Industrial Crops & Products 156 (2020) 112884

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Industrial Crops & Products

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Essential oils from Eucalyptus staigeriana F. Muell. ex Bailey and Eucalyptus

urograndis W. Hill ex Maiden associated to carboxymethylcellulose coating
for the control of Botrytis cinerea Pers. Fr. and Rhizopus stolonifer (Ehrenb.:
Fr.) Vuill. in strawberries
Paula Porrelli Moreira da Silva a, *, Jacqueline de Oliveira a, Anaíle dos Mares Biazotto a,
Marise Martins Parisi b, Eduardo Micoti da Glória c, Marta Helena Fillet Spoto a
a
Department of Agri-food Industry, Food and Nutrition, “Luiz de Queiroz” College of Agriculture, University of São Paulo, Piracicaba, São Paulo, 11 Pádua Dias Avenue,
13418-900, Brazil
b
São Paulo Agribusiness Technology Agency (Center-South Regional Pole), Piracicaba, São Paulo, 127 SP Highway, km 30, 13400-970, Brazil
c
Biological Sciences Department, “Luiz de Queiroz” College of Agriculture, University of São Paulo, Piracicaba, São Paulo, 11 Pádua Dias Avenue, 13418-900, Brazil

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

Keywords: The fungi Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. (R. stolonifer (Ehrenb.:Fr.) Vuill.) and Botrytis cinerea Pers. Fr.
Minimal Fungicidal Concentration (B. cinerea Pers. Fr.) are causal agents of important post-harvest diseases in strawberries. For their control, the
Edible Coatings essential oil (EO) extracted from the leaves of Eucalyptus staigeriana Muell. ex Bailey (E. staigeriana F. Muell. ex
Median Effective Concentration
Bailey) and Eucalyptus urograndis W. Hill ex Maiden (E. urograndis W. Hill ex Maiden) can be a good alternative to
Scanning Electron Microscopy
Carboxymethylcellulose
the synthetic fungicides. Thus, the antifungal activity of the EOs from E. staigeriana F. Muell. ex Bailey and from
E. urograndis W. Hill ex Maiden was evaluated in vitro on the fungi B. cinerea Pers. Fr. and R. stolonifer (Ehrenb.:
Fr.) Vuill.. The effects on pathogen morphology and the in vivo antifungal activity of the EO that presented the
highest activity in vitro, associated with carboxymethylcellulose (CMC), were also verified. The chemical
composition of the essential oils evaluated was determined. The EO from E. staigeriana F. Muell. ex Bailey was the
most efficient in the in vitro assays on the two fungi. This EO, whose major compound is limonene, caused
dehydration and rupture of the fungal hyphae, suggesting its action is on the cell wall and membrane. The
incorporation of this EO to CMC in the curative way reduced the severity of soft rot, caused by R. stolonifer
(Ehrenb.:Fr.) Vuill.; nevertheless, it did not present an effect on the inhibition of B. cinerea Pers. Fr.. These results
suggest that E. staigeriana F. Muell. ex Bailey EO, associated to CMC coating, can be an alternative to the use of
synthetic fungicides for the control of R. stolonifer (Ehrenb.:Fr.) Vuill., mainly.

1. Introduction For the control of deteriorating or pathogenic microorganisms in

Essential Oils (EOs) are secondary metabolites produced in several
parts of the plant in response to some kind of stress (physiological, attack
of pathogens or ecological factors) (Duarte et al., 2018). In nature, they
are identified as plant defense compounds, being attractive for pollina­
tors, which facilitates plant reproduction. They are characterized as
volatile oily liquids, with strong aroma, limpid and rarely colorful, sol­
uble in organic solvents and with density usually below that of water
(Bakkali et al., 2008; Duarte et al., 2018{Bakkali et al., 2008, Biological
effects of essential oils - A review}).
foods, the EOs are generally advantageous when compared with the use
of synthetic antimicrobial agents, since they present low toxicity to
mammals, they are biodegradable and non-persistent in the environ­
ment, besides presenting low production cost (Rehman et al., 2016).
Researchers have reported that 60% of the plant EOs, or of their de­
rivatives, have inhibitory effect against a wide range of fungi, with the
possibility of being used to control fungal rots in fruits and vegetables,
extending their shelf-life (Meepagala et al., 2020; Bertoli et al., 2011). In
this scenario, studies have proved shelf-life extension in either in natura
or processed foods in which EOs with antimicrobial activity were

* Corresponding author.
E-mail addresses: pporrelli@usp.br (P.P.M. da Silva), jacquelineot@hotmail.com (J. de Oliveira), anaile.biazotto@gmail.com (A.M. Biazotto), marisemartins@
yahoo.com.br (M.M. Parisi), emgloria@usp.br (E.M. da Glória), martaspoto@usp.br (M.H.F. Spoto).

