mg T 5
Where Vtotal is the total volume of enzymatic reaction
(mL), Vsample is the volume of the sample (mL),
Cprotein is the protein concentration (mg. mL−1) and
T is the reaction time (min).
Total phenolic compounds and total free radical
scavenging activity
The protocol described by Saavedra et al. (2017) using
the Folin-Ciocalteu reagent was used and absorbance
was read at 765 nm. Total phenolic compounds were
calculated using an external standard curve of gallic
acid (50–200 μg mL−1, y = 0.0039x-0.012, r2 = 0.999)
and expressed in μg mg−1 of dry basis (db).
4 V. G. UARROTA ET AL.
Total antioxidant capacity was determined with the
DPPH method and absorbance was read at 517 nm.
Antioxidant capacity was expressed as inhibition per-
centage (%) and as trolox equivalents per mg using an
external standard curve of Trolox (20–140 μM mL−1,
y = 0.25x -1.039, r2 = 0.998).
Ultra-high-performance liquid chromatography
with photodiode array detector (UPLC-PAD) of
epicatechin, its derivatives and other phenolics
The procedure of Adamson et al. (1999) was used
for sample preparation with small modifications.
Polyphenolic compounds were analyzed using an
UPLC system composed of an Aquity HClass separation
module (Waters, Milford, USA) equipped with an auto-
injector, an Aquity photodiode array detector (PDA eλ
detector) and the Empower software. The column used
for UPLC separation was an Acquity BEH C 18 (1.7 μm,
100 × 2.1 mm) (Waters, Milford, USA) with an Acquity
VandGuard BEH C 18 pre-column (1.7 μm, 5 × 2.1 mm),
operated at 30°C. The mobile phase consisted in 0.1%
formic acid in water (solvent A) and 0.1% formic acid in
acetonitrile (solvent B). The gradient was as follows: 2%
B for 2 min, 2 to 7% B in 2 min, 7 to 12% B in 11 min, 12
to 26% B in 5 min, 26 to 55% B in 5 min, 55 to 95% B in
1 min and 95% B for 3 min. The flow rate and sample
injection used were 0.2 mL min−1 and 2.0 µL, respec-
tively. Spectral data were recorded from 200 nm to
700 nm during the whole run. Phenolic compounds
were identified and quantified by comparing their reten-
tion time and UV-visible spectral data to known pre-
viously injected standards (280, 320 and 360 nm). The
results were expressed in mg .g−1 of dry basis (db).
Statistics and data mining
All data were summarized, tested for their normality
and homogeneity of variances, submitted to univariate
analysis of variance and multivariate statistical
Figure 1. Percentage of black spot incidence in fruits stored during 25 and 40 days for early (a) and late (b) harvest. Orchards A,
B and C belongs to interior zone, D, E and F are from intermediate zone and G, H, I and J from coastal zone.
techniques aiming to find important factors related
to blackspot disorder. All analyses were performed in
R Software (R Core Team, 2019 version 3.6.1).
Results and discussion
External quality parameters
Results of blackspot incidence in fruits are presented
in Figure 1(a) (early harvest) and 1B (late harvest)
while the typical symptoms of this physiological dis-
order are presented in Photography 1. For early har-
vest, blackspot incidence was observed only in orchard
‘A’ and ‘H’ and for late harvest in addition to those
orchards, blackspot was also observed in ‘G’, ‘J’, ‘B’, ‘D’
and ‘I’.
Results of other external quality parameters can be
found in the supplementary data, (Supplementary
Figures S1 to S9). In this research, we discriminate
between blackspot (physiological disorder) from
anthracnosis (pathological disorder). The results
shows that blackspot incidence is positively related to
russet incidence in fruits, relative humidity of the
production region, lenticelosis, minimum tempera-
ture, fruit weight at harvest, at cold chamber outlet
and at ready to eat state (RTE) and weight loss during
cold storage (Figure 2). According to Everett et al.
(2015), the cause of the blackspot symptoms can be
pathological, physical or physiological in origin.
Everett, Boyd, and Pak (2007) reported that daily
temperatures and total rainfall before harvest affect
the quality of the fruits.
Dry matter (DM) is an important parameter of
avocado. Its content can be directly associated with
fruit resistance to shrivelling (Ncama et al., 2018).
Results of one-way ANOVA of dry matter (p < 0.05,
Scott-Knott test) for early and late harvest data sets
showed differences between orchards (Supplementary
 THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY 5

Photography 1. Hass avocado fruits (a) without and (b) with the presence of black spots in the epidermis.

