Postharvest Biology and Technology 124 (2017) 85–90

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Postharvest Biology and Technology

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

Application of a hydrothermal-calcium chloride treatment to inhibit

postharvest anthracnose development in papaya
Lidia Elena Ayón-Reynaa , Arturo González-Roblesb , José Guadalupe Rendón-Maldonadoa ,
María Elena Báez-Floresa , Martha Edith López-Lópeza , Misael Odín Vega-Garcíaa,*
a
Doctorado Regional en Biotecnología, Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Sinaloa, Cd. Universitaria, Av. de las Américas y
Josefa Ortiz S/N, Culiacán, Sinaloa, 80010, Mexico
b
Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados, Av. Instituto Politécnico Nacional 2508, San Pedro
Zacatenco, México D.F., 07360, Mexico

A R T I C L E I N F O A B S T R A C T

Article history:
Received 3 May 2016 Anthracnose is considered an important postharvest disease in papaya. The hydrothermal treatment (HT)
Received in revised form 27 September 2016 and calcium chloride (Ca) have been shown to be effective to inhibit anthracnose. The objective of this
Accepted 13 October 2016 study was to investigate the effect of the combination HT-Ca on the development of anthracnose in
Available online 19 October 2016 papaya. Fruit were inoculated with Colletotrichum gloeosporioides by immersion in a spore suspension
and then were divided into two groups: one received a HT treatment (48
C, 20 min) combined with Ca
Keywords: (1% w/v, 20 min) and the other was used as control. Afterwards, fruit were stored during 20 days at 12
C
Carica papaya to allow the development of the fungal infection. Anthracnose incidence and severity were estimated
Colletotrichum gloeosporioides
visually while the development of the disease was analyzed by light and electron microscopy. HT-Ca
Hot water-Ca immersion
reduced anthracnose incidence and severity compared with the control. Microscopy analysis showed
Microscopy analysis
that HT-Ca melted the epicuticular wax, which covered most of the stomata; this resulted in a lower
mycelial growth in HT-Ca fruit with respect to the control samples. HT-Ca also induced the formation of
round shaped vesicles, which corresponded with the greater accumulation of total phenolics observed in
treated fruit. HT-Ca was effective to delay the symptoms of anthracnose up to 10 days during storage of
papaya at 12
C.
ã 2016 Elsevier B.V. All rights reserved.

1. Introduction production and marketing of papaya (Sripong et al., 2015). The

Papaya (Carica papaya L.) is an important fruit cultivated in
tropical and subtropical areas (Sharma, 2015); India is the main
papaya producing country, while Mexico occupies the sixth place,
providing 37.1% and 5.9% of the total production, respectively
(FAOSTAT, 2014). This fruit has an important antioxidant activity
and its consumption provides health benefits, particularly by its
medicinal properties, and improved digestion (Chutichudet and
Chutichudet, 2014). Because papaya is a climacteric fruit, it suffers
some problems such as rapid ripening and susceptibility to biotic
and abiotic stresses (Zhu et al., 2013). Softening related with
ripening makes this kind of fruit vulnerable to a wide range of
postharvest diseases including anthracnose (Ong et al., 2013), a
disease caused by Colletotrichum gloeosporioides (Penz.) Penz &
Sacc. that causes great economic losses because it affects the
* Corresponding author.
E-mail address: mvega6@yahoo.com (M.O. Vega-García).
symptoms of anthracnose in papaya are characterized by round
brownish depressed lesions, and in some cases, salmon-colored
areas formed by the conidial masses that cover the lesion (Gomes-
Moraes et al., 2013). Colletotrichum species can directly penetrate
the fruit skin or throughout openings like stomata and wounds
(Gomes-Moraes et al., 2013). The incidence of anthracnose is
favored by temperatures about 25–28
C. Nevertheless, the
symptoms may also appear at temperatures below 12
C if the
periods of wetness are long enough (Ferrari-Rockenbach et al.,
2015).
The control of anthracnose in fresh produce is currently based
on the application of chemical fungicides (Sripong et al., 2015).
Nevertheless, the use of fungicides at high concentrations for long
periods of time can induce resistance in the pathogen and favor
environmental contamination (Shi et al., 2012). Moreover, there is
concern that the fruit may be harmful to the consumers if they
have fungicide residues (Ong et al., 2013). At this respect, the
alternatives are expected to include both reduction of fungicides
resistance and lessen the risk related to the use and abuse of

http://dx.doi.org/10.1016/j.postharvbio.2016.10.009
0925-5214/ã 2016 Elsevier B.V. All rights reserved.