https://doi.org/10.1016/j.indcrop.2020.112884
Received 28 April 2020; Received in revised form 3 July 2020; Accepted 18 August 2020
Available online 25 August 2020
0926-6690/© 2020 Elsevier B.V. All rights reserved.
P.P.M. da Silva et al.
applied (Noshirvani and Fasihi, 2018; Sarkhosh et al., 2018; Zillo et al.,
2018; An et al., 2019).
Another technique used to extend the shelf-life of fruits is the
application of edible coatings, which are made from biodegradable in­
gredients, and form a thin layer of this material on the fruit. Edible
coatings act as a selective barrier for gas and moisture transfer, and
prevent the attack of microorganisms (Vargas et al., 2008). Carboxy­
methylcellulose (CMC) is one of the most common cellulose derivatives,
and is widely used in the preparation of edible coatings (Tongdee­
soontorn et al., 2011). EOs can be associated to CMC to avoid being
applied directly to the fruit, which could damage its sensory charac­
teristics. Another advantage of this technique is that EOs may become
more efficient, since the coating will allow the release of active agents
for a longer period (Wattanasatcha et al., 2012).
The genus Eucalyptus comprises more than 700 species distributed
around the world, of which more than 300 contain volatile oils in their
leaves (Pino et al., 2002; Ishnava et al., 2013). Researches have
demonstrated antioxidant and antimicrobial properties of eucalyptus
EOs against a wide range of microorganisms (Tyagi and Malik, 2011;
Dos Santos et al., 2012). Nonetheless, studies on the antifungal activity
of the EOs extracted from the leaves of E. staigeriana F. Muell. ex Bailey
and E. urograndis W. Hill ex Maiden are scarce or missing. Researches
have been proving the antimicrobial properties of these EOs against
bacteria, filamentous fungi and food-deteriorating yeasts, as well as
bacteria that are pathogenic to the man (Goldbeck et al., 2014; Hercu­
lano et al., 2015), besides the insecticidal property against insets
considered pests in agriculture (Gomes and Favero, 2013; De Souza and
Favero, 2015; Cruz et al., 2017). Therefore, there is a gap for researches
aiming at evaluating the antimicrobial capacity of these eucalyptus EOs
against fungi with importance in the post-harvest of fruits, such as
strawberry, which presents high perishability because of the contami­
nation by deteriorating fungi, with consequent damage in its commer­
cialization and shelf-life.
Gray mold, caused by Botrytis cinerea Pers. Fr., and soft rot, caused by
species of Rhizopus and Mucor, are fungal diseases with great incidence
in strawberry in the post-harvest, leading to considerable losses. Gray
mold causes light brown spots that rapidly increase and take the whole
fruit, resulting in rot and a dry and firm aspect, with a gray coating that
is composed of the fungal structures, thereby originating the name of the
disease. On the other hand, the characteristic symptom of soft rot caused
by Rhizopus stolonifer (Ehrenb.:Fr.) Vuill., as indicated by the name itself,
is a soft and watery rot, with juice leakage, which, under high moisture,
is covered by mycelium, interspersed with pathogen structures (Yang
and Jiang, 2015; Parisi et al., 2018). Currently, the control of these
pathogens is dependent on the use of synthetic fungicides, which are
many times applied incorrectly and excessively, leading to contamina­
tion problems, besides promoting the appearance of resistance to active
principles in the fungal populations (Ghini and Kimati, 2000). Thus,
eucalyptus EOs can be a good alternative for the producers and mer­
chants of strawberry in natura, adding more quality and sustainability to
the production chain.
Therefore, the purposes of this work were to investigate if the EOs
from E. staigeriana F. Muell. ex Bailey and from E. urograndis W. Hill ex
Maiden present antifungal action in vitro on the fungi B. cinerea Pers. Fr.
and R. stolonifer (Ehrenb.:Fr.) Vuill. isolated from strawberry and to
determine the chemical composition of these EOs. Another goal was to
verify whether the most efficient EO in the assays in vitro causes any
effects on pathogen morphology and if, when combined with the
carboxymethylcellulose-based coating, controls in vivo the diseases gray
mold and soft rot in strawberries, considering the preventive and cura­
tive applications. The carboxymethylcellulose was used in this work
because, besides being widely used in fruits, is one of the most common
derivatives of cellulose, easy solubility in water and is non-toxic and
non-allergenic (Tongdeesoontorn et al., 2011), being a sustainable
alternative and low cost.
2
Industrial Crops & Products 156 (2020) 112884
2. Material and Methods
2.1. Extraction and analysis of the chemical composition of the essential
oils from Eucalyptus staigeriana F. Muell. ex Bailey and from Eucalyptus
urograndis W. Hill ex Maiden
The leaves from Eucalyptus staigeriana F. Muell. ex Bailey and from
E. urograndis W. Hill ex Maiden were collected in Itatinga (SP, Brazil,
24◦ 59’34.8"S and 48◦ 41’14.4"W), in May and June of 2017 (autumn in
the Southern Hemisphere). The EOs from each species were obtained by
hydrodistillation in equipment of the Clevenger type, for four hours, at
the maximum temperature of 100 ◦ C, using a proportion of plant mass
and water of 1:5. After distillation, the EOs were dried in anhydrous
sodium sulfate and stored at 5 ◦ C in an amber glass bottle (Oliveira et al.,
2019a). The extraction yield was 2.74% for E. staigeriana F. Muell. ex
Bailey and 1.03% for E. urograndis W. Hill ex Maiden OEs (average
values for three extractions).
The chemical composition of the EOs from E. staigeriana F. Muell. ex
Bailey and from E. urograndis W. Hill ex Maiden was determined by gas
chromatography coupled to mass spectrometry, using the a CGMS 2010
(SHIMADZU), equipped with the mass detector QP 2010 Plus (SHI­
MADZU) and capillary column gas chromatography diphenyl dime­
thylpolysiloxane (5% diphenyl and 95% dimethylpolysiloxane). The
identification of the chemical compounds occurred by the calculation of
the Linear Retention Index (LRI), and they were compared with data
published in the literature (Adams, 2007) and with the existing mass
spectra libraries (NIST, WebBook, NIST 07 and WILEY 8).
2.2. In vitro antifungal activity of the EOs on Botrytis cinerea Pers. Fr.
and Rhizopus stolonifer (Ehrenb.:Fr.) Vuill
The pathogens were obtained by the direct isolation of fungal
structures from the infected strawberries, and molecularly characterized
as the species B. cinerea Pers. Fr. and R. stolonifer (Ehrenb.:Fr.) Vuill.,
according to the CTAB method (Doyle and Doyle, 1987), previously
described by Oliveira (2019).
The in vitro antifungal activity of the EOs was evaluated by the pa­
rameters: Percentage Growth Inhibition (PI), Minimal Inhibitory Con­
centration (MIC), Minimal Fungicidal Concentration (MFC) and Median
Effective Concentration (EC50). For this, the method of microdilution
was adapted with potato broth in 96-well plates, proposed by Elshafie
et al. (2016).
For each EO eight concentrations were evaluated, in which the
choice was based on previous tests, being initially emulsified in
Dimethyl Sulfoxide (DMSO) at the proportion 1:1 (EO:DMSO). Stock
solutions of each EO were prepared in potato nutrient liquid medium
(PDB) at different concentrations: 750; 1,000; 1,500; 2,000; 3,000;
4,000; 6,000 and 7,000 μL L-1 in the case of B. cinerea Pers. Fr.; and 750;
1,500; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000 μL L-1 in the case of
R. stolonifer (Ehrenb.:Fr.) Vuill.. As inoculum, a suspension of spores in
PDB was employed, standardized to 105 spores mL-1, prepared from
colonies with 7 days of growth in the culture medium potato-dextrose-
agar (PDA).
To compose the treatments, each well of the microplate was
completed with 150 μL of the stock solution and 50 μL of the spore
suspension. In order to compose the control (T1) the wells were
completed with the DMSO solution, at the same concentration of EO, in
PDB (150 μL) with the addition of the spore suspension (50 μL). As
second treatment (T2) the wells were completed with DMSO solution, at
the same concentration of EO, in PDB (200 μL). And as third treatment
(T3) the wells were completed with the EO stock solutions (200 μL). The
fourth treatment (T4) consisted of the stock solutions at the different
concentrations of EO in wich the spore suspensions that were added.
The microplates for the test with B. cinerea Pers. Fr. were incubated
