Postharvest Biology and Technology 163 (2020) 111123

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Postharvest Biology and Technology

journal homepage: www.elsevier.com/locate/postharvbio

Sodium alginate coatings added with Meyerozyma caribbica: Postharvest T

biocontrol of Colletotrichum gloeosporioides in avocado (Persea americana
Mill. cv. Hass)
Maricarmen Iñiguez-Moreno, Juan Arturo Ragazzo-Sánchez, Julio César Barros-Castillo,
Teresa Sandoval-Contreras, Montserrat Calderón-Santoyo*
Laboratorio Integral de Investigación en Alimentos, Tecnológico Nacional de México/Instituto Tecnológico de Tepic, Av. Tecnológico 2595, Tepic, Nayarit, 63175, Mexico

A R T I C LE I N FO A B S T R A C T

Keywords: Edible coatings have been used as a medium for the incorporation of functional compounds and biocontrol
Biocontrol agents agents to postharvest diseases control on fruits and to maintain their quality parameters. However, there are few
Postharvest pathogens reports about the use of biocontrol agents entrapped in polymeric matrices; to our knowledge, there is no report
Subtropical fruits of their application to anthracnose control in avocado fruit. Hence the aims of this study were to investigate the
Edible coatings
production of volatile organic compounds (VOCs) by Meyerozyma caribbica and the ability of the yeast entrapped
in sodium alginate (SA) coatings to control anthracnose caused by Colletotrichum gloeosporioides Pa14 in avocado
fruit. The yeast viability, biocontrol activity, effect on weight loss as well as the efficacy of the bioactive coatings
to preserve postharvest quality by prevention or cure of C. gloeosporioides infection were assessed. The main
VOCs identified were alcohols (1-Butanol, 3-methyl- and phenethyl alcohol) and esters (ethyl acetate). Results
revealed SA as a matrix able to maintain the yeast viability during the storage on coated avocado fruit, with
minimal reductions between 0.39 and 1 Log10 CFU mL−1, depending on storage conditions. Moreover, it was
demonstrated the ability of the yeast to increase its population during the ripening of avocado fruit previously-
stored at 6 °C. Films with M. caribbica were capable to inhibit C. gloeosporioides growth in vitro. Meanwhile, in
vivo, the preventive treatments were more effective than curative treatments in anthracnose controlling; at 25 °C
the severity was halved, while at 6 °C the reduction reached 100 %. Additionally, in avocado fruit with the
bioactive coatings, the weight loss was reduced by 2–3.7 % respect to the control. In conclusion, this study
demonstrates the ability of M. caribbica to produce VOCs as a mechanism of action against C. gloeosporioides;
additionally, SA coatings with M. caribbica were effective to reduce the weight loss and its potential as an
alternative to control of postharvest diseases in avocado fruit.

1. Introduction
Mexico is the main producer of avocado fruit (Persea americana) in
the world. During 2017, Mexico produced 2.029.886 tons of this fruit
(FAOSTAT, 2019) and exported 985.653 tons with a value of more than
2.000 million dollars (SAGARPA, 2018). Avocado fruit has high eco-
nomic value in the world; however, this fruit is highly susceptible to
mechanical injury (especially bruising), physiological deterioration,
and microbiological decay during the postharvest stage (Bill et al.,
2014a, 2014b). Major postharvest losses are encountered through the
supply chain typically due to anthracnose disease caused by Colleto-
trichum gloeosporioides, whose infections may often begin in the pre-
harvest stage (Aiello et al., 2015; Benyahia et al., 2003). Anthracnose
occurs in fruit and vegetable crops worldwide (Bill et al., 2014a); in
avocado fruit the losses due to this disease can reach the 100 %, de-
pending on environmental conditions (temperature and relative hu-
midity) during the postharvest stage (Landero-Valenzuela et al., 2016).
Conventionally, anthracnose control is achieved using chemical
fungicides, such as benomyl, carbendazim, mancozeb, and prochloraz
(Zhou et al., 2016). In Mexico, the Maximum Residue Limits (MRL) are
regulated in function to the Codex Alimentarius (FAO/WHO, 2018),
however, the avocado fruit producers must to considerer the norma-
tivity of the country at which the avocado fruit will be exported; being
the USA the main consumer (SAGARPA, 2018). Recent studies have
shown that C. gloeosporioides has developed middle to high levels of
resistance against many different fungicidal active ingredients,

⁎
Corresponding author.
E-mail address: mcalderon@ittepic.edu.mx (M. Calderón-Santoyo).

