Scientia Horticulturae 214 (2017) 273–279

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Effect of candelilla wax edible coatings combined with biocontrol

bacteria on strawberry quality during the shelf-life
Ernesto Oregel-Zamudio a , M. Valentina Angoa-Pérez a , Guadalupe Oyoque-Salcedo a ,
Cristóbal Noé Aguilar-González b , Hortencia Gabriela Mena-Violante a,∗
a
Instituto Politécnico Nacional, CIIDIR IPN Unidad Michoacán„ Justo Sierra 28, Centro, Jiquilpan, Michoacán, C.P. 59510, Mexico
b
Department of Food Science and Technology, Universidad Autónoma de Coahuila, Saltillo 25280, Coahuila, Mexico

a r t i c l e i n f o a b s t r a c t

Article history: The effect of edible films of candelilla wax alone or in combination with a Bacillus subtilis HFC103 strain
Received 7 July 2016 on shelf life of strawberry, has been assayed. Treatments were: control, film (edible coating), bacteria
Received in revised form 9 November 2016 (inoculated with B. subtilis HFC103) and film + bacteria (edible coating + B. subtilis HFC103). Fruits were
Accepted 28 November 2016
treated and stored at 25 ◦ C for six d. Decay percentage and weight loss were assayed daily, while pH and
total soluble solids (TSS) were determined at the end of the assay. The film, bacteria and film + bacteria
Keywords:
treatments significantly reduced the decay percentage and weight loss with respect to control since day
Bacillus subtilis
3. Film + bacteria treatment reduced decay around 100% with respect to control on day 6. Film treatment
Fragaria × ananassa
Rhizopus stolonifer
kept a low weight loss during the assay. There were no changes of pH and TSS as effect of the treatments.
Postharvest Additionally, the effects of the treatments described on the severity of the damage caused by Rhizopus
Decay percentage stolonifer were assayed. All the treatments were inoculated with a spore suspension of such pathogen,
stored at 25 ◦ C for 6 d, and the severity index was recorded every 24 h. Film, bacteria and film + bacteria
treatments significantly reduced the severity index with respect to control since day 2. The Bacteria
treatment was the most effective to control R. stolonifer. However, it did not show the same efficacy
to control weight loss or to prolong shelf life of strawberry. Results showed that the combination of
candelilla wax edible films and the inoculation with B. subtilis HFC103 is an innovative strategy with the
potential to prolong shelf life of strawberry.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction The trend is to develop safer and environmentally friendlier

Around 30% postharvest loss of fruits and vegetables in develop-
ing countries is due to microorganisms attack (Hodges et al., 2011).
Significant losses can be attributed to fungal attack during trans-
port and storage (Liu et al., 2013). A common practice to prevent
postharvest decay of fruits is the application of synthetic fungicides
(Feliziani and Romanazzi, 2013). However, many pathogens show
resistance to the pesticides used nowadays (Consortium, 2013).
Moreover, customers demand natural, free of chemical residues
and minimally processed foods (Valencia-Chamorro et al., 2011).
This is why it is necessary to search for preservation alternatives
with the purpose of reducing the negative effects during storage
and improving the quality of food products (Saucedo-Pompa et al.,
2007).
∗ Corresponding author.
E-mail addresses: tenchisgmv@hotmail.com, hmena@ipn.mx
(H.G. Mena-Violante).
technologies in order to reduce postharvest decay and to prolong
shelf life. One of the methods proposed to prolong shelf life of sev-
eral fruits is the application of edible coatings, especially on highly
perishable products (Jiménez et al., 2012; Kester and Fennema,
1986). Their function is to protect the product against mechani-
cal, physical and biological damage during transport and storage
(Campos et al., 2011). The edible coatings are a semipermeable bar-
rier to oxygen, carbon dioxide (CO2), moisture and some solutes,
which implies the reduction of: respiration, oxidation reactions
and water loss, promoting the foods preservation (Falguera et al.,
2011). Furthermore, the edible coatings can be used as carrier of
bioactive compounds (e.g. antioxidants, antimicrobials, probiotics)
to improve the quality of food products (Quirós-Sauceda et al.,
2014). Proteins, lipids and polysaccharides are the main compo-
nents of edible coatings. Maize zein, soy protein, albumen, milk
proteins are some of the most commonly used proteins (Wihodo
and Moraru, 2013). While some of the most commonly studied
polysaccharides to obtain edible coatings have been guar gum,
alginate, pectines, starch, cellulose and its derivatives (Dhanapal

http://dx.doi.org/10.1016/j.scienta.2016.11.038
0304-4238/© 2016 Elsevier B.V. All rights reserved.