86 L.E. Ayón-Reyna et al. / Postharvest Biology and Technology 124 (2017) 85–90
fungicides to humans, animals, and the environment (Hasan et al.,
2012). One strategy to improve the safety, quality and shelf-life of
fruit is to identify treatments that are non-toxic, safe, biodegrad-
able, and effective as antimicrobial agents while retaining the
physical and nutritional quality (Ong et al., 2013).
There are several approved quarantine treatments for impor-
tation of papaya into the United States, such as cold treatment,
vapor heat treatment, steam sterilization hot air, irradiation, and
hot water immersion (USDA-APHIS, 2015). As a protocol for
exportation, papayas are dipped in hot water at 48
C for 20 min to
control postharvest diseases, since hot water immersion (also
called hydrothermal treatment) could directly inhibit fungal
germination and growth, or even kill the fungus (Li et al., 2013b;
USDA-APHIS, 2015). Li et al. (2013a) reported that hot water
treatment (54
C, 4 min) in papaya fruit reduced anthracnose
incidence.
Another approach to control postharvest diseases is the use of
calcium salts. It has been reported that calcium maintains cell wall
structure by interacting with pectin to produce calcium pectate
complexes, which can maintain fruit firmness and reduce the
accessibility of fungal pathogens (Ayón-Reyna et al., 2015). Some
reports have shown that calcium is antagonistic to C. gloeospor-
ioides and it may be used as an alternative treatment for disease
control (Ghani et al., 2011; Madani et al., 2014).
Some studies have evaluated the combination of different
methods, such as hot water and calcium salts. It has been reported
that hydrothermal treatment combined with calcium salts was
effective to suppress microbial growth and to maintain the quality
in some fresh products, such as fresh-cut papaya (Ayón-Reyna
et al., 2015), fresh-cut melon (Silveira et al., 2011), apple (Sharma
et al., 2013), and kiwifruit (Shahkoomahally and Ramezanian,
2013). Meanwhile, Dessalegn et al. (2013) found lower anthracnose
incidence in mango fruit treated with a combination of hot water
and calcium than with treatments alone.
The objective of the present study was to determine the effect of
the combination of hot water and calcium chloride on the
development of papaya anthracnose.
2. Materials and methods
2.1. Plant material
Maradol papaya fruit were harvested at ripening stage 4 (skin
slightly orange with green stripe and pulp completely orange)
(Santamaría-Basulto et al., 2009). Fruit were obtained from a local
commercial plantation close to Culiacan City, State of Sinaloa,
Mexico (located at 24
520 56.800 N latitude and 107
260 52.100 W
longitude) and were selected based on absence of physical damage
and an average weight of 1 kg. Food-grade calcium chloride
(CCFO21–00 Fabpsa, Mexico) was used for the HT-Ca treatment
solution (1% w/v).
2.2. Isolation and identification of fungal pathogen
Colletotrichum gloeosporioides was isolated from cv. Maradol
fruit. To isolate the pathogen, infected tissue pieces were taken and
placed in the center of Petri dishes containing potato dextrose agar
(PDA) (MCD LAB, Tlalnepantla, Estado de Mexico, Mexico) and
incubated at room temperature (25  2
C). Once mycelial growth
was observed, the fungus was re-isolated on fresh PDA to obtain
pure cultures (Casarrubias-Carrillo et al., 2002).
The isolates were identified on the basis of their morphological
and cultural characteristics by lactophenol blue stains and
according to the keys of Barnett and Hunter (1972). The fungus
identity was confirmed by molecular techniques using the internal
transcribed spacers ITS1-ITS2 (White et al., 1990).
2.3. Inoculum preparation
Spores were scraped from a two-week culture and placed in
sterilized distilled water. Conidial suspension was adjusted to
1.5  105 conidia mL1 and added with 0.5% Tween 801 to prevent
spore clumping. Conidia were counted using a hemacytometer
(Hasan et al., 2012).
2.4. Treatments application
Papaya fruit were surface-sanitized by dipping in 1% sodium
hypochlorite solution for 5 min, rinsed in sterile distilled water,
and inoculated by dipping into the spore suspension for 5 min
(Ademe et al., 2013).