at 23 ◦ C and with R. stolonifer (Ehrenb.:Fr.) Vuill. at 25 ◦ C, both under a
photoperiod of 12 hours. The absorbance was read at λ 600 nm in a plate
P.P.M. da Silva et al.
reader equipment (VictorX, Perkin Elmer) after 72 h of incubation for
B. cinerea Pers. Fr. and after 48 h for R. stolonifer (Ehrenb.:Fr.) Vuill..
These incubation periods were based on previous experiments. All
treatments were evaluated in triplicate, and each assay was installed
three times. The EO which obtained the highest antifungal activity was
employed in the following assays.
The percentage of fungal growth inhibition was calculated from the
absorbance values of each treatment, using the formula: PI (%) = [(T1-
T2)-(T4-T3)/(T1-T2)]*100.
MIC was assumed as the EO concentration with reduction of
approximately 95% of fungal growth (Eckert and Ogawa, 1988). To
evaluate the fungicidal effect of the substances, a new fungal inoculation
was performed in Petri dishes containing solidified PDA medium from
an aliquot of 100 μL of the samples which presented PI above 90%.
These dishes were maintained for 72 h at the appropriate conditions for
each fungus, and the Minimal Fungicidal Concentration (MFC) was
regarded as the lowest EO concentration at which the total inactivation
of fungal multiplication capacity occurred in comparison with the con­
trol (Suwanamornlert et al., 2018).
From the results of PI (%), the Median Effective Concentration
(EC50), and its confidence limits, were calculated, which correspond to
the concentration of substance that causes 50% of reduction in the
fungal mycelial growth in relation to the control.
2.3. Effect of the essential oil from Eucalyptus staigeriana F. Muell. ex
Bailey and from Eucalyptus urograndis W. Hill ex Maiden on fungal
morphology
The alterations on the morphology of the fungi caused by the action
of the EO that presented the highest antifungal activity in the evaluation
in vitro (subitem 2.2) were analyzed by Scanning Electron Microscopy
(SEM).
The samples were prepared according to Yu et al. (2015) with some
modifications. From the colonies of the fungi grown in PDA medium, a
spore suspension was prepared at the concentration of 106 spores mL-1.
An aliquot of 250 μL of this suspension was added to 25 mL of potato
broth, with further incubation for five days at 23 ◦ C for B. cinerea Pers.
Fr. and three days at 25 ◦ C for R. stolonifer (Ehrenb.:Fr.) Vuill.. The EO,
emulsified with DMSO (1:1) at the same concentration indicated by the
MIC of each fungus, was added to the potato broth containing the
pathogen after the period of incubation, waiting for further 6 h. Control
samples, without the addition of the EO to the potato broth, were also
prepared. Two installations of this experiment (for each fungus) were
set, with samples in duplicate. The preparation of the material for
evaluation by SEM occurred according to Escanferla et al. (2009) from
the dehydration of the fungal mycelium with further observation at a
Scanning Electron Microscope LEO 435 (Zeiss, England).
2.4. In vivo antifungal activity of the essential oil from Eucalyptus
staigeriana F. Muell. ex Bailey and from Eucalyptus urograndis W. Hill ex
Maiden incorporated to carboxymethylcellulose
This step aimed at evaluating the efficiency of the application in
strawberries (Fragaria spp, in vivo) of the incorporation of the EO which
presented the highest antifungal activity in the experiment in vitro to the
carboxymethylcellulose (CMC) edible coating.
2.4.1. Raw materials and experimental procedures
The EO was evaluated in vivo both preventively and curatively
regarding the infection of the fungi B. cinerea Pers. Fr. and R. stolonifer
(Ehrenb.:Fr.) Vuill., either in association or not to the CMC coating,
which resulted in five treatments: C = fruit without the application of
CMC and EO (control); CMC_prev = fruit treated preventively with only
CMC; CMC_EO_prev = fruit treated preventively with CMC with the
addition of EO; CMC_cur = fruit treated curatively with only CMC and
CMC_EO_cur = fruit treated curatively with CMC with the addition of
3
Industrial Crops & Products 156 (2020) 112884
EO.
CMC presented 99.8% of purity (dry basis), 7.6% of moisture, pH
equal to 7.0 with 340 cP (1% solution at 25 ◦ C) and degree of substitu­
tion of 0.86. CMC was employed at the concentration of 1% and its
preparation was performed by the dilution in distilled water at 60 ◦ C
under stirring on a mechanical stirrer (Fisatom - 713D), at 1,000 rpm, for
15 minutes. Subsequently, glycerol was added at a concentration of
0.5%, as a plasticizing agent (Dashipour et al., 2015), following stirring
for further 20 minutes.
The strawberries were derived from an organic crop located in the
municipality of Cambuí (MG, Brazil, 22◦ 36’ 43" S and 46◦ 03’ 28" W).
Those with a ripe appearance and not presenting, at naked eye, physical
damages or derived from microorganisms, were selected, being then
sanitized by immersion on a 2.5% chlorine solution, for two minutes.
To compose the treatments containing EO (CMC_EO_prev and
CMC_EO_cur), the EO previously emulsified with Tween-80 was added
to the CMC (at 25 ◦ C) at the proportion 2:1 (v/v), due to the toxicity
presented by DMSO in humans. The concentration of EO incorporated in
this coating was extrapolated to the concentration 6,000 μL L-1, which
corresponds to three times the MIC for B. cinerea Pers. Fr. and four times
for R. stolonifer (Ehrenb.:Fr.) Vuill., considering the last as more resil­
ient, since when comparing tests in vivo with tests in vitro, the action of
the essential oils is different among them, making it necessary to in­
crease EO concentration in tests in vivo (Hyldgaard et al., 2012).
For the evaluation in the preventive mode of action, the strawberries
were immersed in their respective treatments for two minutes. After
natural drying, at room temperature, the strawberries were individually
placed in Polyvinyl Chloride (PVC) trays. The fungi (B. cinerea Pers. Fr.
or R. stolonifer (Ehrenb.:Fr.) Vuill.) were inoculated in the strawberries
by the deposition of 30 μL of an inoculum suspension containing 105
spores mL-1, of each pathogen, on a 3-mm deep wound, made with an
insulin needle of 0.25 mm in diameter (BD Ultra-FineTM). The fruit were
placed in a wet chamber at 95% of relative humidity, maintained at
23 ◦ C for B. cinerea Pers. Fr. and at 25 ◦ C for R. stolonifer (Ehrenb.:Fr.)
Vuill., both under a photoperiod of 12 h. After 24 h, the wet chambers
were removed and the fruit remained for up to seven days at the con­
ditions of temperature and photoperiod described.
To evaluate the curative mode of action, the inoculation of the fungi
occurred 24 h before CMC application, with or without EO. The further
procedures were the same described for the preventive mode of action.
The control treatment (C) was composed of strawberries immersed into
sterilized distilled water, for two minutes. Thus, the experimental design
for B. cinerea Pers. Fr. was in a 5 × 7 factorial scheme, involving five
treatments (C, CMC_prev, CMC_EO_prev, CMC_cur and CMC_EO_cur)
and seven consecutive days of evaluation; and for R. stolonifer (Ehrenb.:
Fr.) Vuill. 5 × 3, involving the same treatments and three consecutive
days of evaluation. Five repetitions were used for each treatment, each
repetition composed of 12 strawberries. Each installation of the exper­
iment was performed twice, with an interval of one year.
2.4.2. Evaluation of the antifungal efficiency
The strawberries were evaluated every 24 h by a scale of six levels of
disease severity (size of the area wounded by the fungus), considering:
0 = absence of symptoms; 1 = 1 to 20%; 2 = 21 to 40%; 3 = 41 to 60%;
4 = 61 to 80% and 5 = above 81%, proposed by Oliveira et al. (2019b).
The results were expressed as Disease Index (DI), according to Cia et al.
(2010): DI (%) = {[(1*n1)+(2*n2)+(3*n3)+(4*n4)+(5*n5)]
*100}/(5*N), in which ni is the number of fruit infected in each level of
the scale of scores and N is the total number of fruit. From the DI (%) of
each treatment, the Area Under the Disease Progress Curve (AUDPC)
was calculated, as proposed by Campbell and Madden (1990): AUDPC =
∑
[(yi+yi+1)/2*(ti+1-ti)], in which yi = DI at time ti; yi+1 = DI at time
ti+1; ti = initial reading time and ti+1 = time in days of each read.
The value of the AUDPC summarizes quantitatively the intensity of
the disease over time, combining multiple observations of the disease
progress in a single value (Simko and Piepho, 2012).
P.P.M. da Silva et al. Industrial Crops & Products 156 (2020) 112884