274 E. Oregel-Zamudio et al. / Scientia Horticulturae 214 (2017) 273–279
et al., 2012). Particularly guar gum shows emulsifying action and
can create synergy with materials like xanthan, agar, carrageenan
and starches (Albuquerque et al., 2014). On the other hand, lipids
include the use of waxes such as caranauba wax, beeswax, and
candelilla wax, and others that according to their concentration
regulate the permeability of edible films. Likewise, plasticizers like
glycerol, sorbitol and polyethylene glycol are added (Jiménez et al.,
2013). Glycerol performs as a tensoactive agent and it is an alter-
native to provide structure and texture (Resa et al., 2014). Other
components added to some edible coatings in order to prevent
deterioration and to reduce the risk of saprophytes and pathogens
growth are the antioxidants and antimicrobials. For instance, gal-
lic acid (Campos et al., 2011), which also enhances the mechanical
properties in some edible films (Duan et al., 2011). Even though the
available information for the production of edible films is broad, it
is not applicable to all the products (Vieira et al., 2011). it is still
necessary to develop alternatives for strawberry. In this regard,
films made of polymers, such as candelilla wax, guar gum and glyc-
erol, can be used to control appearance changes, weight loss, water
activity and firmness during storage (De León-Zapata et al., 2015;
Saucedo-Pompa et al., 2007).
There are also biological alternatives for the preservation of
fruits (Ramos et al., 2013). Diseases and postharvest deterioration of
fruits can be fought with certain antagonistic microorganisms such
as fungi, yeasts and bacteria, through several mechanisms that can
work simultaneously, such as the competition for nutrients and
niches, antibiosis, lytic enzymes, volatile inhibitory metabolites,
pH decrease, parasitism and induction of defense responses from
the harvested plant product and others (Montesinos et al., 2015).
The postharvest application of biocontrol agents seems to be an
appropriate strategy for strawberries that are easily damaged or
that cannot be exposed to water-based treatments. In this regard,
Bacillus subtilis is a biological option for the control of several straw-
berry diseases. Indeed, during the postharvest stage Bacillus subtilis
inhibits the fruits rottenness (Punja et al., 2016), and shows anti-
fungal activity and a positive effect on the useful life extension,
this indicates multiple attributes through which this microorgan-
ism can be an alternative of postharvest application (Shternshis
et al., 2015).
Even though both edible coatings and biocontrol have shown
their potential separately, their combination has not yet been stud-
ied, consequently, the objective of this work was to develop an
edible film made of candelilla wax added with biocontrol bacteria
in order to prolong the shelf life of strawberry.
2. Materials and methods
2.1. Strawberry
Strawberry fruit (Fragaria × ananassa Duch, cv. Albion) of uni-
form size, shape, weight, color, and free of physical damage as well
as microorganisms infection, were supplied by regional producers
(Jacona, Michoacán, México).
2.2. Biocontrol bacteria
Bacillus subtilis HFC103 isolated from commercial strawberry
were supplied by the bacterial strain collection of CIIDIR IPN
Unit Michoacán. Identification of bacteria was based on the ITS
sequences; ITS-PCR products generated by universal primers to the
16S were sent to Macrogen USA for sequencing. Sequences were
compared to the database (BLAST, nr/nt) and it showed that HFC103
(913p bp) matched the Bacillus subtilis 16S ribosomal RNA 1NT.