https://doi.org/10.1016/j.postharvbio.2020.111123
Received 10 April 2019; Received in revised form 29 December 2019; Accepted 14 January 2020
0925-5214/ © 2020 Elsevier B.V. All rights reserved.
M. Iñiguez-Moreno, et al.
including benzinidazoles (Chung et al., 2010), dithiocarbamate and
azoles (Osorio et al., 2014; Xu et al., 2014). Hence, an increase in the
concentration fungicides uses, increase the risks to environmental,
phytosanitary and human health, consequently, the development of
novel methods for the control of postharvest decay is required (Mari
et al., 2016). For these reasons, antagonistic microorganisms have been
used as an effective disease management strategy aimed to reduce the
concentration of chemicals agents and to prevent the appearance of
resistant phytopathogens (Moretto et al., 2014). Bacteria and yeasts are
mainly chosen due to having a lower replication time hence they can
colonize the epicarp of the fruit more efficiently than phytopathogenic
fungi (Carmona-Hernandez et al., 2019). Avogreen is a product devel-
oped by the University of Pretoria based on Bacillus subtilis to Cercospora
spp. and Colletotrichum spp. control in avocado fruit during preharvest
or postharvest stage (Demoz and Korsten, 2006). However, actually is
not in use due to this product generate a honeydew on the fruit and
leaves, making to the fruit more susceptible to attack of insects favoring
colonization and decay caused by phytopathogenic fungi, affecting the
avocado fruit quality (Zhang et al., 2018). In relation to the yeast use,
previous studies have reported the antagonistic activity of Debar-
yomyces hansenii, Rhodosporidium fluviale, Meyerozyma caribbica and
Wickerhamomyces anomalus against phytopathogenic fungus on fruit
such as papaya (Hernandez-Montiel et al., 2018), apples (Sansone et al.,
2018) mangoes (Aguirre-Güitrón et al., 2018; Bautista-Rosales et al.,
2013), and avocado fruit (Campos-Martínez et al., 2016); respectively.
M. caribbica have different antagonistic mechanisms against C. gloeos-
porioides (Bautista-Rosales et al., 2013); however, volatile organic
compounds (VOCs) as a mechanism of action against phytopathogenic
fungus has not been studied. These compounds are produced by yeasts,
molds, and bacteria during their primary and secondary metabolism
(Korpi et al., 2009). VOCs are compounds of low molecular weight
(< 300 Da), characterized by low polarity and high vapor pressure,
whose production is strongly influenced by the microbial species, the
growth phase and the environmental conditions (Korpi et al., 2009).
VOCs could be considered as ideal antimicrobials since the contact
between the biocontrol agent and the pathogen agent is not required to
perform their activity (Contarino et al., 2019). Previously, it has been
reported the in vitro activity of 2-methyl-1-butanol and isobutyric acid
produced by the fungus Muscodor albus against diverse phytopathogen
fungi (Mercier and Jiménez, 2004). Additionality, B. subtilis and Bacillus
amyloliquefaciens can produce 21 VOCs with antagonistic activity
against Penicillium digitatum Sacc., Penicillium italicum Wehmer and Pe-
nicillium crustosum Thom (Arrebola et al., 2010).
Furthermore, to improve the yeast establishment and maintenance
on the fruit surface it is necessary the use of a polymeric matrix. There
is a diversity of polymers with a potential to be used as coatings, such as
pectin, starch, locus bean gum and sodium alginate (González-Estrada
et al., 2017; Marín et al., 2017). Sodium alginate (SA) is a poly-
saccharide quite abundant in nature; structural component in marine
brown algae (Phaeophyceae, mainly Laminaria) (Parreidt et al., 2018;
Rhim, 2004), recognized as “Generally Recognized as Safe” (GRAS,
E401) and used as a texturizer, emulsifier, stabilizer, thickener, and
gelling agent in the food industry (FDA, 2018). Additionally, studies
have demonstrated that SA can trap volatile compounds such as es-
sential oils, because according to increased concentrations (1–2 %) of
the biopolymer, the viscosity increment, favoring the compounds re-
tention and making the volatilization more difficult during the film
storage (Aloui et al., 2014; Liakos et al., 2014). The use of edible
coatings to carry antagonistic microorganisms, to be used at both pre-
and postharvest conditions, is an area less explored (Marín et al., 2017).
To our knowledge, there is no report about the application of a bioac-
tive coating based on the use of biopolymer of low cost such as SA with
yeast to anthracnose control in avocado fruit during the postharvest
stage, having a promising future in the avocado fruit industry. Hence,
the objectives of this work were to i) maintain M. caribbica viability
incorporated in films and coatings of SA, ii) determinate the in vitro
2
Postharvest Biology and Technology 163 (2020) 111123
antagonistic activity of M. caribbica against C. gloeosporioides isolated
from avocado fruit iii) identify the VOCs produced by M. caribbica and
iv) ascertain M. caribbica potential ability in SA coatings to control of
anthracnose caused by C. gloeosporioides in avocado fruit during the
postharvest stage.
2. Materials and methods
2.1. Plant materials
Healthy and size homogeneous avocado fruit (Persea americana Mill.
cv. Hass) at the second stage of maturity (slightly soft just started to
ripen) (Sellamuthu et al., 2013) without mechanical injury were se-
lected. Fruit was obtained from Leb Trading Products (Guacamodely) S.
de R.L. de C.V. (21°43′ N, -104°90′ W) located in Xalisco, Nayarit