2.5. Statistical analysis of the results Table 1

Chemical composition of the essential oil extracted from Eucalyptus staigeriana F.
The values of percentage of fungal growth inhibition (PI) of each Muell. ex Bailey leaves.
fungus were subjected to the analysis of variance (ANOVA) for the F Test Compounds1 Area (%) 2 LRI 3
and the means were compared by the Tukey’s Test at the level of sig­ α-Thujene 11.22 930
nificance of 5%. The determination of the Median Effective Concentra­ α-pinene 6.6 937
tion (EC50) of each EO and of its corresponding limits of confidence at β-pinene 3.42 981
95%, for each fungus, was performed by the Probit analysis from Myrcene 0.84 995
α-Phellandrene 1.58 1009
concentration-response log curves (logistic function), with the variable
Terpinene < alpha-> 0.15 1021
being the response to PI (%). Cymene < para> (p-cymene) 0.9 1029
The results of the AUDPC calculation regarding the treatments Limonene 14.93 1034
studied in the in vivo approach of the substances on each fungal species 1,8 cineole 5.89 1037
were subjected to the analysis of variance (ANOVA) for the F Test in cis-β-ocimene 0.41 1042
trans-β-ocimene 0.31 1053
randomized blocks, which corresponded to the two experiments in vivo. γ-terpinene 1.19 1064
The means were compared by the Tukey’s Test at the level of signifi­ Linalool oxide, (furanoid), cis- 0.14 1078
cance of 5%. α-Terpinolene 6.75 1093
The statistical software Statistical Analysis System (SAS) model 9.4 Linalool 1.41 1104
Isopentyl isovalerate 0.22 1109
was employed to conduct the statistical analyses.
trans-Pinocarveol 0.13 1146
cis-Pinocarveol 0.11 1170
3. Results p-mentha-1,5-dien-8-ol 0.58 1173
Terpinen-4-ol 1.51 1183
3.1. Chemical composition of the essential oils from Eucalyptus NI 0.35 1187
p-Cymen-8-ol 0.37 1192
staigeriana F. Muell. ex Bailey and from Eucalyptus urograndis W. Hill ex α-Terpineol 2.66 1197
Maiden Nerol 1.4 1234
Nerol 6.41 1248
The chemical composition of the EOs from E. staigeriana F. Muell. ex Geraniol 5.65 1261
Methyl citronellate 0.3 1265
Bailey and from E. urograndis W. Hill ex Maiden was very distinct. A total
Geraniol 8.34 1278
of 43 compounds were found in the EO from E. staigeriana F. Muell. ex NI 0.27 1292
Bailey, five of which were not identified. Among those identified, Geranyl formate 0.1 1308
Limonene (14.93%), α-Thujene (11.22%) and Geranial (8.34%) were the Methyl geranate 6.09 1330
majoritarian (Table 1). In the EO from E. urograndis W. Hill ex Maiden, exo-2-Hydroxycineole acetate 0.17 1349
Citronellyl acetate 0.49 1358
34 compounds were detected, seven of which were not identified, with
Neryl acetate 1.49 1369
1,8 cineol (41.34%), α-pinene (27.66 %) and α-Terpinyl acetate (7.95%) Geranyl acetate 4.77 1388
being the majoritarian (Table 2). β-Caryophyllene 0.13 1430
The three major compounds present in the EO from E. staigeriana F. Bicyclogermacrene 0.23 1507
NI 0.13 1581
Muell. ex Bailey were not detected in the composition of the EO from
Spathulenol 0.09 1591
E. urograndis W. Hill ex Maiden. These EOs have 18 matching com­ Viridiflorol 1.01 1597
pounds. Among them, 1,8 cineol and α-pinene are the ones in the highest NI 0.74 1606
amount in E. urograndis W. Hill ex Maiden EO composition; nonetheless, Rosifoliol 0.34 1615
in E. staigeriana F. Muell. ex Bailey EO they are present in a low amount, NI 0.19 1636

5.89 and 6.60%, respectively. LRI = Linear Retention Index.