2.3. Fungus
Rhizopus stolonifer strain belonging to the repository of CIIDIR
IPN Unit Michoacán, which was identified with the keys of
(Schipper and Stalpers, 1984), was used to perform the antifun-
gal assay. Koch’s postulates were completed in the laboratory by
inoculating strawberries.
2.4. Raw materials
Refined candelilla wax was supplied by the Department of Food
Research of the Chemical Sciences Faculty at the Autonomous Uni-
versity of Coahuila (Saltillo, Mexico). Guar gum and glycerol were
purchased at the raw materials local market (Saltillo, Mexico). Food
grade gallic acid (Golden Bell Reactives, Zapopan, Jalisco, Mexico)
was purchased from a reactive supplier.
2.5. Edible coating formulation
The edible coating was formulated as follows: Guar gum (0.8%),
Candelilla wax (0.2%), Glycerol (0.3%) and Gallic Acid (0.15%). It
was prepared according to (Saucedo-Pompa et al., 2007), with
some modifications. Guar gum was added to hot distilled water
(80 ◦ C), and dissolved with an homogenizer (model T 25 digital
ULTRA-TURRAX, Janke & Kunkel GmbH & Co., Staufen, Germany)
at 20500 rpm for 10 min. Candelilla wax was added and homog-
enized at 20500 rpm for 10 min. Finally, glycerol was added and
homogenized 10 min more at the same speed. The emulsion was
left to cool at 25 ◦ C. Physicochemical parameters of the edible coat-
ing were: thickness 0.059 cm, density 0.129 cm3 /g, permeation rate
to water vapor 2990.81 (g/h)*m2 , water vapor transmission 0.74
(g/h) m2 *Pa, transparency 13.05%, humidity 33.00% and solubility
12.34%.
2.6. Fruit inoculation with biocontrol bacteria
Bacillus subtilis HFC103 strain was grown in horizontal shak-
ing (120 rpm) for 24 h at 25 ◦ C in modified potato-dextrose-agar
medium (MPDA) in which agar was not added (Johnson and Curl,
1972). Bacterial suspension was adjusted to yield 1 × 106 CFU ml−1
using MPDA. Fruits were immersed in the bacterial suspension for
2 s and were allowed to dry at 25 ◦ C for 15 min.
2.7. Application of the edible coating
Candelilla wax based coatings were applied on fresh straw-
berries, previously inoculated with Bacillus subtilis HFC103, to
evaluate their antifungal efficacy against Rhizopus stolonifer. The
edible coatings were applied according to the methodology used
by (Saucedo-Pompa et al., 2007) with some modifications. Fruits
were immersed in the emulsion at 25 ◦ C for one second and were
allowed to dry at 25 ◦ C for 15 min.
2.8. Shelf life assay
A shelf life assay was carried out to verify the effect of candelilla
wax edible coatings combined with B. subtilis HFC103 on decay
and quality of strawberries. The treatments were: control (without
coating and non-inoculated), film (edible coating), bacteria (inocu-
lated with B. subtilis HFC103) and film + bacteria (edible coating + B.
subtilis HFC103). Each fruit was a single replication and there were
27 replications per treatment (n = 27).
Decay percentage is the percentage of unacceptable fruits, con-
sidering as unacceptable fruit that in which the pulp has lost its
firmness and that has dark spots on more than 15% of its surface
due to the attack of natural microorganisms.
 E. Oregel-Zamudio et al. / Scientia Horticulturae 214 (2017) 273–279
For quality measurements, fruit weight was recorded (model
AQT 600, Adam Equipment Company, Oxford, England) every 24 h
during a 6 d period and the percentage of lost weight was calculated.
Each fruit was a single replication and there were 27 replications
per treatment (n = 27).
The soluble solids content was obtained by measuring the
Refractive Index of the strawberry juice using a manual refractome-
ter (model ATC-1, Atago Co., Tokyo, Japan); fruits were cut in half
and a drop of juice was placed on the lens and the reading was
taken in degree Brix (◦ Bx). This reading gives the% of soluble solids
content in the fruit. The average of ◦ Brix per fruit was recorded. The
average of three fruits was a single replication and there were three
replications per treatment (n = 9).