Weight_loss_outlet

weight_loss_total
Weight_loss_ripe

Weight_harvest

SI_Skinwound
Inc_Blackspot

SI_lenticelosis
Weight_outlet
SI_browning
DaystoRTE

Weight_rte
SI_Russet

Firm_RTE
Firm_harv

Drymatter

Firm_25d

Firm_40d
ColRTE

1
DaystoRTE

Weight_loss_ripe

ColRTE
SI_Russet

Firm_harv

Drymatter

SI_brow ning

Firm_RTE

Inc_Blackspot
0
Weight_harvest

Weight_outlet

Weight_rte

SI_lenticelosis

SI_Skinw ound
Weight_loss_outlet

w eight_loss_total

Firm_25d

Firm_40d
-1

Figure 2. Matrix of correlations (Pearson, p < 0.05) of all external and quality variables.
Abbreviations: DaysRTE – number of days from cold storage outlet to ready to eat state (RTE); Weight_loss_ripe – weight loss from cold storage outlet to RTE
(Shelf life storage); ColRTE – Color of the fruit at RTE state; SI_Russet – Severity index of russet in fruits; Firm_ harv – Firmness of the fruits at harvest;
Firm_RTE – Firmness of the fruits at RTE; Firm_25d and Firm_40d – Firmness of the fruits after 25 and 40 days of cold storage respectively; SI_browning –
Severity index of skin browning; SI_lenticelosis – Severity index of lenticelosis; SI_skinwound – Severity index of skin wound; Weight_harvest, Weight_outlet,
Weight_rte – Weight of the fruits at harvest, at cold storage outlet and at RTE state respectively; Weight_loss_total – Total weight loss from harvest until RTE.

Figure S1). In general average pulp dry matter were found by Mazhar, Joyce, Hofman, and Vu (2018).
increased significantly (p < 0.05) from 22.86 ± 0.41% We found a non-significant positive association of DM
at early to 26.14 ± 0.36% at late harvest. Similar results (p value = 0.254) with blackspot incidence. Pak,
6 V. G. UARROTA ET AL.

Dixon, and Cutting (2003) reported a negative asso- facilitate secondary infection leading to fruit rots
ciation between DM and with incidence of rots and in green fruit (Everett et al., 2008).
vascular strands. In this study, orchards of late harvest
presented more blackspot than those of early harvest.
The firmness of the fruits decreased progressively Enzymatic and non-enzymatic defence system
from harvest until ready-to-eat state (RTE) for all harvests
Total protein content increased from early to late
(Supplementary Figure S2 and S3). Two-way ANOVA
harvest in ‘A’, ‘B’ and ‘C’, orchards belonging to inter-
revealed significant statistical differences between the
ior agro-climatic zone. Interestingly, in orchard ‘A’
orchards and during the storage period (See manuscript
where higher blackspot incidence was observed in
HTML report for details). Positive association of firmness
the late harvest, there was a large accumulation of
at harvest with blackspot was observed (Figure 2) but
proteins (Figure 3(a)). Pearson correlation (p < 0.05)
firmness after 25 and 40 days of storage was negatively
revealed positive association of blackspot incidence
associated with blackspot (Figure 2). The loss of firmness
with total proteins (Figure 4). Blakey, Bower, and
during cold storage is indicative of metabolic reactions
Bertling (2009) studying the trends in proteins and
occurring in the avocado epidermis.
sugar contents during avocado ripening reported
The weight of the fruits at harvest (Supplementary
a variation (i.e. increase in some orchards and
Figure S4) was significantly higher for ‘A’ and ‘D’ during
decrease in others) that may be due to differences in
the two harvests and ‘J’ the lower fruit weight. Significant
production regions and seasons. The increase in total
differences were also observed from early to late harvest
proteins in the orchards of interior agro-climatic zone
except for ‘I’ and ‘C’ where non-significant statistical
(presenting higher blackspot incidence) can be
differences were observed (p < 0.05). During cold cham-
explained due to energy requirements that are needed
ber storage (Supplementary Figure S5), lower weight loss
for enzyme biosynthesis for more mature fruits.
was observed for ‘D’, ‘G’ and ‘F’ without statistical sig-
SOD activity (Figure 3(b)) significantly decreased
nificant differences. In samples of late harvest, the orch-
from early to late harvest for the orchards ‘A’ and ‘I’
ard ‘D’ presented higher weight loss during storage and
(p < 0.05). ROS homeostasis can be mediated by SOD
the orchards ‘E’ and ‘F’ the lowest values. The large water
enzymes (Mittler, 2017, 2002). Superoxide dismutases are
loss characteristic was unique for orchard ‘D’ of late
the major antioxidant defence systems that are very effi-
harvest, possibly fruits suffered extensive metabolic reac-
cient at scavenging ROS (Fukai & Ushio-Fukai, 2011).
tions during storage. Exact reasons remain unknown in
Interestingly, lower SOD activity in the orchard ‘A’ and
this research. From cold chamber outlet to RTE
‘H’ contributed to higher incidence of blackspot disorder
(Supplementary Figure S6), higher fruit weight loss was
in these orchards at late harvest. Differences observed in
observed in the orchard ‘I’ for early and late harvest,
this study may be attributed to differences in orchards
respectively.
(i.e. agro-climatic conditions, management practices,
Pearson correlation (p < 0.05) revealed a posi-
abiotic stressors). Developmental differences have an
tive association of fruit weight (at harvest, at cold
impact in SOD activity. Wang, Zheng, Khuong, and
chamber outlet and at RTE) with blackspot and
Lovatt (2016) observed a decrease and then an increase
negative association with total weight loss with
of SOD activity during fruit developmental stages.
and firmness of fruits (Figure 2). Contrarily, the
PPO activity (Figure 3(c)) presented large variabil-
weight loss during ripening at 20°C was negatively
ity between the sampled orchards and during the two
associated with blackspot incidence. Escobar,
harvests. Orchards ‘A’, ‘B’, ‘C’ and ‘I’ showed lower
Rodriguez, Cortes, and Correa (2019) reported
activity of this enzyme at late harvest and all of them
that fruit weight and the dynamics of fruit weight
presented incidence of blackspot disorder. Wang et al.
loss are also dependent on production and agro-
(2016) also observed a variation in PPO according to
climatic conditions.
the fruit developmental stage and type of Hass avo-
Results of lenticelosis, russet, and skin wound are
cado phenotype. Negative association of PPO with
presented as Supplementary Figures S7, S8 and S9,
blackspot disorder was found.
respectively. Positive association of lenticelosis,
CAT activity (Figure 3(d)) significantly decreased
with blackspot incidence was found in the study
from early to late harvest in the orchards ‘A’,”B” and
(Figure 2). The results are in agreement with those
‘F’. The orchards ‘A’ and ‘B’ presented incidences of
reported by Escobar et al. (2019), who states that
blackspot disorder at late harvest. Wang et al. (2016)
lenticelosis and russet in fruits can promote fruit
reported an increase of CAT during fruit development
damage and weight loss during cold storage and
and a decrease at the final stages in Hass avocado. CAT
induce premature ripening of the fruits due to epi-
in this study was negatively associated with incidence
dermal cell death. Lenticelosis is typically apparent
of blackspot and differences observed between the
at harvest as small dark spots and is of concern
orchards may be attributed to differences in stress
because of the adverse effect on fruit appearance
conditions subjected to each orchard.
upon arrival in the marketplace, and possibly
 THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY 7