Fruit were randomly divided into two groups. One of them did
not receive a treatment and was used as control, and the other
group was immersed for 20 min in hot water (48
C) containing 1%
(w/v) calcium chloride, using a water bath with a temperature
controller (Model 1266–02; Cole Parmer, Ill., U.S.A.). Calcium
chloride concentration and hydrothermal treatment conditions
were based on previous reports (Couey et al., 1984; Ayón-Reyna
et al., 2015). After the application of the treatments, fruit were
placed into unsealed plastic bags and stored at 12
C for 20 days
with a 90–95% relative humidity. Three replicates per treatment
were performed and each treatment included 12 fruit.
2.5. Disease incidence and severity
Disease incidence was recorded according to Ong and Ali (2015)
based on the anthracnose symptoms on fruit surfaces. The effect of
HT-Ca on disease incidence was evaluated daily until all fruit
showed symptoms. It was expressed as the number of fruit
showing anthracnose out of the total number of fruit in each
treatment.
Disease severity was scored at 4 day intervals following the
method of Ademe et al. (2013). Disease severity was rated using a 1
to 5 scale, where 1 represented no sign of anthracnose disease on
the fruit surface. A rating of 2 was scored when up to a quarter (1–
25%) of the fruit surface was rotten. A rating of 3 was recorded
when 26–50% of the fruit surface was infected with anthracnose. If
51–75% of the fruit surface exhibited anthracnose symptoms, a
rating of 4 was scored. If anthracnose symptoms appeared on more
than 76% of the fruit surface, then a rating of 5 was scored.
2.6. Light and transmission electron microscopy
Papaya samples were prepared for light and transmission
electron microscopy as described by Phothiset and Charoenrein
(2013). Sections of about 2  2  2 mm were excided from the fruit
surface at 0, 12, and 24 h, and at 2, 4, 5, 6, 8, 10 and 12 days after the
treatments were applied and then transferred to 1.5 mL microtubes
containing 2.5% glutaraldehyde in 0.1 mol L1 sodium cacodylate
buffer (pH 7.4), followed by incubation for 12 h at room
temperature. Samples were rinsed three times with the same
buffer for 20 min and post-fixed with 1% v/v osmium tetroxide in
0.1 mol L1 sodium cacodylate buffer (pH 7.4) for 2 h at room
temperature. Samples were dehydrated in an ascending ethanol
series (30, 50, 70, 80, 90, and 100%) for 20 min each. To complete
the dehydration, they were immersed twice in absolute ethanol for
20 min each. Afterwards, dehydrated samples were rinsed in
propylene oxide (three times, 20 min each), followed by infiltration
overnight in a mixture of propylene oxide–polybed resin in a 1:1
ratio, then they were transferred to propylene oxide–polybed resin
mixture in a 1:2 ratio followed by an infiltration in pure polybed
resin three times, for 1.5 h each. Later on, for polymerization, they
were placed in an oven at 60
C for 48 h and sectioned with a
 L.E. Ayón-Reyna et al. / Postharvest Biology and Technology 124 (2017) 85–90
Reichert-Jung Ultracut (Wien, Austria) ultramicrotome using a
glass knife at 1 mm thickness and stained with 1% (w/v) toluidine
blue for observation under a light microscope (Axiophot photo-
microscope Carl Zeiss, Germany). Photographs were obtained with
a Zeiss AxioCam MRc digital camera (Carl Zeiss Vision GmbH,
Germany). Ultra-thin sections (80 nm) were achieved using a
diamond knife, stained with 5% uranyl acetate and lead citrate.
Thin sections were observed in a JEOL JEM-1011 transmission
electron microscope (Tokio, Japan) at 80 kV.
2.7. Scanning electron microscopy (SEM)
For the SEM studies, tissue sections of 5  5  5 mm were fixed
and dehydrated as previously described for light microscopy.
Samples were then dried to the critical point by immersing
fragments in liquid carbon dioxide in a Samdry-780 Critical Point
Dryer (Rockville, Maryland USA), at 1200 psi and 31
C. Dried
samples were mounted on aluminum stubs and coated with gold at
5 mA and 1.5 kV using a sputter coater (Ion Sputter JFC-1100, Tokio,
Japan), for 5 min. Observations were made using a JSM-7100F
Scanning Electron Microscope (Tokio, Japan) at 5 kV.
