Industrial Crops & Products 171 (2021) 113932

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Industrial Crops & Products

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

Agroecological approach to seed protection using basil essential oil

Rafael Torre a, Elisabeth Alves Duarte Pereira a, Rayssa Vicente Nascimento a,
Thayna Ferreira Guedes b, Paulo Ricardo de Souza Faria c, Marcela de Souza Alves a, Marco
Andre Alves de Souza d, *
a
Postgraduate Program in Chemistry, Institute of Chemistry, Federal Rural University of Rio de Janeiro, BR 465, Km7, CEP 23897-000, Seropédica, Rio de Janeiro,
Brazil
b
Course of Agronomy, Institute of Agronomy, Federal Rural University of Rio de Janeiro, BR 465, Km7, CEP 23897-000, Seropédica, Rio de Janeiro, Brazil
c
Course of Biological Sciences, CEDERJ/Federal University of Rio de Janeiro, rua Roberto Silveira, 86, CEP 27175-000, Piraí, Rio de Janeiro, Brazil
d
Department of Biochemistry, Institute of Chemistry, Federal Rural University of Rio de Janeiro, BR 465, Km 7, CEP 23897-000, Seropédica, Rio de Janeiro, Brazil

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

Keywords: Essential oils have been widely studied for the protection of crops and their products against pests and diseases,
Ocimum basilicum as an alternative to the use of synthetic pesticides, mainly because they are less harmful to the environment and
Fusarium oxysporum human health. Basil essential oil (Ocimum basilicum L.) is known for its antimicrobial properties. In this study,
Penicillium spp.
basil aromatic oil was obtained by hydrodistillation for different times (20, 40, 60, 90 and 120 min), to estimate
Colletotrichum gloeosporioides
Seed germination
and evaluate variations in chemical composition. Basil essential oil and linalool, the major substance in the
essential oil, were submitted to toxicity tests against three common storage fungi (Fusarium oxysporum, Penicil­
lium spp. and Colletotrichum gloeosporioides) and for their effect on the germination of commercial seeds (lettuce
and tomatoes). Considering the estimates based on the extraction kinetics, hydrodistillation times longer than 60
min proved to be disadvantageous, considering the variations observed in the quantity and quality of essential
oil, which did not constitute a considerable gain in essential oil mass, as well as, a different chemical profile. The
basil essential oil and linalool showed toxicity against the fungi F. oxysporum (median inhibitory concentration
(IC50) = 13.18 and 12.60 μg/mL), Penicillium spp. (IC50 = 17.5 and 8.77 μg/mL) and C. gloeosporioides (IC50 =
12.91 and 9.15 μg/mL), respectively. The tomato and lettuce seeds’ germination were not significantly affected
by the essential oils. The results indicate the potential use of basil essential oil for the protection of tomato and
lettuce seeds.

1. Introduction and bacteria of agricultural interest (Alonso-Gato et al., 2021), as al­

The use of pesticides has contributed to environmental contamina­
tion, including negative effects on soil microorganisms (Karas et al.,
2018) and insects such as bees (Chmiel et al., 2020) that are important
for environmental sustainability. In addition, there is a risk to human
health due to inadequate handling of pesticides and consumption of
contaminated food and water (Nieder et al., 2018).
In contrast to conventional agriculture, organic agriculture pre­
supposes pesticide-free production, but allows the use of natural prod­
ucts to control pests and diseases (Meena and Mishra, 2020). In this
context, the use of essential oils has been widely studied for the control
of stored grain insects (Chaubey and Chaubey, 2019), as well as fungi
ternatives to the use of pesticides (Alves et al., 2019; Cardiet et al.,
2012).
Vegetables, in general, are susceptible to fungi either in the field or
post-harvest. Some of these fungi are difficult to chemically control and
are easily dispersed throughout the environment, requiring strict phy­
tosanitary control of agricultural implements and practices, including
during seed handling and storage (Ul Haq and Ijaz, 2020).
The commercialization of seeds for the agricultural sector involves
guaranteeing the health and viability of these seeds, in order to prevent
the spread of diseases (Lamichhane et al., 2020), such as fusariosis and
anthracnose. Coating seeds with essential oils is an interesting proposal
for the organic agriculture segment, considering the restriction on the

* Corresponding author.
E-mail addresses: rafatorre2502@gmail.com (R. Torre), bethadp@hotmail.com (E.A.D. Pereira), rayssa_vn2@hotmail.com (R.V. Nascimento), thayna.guedes21@
hotmail.com (T.F. Guedes), paulo.ricardo.souza.faria@gmail.com (P.R.S. Faria), marcelaprofbio@hotmail.com (M.S. Alves), decoerej@ufrrj.br, decoerej@yahoo.
com.br (M.A.A. Souza).