(Mexico) and transported to the Food Microbiology Laboratory at In-
stituto Tecnológico de Tepic in polypropylene boxes to avoid mechan-
ical damage. Sodium alginate (SA; Sigma Aldrich, St Louis, USA) and
glycerol (99.5 % purity; Jalmek; Nuevo León, Mexico) was used as a
plasticizer.
2.2. Pathogen and antagonistic microorganism
Colletotrichum gloeosporioides Pa14 (GenBank ID: MN477464) was
isolated from decayed avocado fruit in Nayarit, Mexico and stored onto
PDA medium at 4 °C. The conidial suspension was prepared using 10 d
old fungal cultures in PDA Petri dishes, 15 mL of sterile distilled water
(SDW) containing 0.1 % (v/v) Tween 80 (Sigma Aldrich, Saint Quentin
Fallavier, France) and 0.85 % (w/v) sodium chloride (NaCl; Sigma
Aldrich, St. Louis, USA) were added to the cultures and Petri dishes
were scraped using a sterile loop. The liquid was filtered in sterile gauze
and recovered in a dilution flask. Spore concentration was determined
by microscopic counting in a hemocytometer (LO - Laboroptik Ltd;
Lancing, UK) (González-Estrada et al., 2017). M. caribbica (GenBank ID:
JQ398674) was previously isolated from “Ataulfo” mangoes (Bautista-
Rosales et al., 2013). The yeast was stored in yeast extract-peptone-
dextrose (YPD) broth (yeast extract 10 g, peptone 20 g, dextrose 20 g,
agar 15 g, dissolved in 1 L of distilled water; Oxoid, Basingstoke, UK)
with 40 % (v/v) glycerol (Sigma-Aldrich, Steinheim, Germany) at -80
°C; and was reactivated in YPD broth and 10 % v/v autoclaved myce-
lium of C. gloeosporioides was added as a substrate to induce the
synthesis of β-1,3-glucanase and chitinase; was incubated at 28 °C for
70 h as described by Bautista-Rosales et al. (2013).
2.3. In vitro antagonistic activity
2.3.1. Evaluation in vitro of M. caribbica antagonistic activity
The determination of the antagonistic activity of M. caribbica
against C. gloeosporioides was performed on Potato Dextrose Agar (PDA,
Oxoid, Basingstoke, UK) Petri dishes. Each plate was streaked with 100
μL of yeast suspension (1 × 109 cells mL−1) and was incubated for 1 h
at 25 °C. Then were inoculated in the center with a mycelium plug (7
mm) from a 7 d culture of the fungus in PDA or with 50 μL of spore
suspension (1 × 105 spores mL−1). Finally, the inoculated medium was
incubated at 6 and 25 °C (storage temperature for avocado fruit and
room temperature, respectively). Plates were covered with parafilm to
avoid dehydration. The growth diameter was registered during 7 d (25
°C) and 20 d (6 °C). Petri dishes with fungus plug were included as a
positive control for C. gloeosporioides growth. The percentage of in-
hibition of mycelial growth was calculated according to Eq. 1:
dc − dt ⎞
Inhibition (%) = ⎛ 100
⎝ dc ⎠ (1)
where dc (cm) is the mean of colony diameter for the control and dt
(cm) is the mean of colony diameter for the treatment. Three replicates
were used for each treatment and the experiment was repeated once.
M. Iñiguez-Moreno, et al.
2.3.2. Volatile organic compounds (VOCs)
The antifungal activity of VOCs was determined in vitro by the
method proposed by Hernandez-Montiel et al. (2018). Each Petri dish
with PDA was inoculated in the center with a plug from a 7 d culture of
the fungus in PDA or with 50 μL of spore suspension (1 × 105 spores
mL−1). At the same time, 100 μL of the yeast suspension (1 × 109 cells
mL−1) were streaked in other plates containing PDA and were let to dry
for 1 h at 25 °C. Each inoculated plate was placed mouth-to-mouth,
sealed with three laps of parafilm and incubated for 7 d at 25 °C and 20
d at 6 °C. Petri dishes with mycelial plug and other with spores, both in
yeast absence were included as a positive control for C. gloeosporioides
growth. The percentage of inhibition of mycelial growth was calculated
according to Eq. 1. Three replicates were used for each treatment and
the experiment was repeated once.
2.4. Identification of volatile organic compounds (VOCs) produced by M.
caribbica
The identification of VOCs produced by M. caribbica was performed
using headspace solid-phase microextraction coupled to gas chroma-
tography with mass spectrometry (SPME-GC–MS) analyses. The cul-
tures were obtained such above and were incubated at 6 °C for 20 d and
at 25 °C for 7 d. At the end of the incubation period, the sealed Petri
dishes were kept for 15 min at 30 °C to achieve equilibrium of VOCs in
the headspace, then a syringe was manually inserted into a small hole in
the parafilm, to trapping the VOCs produced, a fiber coated with 50/30
μm divinylbenzene/polydimethylsiloxane/carboxen (DVB/PDMS/CAR)
(Supelco: Bellefonte, USA) was employed. The fiber was exposed to the
air space for 15 min and thermally desorbed into the hot injector of the
GC (Model 7890A, Agilent Technologies; Palo Alto, USA) coupled to a
MS (Model 270; Agilent technologies, Palo Alto, USA) at 250 °C using
split injection, equipped with a VF-5MS capillary column (30 m ×0.25
mm ×0.25 μm) coated with 5 % diphenyl 95 % dimethylpolysiloxane.
The oven temperature was set at 40 °C for 3 min and then programmed
to rise from 40 to 90 °C at 10 °C min−1, from 90 °C to 180 °C at 5 °C
min−1 and from 180 °C to 260 °C at 20 °C min−1. The transfer line was
heated at 250 °C and the ion source at 220 °C. Helium carrier gas had a
flow of 1 mL min−1. The mass spectrometer was operated in electron
impact mode at 70 eV, scanning the range of 35/500 m/z, in a full scan
acquisition mode (Di Francesco et al., 2015). To identify the VOCs the
GC retention times and mass spectra were cross-referenced with Na-