a b c

d e f

Figure 3. (a) Total soluble proteins, (b) SOD activity, (c) Polyphenol oxidase, (d) Peroxidase activity, (e) Catalase activity and (f)
Phenylalanine ammonia lyase of all orchards measured at harvest. Dotted lines correspond from left to right: interior, intermediate
and coastal orchards.

POD activity (Figure 3(e)) significantly decreased
from early to late harvest in the orchards ‘A’, ‘C’ and
‘F’ (p < 0.05, Scott-Knott test). These last two orchards
did not present incidence of blackspot disorder at all
harvests. POD reduces H2O2 to water while oxidizing
a variety of substrates. Together with PPO, are the two
well-known enzymes involved in the browning pro-
cesses (i.e. oxidative degradation of phenolic com-
pounds) of fruits and vegetables (Nokthai, Lee, &
Shank, 2010). Wang et al. (2016) studying the harvest
date on quality attributes of Hass avocados observed
a variation of POD activity during fruit development
and a typical trend was not found, but a synergetic
association of POD and PPO was reported. Such syner-
gism of these two enzymes was also found in this study.
POD activity was negatively associated with blackspot
disorder in Hass avocado fruits, indicating that this
enzyme plays also a crucial role as antioxidant enzyme.
A decrease in PAL activity (Figure 3(f)) from early to
late harvest was significant for the orchards ‘A’ and ‘I’, all
of them presented blackspot disorder at late harvest. PAL
was negatively associated with blackspot disorder in Hass
avocado fruits. Positive associations between PAL with
PPO, CAT, SOD, POD and phenolics was observed in
this study, results which also corroborated with those
previously reported by Aydaş, Ozturk, and Aslım (2013).
Total phenolic content of all orchards (Figure 4(a))
decreased from early to late harvest. Such observation
was also found for antioxidant scavenging activity of the
phenolic extracts of those samples (Figure 4(b)).

Figure 4. (a) Total phenolic contents, (b) ABTS % inhibition and (c) Epicatechin content of all orchards measured at harvest. Dotted
lines correspond from left to right: interior, intermediate and coastal orchards.
8 V. G. UARROTA ET AL.