1
Identified by CG/MS, listed increasingly according to the LRI.
2
3.2. In vitro antifungal activity of the EOs from Eucalyptus staigeriana F. Relative amounts of the compounds identified based on the area of each peak
Muell. ex Bailey and Eucalyptus urograndis W. Hill ex Maiden on Botrytis in the total area of the chromatogram.
3
cinerea Pers. Fr. and Rhizopus stolonifer (Ehrenb.:Fr.) Vuill Calculated linear retention indices.

The EOs inhibited the development of B. cinerea Pers. Fr. and
R. stolonifer (Ehrenb.:Fr.) Vuill., presenting a dose-dependent antifungal
activity (Fig. 1). In general, the EO from E. staigeriana F. Muell. ex Bailey
exhibited the highest antifungal activity in vitro on the two fungal spe­
cies evaluated.
The MIC and the MFC of E. staigeriana F. Muell. ex Bailey EO for the
control of B. cinerea Pers. Fr. presented the same concentration of
2,000 μL L-1 (Table 3). In contrast, E. urograndis W. Hill ex Maiden EO
presented an unsatisfactory antifungal action, since its MIC was not
observed even for the highest dose evaluated (4,000 μL L-1), with its MFC
corresponding to 6,000 μL L-1.
The EO from E. staigeriana F. Muell. ex Bailey also exhibited the
highest antifungal capacity against R. stolonifer (Ehrenb.:Fr.) Vuill.,
since it inhibited the development of this fungus at the concentration of
1,500 μL L-1 (Table 3). Nevertheless, the concentration corresponding to
its fungicidal action was above that of the MIC, since the MFC for this EO
was not observed even for the highest dose evaluated (7,000 μL L-1).
Conversely, the EO from E. urograndis W. Hill ex Maiden demonstrated
antifungal capacity inferior to that of E. staigeriana F. Muell. ex Bailey,
since it could not inhibit fungal growth in any of the concentrations
evaluated.
From these results, it was verified that the sensitivity of the fungi to
the essential oils was different, with R. stolonifer (Ehrenb.:Fr.) Vuill.
being more resistant to the action of the EOs, being necessary higher
concentrations of any of the EOs tested to have its development fully
inhibited.
Regarding the calculation of the EC50 of the EOs from E. staigeriana F.
Muell. ex Bailey and from E. urograndis W. Hill ex Maiden, there are no
studies in the literature that have addressed the EC50 values for these
EOs, on any fungi that attack fruits in the post-harvest. E. staigeriana F.
Muell. ex Bailey EO was proven as the most efficient compound against
the fungi tested, since the EC50 values verified for this EO were 92.8 and
226.9 (μL L-1) for B. cinerea Pers. Fr. and R. stolonifer (Ehrenb.:Fr.) Vuill.,
respectively (Table 4). On the contrary, E. urograndis W. Hill ex Maiden
EO presented the highest EC50 values for both fungi, corresponding to
944 for B. cinerea Pers. Fr. and 1763 for R. stolonifer (Ehrenb.:Fr.) Vuill.,
confirming that this EO is not effective on the reduction of the devel­
opment of the fungi.
As observed in the results of MIC and MFC, R. stolonifer (Ehrenb.:Fr.)
Vuill. exhibited the highest EC50 values, considering the two EOs, which