tional Institute of Standards and Technology (NIST 11 MS Library).
Controls consisted of i) Petri dishes with PDA and ii) Petri dishes con-
taining the phytopathogenic fungi in the absence of yeast. Controls
were performed under the same conditions as the sample in order to
exclude interfering substance. All measurements were made with three
replicates, each replicate representing the analysis of a different Petri
dish.
2.5. Preparation of films and coatings solution
Films and coatings were prepared with 1.5 % SA (w/v) and 20 %
glycerol (w/w) in relation to dry SA (Aloui et al., 2015); the solution
was stirred at a controlled temperature of 80 °C until the mixture be-
came clear. Additionally, M. caribbica was added to give a final con-
centration of 1 × 109 cells mL−1. The films were left to dry for 20 h at
25 °C in a biosecurity cabinet (Novatech, Model CFLV-120, Kingwood,
USA). Before the application of coating-forming solutions, avocado fruit
was surface-disinfected by dipping in 2 % sodium hypochlorite solution
(v/v) for 2 min, and then were washed with SDW. For coating treat-
ment, the casting method was used, the avocado fruit were dipped for 1
min in the coating-forming solution; then, the fruit was left to dry for 1
h at 25 °C and 45 % RH in a biosafety cabinet (González-Estrada et al.,
2017). Fruit was stored during 15 d for evaluation of weight loss.
3
Postharvest Biology and Technology 163 (2020) 111123
2.6. Evaluation of properties of SA films
2.6.1. Viability of M. caribbica during films storage
The viability of the antagonistic yeast incorporated into films and
stored at 6 and 25 °C was assessed as follows: films were aseptically
placed into a sterile flask with 100 mL of sterile saline solution (0.85
%). Then, the solution containing the film was stirred at 100 rpm at 25
°C for 1 h to dissolve the film and allow microorganisms to release into
the solution. Serial dilutions were prepared and 100 μL of the sample
was spread on YPD. Counts for M. caribbica were performed after 48 h
incubation at 28 °C. For each experiment were used three replicates for
each treatment and the experiment was repeated once.
2.6.2. In vitro antifungal properties of SA films
Prior to the experiment, C. gloeosporioides was pre-cultured on PDA
for 10 d at 25 °C. Conidial suspensions were obtained as indicated
above. Spore concentration was adjusted to 1 × 105 spores mL−1 by
microscopic counting in a hemocytometer. Fifty mL of fungal spores
suspension (1 × 105 spores mL−1) were spread onto PDA disks (1.4 cm)
and then let to dry for 10 min in a biosafety cabinet. After that, the disks
were covered with films containing or not the antagonistic yeast.
Subsequently, the Petri dishes were incubated at 6 and 25 °C. After
incubation time, the films were aseptically removed to determine the
germination rate of C. gloeosporioides by microscopy. The percentage of
germinated spores was measured after 6 (25 °C) and 44 h (6 °C) in
samples of approximately 200 spores (González-Estrada et al., 2017). A
spore was considered germinated when its germ tube was longer than
half the length of the spore (Fernando et al., 2000). The determination
of the antifungal performance of films against C. gloeosporioides growth
was made on PDA plugs (7 mm) with mycelium (growth 7 d at 25 °C).
The plugs were aseptically placed onto PDA medium without fungus
and with 50 μL of spore suspension (1 × 105 spores mL−1). Then, the
inoculated medium was covered with films containing or not yeasts.
Finally, the inoculated medium covered by films was incubated at 6 and
25 °C. Plates were covered with parafilm to avoid dehydration. The
growth diameter was registered during 7 (25 °C) and 20 d (6 °C). In-
oculated but uncoated PDA Petri dishes were used as a positive control
to C. gloeosporioides growth (González-Estrada et al., 2017). The in-
hibition percentage of mycelial growth was calculated according to Eq.
1. For each experiment were used three replicates for each treatment
and the experiment was repeated once.
2.7. Evaluation of SA coatings with yeast applied on avocado fruit
2.7.1. Viability of M. caribbica in coatings during avocado fruit storage
The viability of yeast in coatings was evaluated under three storage
conditions 1) 6 °C for 15 d; 2) 25 °C for 15 d and 3) 6 °C for 10 d and
ripened at 25 °C for 5 d, to mimic the conditions at the retail level.
Individually, the fruit were placed into sterile stomacher bags (Seward,
BA6041/CLR) containing 200 mL of sterile physiological saline (0.85 %
NaCl), the yeasts were removed by sonication (90 s, KENDAL, Model CD
48-20 operating at 50/60 Hz, Middletown, USA). Then, the solution
containing only the coating was homogenized for 30 s at 260 rpm, using
a stomacher blender (Stomacher 400, Circulator) to allow micro-
organisms to release into the solution. Serial dilutions were prepared
and 100 μL samples of appropriate dilutions were spread on YPD.
Colonies were counted after incubation for 48 h at 28 °C. For each
experiment, three replicates were used and the experiment was re-
peated once.
2.7.2. Curative and preventive treatments using SA coatings with M.
caribbica
Avocado fruit was wounded (2 holes per fruit, near to peduncle and
in the equatorial zone) with a sterile needle (3 mm deep and 3 mm
wide), and inoculated with 10 μL of a spore suspension of C. gloeos-
porioides (105 spores mL−1), and left to air-dry. Curative treatments
M. Iñiguez-Moreno, et al. Postharvest Biology and Technology 163 (2020) 111123