Inc_Blackspot
Epicatechin
HR_prom

Fenolicos
HR_max
Proteins

HR_min

T_prom
SumEpi

T_max
Der_1
Der_2

Der_5
Der_3
Der_4

T_min

SOD

POD
AOX

PPO

CAT
PAL
1
Proteins
HR_max
HR_min
HR_prom
Epicatechin
Der_1
Der_2
SumEpi
Der_5
Der_3
Der_4
0
Fenolicos
AOX
T_max
T_prom
T_min
Inc_Blackspot
PPO
PAL
SOD
CAT
POD
-1

Figure 5. Matrix of correlations (Pearson correlation, p < 0.05) of all enzymatic and non-enzymatic variables.
Abbreviations: HR_max – Máximum relative humidity; HR_min – Mínimum relative humidity; HR_prom – Average relative humidity; Der_1, 2, 3, 4 and 5 –
Epicatechin derivative 1, 2, 3, 4 and 5 respectively; SumEpi – Total epicatechin content; Fenolicos – Total phenolic content; AOX – Antioxidant radical
scavenging activity of phenolic extracts; T_min, T_max and T_prom – Mínimum, máximum and average temperature respectively; Inc_Blackspot –
Blackspot incidence; PPO – Polyphenol oxidase; PAL- Phenylalanine ammonia lyase; SOD – Superoxide dismutase; CAT- Catalase and POD-Peroxidase
activity

Phenolics work in concert with antioxidant enzymes to important role in plant defence mechanisms by delaying
tolerate the intracellular generation of ROS, and help in fungal decay via lowering the activity of lipoxygenase
promoting plant growth and or development, sometimes activity (Glowacz, Bill, Tinyane, & Sivakumar, 2017).
also in strengthening the responses to abiotic/biotic Obianom, Romanazzi, and Sivakumar (2019) and Bill,
environmental stressors (Gupta, Palma, & Corpas, Korsten, Remize, Glowacz, and Sivakumar (2017)
2018). Increases in the phenolic content under stress observed an increase in epicatechin of diseased avocado
conditions may support the role of this group of com- fruits. Epicatechin was also found to increase in avocado
pounds plays in alleviating oxidative stress (Maksimović fruits after application of jasmonate, methyl salicilate and
& Živanović, 2012; Uarrota et al., 2019a). It is also impor- thyme oil vapour (Glowacz et al., 2017). The results of this
tant to remember that phenolic compounds may be study corroborate with previous reports, as it was found
affected by an array of factors at genetic and environ- a negative association of these compounds with blackspot
mental levels (Gobbo-Neto & Lopes, 2007; Uarrota et al., disorder in Hass avocado fruits (Figure 5).
2019b; Uarrota, Severino, & Maraschin, 2011; Yang et al.,
2018). The decrease in phenolics observed from early to
Principal component analysis (PCA) and Linear
late harvest fruits in this study contributed to the inci-
discriminant analysis (LDA) applied to the external
dence of blackspot disorder in the orchards ‘J’, ‘G’ and ‘H’
quality parameters dataset
(Figure 4(a)).
A decrease in epicatechin (Figure 4(c)) and its deriva- The score plot of PCA analysis taking the orchard,
tives from early to late harvest was found in this study. harvest season, days of cold storage and agro-climatic
Epicatechin and derivatives have been reported to play an zone are presented as Supplementary Figure S10 and
 THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY
the contribution of the variables as Supplementary
Figure S11. PCA analysis only captured 35.8% of total
variance explained by the dataset. Samples were also
better classified according to season of harvest and zone
of production. Fruits of coastal zone were distinct due
to blackspot incidence, dry matter and incidence of
russet and external browning.
LDA has been used to predict the probability of
belonging to a given class (or category) based on one or
multiple predictor variables. LDA captured 89% of the
total variance being 75% and 14% for latent variable 1
(LD1) and 2, respectively (data not shown). The accuracy
of the model was 92.5%. Samples in the LD1 (+, i.e.
positive side of the axis) are associated with the weight
of the fruits at harvest, incidence of russet and total weight
loss while those in LD1(-) are highly correlated with
weight at cold chamber outlet, lenticelosis, and presence
of skin wound in the fruits. LD2 was positively associated
with the weight of the fruits at cold chamber outlet, total
weight loss, presence of blackspot, lenticelosis, russet,
external browning and skin wound.
PCA, Partial least squares regression (PLSR) and
Decision tree model (DTs) applied to antioxidant
defence system dataset
PCA of antioxidant defence system dataset captured
57% of total variance. PCA taking the orchard as
a factor clearly showed differences in fruits harvested
from ‘A’,”B”, ‘D’, ‘F’ from others (Figure 6(a)).
Orchards from coastal zone showed to be different
from others. Fruits of the orchard ‘A’ were most influ-
enced by the incidence of blackspot disorder. The
squared loadings (Figure 6(b)) shows that epicatechin
and its derivatives followed by temperature and rela-
tive humidity highly contributed to the data variability
Figure 6. (a) Biplot of PCA according to the orchards as factors and (b) The squared loadings (i.e. contribution of the variables) in
the total variance captured by PCA.
9
in PC1 while PC2 is mainly contributed by SOD, PAL,
proteins, PPO and CAT.
Supplementary Table S1 presents the statistics for
each tested PLS-R model and Supplementary Figure
S12 shows the variables selected by variable impor-
tance for projection (VIP) and selectivity ratio (SR)
algorithms. By using SR, the model performed well
and it was selected to build the predictive model. The
variance captured by the model is improved from
26.69% to 84.94%, being 45.92% and 39.02% of the
variance for latent variable 1 and 2 respectively after
variable selection (Supplementary Figure S13-S14).
The model found positive correlation of blackspot
with the minimum temperature and negative correla-
tion with maximum relative humidity, maximum and
average temperature.
Figure 7, presents the classification decision tree
(DTs) performed. Maximum temperature was selected
as first important variable. With temperatures above
33°C, the probability of the blackspot incidence is
lower (i.e. 75% absence of blackspot), contrarily, 25%
of moderate blackspot incidence can be observed. In
the second node, the epicatechin derivate 2 and mini-
mum temperature are main variables related to black-
spot incidence. If epicatechin derivate 2 is superior to
6.9, it is predicted 86% of the probability of absence of
blackspot disorder. Minimum temperatures lower
than 5°C may drive blackspot disorder in Hass avo-
cado fruits. Epicatechin derivative 4 is other important
compound that may contribute in alleviating the dis-
order in fruits. The model performed well and pre-
sented 95% of accuracy with the Cohen’s kappa of
0.913, 100% of sensitivity and specificity except for
class ‘severe blackspot’ which presented 94% of speci-
ficity due to the misclassification rate observed in the
confusion matrix.
10 V. G. UARROTA ET AL.