For pH measurements three fruits samples were homogenized
with a kitchen mixer for 5 min. The pH of the mix was measured
with a pH meter (model Thermo Orion 920A, Thermo Fisher Scien-
tific Inc., Boston MA, USA). The average of three fruits was a single
replication and there were three replications per treatment (n = 9).
2.9. Antifungal assay with Rhizopus stolonifer
A shelf life assay was conducted to verify the effect of can-
delilla wax edible coatings combined with B. subtilis on damage
caused by R. stolonifer on strawberries fruits, during 6 d of storage
at 25 ◦ C. The fruits were set into four groups with nine strawberry
fruits per group and there were three replications: (i) control (with-
out candelilla wax edible coating and non-inoculated); (ii) film
(candelilla wax edible coating); (iii) bacteria (inoculated with B.
subtilis HFC103); (iv) film + bacteria (candelilla wax edible coating
and inoculated with B. subtilis HFC103). Fruits were placed on ster-
ile polyethylene boxes. The fungal suspensions (1 × 106 spores per
ml) were sprayed onto fresh strawberry fruits in sterile conditions
at 25 ◦ C. Each side of each fruit was sprayed twice. After spray-
ing, fruits were allowed to dry for 15 min under sterile conditions.
Severity index was calculated during storage, with five degrees of
damage based on a scale with the following categories 1 = 0–24,
2 = 25–49, 3 = 50–74, 4 = 75–99, 5 = 100% of visual damage per fruit.
The equation used was Eq. (1).
Severityindex = (Xi(1) + Xi(2) + Xi(3) + Xi(4) + Xi(5))/N
Where: Xi = Number of diseased fruits for every degree of damage;
1, 2, 3, 4, 5 = degree of damage on the scale, N = Total number of
fruits per experimental unit.
2.10. Statistical analysis
For each measurement, three replicates were tested. Analysis of
variance and Tukey test were used to perform statistical analysis
®
on all the results, using SAS System for Windows 9.0 version. Dif-
ferences between means were considered to be significant when
p ≤ 0.05.
3. Results and discussion
3.1. Shelf life assays
Edible coatings and postharvest biocontrol have been widely
studied, however, there are no reports about their combination
to form edible films applied to fruits. According to the results
obtained in this work, the combination of both preservation meth-
ods, showed the potential to preserve strawberry.
During the shelf life assay with edible coatings made of can-
delilla wax and B. subtilis HFC103, separate or combined, all the
treatments showed 100% of acceptable fruits on day 1; on the sec-
ond storage day only the bacteria and film + bacteria treatments
275
significantly reduced the decay percentage (≈ 8 and 13%) with
respect to control. While the film, bacteria and film + bacteria treat-
ments significantly reduced the decay percentage with respect to
the control from day three (≈81, 52 and 92% respectively) to the end
of the assay. Interestingly, the film + bacteria treatment achieved
the reduction of decay almost 100% with respect to the control on
day 6 (Fig. 1).
During the shelf life assay, all the treatments constituted a
protecting coating that delayed strawberry decay. In the case of
treatments based on candelilla wax, this might have been due to
the generation of microenvironments with conditions that did not
favor the growth of saprophytic microorganisms, which contribute
to postharvest deterioration, attacking the fleshy surface of the fruit
when it is ripe (Peretto et al., 2014). In this regard, it is known that
during the postharvest stage, multiple and complex mechanisms
are activated in the fruits as a response to the attack from pathogens
(Prusky et al., 2014). Additionally, it has been reported that it is
possible to stimulate response mechanisms to delay the deteriora-
tion processes through biocontrol microorganisms (Parafati et al.,
2015). Such mechanisms include the activation of ethylene biosyn-
thesis, cuticular changes, changes in the cell wall and others (Prusky
et al., 2014), which could be induced as a result of the B. subtilis
activity, however, more research is required.