https://doi.org/10.1016/j.indcrop.2021.113932
Received 30 April 2021; Received in revised form 9 August 2021; Accepted 11 August 2021
Available online 31 August 2021
0926-6690/© 2021 Elsevier B.V. All rights reserved.
R. Torre et al.
use of pesticides (van Bruggen and Finckh, 2016). In this context, it is
necessary to evaluate whether essential oils provide protection to seeds
and whether coating with essential oil affects seed viability.
A similar strategy to seed protection has been developed for plant
protection (Qadri et al., 2020). The increasing restrictions on the use of
pesticides for the agricultural food production indicate the need for al­
ternatives, and some of them have been the application of essential oils
as a way to increase the resistance of orange fruits against environ­
mental fungi, such as Penicillium, which causes mold and rot (Bakh­
tiarizade and Souri, 2019).
Basil (O. basilicum L.) is an annual cosmopolitan herbaceous plant
with different uses (Corrado et al., 2020). The content (%) of essential oil
depends on the characteristics of each basil genotype and the plant
management conditions, such as the nutrients supply (Souri et al.,
2019), but in general the content varies between 0.07 and 1.9 % based
on the dry mass (Zheljazkov et al., 2008). Chemical polymorphism exists
in relation to the essential oil composition, but the most common che­
motypes are those rich in 1,8-cineole, linalool, methyl chavicol, eugenol,
methyl cinnamate and trans-α-bergamotene, although other chemotypes
can be found (Varga et al., 2017).
Factors such as distillation time can influence both the production
and the chemical quality of essential oils (Cavalcanti et al., 2015; Nas­
cimento et al., 2020), hence also affecting their biological properties.
For this reason, in this work, before the biological tests, the extraction
kinetics were analyzed based on different distillation times to verify
whether the extraction time affects the chemical composition. Then the
essential oils were evaluated for toxicity against fungi of agricultural
interest and for the germination of seeds (tomatoes and lettuce), two
species important in organic agriculture.
2. Material and methods
2.1. Material
The PDA solid medium was prepared based on a 10: 1: 2 ratios
(potato: dextrose: agar-agar). Colonies of F. oxysporum (id: C.110, to­
mato isolate) and C. gloeosporioides (id: C.103, papaya isolate) were
provided by the Laboratory of Phytopathology (ICBS/UFRRJ). Penicil­
lium spp. (without id.) was isolated by the Laboratory of Aromatic and
Medicinal Plants (IQ/UFRRJ). All fungi were propagated and main­
tained in a BDA medium by the Laboratory of Aromatic and Medicinal
Plants (IQ/UFRRJ), since then. Linalool (id: 62139) and other reagents
were purchased from Vetec and Sigma-Aldrich (Brazil). Commercial
seeds of O. basilicum L. (short-leaf basil) were purchased in the market,
then and cultivated in the experimental area of the Setor de Grandes
Culturas (IA/UFRRJ). The leaves of several basil plants were collected,
dried at room temperature, protected from light and moisture and then
stored until the time of distillation.
2.2. Essential oil
Basil dry leaves samples were subjected to hydrodistillation by
triplicate, using the Clevenger apparatus for two consecutive hours. The
essential oil was separated by phase difference, the moisture removed
with anhydrous sodium sulfate and then stored in amber glass bottles at
− 20 ◦ C for chemical analysis and biological activity tests.
2.3. Extraction kinetics
Basil dry leaves samples were subjected to hydrodistillation by
triplicate, using the Clevenger apparatus for 2 h. About 15 mL of the
hydrolate samples were collected after different time periods of distil­
lation (20, 40, 60, 90 and 120 min) and then individually partitioned
with 3 × 5 mL of dichloromethane. The less polar phase was dried over
anhydrous sodium sulfate, filtered and concentrated with nitrogen gas at
room temperature until constant weight. Gravimetric measurements
2
Industrial Crops & Products 171 (2021) 113932
were performed based on the dry weight of leaves and converted to
volatile oil percentage (w/w). Then, the samples were stored in amber
glass bottles at − 20 ◦ C for analysis.
2.4. Chemical analysis
Gas chromatographic (GC) analysis was carried out with a Hewlett-
Packard 5890 II apparatus (Palo Alto, USA) equipped with a flame
ionization detector (FID), and a split/splitless injector at a 1:20 split
ratio was used to separate and detect volatile oil constituents. Sub­
stances were separated into a VF-5 ms fused silica capillary column (30
m ×0.25 mm i.d., film thickness 0.25 μm, Agilent J&W). The oven,
injector and detector temperatures were programmed as reported by
Adams (2007). The carrier gas used was He (1 mL/min). Injected volume
was 1 μL at a 1:20 split ratio. Percentage of volatile oil compounds was
calculated from the relative area of each peak analyzed by FID. Volatile
oils were also analyzed with a GC/MS QP-2010 Plus instrument (Shi­
madzu, Japan). Carrier gas flow, capillary column and temperature
conditions for GC/MS analysis were the same as those described for
GC/FID and reported by Adams (2007). Mass spectrometer operating
conditions were ionization voltage of 70 eV, mass range of 40− 400 m/z
and 0.5 scan/s. The compounds’ retention indexes were calculated
based on co-injection of samples with a C8-C20 hydrocarbon mixture, as
reported by van Den Dool and Dec. Kratz (1963). Constituents were
identified by comparison of their mass spectra with the National Insti­
tute of Standards and Technology (NIST-2008) library and with those
reported by Adams (2007).
2.5. Fungicidal activity assay
The dilution technique in solid culture medium was used. Disks (0.6
cm in diameter) containing reproductive structures of F. oxysporum,
Penicillium spp. and C. gloeosporioides were transferred to the center of
Petri plates, containing culture PDA medium previously transferred.
Then, the Petri plates were closed, sealed with PVC film and incubated in
a controlled temperature chamber (25 +/- 1 ◦ C) for 72 h. The treatments
consisted of adding Dimethyl sulfoxide (DMSO) solutions plus essential
oil or linalool in the potato dextrose agar (PDA) medium. The concen­
trations of essential oil and linalool in the nutrient medium were 1, 10,
100, 1000 and 10,000 μg/mL and DMSO was 8 μL/mL. Two negative
controls were prepared, one with only the PDA medium and the other
with 0.8 % DMSO. Positive control was prepared with direct addition of
the commercial fungicide Manzate Folicur (20 % tebuconazole) to the
PDA medium, at a concentration of 1 μL/mL. Each treatment consisted
of five sample units (n = 5). After 72 h of incubation, the Petri plates
were digitized and the images processed using the ImageJ v. 1.49 (Na­
tional Institute of Health, USA), using the functions: "set scale", "make
binary", "wand (tracing) tool" and "measure" for the acquisition of the
fungal halo growth area. The inhibition was obtained through the
equation: I(%) = 100 - (EOTA × 100/CA). Where: I(%), percentage of
inhibition; EOTA, essential oil treatments area and CA, control area.
2.6. Germination activity assay
For the biological assay, the method of germination on filter paper in
Petri plates was used according to the Seed Analysis Rule (Brasil, 2009).
Samples of lettuce and tomato seeds were previously coated with
essential oil after application of alcohol-containing solution (absolute
ethanol) plus essential oil in concentrations of 1, 10, 100, 1000 and 10,
000 μg/mL. The control consisted of seeds treated with ethanol only (It
was previously confirmed that seeds treated with alcohol-containing
solution not differ statistically from the control with distilled water
alone). For this, 125 seeds of each species were submerged in 5 mL of the
respective solutions until the complete elimination of the solvent, on
negative pressure in the rotary evaporator. Then, 25 seeds were
distributed per plate, on filter paper moistened with 2 mL of distilled
R. Torre et al. Industrial Crops & Products 171 (2021) 113932