1
Absence_of _BS
.55 .15 .15 .15
100%
yes T_max >= 33 no

2 3

Absence_of _BS Moderate_BS

.75 .25 .00 .00 .25 .00 .37 .37
60% 40%
D er _2 >= 6. 9 T_mi n >= 5

6
Sev ere_BS
.33 .00 .17 .50
30%
Der_4 >= 8

13
Sev ere_BS
.05 .00 .24 .71
21%
Epicatechin < 13

4 5 12 26 27 7

Absence_of _BS Lower_BS Absence_of _BS Moderate_BS Sev ere_BS Moderate_BS

.86 .14 .00 .00 .27 .73 .00 .00 1.00 .00 .00 .00 .12 .00 .62 .25 .00 .00 .00 1.00 .00 .00 1.00 .00
49% 11% 9% 8% 13% 10%

Classification Decision tree model for Blackspot Incidence

Figure 7. Classification decision tree of the blackspot incidence and the main variables selected by the machine leaning
technique.

Conclusions
The results presented prompt us to conclude that
the weight of the fruits at harvest and weight loss
during the storage period is involved in blackspot
disorder development. Large incidence of lentice-
losis may drive blackspot spot development during
cold storage. Higher blackspot incidence in the
orchards was characterized by decrease in SOD,
CAT, POD, PAL activities, phenolics and epicate-
chin levels from early to late harvest. PCA analysis
showed epicatechin and certain derivatives, tem-
perature, relative humidity and enzymes as impor-
tant specific variables that affect the blackspot
disorder while PLS-R predictive model showed
positive correlation between minimum tempera-
tures and blackspot disorder. Beside such preli-
minary results, more research must be conducted
combining quality parameter variables, agro-
climatic conditions, antioxidant defence system
with transcriptomics for better understanding the
potential biomarkers related to blackspot disorder
in Hass avocado.
Acknowledgments
This research was funded by the Comité de Paltas Chile
and associated producers and exporters (Santa Cruz, El
Parque, Jorge Schmidt, Baika, Subsole). The authors also
acknowledge Sociedad Gardiazabal y Mena Ltda and
Instituto de Investigaciones Agropecuarias (INIA) La
Platina. The first author (Virgílio Uarrota) and corre-
sponding author (Romina Pedreschi) thank CONICYT-
FONDECYT (Chile), projects 3190055 and 1180303 for
sponsorship and Vicerrectoria de Investigación
y Estudios avanzados (VRIEA) of Pontificia Universidad
Catolica de Valparaiso for all research facilities provided.
Disclosure statement
No potential conflict of interest was reported by the
authors.
Funding
This work was supported by the Comité de Paltas Chile and
associated producers and exporters (Santa Cruz, El Parque,
Jorge Schmidt, Baika, Subsole) [NA].
 THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY
ORCID
Virgílio Gavicho Uarrota http://orcid.org/0000-0002-
2974-1625
Claudio Zulueta http://orcid.org/0000-0002-9232-9705
Rosana Chirinos http://orcid.org/0000-0002-9045-7471
David Campos http://orcid.org/0000-0003-1722-1187
Romina Pedreschi http://orcid.org/0000-0001-9927-
2919
References
Adamson, G., Lazarus, S., Mitchell, A., Prior, R., Cao, G.,
Jacobs, P., . . . Schmitz, H. (1999). HPLC method for the
quantification of procyanidins in cocoa and chocolate
samples and correlation to total antioxidant capacity.
Journal of Agricultural and Food Chemistry, 47(10),
4184–4188. doi:10.1021/jf990317m
Aebi, H. (1984). Catalase in vitro. Methods in Enzymology,
105, 121–126.
Assis, J., Maldonado, R., Muñoz, T., Escribano, M., &
Merodio, C. (2001). Effect of high carbon dioxide con-
centration on PAL activity and phenolic contents in
ripening cherimoya fruit. Postharvest Biology and
Technology, 23(1), 33–39. doi:10.1016/S0925-5214(01)
00100-4
Aydaş, S., Ozturk, S., & Aslım, B. (2013). Phenylalanine ammo-
nia lyase (PAL) enzyme activity and antioxidant properties
of some cyanobacteria isolates. Food Chemistry, 136,