Microorganisms like biocontrol agents have some limitations,
for example, they depend on the environmental conditions, gener-
ally they only affect one target organism, however, the biocontrol
agent may be fed by a beneficial microorganism, besides this, not all
the diseases can be controlled by biological agents, finally, biocon-
trol does not eliminate the target population completely (Waage
et al., 2012). These limitations can be resolved through the manip-
ulation of formulations, or in some other ways (Ilhan and Karabulut,
2013). Besides, biocontrol can be integrated with other alternative
methods that do not provide an appropriate protection by them-
selves, but when they are combined with biocontrol they may have
a synergic effect (Grosch et al., 2012). This was evidenced with the
treatment that combined the coating with B. subtilis HFC103 and the
candelilla wax edible coating. This treatment surpassed the effect
of individual treatments by keeping a greater amount of acceptable
fruit during the shelf life assay, reducing decay almost 100% with
(1) respect to the control on day 6 (Fig. 1). Just as expected, the can-
delilla wax coatings fulfilled their protection function, by covering
the fruits and performing as a semipermeable barrier (Kester and
Fennema, 1986), while B. subtilis HFC103 might have contributed to
the decrease of decay percentage through one or more of its multi-
ple biocontrol mechanisms reported previously, such as antibiotics
production (Stein, 2005), siderophores (Yu et al., 2011), among oth-
ers. Another B. subtilis HFC103 protection mechanism could be the
biofilms formation (Bais et al., 2004), contributing with an addi-
tional semipermeable barrier.
Postharvest weight loss is an important indicator of the
freshness of fruits, and its control is a challenge for their commer-
cialization. It is known that the water migration from the fruit to
the environment is the main cause of weight loss during storage
(Duan et al., 2011). Just as expected, the results showed that the
weight loss percentage increased during storage in all the treat-
ments (Elsabee and Abdou, 2013). However, all the treatments
with candelilla wax and/or B. subtilis HFC103 coatings showed sig-
nificantly lower weight loss than control (Fig. 2). Interestingly,
the bacteria treatment significantly reduced the weight loss with
respect to the control on the days 1, 3, 4, 5 and 6 by about 4% 13% 16%
14% and 16%, respectively. It is noteworthy that the film treatment
along the assay kept a lower weight loss with respect to the rest of
the treatments, and reduced it significantly with respect to control
on the days 1, 2, 3, 4, 5 and 6 9, 13, 31, 31, 26 and 30%, respectively
(Fig. 2). This could be attributed to the implicit characteristics that
the coating provides as an additional semipermeable layer, which
276 E. Oregel-Zamudio et al. / Scientia Horticulturae 214 (2017) 273–279

Fig. 1. Evolution of the decay percentage on strawberry fruits during 6 d of storage at 25 ◦ C. Different letters within each storage day indicate significant differences based
on Tukey test (P < 0.05, n = 27).

Fig. 2. Weight loss (%) of strawberry fruits during 6 d of storage at 25 ◦ C. Different letters within each storage day indicate significant differences based on Tukey test (P < 0.05,
n = 27).
 E. Oregel-Zamudio et al. / Scientia Horticulturae 214 (2017) 273–279
Table 1
Physicochemical parameters.
Day 0
pH Control 3.514 ± 0.009a
Film 3.514 ± 0.009a
Bacteria 3.512 ± 0.011a
Film + Bacteria 3.510 ± 0.012a
soluble Control 7.1 ± 0.1a
solids (◦ Brix) Film 7.1 ± 0.1a
Bacteria 7.1 ± 0.1a
Film + Bacteria 7.1 ± 0.1a
restricts the transport of water vapor from the interior, with con-
sequent reduction of the transpiration and the possible microbial
activity (Bisen et al., 2012), as it has been shown on a wide variety
of fruits (Díaz-Mula et al., 2009). On the other hand, the differences
in the capacities among the different treatments to reduce weight
loss, could be attributed to the different permeability to water vapor
on the part of polysaccharides and the compounds present on the
formulation of the edible coatings and the biofilm made of bacteria
(Al-Hassan and Norziah, 2012).