4
P.P.M. da Silva et al. Industrial Crops & Products 156 (2020) 112884

Table 2 values of these treatments did not differ significantly (P < 0.05) from the
Chemical composition of the essential oil extracted from Eucalyptus urograndis control treatment (C) and from the fruit treated preventively with only
W. Hill ex Maiden leaves. carboxymethylcellulose (CMC_prev) (Table 5).
Compounds 1 Area (%) 2 LRI 3 Regarding the assay with R. stolonifer (Ehrenb.:Fr.) Vuill. inocula­
α-pinene 27.66 939
tion, the severity of the disease soft rot was the lowest (P < 0.05) in the
α-fenchene 0.12 951 fruit treated curatively with the CMC coating incorporated with
Camphene 0.46 953 E. staigeriana F. Muell. ex Bailey EO (CMC_EO_cur), indicating that this
β-pinene 0.18 982 treatment reduced the progress of the disease (Table 5). On the other
α-Phellandrene 0.3 1009
hand, the fruit treated preventively presented the highest disease
3-Methylbutyl 2-methylpropanoate 0.18 1017
Cymene < para> (p-cymene) 0.77 1030 severity, being similar (P < 0.05) to the control treatment. These results
1,8 cineole 41.34 1038 confirm the antifungal potential of the EO from E. staigeriana F. Muell. ex
cis-β-ocimene 4.83 1042 Bailey which, when applied curatively in association with CMC, can
trans-β-ocimene 0.22 1053 control R. stolonifer (Ehrenb.:Fr.) Vuill.. Moreover, these results are in
0.25 1064
agreement with observed in the in vitro test, which indicates that a lower
γ-terpinene
α-Terpinolene 0.71 1094
Linalool 0.22 1105 concentration of OE (1,500 μL L-1) is able to inhibit the mycelial growth
Fenchol 0.54 1120 of R. stolonifer (Ehrenb.:Fr.) Vuill.. Nevertheless, regarding B. cinerea
α-Campholenal 0.18 1133 Pers. Fr. it was not possible to observe a significant reduction in disease
Pinocarveol-trans 0.27 1146
progress when the EO was incorporated, this fact being observed in the
Borneol 1.44 1173
Terpinen-4-ol 0.8 1184 preventive application of CMC.
NI 0.14 1193
α-Terpineol 6.9 1198 4. Discussion
exo-2-Hydroxycineole acetate 0.1 1350
α-Terpinyl acetate 7.95 1357
β-Caryophyllene 0.58 1431
The main components of most of the essential oils are monoterpenes
NI 0.57 1469 (Vintilă et al., 2018), which belong to a class of secondary metabolites of
Bicyclogermacrene 0.21 1508 the plants, to which the efficient antifungal activity of the essential oils is
Geranyl isobutyrate 0.31 1519 attributed (Marei and Abdelgaleil, 2018; Zillo et al., 2018; Wang et al.,
NI 0.12 1534
2018; Oliveira et al., 2019a; Xing et al., 2019). Therefore, the inhibitory
Spathulenol 0.38 1591
Viridiflorol 0.68 1598 action of the EOs from E. staigeriana F. Muell. ex Bailey and from
NI 0.53 1606 E. urograndis W. Hill ex Maiden on the phytopathogenic fungi B. cinerea
Rosifoliol 0.16 1616 Pers. Fr. and R. stolonifer (Ehrenb.:Fr.) Vuill. is justified, since the
NI 0.45 1629 chemical composition of these EOs is mainly composed of mono­
NI 0.15 1637
NI 0.33 1642
terpenes, such as Limonene, α-Thujene, α-Terpinolene, α-pinene, 1,8
cineole, α-pinene, α-Terpinyl acetate, among others. Substances rich in
LRI = Linear Retention Index. terpenoids are natural antifungals because they are apolar chemical
1
Identified by CG/MS, listed increasingly according to the LRI.
2 substances with hydrophobic and lipophilic characteristics that facili­
Relative amounts of the compounds identified based on the area of each peak
tate interaction with the membrane elements fungal cell, interfering in
in the total area of the chromatogram.
3
Calculated linear retention indices.
energy homeostasis and caused cell membrane damage (Mello et al.,
2006; Viriato, 2014).
It is known that the production and composition of the EOs of a single
confirms its superior resistance to these antifungal compounds.
species are variable, depending on factors such as the type of material
Given the fact that E. staigeriana F. Muell. ex Bailey EO presented the
used for EO extraction (leaves, flowers, roots, etc.), seasonal variation,
lowest MIC, MFC and EC50 for the fungi evaluated, it was selected for
cultivation environment (climate, pollution, presence of diseases and
evaluation regarding its action on the morphology of the pathogens and
pests, conditions and type of the soil and rainfall), harvest time,
in the experiments in vivo.
geographic variation, method of extraction and storage of the EO (Fig­
ueiredo et al., 2008; Herculano et al., 2015; Dhouioui et al., 2016). The
3.3. Effect of the essential oil from Eucalyptus staigeriana F. Muell. ex antifungal action demonstrated by them is related to the chemical
Bailey on the morphology of the fungi composition of each one and with the amount of each compound iden­
tified, the antifungal action not being limited to only a single compound
The exposure of the fungi to the EO from E. staigeriana F. Muell. ex (Sharifi et al., 2008; Vilela et al., 2009). Furthermore, a possible syn­
Bailey caused structural alterations on their hyphae, suggesting its ac­ ergistic effect must be considered among the major compounds of the EO
tion on the cell wall. In the absence of the EO, the hyphae of the fungus on the antifungal action (Kazemi, 2014).
B. cinerea Pers. Fr. were turgid and elongated, with some branches E. staigeriana F. Muell. ex Bailey EO presented a similar chemical
(Fig. 2A). Nevertheless, the application of the EO caused the formation composition to that observed by other authors. Cruz et al. (2017)
of wrinkles, torsion, peeling and dehydration of the hyphae (Fig. 2B). observed the major compounds Limonene (28.73%), Geraniol (15.20%)
As exposed for B. cinerea Pers. Fr., the EO from E. staigeriana F. Muell. and Nerol (12.16%). Other works have also demonstrated that Limonene
ex Bailey caused dehydrations, fragmentations and torsions on the is predominant in E. staigeriana F. Muell. ex Bailey EO, with amounts
structure of R. stolonifer (Ehrenb.:Fr.) Vuill. hyphae (Fig. 2C). The hy­ varying between 16.85 and 72.9% of the total (Ribeiro et al., 2013;
phae remained turgid, elongated and with the septa defined in the Herculano et al., 2015; Tomazoni et al., 2017). Besides them, the com­
absence of this EO (Fig. 2D). pound 1,8 cineole emerged as majoritarian in some studies, with its
content varying between 9.5 and 34.8% (Gilles et al., 2010; Ribeiro
3.4. In vivo antifungal activity of the essential oil from Eucalyptus et al., 2013; Pedrotti et al., 2019), which differs from the amount
staigeriana F. Muell. ex Bailey incorporated to carboxymethylcellulose exhibited in this study for this compound (5.89%).
The chemical composition of E. urograndis W. Hill ex Maiden EO is
The application of CMC associated or not with E. staigeriana F. Muell. little reported. Nevertheless, in the few existing reports, the compound
ex Bailey EO on strawberries inoculated with B. cinerea Pers. Fr. did not 1,8 cineole is always above 25% of the compounds (Araújo et al., 2010;
have an effect on the severity of the disease gray mold, since the AUDPC Goldbeck et al., 2014). Comparing our study with the one of Goldbeck

5
P.P.M. da Silva et al. Industrial Crops & Products 156 (2020) 112884

Fig. 1. Mycelial growth inhibition (%) of Botrytis cinerea (A) and

Rhizopus stolonifer (B), isolated from strawberry, in potato nutrient
medium, with the addition of different concentrations of the
essential oils from Eucalyptus staigeriana and E. urograndis. The
vertical bars indicate the Standard Deviation of the mean, and the
different letters between the EOs indicate mean values signifi­
cantly different at P < 0.05, according to the Tukey’s test. Mean of
three installations, each one with three repetitions per treatment.

Table 3 Table 4
Minimal Inhibitory Concentration (MIC) and Minimal Fungicidal Concentration Median Effective Concentration (EC50) and its limits of confidence at 95%, of the
(MFC) of the essential oils from Eucalyptus staigeriana F. Muell. ex Bailey and essential oils from Eucalyptus staigeriana F. Muell. ex Bailey and E. urograndis W.
E. urograndis W. Hill ex Maiden on Botrytis cinerea Pers. Fr. and Rhizopus stolonifer Hill ex Maiden on Botrytis cinerea Pers. Fr. and Rhizopus stolonifer (Ehrenb.:Fr.)
(Ehrenb.:Fr.) Vuill., isolated from strawberries. Mean of three installations, each Vuill., strawberry pathogens. Mean of three installations, each one with three
one with three repetitions per treatment. repetitions per treatment.
[μL L- Fungus E. staigeriana F. Muell. E. urograndis W. Hill EC50 (μL L-1)
1
] ex Bailey ex Maiden
Pathogen E. staigeriana F. Muell. ex E. urograndis W. Hill ex
B. cinerea Pers. Fr. 2,000 4,000 Bailey Maiden
MIC R. stolonifer (Ehrenb.: B. cinerea Pers. Fr. 92.8 (30.0-176.0) 944.0 (873.1 – 1011.0)
1,500 > 7,000
Fr.) Vuill. R. stolonifer (Ehrenb.:Fr.) 226.9 (132.2 - 322.3) 1,763.0 (1,623.0 –
B. cinerea Pers. Fr. 2,000 6,000 Vuill. 1,900.0)
MFC R. stolonifer (Ehrenb.:
7,000 > 7,000
Fr.) Vuill.
17.45%, and also in the case of α-Terpinyl acetate, which appeared at
the same percentage here and in the other report, representing 7.95% of
et al. (2014) we verified that the high incidences of some other com­ the compounds.
pounds are coincident, such as in the case of α-pinene, which in our EO The results of the evaluation of the antifungal activity in vitro prove
was representing 27.66% of the compounds, and in the other work that the EOs of the species of eucalyptus could reduce the growth of the