164–169. doi:10.1016/j.foodchem.2012.07.119
Bailly, C., & Kranner, I. (2011). Analyses of reactive oxygen
species and antioxidants in relation to seed longevity and
germination. Seed Dormancy, 773, 343–367.
Baumgartner, M., Keller, E.R., & Schwendimann, F. (1983).
Versuch einer Charakteriesierung von blaustabilität und
blaulabilität bei der kartoffel durch knolleneigenschaften
(An investigation into the stability and instability of the
‘blue reaction’in potato by an examination of tuber char-
acteristics). Potato Research, 26, 15–30. doi:10.1007/
BF02357370
Bi, X., Hemar, Y., Balaban, M., & Liao, X. (2015). The effect
of ultrasound on particle size, color, viscosity and poly-
phenol oxidase activity of diluted avocado puree.
Ultrasonics Sonochemistry, 27, 567–575. doi:10.1016/j.
ultsonch.2015.04.011
Bill, M., Korsten, L., Remize, F., Glowacz, M., & Sivakumar, D.
(2017). Effect of thyme oil vapours exposure on phenylala-
nine ammonia-lyase (PAL) and lipoxygenase (LOX) genes
expression, and control of anthracnose in ‘Hass’ and ‘Ryan’
avocado fruit. Scientia Horticulturae, 224, 232–237.
doi:10.1016/j.scienta.2017.06.026
Blakey, R.J., Bower, J.P., & Bertling, I. (2009). Protein and
sugar trends during the avocado season. South African
Avocado Growers’ Association Yearbook, 32, 18–21.
Bradford, M.M. (1976). A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Analytical Biochemistry,
72, 248–254. doi:10.1016/0003-2697(76)90527-3
California Avocado Commission. (2020). Effects of wind on
California avocado trees. Retrieved from https://www.cali
forniaavocadogrowers.com/cultural-management-library
/effects-wind-california-avocado-trees
Carpenter, C.W. (1931). Diseases of the avocado. California
Avocado Association 1931 Yearbook, 16, 102–106.
Cheng, G., & Breen, P. (1991). Activity of phenylalanine
ammonia-Lyase (PAL) and concentrations of anthocyanins
11
and phenolics in developing strawberry fruit. Journal of the
American Society for Horticultural Science, 116(5), 865–869.
doi:10.21273/JASHS.116.5.865
Decros, G., Baldet, P., Beauvoit, B., Stevens, R., Flandin, A.,
Colombie, S., . . . Petriarcq, P. (2019). Get the balance right:
ROS homeostasis and redox signaling in fruit. Frontiers in
Plant Science, 10, e1091. doi:10.3389/fpls.2019.01091
Dreher, M., & Davenport, A. (2013). Hass avocado compo-
sition and potential health effects. Critical Reviews in
Food Science and Nutrition, 53, 738–750. doi:10.1080/
10408398.2011.556759
Escobar, J., Rodriguez, P., Cortes, M., & Correa, G. (2019).
Influencia de la materia seca como Índice de madurez de
cosecha y tiempo de almacenamiento en frío sobre la
calidad del aguacate cv. Hass producido en la región del
trópico alto. Información Tecnológica, 30, 199–210.
doi:10.4067/S0718-07642019000300199
Everett, K., Boyd, L., & Pak, H. (2007). Cutting J. Calcium,
fungicide sprays and canopy density influence posthar-
vest rots of avocado. Australasian Plant Pathology, 36, 22.
doi:10.1071/AP06076
Everett, K., Hallett, I., Rees-George, J., Chynoweth, R., &
Pak, H. (2008). Avocado lenticel damage: The cause and
the effect on fruit quality. Postharvest Biology and
Technology, 48, 383–390. doi:10.1016/j.postharvbio.2007.
09.008
Everett, K.R., Pushparajah, I.P.S., Woolf, A.B., Burdon, J.N.,
Escobedo, V., & Vasquez, K. (2015). Investigation of the
cause of ‘Black spot’ disorder of avocado fruit in Peru. In
Proceedings of VIII Congreso Mundial de la Palta (pp.
436–440). Lima, Perú.
FAOSTAT. (2018). Food and agriculture statistical division.
Retrieved from www.fao.org/faostat/en/#data/TP
FreshPlaza. (2019). The world’s 10 largest avocado exporters.
Retrieved from https://www.freshplaza.com/article/