Previous works have shown a decrease of weight loss of straw-
berry dependent on the coatings composition, that perform as a
semipermeable barrier against moisture loss (Gol et al., 2013).
However, there are no reports in which biocontrol microorgan-
isms are used to decrease the weight loss percentage. One possible
action mechanism that explains the behavior of the weight loss
percentage decrease in treatments with bacteria could be that B.
subtilis HFC103 develops structurally complex multicellular matri-
ces named biofilms. Genetic, biochemical and cytological evidence
has shown that they are mainly made of a protein component and
a polysaccharide component (Branda et al., 2006), whose nature is
the same of those commonly used as ingredients for the elabora-
tion of edible coatings, and they could be acting as an additional
barrier over the layer of epidermis cells of fruits.
It is worth stressing that none of the treatments affected nega-
tively the quality parameters of the fruits during the shelf life assay.
The treatments applied did not affect the pH of fruits. However,
a significant decrease of this parameter with all the treatments
between day 4 and day 6 was observed (Table 1). Sugars just as
much as the organic acids present on the ripe fruits, contribute sig-
nificantly to their flavor. Even though the patterns of accumulation
of organic acids are frequently different when considering the total
amount of this compounds per fruit, in the pulp of several fruits
there is an increase in the content of organic acids during the great-
est part of their development and ripening reviewed by Famiani
et al. (2015). Several mechanisms dependent on the cultivar, can
use a series of metabolic pathways that determine such patterns
(Famiani et al., 2015). It has been reported that the main organic
acid that accumulates in ripe strawberries, is citric acid (Famiani
et al., 2005), which may be involved in the decrease of pH observed
during the shelf life assay in all treatments.
It has been reported that total soluble solids in strawberry,
increase during development and ripening of the fruits, given that
the abundance of soluble sugars (glucose, fructose and sucrose)
grows per fruit as well as per gram of fresh tissue (Famiani et al.,
2005), reaching values that oscillate between 4.6 and 11.9◦ Brix
during red ripe stage (Famiani et al., 2005; Kader, 1991; Sharma
and Shyan, 2009). In the present work, the fruits showed ◦ Brix val-
ues within the range above mentioned (≈7.0), and the differences
found among several studies may be attributed to the cultivar used
(Sharma and Thakur, 2008). Even though a significant increase of
◦ Brix has been reported for several fruits during postharvest stor-
age, given that their metabolism continues through the conversion
of starch and acid to sugars (Cao et al., 2015); ◦ Brix did not show
277
Day 2 Day 4 Day 6
3.514 ± 0.009a 3.472 ± 0.008b 3.464 ± 0.005b
3.520 ± 0.010a 3.480 ± 0.007b 3.456 ± 0.005c
3.508 ± 0.004a 3.476 ± 0.021b 3.438 ± 0.013c
3.512 ± 0.008a 3.452 ± 0.008b 3.450 ± 0.007b
7.2 ± 0.1a 7.1 ± 0.1a 7.2 ± 0.1a
7.1 ± 0.1a 7.2 ± 0.1a 7.2 ± 0.1a
7.1 ± 0.1a 7.1 ± 0.1a 7.2 ± 0.1a
7.1 ± 0.1a 7.1 ± 0.1a 7.2 ± 0.1a
any significant change during the shelf life assay (Table 1). On the
other hand, no significant effect was found on the accumulation
of sugars in strawberry fruits regarding the application of coatings
made with candelilla wax and biocontrol bacteria, during the shelf
life assay.
Means are followed by standard deviations. Different letters
within each row indicate significant differences among days of
storage based on Tukey test (P < 0.05, n = 3).