were performed by first inoculating the fruit with the spore suspension
and leaving to dry 6 h, allowing the pathogen infection before applying
the appropriate treatment. Preventive treatments were done by first
applying the treatment to wounded fruit, leaving the fruit to dry 6 h at
25 °C and then exposing treated fruit to the pathogen by inoculating
with the spore suspension. Avocado fruit inoculated before or after
immersion treatment in SDW were used as positive controls. Fruit were
stored in a camera (Novatech, Model CA-550; Kingwood, USA) with
humidifiers (Vitallys Plus, Model VUH-3, Marlborough, USA) to ensure
high relative humidity under two conditions 1) 25 °C during 15 d (75 %
RH) and 2) 6 °C (95 % RH) during 10 d and ripened at 25 °C (75 % RH)
during 5 d. Disease incidence (percentage of infected fruit), infected
wounds (percentage) and severity (lesion diameter, mm) were eval-
uated. An avocado fruit was considered decayed when at least one of
the inoculated wounds was infected. At the end of storage, the avocado
fruit were cut and the damage in the flesh was classified according to
Guarnaccia et al. (2016) scale. The treatments applied were (1) positive
controls (2) SA (1.5 %, w/v) without yeast, (3) yeast (1 × 109 CFU
mL−1), and (4) SA (1.5 %, w/v) with yeast (1 × 109 CFU mL−1). Each Fig. 1. Viability of Meyerozyma caribbica entrapped in sodium alginate
treatment was replicated twice with 10 avocado fruit per treatment. films. The graph showed the yeast viability over 20 d of storage at 6 and 25 °C.
Each point represents the mean ± standard deviation (n = 6).
2.8. Statistical analysis
phenethyl alcohol) esters (ethyl acetate) were found to be the main
Data were processed by one-way analysis of variance (ANOVA). VOCs produced. According to the incubation conditions, isoamyl
Prior to ANOVA, the percentages data were arcsine-square-root trans- acetate was detected at 6 °C but not at 25 °C (Table 2).
formed. The statistical data analysis was performed using the software
Statgraphics Centurion XV (Statpoint Technologies, Inc., Warrenton,
3.3. Evaluation of M. caribbica in SA coatings applied on avocado fruit
USA), the post-hoc least significant difference (LDS) Fisher test
(p < 0.05) was used for comparison of means. The averages per treat-
3.3.1. Viability of M. caribbica in coatings during storage
ment for the disease incidence, infected wounds, and severity are re-
Fig. 2 showed the viability of M. caribbica in SA coatings applied on
ported as their untransformed values.
avocado fruit and stored at three different conditions. At 6 °C, the cell
density had a decrement after the 6th d of storage and maintain their
3. Results
population until the 15th d, with a final reduction of 1 Log10 CFU
fruit−1. Otherwise, at 25 °C the yeast density had a decrement at the
3.1. Properties of SA films
beginning of the storage, then cell density increased between the 6th
and 12th d; to the end of the experiment, the density was similar to that
3.1.1. Viability of M. caribbica during the storage of films
obtained after 3 d of storage (p > 0.05). However, the viability on
The viability of M. caribbica incorporated in SA films was tested for
avocado fruit stored at 6 °C during 10 d and ripened at 25 °C, experi-
20 d at 6 and 25 °C in order to determine if the polymeric matrix was
mented a decrement, but with the change of temperature, the yeast
able to maintain the yeast survival under both conditions. After storage
counts increased until no significant difference (p < 0.05) with the
time, reductions of 0.57 and 0.81 Log10 CFU mL−1 (p < 0.05) were
counts at 25 °C was observed (Fig. 2).
obtained at 6 and 25 °C, respectively. At 6 °C the reduction was sig-
nificant after 12 d of storage (p < 0.05). Otherwise, at 25 °C the re-
duction was significant after 8 d (p < 0.05) and between 10 and 20 d of 3.3.2. Postharvest protection of avocado fruit with SA coatings (preventive
storage the cell density in the films did not change (p > 0.05) (Fig. 1). and curative treatments)
In this investigation, we proposed the use of SA coating with M.
3.1.2. Inhibition in vitro of C. gloeosporioides by M. caribbica caribbica to anthracnose control in avocado fruit. The results of pre-
The results of the inhibition of C. gloeosporioides by M. caribbica are ventive and curative treatments against anthracnose caused by C.
shown in Table 1. In the sealed plate assay, the microorganisms had no gloeosporioides in avocado fruit during the storage at 6 and 25 °C are
physical contact, hence the VOCs produced by M. caribbica can reduce shown in Fig. 3. The higher infection level was observed after 15 d of
the mycelial growth of C. gloeosporioides in 45 % at 25 °C and in 67 % at storage in the controls (100 % incidence). The level of anthracnose
6 °C (p < 0.05). However, when the yeast is directly in contact with the protection (incidence and infected wounds) was higher at 6 °C than at
fungus, they showed a 97–100 % of germination inhibition at 6 and 25 25 °C (p < 0.05) (Fig. 3). At 25 °C, the infected wounds and anthracnose
°C, and a 96 % inhibition of mycelial growth. The use of SA films were observed in all the treatments (100 %) except with SA + Yeast,
without yeast did not have an inhibitory effect on the germination and which had a 10 % reduction both on the incidence and infected wounds
mycelial growth at 25 °C; while, at 6 °C, these films inhibited the ger- in comparison to the control (p < 0.05). However, the severity was
mination in 50.6 %. The SA films with yeast had a great effect on reduced in 30–40 % with the SA or yeast application, and 50 % of
germination and mycelial growth inhibition (99–100 %) under tested reduction with the application of SA+Yeast in comparison to the
conditions (Table 1). control (p < 0.05) (Fig. 3B, 4A-D). After the storage, the avocado fruit
was cut to observe the flesh appearance, the flesh had more than 75 %
3.2. VOCs identification of damage in the controls (grade 4; Fig. 4E), with SA or Yeast treat-
ments the damage was grade 3; however, the obtained damage was less
In the present study the VOCs produced by M. caribbica, which were than 40 % with SA + Yeast treatment (Fig. 4F).
effective against C. gloeosporioides isolated from avocado fruit under Moreover, at 6 °C we observed a significant reduction in the in-
both tested conditions (6 °C during 20 d and 25 °C during 7 d), were cidence of anthracnose in comparison to infected wounds (20–40 %) in
identified by SPME-GC–MS. Alcohols (1-Butanol, 3-methyl- and avocado fruit coated with SA and Yeast treatments (p < 0.05).