9165959/the-world-s-10-largest-avocado-exporters/
Fukai, T., & Ushio-Fukai, M. (2011). Superoxide dismutases:
Role in Redox Signaling, vascular function and diseases.
Antioxidant and Redox Signaling, 15, 1583–1606.
doi:10.1089/ars.2011.3999
Gancarz, M. (2018). At harvest prediction of the suscept-
ibility of potato varieties to blackspot after impact over
long-term storage. Postharvest Biology and Technology,
142, 93–98. doi:10.1016/j.postharvbio.2018.01.009
Genga, A., Mattana, M., Corragio, I., Locatteli, F.,
Piffanelli, P., & Consonni, R. (2011). Plant metabolomics:
A characterization of plant responses to abiotic stresses.
In: A. Shanker Prof. (Ed.), Abiotic stress in plants -
Mechanisms and adaptations. ISBN: 978-953-307-394-1,
InTech. doi:10.5772/23844
Gerasimova, N., Pridvorova, S., & Ozeretskovskaya, O.
(2005). Role of L -Phenylalanine ammonia Lyase in the
induced resistance and susceptibility of sotato plants.
Applied Biochemistry and Microbiology, 41, 103–105.
Giannopolitis, C., & Ries, S. (1977a). Superoxide dismutases:
I. Occurrence in higher plants. Plant Physiology, 59,
309–314. doi:10.1104/pp.59.2.309
Giannopolitis, C., & Ries, S. (1977b). Superoxide dismu-
tases. II. Purification and quantitative relationship with
water-soluble protein in seedlings. Plant Physiology, 59
(2), 315–318. doi:10.1104/pp.59.2.315
Glowacz, M., Bill, M., Tinyane, P., & Sivakumar, D. (2017).
Maintaining postharvest quality of cold stored ‘Hass’ avoca-
dos by altering the fatty acids content and composition with
the use of natural volatile compounds-methyl jasmonate and
methyl salicylate. Journal of the Science of Food and
Agriculture, 97, 5186–5193. doi:10.1002/jsfa.8400
12 V. G. UARROTA ET AL.
Gobbo-Neto, L., & Lopes, N. (2007). Plantas medicinais:
Fatores de influência no conteúdo de metabólitos
secundários. Química Nova, 30(2), 374–381.
doi:10.1590/S0100-40422007000200026
Guetsky, R., Kobiler, I., Wang, X., Perlman, N., Gollop, N.,
Avila-Quezada, G., . . . Prusky, D. (2005). Metabolism of
the flavonoid epicatechin by laccase of Colletotrichum gloeos-
porioides and its effect on pathogenicity on avocado fruits.
Phytopathology, 95, 1341–1348. doi:10.1094/PHYTO-95-
1341
Gupta, D.K., Palma, J.M., & Corpas, F.J. (2018).
Antioxidants and antioxidant enzymes in higher plants
(1st ed.). Springer International Publishing. ISBN: 978-
3-319-75088-0. doi:10.1007/978-3-319-75088-0
Lewis, C.E. (1978). The maturity of avocados-a general
review. Journal of the Science of Food and Agriculture,
29, 857–866. doi:10.1002/jsfa.2740291007
Maksimović, J., & Živanović, B. (2012). Quantification of
the antioxidant activity in salt-stressed tissues. In:
Shabala S., Cuin T. (eds) Plant salt tolerance. Methods
in molecular biology (methods and protocols), vol 913.
Humana Press, Totowa, NJ. Plant Salt Tolerance, 913,
237–250.
Mazhar, M., Joyce, D., Hofman, P., & Vu, N. (2018). Factors
contributing to increased bruise expression in avocado
(Persea americana Mill.) cv. ‘Hass’ fruit. Postharvest
Biology And Technology, 143, 58–67. doi:10.1016/j.
postharvbio.2018.04.015
Mittler, R. (2002). Oxidative stress, antioxidants and stress
tolerance. Trends in Plant Science, 7(9), 405–410.
doi:10.1016/S1360-1385(02)02312-9
Mittler, R. (2017). ROS are good. Trends in Plant Science, 22
(1), 11–19. doi:10.1016/j.tplants.2016.08.002
Ncama, K., Magwaza, L., Poblete-Echeverría, C.,
Nieuwoudt, H., Tesfay, S., & Mditshwa, A. (2018). On-tree
indexing of ‘Hass’ avocado fruit by non-destructive assess-
ment of pulp dry matter and oil content. Biosystems
Engineering, 174, 41–49. doi:10.1016/j.biosystemseng.2018.
06.011
Nokthai, P., Lee, V., & Shank, L. (2010). Molecular modeling of