water. The Petri plates were identified and sealed with plastic film. Each
treatment consisted of five sample units (n = 5). Finally, the Petri plates
were incubated in the chamber at a 12 h photoperiod and temperature of
25 ◦ C. The plates containing the lettuce seeds were opened for counting
germinated seeds after seven days of incubation and the tomato plates
after 14 days. The seeds that gave rise to healthy seedlings, containing
developed root and free primary leaves, were considered germinated.
2.7. Statistical analysis
The aromatic oil samples in different time periods were obtained in
triplicate (n = 3) and the percentages of their compounds were
determined by GC-FID analysis from mixed samples. No constraints
were placed on data analyses. To ascertain the model with best-fit to the
experimental values, the following parameters were evaluated: good­
ness of fit, according to the r-squared value; data normality, based on the
D’Agostino-Pearson and Shapiro-Wilk tests; and homoscedasticity of
variances. The median inhibitory concentration (IC50) was evaluated
based on probit analysis by equation y=Bottom+(Top-Bottom)/(1+10^
((LogIC50-x))), and the normal distribution of the residual was
confirmed by same test described above. Data were expressed as arith­
metic means ± standard deviation (SD) and submitted to analysis of
variance (ANOVA), and differences between means were determined
using the Tukey test at P = 0.05. All test were performed using the

Table 1
Basil (O. basilicum) volatile oil composition after 2 h of continuous hydrodistillation and at different sampling times (20, 50, 70, 90 and 120 min).
Concentration (%)2