3.2. Assays with Rhizopus stolonifer
R. stolonifer is considered one of the main phytopathogens that
cause postharvest disease; it causes postharvest rots in agricultural
products, it is responsible for important economic losses. It is a cos-
mopolitan minor filamentous fungi, widely distributed in nature,
and it survives in a saprophytic form in the soil and in organic waste
with the potential to invade plant tissues. It occurs during storage,
transport and commercialization of various agricultural products,
such is the case of strawberry (Bautista-Baños et al., 2014). The fun-
gus inoculum is present on the field, and stays suppressed during
the fruit growth, until a particular phase during storage and if the
conditions allow it, the pathogens change to a necrophytic form of
life (Byrde and Willetts, 2013).
The postharvest application of the treatments with edible coat-
ings and bacterial antagonists resulted in a significant reduction
of R. stolonifer severity index. All the treatments applied, reduced
significantly the severity index regarding the control from day 2 to
the end of the assay (Fig. 3). The film treatment reduced the sever-
ity index regarding the control on days 2, 3, 4, 5 and 6by about 13,
30, 37, 46 and 47% respectively. This reduction could be attributed
to the action of the coatings that promoted non favorable condi-
tions for the fungi occurrence, delaying the damage caused by fungi
(Peretto et al., 2014). The film + bacteria treatment also reduced
significantly the severity index regarding the control on days 2,
3, 4, 5 and 6 (≈21, 41, 47, 54 and 56%, respectively). The major
impacts of protection against R. stolonifer, were achieved with B.
subtilis HFC103 applied individually, which reduced significantly
the severity index regarding the control on days 2, 3, 4, 5 and 6 by
about27, 52, 64, 74 and 78%, respectively.
Several modes of action have been proposed to explain the
microbial antagonists biocontrol activity (Zhao et al., 2010). The
competition between the pathogen and the antagonist for nutri-
ents and space is considered one of the main mechanisms through
which antimicrobial agents control pathogens that cause posthar-
vest damage, besides the production of antibiotics and induced
resistance, among others (Sharma et al., 2009). B. subtilis is known to
act through multiple mechanisms of biological control altogether
(Li et al., 2013), and probably more than one mechanism acts to
inhibit R. stolonifer growth. A large number of studies on the use of
microbial antagonists have been carried out (Sharma et al., 2009).
There is evidence that certain B. subtilis strains inhibit the growth
of R. stolonifer (Wang et al., 2013). However, the mechanism or
278 E. Oregel-Zamudio et al. / Scientia Horticulturae 214 (2017) 273–279
Fig. 3. Evolution of severity index of Rhizopus stolonifer fungi on strawberry fruits during 6 d of storage at 25 ◦ C. Different letters within each storage day indicate significant
differences based on Tukey test (P < 0.05, n = 27).
mechanisms through which biocontrol microorganisms influence
the pathogens has not been yet completely elucidated.
According to the results obtained in this work, we could spec-
ulate that, the competition for space and nutrients could play an
important role to reduce the deterioration caused by R. Stolonifer.
Moreover, it is known that as a response to fruit infection with
pathogens in early stages of harvest, the fruit starts defensive sig-
nal transduction cascades that culminate with the accumulation
of antifungal proteins that limit the growth and development of
fungi (Prusky et al., 2014). B. subtilis could contribute to the induc-
tion of such signals as it has been reported with other organs like
the root (Li et al., 2013). On the other hand, it is known that B.
subtilis controls pathogens through the production of antibiotics
(iturine), which is known as the second most important mecha-
nism through which microbial antagonists remove pathogens from
fruits and vegetables after harvest (Gueldner et al., 1988). As it was
expected, the treatments with B. subtilis HFC103 were quite effec-
tive in the reduction of soft rot of strawberry caused by R. stolonifer,
since in previous studies the strain was selected for such purpose.
However, the mechanism or mechanisms through which this effect
is exerted are still unknown and further research is required to
elucidate them.
4. Conclusions
The results showed that the combination of candelilla wax
edible films and biocontrol bacteria (B. subtilis) is a promising alter-
native for the reduction of postharvest deterioration of strawberry.
These both tools preserve the desired quality parameters such as
total soluble solids and pH, besides reducing the fruit decay and
providing significant protection against R. stolonifer, which is an
important postharvest phytopathogen.
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