peroxidase and polyphenol oxidase: Substrate specificity and
active site comparison. International Journal of Molecular
Sciences, 11, 3266–3276. doi:10.3390/ijms11093266
Obianom, C., Romanazzi, G., & Sivakumar, D. (2019).
Effects of chitosan treatment on avocado postharvest
diseases and expression of phenylalanine
ammonia-lyase, chitinase and lipoxygenase genes.
Postharvest Biology and Technology, 147, 214–221.
doi:10.1016/j.postharvbio.2018.10.004
Ochoa-Ascencio, S., Hertog, M., & Nicolaï, B. (2009).
Modelling the transient effect of 1-MCP on ‘Hass’ avo-
cado softening: A Mexican comparative study.
Postharvest Biology and Technology, 51(1), 62–72.
doi:10.1016/j.postharvbio.2008.06.002
Pak, H.A., Dixon, J., & Cutting, J.G.M. (2003). Influence of
early season maturity on fruit quality in New Zealand
‘Hass’ avocados. New Zealand avocado grower’s associa-
tion annual research report. Tauranga, 3, 54–59.
Pedreschi, R., Uarrota, V., Fuentealba, C., Alvaro, J.,
Olmedo, P., Defilippi, B., . . . Campos-Vargas, R. (2019).
Primary metabolism in avocado fruit. Frontiers in Plant
Science, 10, article 795. doi:10.3389/fpls.2019.00795
Popović, B., Štajner, D., Ždero-Pavlović, R., Tumbas-
Šaponjac, V., Čanadanović-Brunet, J., & Orlović, S.
(2016). Water stress induces changes in polyphenol pro-
file and antioxidant capacity in poplar plants (Populus
spp.). Plant Physiology and Biochemistry, 105, 242–250.
doi:10.1016/j.plaphy.2016.04.036
R Core Team. (2019). R: A language and environment for
statistical computing. Vienna, Austria: R Foundation for
Statistical Computing. Retrieved from https://www.
R-project.org/
Ramírez-Gil, J.G., López, J.H., & Henao-Rojas, J.C. (2020).
Causes of Hass avocado fruit rejection in preharvest,
harvest, and packinghouse: Economic losses and asso-
ciated variables. Agronomy, 10, 1–13.
Saavedra, J., Córdova, A., Navarro, R., Díaz-Calderón, P.,
Fuentealba, C., Astudillo-Castro, C., & Galvez, L. (2017).
Industrial avocado waste: functional compounds preservation
by convective drying process. Journal Of Food Engineering, 198,
81–90. doi: 10.1016/j.jfoodeng.2016.11.018
Uarrota, V., Severino, R., & Maraschin, M. (2011). Maize
Landraces (Zea mays L.): A new prospective source for
secondary metabolite production. International Journal of
Agricultural Research, 6(3), 218–226. doi:10.3923/
ijar.2011.218.226
Uarrota, V.G., Fuentealba, C., Hernández, I., Defilippi-
Bruzzone, B., Meneses, C., Campos-Vargas, R., . . .
Pedreschi, R. (2019a). Integration of proteomics and
metabolomics data of early and middle season Hass avo-
cados under heat treatment. Food Chemistry, 289,
512–521. doi:10.1016/j.foodchem.2019.03.090
Uarrota, V.G., Segatto, C., Voytena, A., Maraschin, M.,
Avila, L., Kazama, D., . . . Souza, C.A. (2019b).
Metabolic fingerprinting of water-stressed soybean culti-
vars by gas chromatography, near-infrared and
UV-visible spectroscopy combined with chemometrics.
Journal of Agronomy and Crop Science, 205(2), 141–156.
doi:10.1111/jac.12311
Wang, M., Zheng, Y., Khuong, T., & Lovatt, C. (2016).
Developmental differences in antioxidant compounds and
systems in normal and small-phenotype fruit of ‘Hass’ avo-
cado (Persea americana Mill.). Scientia Horticulturae, 206,
15–23. doi:10.1016/j.scienta.2016.04.029
Woolf, A., Wibisono, R., Farr, J., Hallett, I., Richter, L.,
Oey, I., . . . Requejo-Jackman, C. (2013). Effect of high
pressure processing on avocado slices. Innovative Food
Science and Emerging Technologies, 18, 65–73.
doi:10.1016/j.ifset.2013.02.011
Yang, L., Wen, K., Ruan, X., Zhao, Y., Wei, F., & Wang, Q.
(2018). Response of plant secondary metabolites to envir-
onmental factors. Molecules, 23(4), 762, 1–26.