2.8. Total phenolics
Methanol extracts were obtained following the methodology
reported by Moo-Huchin et al. (2014). A 5 g sample was
homogenized with 10 mL of 100% methanol using an Ultra-Turrax,
sonicated for 30 min, and centrifuged at 400  g for 10 min at 4
C.
The supernatant was recovered and the pellet was homogenized
with 10 mL of 100% methanol. The homogenate was sonicated,
centrifuged again using the same conditions, and the supernatant
was recovered. The supernatants of the 2 extracts were mixed and
the volume was adjusted to 4 mL. Total phenolics content was
determined according to López-Angulo et al. (2014) with some
modifications. A 20 mL aliquot of concentrated methanol extract
was combined with 180 mL of 0.22N Folin-Ciocalteu reagent, and
50 mL of 7% Na2CO3 and allowed to rest for 30 min (25
C/darkness).
The absorbance was measured at 765 nm using a microplate reader
(Synergy HT, BioTek Instruments, Winooski, Vermont, USA). The
control consisted of 100% methanol. Total phenolics content was
obtained using a standard calibration curve of gallic acid and the
results were expressed as g gallic acid equivalent (GAE) per
kilogram of fresh weight (FW). Nine measurements were
performed for each treatment.
2.9. Statistical analysis
A completely randomized experimental design with 3 repli-
cates was performed through multiple analyses of variance using
Statgraphics Plus 5.1 and the means were compared using Fisher’s
least significant difference (LSD) test (p < 0.05). The means with
the corresponding LSD for each level were plotted using SigmaPlot
12.0.
3. Results and discussion
3.1. Disease incidence and severity
The assay of HT-Ca against C. gloeosporioides showed that the
disease incidence was significantly reduced over the control from
day 7 until day 13 of storage at 12
C (Fig. 1A). The symptoms of
anthracnose first appeared in control fruit after 6 days, while in HT-
Ca treated fruit they appeared until the 11th day. HT-Ca treatment
was able to delay the presence of symptoms during 10 days, which
represents a longer time to commercialize the fruit. Disease
incidence for untreated fruit rapidly increased during the storage
87
100
Anthracnose incidence (%)
Control
HT-Ca
80
60
40
20
0
A
A
0 5 6 7 8 9 10 11 12 13 14 15 16
5
Anthracnose severity
4
3
2
1
B
0
0 4 8 12 16 20
Days of storage at 12°C
Fig. 1. Effect of HT-Ca on anthracnose incidence (A) and severity (B) of papaya
inoculated with Colletotrichum gloeosporioides and stored at 12
C for 20 days.
Severity index: 1 = 0%, 2 = 1–25%, 3 = 26–50%, 4 = 52–75%, 5 = 76–100%.
Vertical bars indicate LSD (p < 0.05).
and reached the maximum (100%) at day 13, while in HT-Ca treated
fruit the maximum incidence was observed after 15 days.
During the first 8 days of storage anthracnose symptoms did not
become visible on the surfaces of fruit treated with HT-Ca;
however, control fruit showed symptoms that were more severe
throughout the storage period (Fig. 1B). At day 12, disease severity
for control treatment was 3 and increased to 3.67 and 4 at days 16
and 20, respectively. For HT-Ca treated fruit, the severity index was
2 at day 12 and remained constant until the end of the storage.
Significant differences in anthracnose severity were observed for
control and treated fruit at days 16 and 20 of the storage period;
however, once the symptoms appeared the fruit were not suitable
for export.
HT-Ca treatment was effective in delaying the onset of disease
symptoms as well as their appearance and severity. The fungitoxic
activity of HT-Ca could be attributed to the effect of the heat
treatment on pectin demethylation by pectinmethylesterase to
form anionic carboxyl groups with which calcium ions can form
salt bridge cross-links that avoid the action of cell wall degrading
enzymes, providing greater firmness to the fruit (Aguayo et al.,
2015). Thus, it could give more resistance to the tissue against
fungal attack because it stabilizes or strengthens the cell walls
(Ayón-Reyna et al., 2015), helping to reduce the accessibility of
fungal hydrolases that cause anthracnose. On the other hand,
warm temperatures (40–60
C) can increase the beneficial effects
of the calcium treatment due to higher calcium retention inside the
fruit (Silveira et al., 2011). Also, microorganisms act mainly on skin
and the first layers of parenchymal cells of the fruit. In these cases,
high temperatures exert either lethal or sublethal effects to inhibit
fungal germination and growth (Ayón-Reyna et al., 2015). Madani
et al. (2014) suggested that calcium reduces anthracnose in papaya
88 L.E. Ayón-Reyna et al. / Postharvest Biology and Technology 124 (2017) 85–90
due to a toxic effect, because high concentrations of extracellular
calcium may increase calcium in the cytosol to toxic level, affecting
the osmotic balance in fungal cells.