6
P.P.M. da Silva et al. Industrial Crops & Products 156 (2020) 112884

Fig. 2. Action of the essential oil (EO) from Eucalyptus staigeriana on the morphology of the hyphae of Botrytis cinerea and Rhizopus stolonifer, both isolated from
strawberries, visualized under Scanning Electron Microscopy. B. cinerea in the absence of EO (A) and subjected to 2000 μL L-1 of EO for 6 h of contact (B). R. stolonifer
in the absence of EO (C) and subjected to 2000 μL L-1 of EO for 6 h of contact (D). The arrows demonstrate the main points of destruction caused by the EO on the
hyphae of the fungus.

possible source of active principles for the development of a product for

Table 5
the natural control of these pathogens.
Area Under the Disease Progress Curve (AUDPC) referring to the severity of gray
In this sense, the EO from E. staigeriana F. Muell. ex Bailey was also
mold and soft rot, caused by Botrytis cinerea Pers. Fr. and Rhizopus stolonifer
(Ehrenb.:Fr.) Vuill., respectively, in “Oso Grande” strawberries subjected to
efficient in the control of Alternaria solani (Ellis and Martin) Sorauer
treatments with or without the essential oil from Eucalyptus staigeriana (mean isolated from tomatoes (Tomazoni et al., 2017), in the control of Colle­
values, ± SD, no. = 60). totrichum gloesporioides (Penz) from papaya (Zillo et al., 2018), and of
B. cinerea Pers. Fr. from wine grapes (Pedrotti et al., 2019). In straw­
Treatments Botrytis cinerea Pers. Fr. Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. 1
berries, this EO demonstrated activity against B. cinerea Pers. Fr. and
C 189.74 ± 44.60 A 141.25 ± 44.76 AB
R. stolonifer (Ehrenb.:Fr.) Vuill., but this activity was more reduced than
CMC_prev 146.19 ± 44.19 BC 137.92 ± 18.20 AB
CMC_EO_prev 148.71 ± 28.73 BC 156.41 ± 33.22 A
the EO from Lippia sidoides Cham. also evaluated in the study (Oliveira
CMC_cur 125.87 ± 77.19 C 117.51 ± 21.44 B et al., 2019b). Regarding E. urograndis W. Hill ex Maiden EO, there are
CMC_EO_cur 175.45 ± 63.91 AB 79.92 ± 14.20 C no reports in the literature on its antifungal activity on any type of
Distinct letters on the columns represent a significant difference among the filamentous fungi.
treatments by the Tukey’s test (P < 0.05). SD = Standard deviation of the mean, In this study, the MFC values of the EO from E. staigeriana F. Muell. ex
no. = number of fruit used per treatment; C: fruit without the application of Bailey were equal to the value observed for the MIC on B. cinerea Pers.
carboxymethylcellulose and essential oil (control); CMC_prev: fruit treated Fr. and 4.6 times higher than that observed on R. stolonifer (Ehrenb.:Fr.)
preventively with only carboxymethylcellulose; CMC_EO_prev: fruit treated Vuill.. For E. urograndis W. Hill ex Maiden EO, the MFC value was 1.5
preventively with carboxymethylcellulose and essential oil; CMC_cur: fruit times higher than that of MIC for B. cinerea Pers. Fr. and equal for
treated curatively with only carboxymethylcellulose; CMC_EO_cur: fruit treated R. stolonifer (Ehrenb.:Fr.) Vuill.. Likewise, Farzaneh et al. (2015) verified
curatively with carboxymethylcellulose and essential oil. 1 The original results MFC values twice superior to those of the MIC of Satureja khuzistanica
were transformed by the equation indicated by the test of Shapiro-Wilk (ŷ =
Jamzad EO on the control of the fungi B. cinerea Pers. Fr. and R. stolonifer
log10 y) and subjected to the Tukey’s mean difference test.
(Ehrenb.:Fr.) Vuill., isolated from strawberry. Xing et al. (2019) also
observed equal values of MIC and of MFC in the EO from Bergamot
fungi B. cinerea Pers. Fr. and R. stolonifer (Ehrenb.:Fr.) Vuill., with the EO (Citrus medica L. var. sarcodactylis) on the control of Saccharomyces
from E. staigeriana F. Muell. ex Bailey being the most efficient. These cerevisiae. Our results are coherent, since the fungicide concentrations of
results are important, since the studies on the action of these EOs on the those EOs created an unfavorable condition for the growth of the fungal
fungi studied are scarce (for E. staigeriana F. Muell. ex Bailey EO) or spores, which are more resistant structures than the hyphae. Thus, the
absent (for E. urograndis W. Hill ex Maiden EO). Additionally, the results MFCs probably caused irreversible damages on the spores, such as the
obtained emphasize the EO from E. staigeriana F. Muell. ex Bailey as a breaking of the mitochondrial energy metabolism and the induction of