4
M. Iñiguez-Moreno, et al. Postharvest Biology and Technology 163 (2020) 111123

Table 1
Antifungal activity of Meyerozyma caribbica against Colletotrichum gloeosporioides.
Conditions Percentage of inhibition

Germination Mycelial growth

6 °C 25 °C 6 °C 25 °C
M. caribbica 100 ± 0.0 Aa 98.6 ± 1.2 Bb 96.4 ± 3.9 Ba 96.7 ± 1.1 Ba
VOCs 83.7 ± 0.9 Ba 55.0 ± 4.5 Cb 67.1 ± 2.0 Ca 45.2 ± 2.6 Cb
Sodium alginate films 50.6 ± 0.0 Ca 0.0 ± 0.0 Db 46.3 ± 2.6 Da 0.0 ± 0.0 Db
Sodium alginate films with M. caribbica 100.0 ± 0.0 Aa 99.6 ± 0.4 Ab 99.6 ± 0.4 Aa 99.2 ± 0.7 Aa

Values are expressed as means ± standard deviation (n = 6). Values in the same column followed by different capital-case letter are significantly different according
to Fisher’s LSD test at p < 0.05. Values in the same row, that comparing germination or mycelial growth, followed by different lower-case letter are significantly
different according to Fisher’s LSD test at p < 0.05.

Table 2
Main volatile organic compounds (VOCs) produced by Meyerozyma caribbica during incubation in the presence of Colletotrichum gloeosporioides.
Possible compound Molecular formula Molecular weight Retention time (min) Linear retention index

Ethanol C2H6O 46 1.56 448

Ethyl acetate C4H8O2 88 2.23 578
Isobutyl alcohol C4H10O 74 2.35 626
1-Butanol, 3-methyl- C5H12O 88 3.94 727
1-Butanol, 2-methyl- C5H12O 88 4.02 734
Isoamyl acetatea C7H14O2 130 6.86 875
Phenethyl alcohol C8H10O 122 11.88 1120

a
Detected only in samples incubated at 6 °C during 20 d.

3.3.3. Assessment of coated avocado fruit weight loss

Changes in the weight loss of coated avocado fruit and uncoated
during storage time are shown in Fig. 5. At the three tested conditions,
the weight loss of avocado fruit increased progressively throughout the
storage time, showing a linear behavior. The lowest weight loss was
obtained in the avocado fruit coated with SA + Yeast; with a 2 % of
difference in weight loss after 15 d of storage at 6 °C and 6 °C with
ripening at 25 °C in comparison with the control (p < 0.05). However,
there was not a difference in comparison with the avocado fruit coated
with SA and M. caribbica (Fig. 5). Meanwhile, at 25 °C, the control lost
3.7 % more than the avocado fruit coated with SA + Yeast (p < 0.05).
These results suggested that SA+Yeast coatings were effective to de-
crease weight loss during avocado fruit storage. Additionally, the dif-
ferent treatments did not show a significative difference in the weight
loss in avocado fruit stored at 6 °C; however, the storage at 6 °C allows
the reduction of weight loss between 2–3.5 % in comparison to storage
at 25 °C (p < 0.05).

4. Discussion
Fig. 2. Population dynamics of Meyerozyma caribbica in coatings applied
on avocado fruit. The graph shows the yeast viability in coated avocado fruit
stored at 6 and 25 °C during 15 d and at 6 °C for 10 d and ripened at 25 °C for 5
Diverse microorganisms have been used as biocontrol agents
d. Each point represents the mean ± standard deviation (n = 6). The dotted (Carmona-Hernandez et al., 2019; Spadaro and Droby, 2016); however
line indicates the change of temperature during the avocado fruit storage. few studies have been focusing on improving their inhibitory effect
through the use of a polymeric matrix (Marín et al., 2017). Hence, in
this research, we assessed the effect of M. caribbica in SA films and
Moreover, preventive treatments with SA and Yeast reached an an-
coatings against C. gloeosporioides isolated from avocado fruit. In this
thracnose reduction of 80 % and curative treatments produced reduc-
study, the inhibition produced by the SA films without yeast is due to
tions were 30 and 50 %, respectively. However, the severity in curative
the barrier to oxygen generating a depletion in their concentration
treatments did not show a difference in comparison to the control
(Rhim, 2004); besides, the low temperature has a direct adverse effect
(p > 0.05); nonetheless, the preventive treatments revealed a sig-
on spore germination (Everett, 2003). M. caribbica showed high in-
nificative reduction in comparison to the control (p < 0.05). The avo-
hibition percentages obtained under in vitro conditions, this was attri-
cado fruit coated with SA+Yeast in the curative and preventive treat-
butable to its ability to compete for space and nutrients, to biofilm
ments did not show infected wounds, hence the anthracnose disease
development and to the synthesis of β-1,3-glucanase and chitinase
was not developed (Fig. 3A). Therefore, the application of SA + Yeast
(Bautista-Rosales et al., 2013; Liu et al., 2013). Moreover, the inhibition
in combination with the low temperature allowed a reduction of 100 %
of C. gloeosporioides by M. caribbica can be related to the production of
of anthracnose in the avocado fruit (Fig. 3). The flesh appearance of
VOCs. In this study, the inhibition percentages by VOCs were higher
avocado fruit corresponded to 0 grade (Fig. 4G).
than those reached using D. hansenii against C. gloeosporioides (16–36
%) (Hernandez-Montiel et al., 2018). Alcohols and esters were the main
VOCs identified; these compounds were associated before with the