The high antifungal activity of HT-Ca treatment evidenced in
this study was in agreement with previous findings. Sharma et al.
(2013) investigated the effect of the application of calcium chloride
and hot water treatment on apple decay caused by Penicillium
expansum and Botrytis cinerea, showing that fruit treated with the
combination had lesser decay area than the control; they also
reported that the combination of calcium and hot water was more
effective than the individual treatments. Also, Ranjbar et al. (2007)
reported that a combination of calcium chloride and hot water
treatment maintained the quality of pomegranate fruit and
decreased fungi infection.
3.2. Light and transmission electron microscopy
Light microscopy showed that after inoculation the control and
HT-Ca treated fruit showed well defined cell walls (Fig. 2A and B). It
also revealed that the HT-Ca treatment melted the epicuticular
wax, which plugged partially or completely most of the stomata,
covering the guard cells outside, thus acting as a mechanical
barrier against pathogens (Fig. 2B). However, after 10 days of
inoculation, cross-section photomicrographs of tissue stained with
toluidine blue revealed an intense blue color in the epidermal cells
of control papayas (Fig. 2C), which could be attributed to an
increase in the synthesis of phenolic compounds and lignin
because these processes can occur in plants as a response to
pathogen infection (Ferrari-Rockenbach et al., 2015). On the other
hand, some round shaped structures were revealed inside the first
layers of parenchyma cells in HT-Ca treated fruit (Fig. 2D) which
were not observed in the control fruit (Fig. 2C). Transmission
electron microscopy confirmed the absence of these vesicles in
control fruit (Fig. 2E) and revealed their presence inside the cells of
the HT-Ca treated fruit (Fig. 2F), which could indicate that the
Fig. 2. Light micrographs of transversal section of untreated (A) and HT-Ca treated
papayas (B) at the beginning of the storage showing the cell walls and guard cells of
stomata covered by molten wax. Untreated (C) and HT-Ca treated papayas (D) after
10 days of storage showing phenolics and lignin in the epidermal cells, and round
shaped structures like vesicles inside the first layers of parenchyma cells,
respectively. Bars = 20 mm. Transmission electron micrographs of transversal
section of untreated papayas after 10 days of storage showing the absence of
round shaped structures inside the cells (E) and HT-Ca treated papayas showing
vesicles inside of papaya cells (F).
CW = Cell wall, MW = Molten wax, S = Stomata, GC = Guard cell, V = Vesicle.
synthesis and accumulation of phenolic compounds and lignin
inside of the vesicles were induced by the heat stress (Fallik, 2004),
which was confirmed when total phenolic concentration was
measured.
3.3. Scanning electron microscopy
Several natural openings or stomata were shown on the fruit
surface, which could allow the entrance of C. gloeosporioides.
However, the results of scanning electron microscopy clearly
illustrated that stomata of untreated fruit remained open
throughout the storage (Fig. 3A and C), while the application of
the HT-Ca treatment caused that the stomata were partially or
entirely sealed with molten wax (Fig. 3B and D). The results
reported by Pereira-Kechinski et al. (2012) largely support the
findings from this study because they observed that the hot water
treatment resulted in wax-covered stomata on the papaya surface.
It was proposed that if the natural openings were covered by the
molten wax, this could physically prevent the invasion of the
pathogen, and therefore reduce disease incidence.
The SEM photomicrographs of control samples show the typical
surface morphology of papaya fruit (Fig. 3E), where cracked layers
were found in the untreated fruit. These fissured layers were thick,
crusty coverings. On the other hand, HT-Ca modified the natural
wax layer of the papayas, changing the wax arrangement on the
fruit surface and producing a more uniform covering with fewer
cracks, which appeared to be smoother and less granular than the
control (Fig. 3F). These results are in agreement with those
Fig. 3. Scanning electron photomicrograph of the stomata of cuticular surface of
untreated papaya at 0 (A) and 6 (C) days of storage where it is possible to observe
that stomata remained open throughout the storage. SEM of the surface of papaya
treated with HT-Ca after 0 (B) and 6 (D) days of storage where it can be observed that
the stomata are almost completely covered by the molten wax. SEM images of
untreated (E) and HT-Ca treated papaya surfaces (F) at the beginning of storage. The
control samples of the papaya cuticles illustrate the typical surface morphology of
the fruit, showing fissured layers, and crusty coverings. HT-Ca melted the wax,
modifying the natural wax layer of the papayas and producing a smoother covering.