7
P.P.M. da Silva et al.
early apoptosis (Ma et al., 2019).
The EC50 values of the EOs also indicated a more efficient action of
E. staigeriana F. Muell. ex Bailey EO. Badawy and Abdelgaleil (2014)
observed that Artemisia monosperma Del. EO had an EC50 value for
B. cinerea Pers. Fr. (111 μL L-1) approximately five times lower to the
EC50 value of E. staigeriana F. Muell. ex Bailey EO (575.70 μL L-1) on the
same fungus. Geng et al. (2016) found an EC50 value superior to ours for
the EO of the dried apricot kernel powder (Armeniaca sibirica L. Lam.)
against B. cinerea Pers. Fr. (217.0 μg mL-1).
The results obtained in this work show different sensitivity of the
fungal species to the EOs evaluated. The fungus B. cinerea Pers. Fr. was
more sensitive than R. stolonifer (Ehrenb.:Fr.) Vuill., since more reduced
concentrations of the EOs showed a more pronounced antifungal action
on that fungus. The in vivo antifungal activity of the EOs depend on the
nature and type of the fungi, as well as the physical and biochemical
characteristics of the substrate used (Yilmaz et al., 2016), underpinning
the differences observed among the modes of action of the EO on the
control of each pathogen. Another hypothesis for the higher resistance of
R. stolonifer (Ehrenb.:Fr.) Vuill. may be the fact that during the infection
of the host plant there were cell biological changes, morphogenetic
differentiation and the production of numerous types of effectors used in
the evasion of host defense systems, such as the presence of EO, which
vary according to the fungus involved (Ikeda et al., 2019).
A similar result to ours was verified by Reddy et al. (1998), when
evaluating the antifungal action of the EO from Thimus vulgaris
(200 μL L-1) on B. cinerea Pers. Fr. (inhibition of 90%) and on R. stolonifer
(Ehrenb.:Fr.) Vuill. (inhibition of 66%), both isolated from strawberry.
The discrepancy in the sensitivity between the pathogens to that EO
can also be justified by the fact that the antifungal action is related to the
hydrophobicity of these substances, which is responsible for the increase
in cell permeability and loss of cell compounds (Bevilacqua et al., 2017).
Some authors suggest that it can be related to the rise in the amounts of
lipid peroxides, such as the hydroxyl radicals, mitochondrial fragmen­
tation, inhibition of cell wall synthesis and of the ergosterol pathway,
and inhibition of respiratory enzymes of the fungi (Lucini et al., 2006;
Zhang et al., 2014; Escamilla-García et al., 2017), which can vary with
fungal species.
The sensitivity of the fungal species to some of the components of the
EOs evaluated can also justify the difference in sensitivity between
B. cinerea Pers. Fr. and R. stolonifer (Ehrenb.:Fr.) Vuill.. The EO from
E. staigeriana F. Muell. ex Bailey is rich in limonene, which is a com­
pound widely reported in the literature as a growth inhibitor of fila­
mentous fungi (Dambolena et al., 2008).
The EOs can also act on the fungal mycelium, releasing components
of the cytoplasm, which generates a loss of stiffness and integrity of the
hyaline cell wall, resulting in its collapse and mycelium death (Sharma
and Tripathi, 2006). This fact justifies the alterations promoted by the
EO from E. staigeriana F. Muell. ex Bailey on the morphology of the fungi
(rupture, peeling and dehydration) which were observed by SEM.
Similar alterations to these were observed in R. stolonifer (Ehrenb.:Fr.)
Vuill. mycelium when subjected to the treatment with Lippia sidoides EO
at 125 μL L-1 (Oliveira et al., 2019b).
The application of CMC emulsion incorporated with the EO from
E. staigeriana F. Muell. ex Bailey produced differentiated results,
dependent on the fungal species evaluated. Thus, the curative applica­
tion of the emulsion demonstrated the capacity of reducing considerably
only the disease soft rot, caused by R. stolonifer (Ehrenb.:Fr.) Vuill.,
which can be justified by the fact that the process of immersion of the
fruit on the formulation favored the reduction of inoculum pressure.
It is worth noting that the incidence of the diseases was high in all
treatments, given the high inoculum pressure applied in the fruit and the
favorable conditions for the development of the fungus during the tests
in vivo. Furthermore, the in vivo antifungal action of the EOs can be
derived from other factors, such as the fast diffusion of the EOs, which
have a lipophilic characteristic on the plant tissues, with further action
on the mycelia and conidia of the fungi, being important to consider an
8
Industrial Crops & Products 156 (2020) 112884
possible induction of resistance in the fruit (Schwan-Estrada and Stan­
garlin, 2005; Xing et al., 2012).
Our results on the curative application in vivo on R. stolonifer
(Ehrenb.:Fr.) Vuill. are in agreement with those of Sameza et al. (2016)
and Oliveira et al. (2019b), when demonstrating that the curative
application of several EOs reduced the severity of the rot produced by
R. stolonifer (Ehrenb.:Fr.) Vuill. in strawberries and yam, respectively.
Therefore, we conclude that the essential oils from E. staigeriana F.
Muell. ex Bailey and from E. urograndis W. Hill ex Maiden presented in
vitro antifungal activity against the fungi B. cinerea Pers. Fr. and
R. stolonifer (Ehrenb.:Fr.) Vuill.. The EO with the highest antifungal
activity was the EO from E. staigeriana F. Muell. ex Bailey, whose major
compound was limonene, which exhibited action on the membrane and
cell wall of the fungus. The application of the EO from E. staigeriana F.
Muell. ex Bailey incorporated to CMC in vivo on strawberries reduced the
severity of the disease soft rot, caused by R. stolonifer (Ehrenb.:Fr.)
Vuill., with the curative application being the most efficient. There was
no significant reduction on the severity of the disease gray mold, caused
by B. cinerea Pers. Fr., in strawberries.
The novelty of this work is the revelation of the efficiency of the EO
from E. staigeriana F. Muell. ex Bailey as possible natural antifungal
agent for use in strawberries, which is considered a source of low cost,
less aggressive to the environment when compared with the synthetic
fungicides, and with a lower chance of development of resistance by the
microorganisms. Still, the potential of E. urograndis W. Hill ex Maiden
EO must not be discarded, since it also showed an action on the fungi,
but at higher concentrations. Complementary studies to this work must
be performed to optimize the formulation of the product (EOs and CMC),
aiming at a simple and fast application on the strawberries, as well as
studies related to the quality (sensory and physicochemical) of the
strawberries in which the EO was applied.
Funding
This work was supported in part by the Coordenação de Aperfei­
çoamento de Pessoal de Nível Superior - Brazil (CAPES) [Finance Code
001].
CRediT authorship contribution statement
Paula Porrelli Moreira da Silva: Conceptualization, Methodology,
Validation, Formal analysis, Investigation, Data curation, Writing - re­
view & editing, Project administration. Jacqueline de Oliveira:
Conceptualization, Methodology, Writing - review & editing. Anaíle dos
Mares Biazotto: Methodology, Investigation, Writing - review & edit­
ing. Marise Martins Parisi: Conceptualization, Writing - review &
editing. Eduardo Micoti da Glória: Conceptualization, Methodology,
Writing - review & editing, Supervision. Marta Helena Fillet Spoto:
Conceptualization, Resources, Writing - review & editing, Supervision.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
The authors acknowledge Mr. Rildo Moreira, from the Experimental
Station of Forest Sciences of Itatinga (EECFI, ESALQ/USP), for providing
the leaves of E. staigeriana F. Muell. ex Bailey and E. urograndis W. Hill ex
Maiden, Ms. Carmen Milagros Sinche Ambrosio for the support of EOs
chemical characterization, and the company Denver Especialidades
Químicas, for providing with no charge the carboxymethylcellulose used
in this work.
P.P.M. da Silva et al. Industrial Crops & Products 156 (2020) 112884

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