5
M. Iñiguez-Moreno, et al. Postharvest Biology and Technology 163 (2020) 111123

Fig. 3. Curative and preventive treatments against Colletotrichum gloeosporioides applied on avocado fruit. The fruit with curative and preventive treatments
were stored at 6 °C (during 10 d and ripened at 25 °C during 5 d to mimic the conditions at the retail level) and 25 °C for 15 d. Mean values in the same graph followed
by different lower-case letter are significantly different according to Fisher’s LSD test at p < 0.05. Control (sterile distilled water); SA (1.5 %); Yeast (1 × 109 CFU
mL−1) and SA + Yeast (1.5 % + Yeast 1 × 109 CFU mL−1). Each bar represents the mean values ± standard deviation (n = 30).

Fig. 4. Effect of SA coatings in reducing

anthracnose caused by Colletotrichum
gloeosporioides in avocado fruit. The efficacy
of preventive treatments applied on avocado
fruit after 15 d of storage is represented: A)
Control; B) SA coating (1.5 %); C) Yeast (1 ×
109 CFU mL−1); D) SA + Yeast (1.5 % SA +
Yeast 1 × 109 CFU mL−1). The appearance of
flesh in E) Control; F) SA + Yeast at 25 °C and
G) SA + Yeast stored at 6 °C for 10 d and ri-
pening at 25 °C for 5 d.