S = Stomata. Bars = 10 mm.
 L.E. Ayón-Reyna et al. / Postharvest Biology and Technology 124 (2017) 85–90
reported by Roy et al. (1994), Pereira-Kechinski et al. (2012), and
Ong et al. (2013). Also, Lurie et al. (1996) evaluated the effect of
calcium chloride combined with hot water in apples, and found
that the applied treatment melted the epicuticular waxes and filled
the cracks on the fruit surface.
On the other hand, it was observed that conidia were adhered to
the surface of the untreated and HT-Ca treated fruit after the
inoculation (Fig. 4A and B). The surfaces of HT-Ca treated fruit had
spores deposited in the molten wax just after the application of the
treatment. In this context, Schirra et al. (1999) observed conidia
that appeared to be covered by molten wax after heat treatment in
cactus pears. Meanwhile, Li et al. (2013b) reported that conidia
may be encapsulated and inactivated by molten wax, and the
encapsulation is considered as an additional factor in protection of
fruit against decay.
The results obtained of the antifungal activity via SEM clearly
revealed that the growth of C. gloeosporioides was delayed by HT-
Ca. Mycelia were observed growing on the fruit surface in both
treated and untreated fruit. However, extensive mycelial growth
was observed in control fruit after 12 days of storage, showing that
mycelium completely covered their surface (Fig. 4C), while lower
mycelial growth was observed in fruit with HT-Ca treatment,
whose surface was only partially covered (Fig. 4D). Nevertheless,
despite the differences in mycelial growth, the fungus was able to
infect the treated fruit and the symptoms appeared after 11 days of
storage at 12
C.
3.4. Total phenolics
The total phenolics content (TPC) at the beginning of the storage
was 0.23 gGAE kg FW1 and increased as the storage progressed
(Fig. 5). No significant differences in TPC were observed during the
first 8 days; however, after day 12 TPC was significantly higher in
HT-Ca treated fruit than the control. This increase could be due to
the stress caused by the heat treatment because this condition
plays an important role in the activation of the synthesis and
accumulation of phenols (Heredia and Cisneros-Zevallos, 2009).
Also, it has been reported that high concentrations of cytosolic
calcium induce endogenous resistance mechanisms, such as the
synthesis of phenolic compounds (Madani et al., 2014). Similar
results were reported by Sharma et al. (2013) who found that
Fig. 4. Scanning electron microscopy of conidium and hyphae of Colletotrichum
gloeosporioides on Maradol papaya. Fungal conidium on fruit surface of untreated
(A) and HT-Ca treated papayas (B) deposited in the molten wax at the beginning of
the storage. Fungal hyphae grown on untreated (C) and HT-Ca treated papayas (D)
after 12 days of storage. Bars = 10 mm.
89
0.35 Control
Total phenolic content
HT-Ca
(g GAE kgFW )
-1
0.30
0.25
0.20
0 4 8 12 16 20
Days of storage at 12°C
Fig. 5. Effect of HT-Ca on total phenolics of papaya inoculated with Colletotrichum
gloeosporioides and stored at 12
C for 20 days.
untreated apples had lower levels of phenolic compounds than
those treated with hot water and calcium.
4. Conclusion
The application of HT-Ca affected the development of Colleto-
trichum gloeosporioides, decreasing anthracnose incidence and
severity. HT-Ca melted the epicuticular wax forming a barrier that
reduced the mycelial growth on papaya fruit. The formation of
round shaped structures was observed inside the cells of HT-Ca
treated fruit and was related with the greater accumulation of total
phenolics. The combination HT-Ca could be applied to papaya as an
effective fungistatic treatment to delay the anthracnose disease
during postharvest storage.
Acknowledgments
This work was supported by a grant from Universidad
Autonoma de Sinaloa (PROFAPI 2014/223). LEAR was the recipient
of a scholarship from CONACyT-Mexico. Authors wish to acknowl-
edge one anonymous reviewer and Dr. Jose A. Lopez-Valenzuela for
their English edition and critical reviewing of the manuscript.
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