6
M. Iñiguez-Moreno, et al.
Fig. 5. Weight loss of coated avocado fruit during storage. The avocado
fruit were coated with sodium alginate (SA, 1.5 %); Yeast (1 × 109 CFU mL−1);
SA + Yeast (1.5 % SA + Yeast 1 × 109 CFU mL−1) and stored at 6 and 25 °C
for 15 d and at 6 °C for 10 d and ripened at 25 °C for 5 d; and control (SDW).
Values are expressed as means ± standard deviation (n = 20). *: indicates
significant differences between water control and SA + Yeast treatment ac-
cording to Fisher’s LSD test at p < 0.05.
antagonistic activity of W. anomalus, Metschnikowia pulcherrima, Aur-
eobasidium pullulans and Saccharomyces cerevisiae (Contarino et al.,
2019), and fungus as M. albus (Mercier and Jiménez, 2004), while in
bacteria such as B. subtilis the main VOCs is the acetoin (ketone)
(Arrebola et al., 2010). The accepted mechanism of action of the al-
cohols is damage to the plasma membrane and rapid denaturation of
proteins, with subsequent interference with metabolism and cell lysis
(McDonnell and Russell, 1999). Lipophilic compounds as 1-butanol, 2-
7
Postharvest Biology and Technology 163 (2020) 111123
methyl- and 1-butanol, 3-methyl- with high affinity for plasma mem-
brane have higher toxicity than ethanol (Weber and de Bont, 1996).
Otherwise, it has been previously reported the production of ethyl
acetate and 3-methylbutanal by M. caribbica (Zhang et al., 2014). Ethyl
acetate and isoamyl acetate are the main esters produced by the yeasts
(Contarino et al., 2019). Plata et al. (2003) reported the presence of
isoamyl acetate was only detected after 10 d of incubation in Pichia
membranaefaciens and Candida guilliermondii, probably due to the low
level of esterase activity, this was linked to the low temperature of
incubation in our study can explain the presence of isoamyl acetate
when the yeast was incubated at 6 °C during 20 d. This compound
causes damage to cell membranes and inactivated membrane proteins,
impairing respiration (Ando et al., 2015). However, it has been pro-
posed that yeast biocontrol efficacy through the production of VOCs
could be the result of a synergistic effect between VOCs and carbon
dioxide in the environment (Contarino et al., 2019).
Important characteristics of a biocontrol agent-coatings are 1)
water-soluble, 2) able to improve the dispersibility of cell suspension
and maintain or increase cell population when applied in the product
and 3) contain safe ingredients for final consumers (Marín et al., 2017).
Therefore, the viability of M. caribbica in SA films and coatings applied
on avocado fruit was assessed. In films, the highest reduction in the
viability was obtained at 25 °C. This reduction is lower than the pre-
viously reported by Aloui et al. (2015), who assessed the viability of W.
anomalus in 2 % SA films at 25 °C, after 14 d of storage they had a
reduction of 0.95 CFU cm−2. Hence, SA can be considered is a poly-
meric matrix that allows maintaining the viability of M. caribbica at 6
and 25 °C. The difference in the viability obtained under the two tested
temperatures can be related to the yeast optimal temperature growth
(24−28 °C), that contribute to have a higher decrement in their via-
bility at 25 °C, due to its active metabolism and lack of nutrients; even
when M. caribbica is able to use glycerol as a carbon source (Polburee
et al., 2015). The variations in the viability of M. caribbica in coatings
applied on avocado fruit stored at 25 °C and stored at 6 °C during 10 d
and ripened at 25 °C can be associated with a higher moisture loss at 25
°C than at 6 °C and at the differences in the relative humidity 75 and 95
%, respectively (Bill et al., 2014b) in concordance with the results of
weight loss obtained in this study.
Avocado fruit is susceptible to diverse postharvest diseases, being
the anthracnose caused by C. gloeosporioides the most important. A way
to extended the avocado fruit shelf-life is storage between 5−13 °C in
high relative humidity (85–90 %); however, it should be considered
that the temperature at the retail level tends to 25 °C (Bill et al., 2014b).
Then, the use of SA coating with M. caribbica to anthracnose control
was proposed. Overall, preventive treatments were more efficient than
curative treatments under both tested conditions, this in concordance
with González-Estrada et al. (2017), due to allowing the yeast estab-
lishment owing to its ability to colonize the fruit wound faster than
fungus (Bautista-Rosales et al., 2013). The level of anthracnose pro-
tection (infected wounds and incidence of anthracnose) was higher at 6
°C than at 25 °C (p < 0.05) (Fig. 3); due to the low-temperature effect
on the decrease of C. gloeosporioides growth rate, and because optimal
temperature for appressoria formation and anthracnose development
ranged between 25−31 °C (Kenny et al., 2012). Moreover, ripening of
avocado fruit occurs more rapidly at 25 °C than a 6 °C, during the ri-
pening, the pH of exocarp increase (5.2–6.3) and C. gloeosporioides
begins the pectate lyase synthesis (pH 5.8) contributing to fungus es-
tablishment (Yakoby et al., 2000). Campos-Martínez et al. (2016)
evaluated the effect of W. anomalus on the anthracnose caused by C.
gloeosporioides in avocado fruit during 7 d at 28 °C with a reduction of
severity of 34.4 %, in concordance with our results at 15 d at 25 °C.
Therefore, the use of the polymeric matrix contributed to increase the
yeast antagonistic activity.
Otherwise, in avocado fruit coated with SA + Yeast stored at 6 °C
and ripened at 25 °C C. gloeosporioides was not able to grow and cause
anthracnose. In addition to the events explained above, the low
M. Iñiguez-Moreno, et al.
temperature allows avocado fruit to increase the time to reach the ri-
peness state of consumption; the ripening delay contributes to the
maintenance of acetogenins, particularly the persin level, which had
fungitoxic activity (Rodríguez-López et al., 2015). The reduction in the
infected wounds percentage, anthracnose incidence and severity by SA
coating without yeast (Fig. 3) is associated with the O2 and CO2 ex-
change decrease and the aerobic metabolism reduction as a con-
sequence of the formed barrier (Song et al., 2011). However, this effect
was not enough for C. gloeosporioides inhibition.
The weight loss is an important characteristic to considerer in the
use of edible coatings. According to our results, Aloui et al. (2015)
reported no significant difference between the weight loss in coated
Valencia oranges with SA and SA with W. anomalus after 15 d of storage
at 25 °C. Weight loss is directly related first, to the vapor-phase diffu-
sion driven by the vapor pressure between inside and outside cell
compartments of the fruit, and second, to water loss which takes place
through the stomata, stem scar, and cuticle. The amount of water loss
depends on cuticle composition and thickness, which varies for the
cultivars and maturity stage (Bill et al., 2014b; Maftoonazad and
Ramaswamy, 2008).
5. Conclusion
The present study demonstrated, that the production of VOCs plays
an essential role in the antagonistic activity of M. caribbica against C.
gloeosporioides. However, due to the production of VOCs is strongly
influenced by the growth substrates and environmental conditions;
more studies are necessary to understand the effect of each VOCs on the
phytopathogenic fungus, in order to improve the yeast efficacy.
Additionally, the use of SA coatings contributes to maintain the M.
caribbica viability and improves their efficacy to anthracnose control in
avocado fruit. Additionally, SA coatings with M. caribbica reduce sig-
nificantly the weight loss in avocado fruit during storage. According to
these results, SA coatings with M. caribbica can be used as an alternative
to extend shelf-life during the postharvest stage.
Author statement
Maricarmen Iñiguez-Moreno: Perform of the experiments, data/
evidence collection, article redaction
Juan Arturo Ragazzo-Sánchez: Application of statistical, mathe-
matical, computational, or other formal techniques to analyze or syn-
thesize study data
Julio César Barros-Castillo: Perform of the COV’s determination
Teresa Sandoval-Contreras: Perform of the experiments
Montserrat Calderón-SantoyoIdeas: formulation or evolution of
overarching research goals and aims
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
This research was supported by Consejo Nacional de Ciencia y
Tecnología (CONACYT), México, project number 2015-01-1053 to M.
Calderón-Santoyo and postdoctoral grant 2018-000005-01NACV-00746
awarded to M. Iñiguez-Moreno. This research is part of the activities of
the CYTED Network 319RT0576 “Desarrollo sostenible en
agroalimentación y aprovechamiento de residuos industriales”. The
authors thank Leb Trading Products (Guacamodely) S. de R.L. de C.V.
for providing the avocado fruit for this work.
8
Postharvest Biology and Technology 163 (2020) 111123
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.postharvbio.2020.
111123.
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