Compounds1 IAC IAL Sampling time (min) Continuous

0–20 20–50 50–70 70–90 100–120 2h

α-pinene 932 933 – 1.4 – – – 0.1

sabinene 969 972 – 0.3 – – – –
β-pinene 974 976 – 2.0 – – – 0.2
myrcene 988 991 0.3 0.6 – – – 0.1
limonene/ sylvestrene 1024/25 1028 – 0.7 – – – 0.1
eucalyptol 1033 1030 6.2 3.9 1.2 – – 3.4
cis-sabinene hydrate 1065 1067 0.8 – – – – 0.4
linalool 1095 1105 59.4 40.3 30.9 1.3 1.4 63.2
camphor 1141 1143 1.3 0.9 0.5 – – 0.8
δ-terpineol 1162 1167 0.5 – – – – –
4-terpineol 1174 1177 3.7 3.4 2.4 – – 1.8
α-terpineol 1186 1191 1.5 1.8 1.4 – – 1.1
octyl acetate 1211 1213 0.7 0.9 0.7 – – 0.5
neral 1238 1241 0.5 1.5 1.0 – 3.6 0.6
geraniol 1249 1456 2.9 3.4 3.4 – – 1.9
geranial 1264 1271 0.7 2.2 1.9 0.8 5.7 1.1
isobornyl acetate 1283 1286 1.4 1.7 1.2 – – 0.9
NI – 1342 0.4 – – – – 0.2
eugenol 1356 1359 4.8 11.6 17.3 8.6 8.4 5.5
α-ylangene 1373 1375 – – – 0.8 0.5 0.1
geranyl acetate 1379 1385 1.4 1.9 2.0 1.8 1.3 1.2
β-elemene 1389 1392 0.5 0.9 1.3 5.0 4.0 0.9
β-caryophyllene 1417 1418 – – – 0.9 0.8 0.1
α-trans-bergamotene 1432 1436 2.0 2.7 3.5 11.4 9.6 2.3
α-guaiene 1437 1439 0.5 0.8 1.0 3.2 2.9 0.7
α-humulene 1452 1453 – – – 1.7 1.2 0.2
cis-cadina-1 (6), 4-diene 1461 1462 – – – 1.8 1.1 0.2
γ-muurolene 1478 1481 2.1 3.7 3.7 9.6 8.2 2.4
NI – 1485 – – – 1.5 1.3 0.1
bicyclogermacrene 1500 1496 0.3 – 0.6 2.8 2.2 0.3
acifilene 1501 1499 – – – 1.2 0.8 0.1
α-bulnesene 1509 1505 2.1 2.8 3.9 10.7 9.1 2.2
γ-cadinene 1513 1514 1.2 2.0 3.0 8.3 7.7 1.4
δ-cadinene 1522 1524 – – – 2.5 2.0 0.1
chavibetol acetate 1524 1528 – – 1.0 – – –
(E)-nerolidol 1561 1565 – – – 0.7 0.5 –
spathulenol 1577 1577 – – 0.6 – – –
globulol 1590 1591 – – – – 0.5 –
1,10-di-epi-cubenol 1618 1615 0.7 1.3 2.1 3.3 3.1 1.0
epi-α-cadinol 1638 1642 4.2 7.4 12.5 16.2 18.4 4.8
β-eudesmol 1649 1650 – – 1.4 1.6 1.8 0.1
α-cadinol 1652 1655 – – 1.8 3.4 4.0 0.2
elemol acetate 1680 1678 – – – 0.7 – 0
Monoterpene hydrocarbons 0.3 5.0 0 0 0 0.5
Oxygenated monoterpenes 80.2 61.0 45.8 3.9 12.0 76.3
Total monoterpenes 80.5 65.9 45.8 3.9 12.0 76.8
Sesquiterpene hydrocarbons 8.8 12.9 17.0 59.7 50.0 10.9
Oxygenated sesquiterpenes 4.9 8.7 18.3 25.8 28.4 6.1
Total sesquiterpenes 13.7 21.6 35.3 85.4 78.4 17.0
Phenylpropanoids 4.8 11.6 18.3 8.6 8.4 5.5
Total 100 100 100 99.4 100 100
1
The chemical profile was analyzed by GC–MS and is organized in the table in order of elution in the capillary chromatographic column (5% phenyl, 95 %
dimethylpolysiloxane).
2
The concentration (%) was calculated based on the total peak area by GC-FID. AIC and AIL represent the calculated and literature arithmetic retention indices
relative to n-alkanes C8-C20, respectively. Not identified (NI). No content (-).

3
R. Torre et al. Industrial Crops & Products 171 (2021) 113932

GraphPad Prism 6.0 software. Table 2

Growth areas of F. oxysporum, C. gloeosporioides and Penicillium spp. in solid
3. Results nutrient medium (PDA) containing basil essential oil in different concentrations.
Concentration Fusarium Colletotrichum Penicillium
The essential oil obtained after 2 h of hydrodistillation showed a (μg/mL) mean ± SD mean ± SD mean ± SD
chemical profile rich in oxygenated monoterpenes (76.3 %), with Growth areas (cm2)
linalool (63.2 %) being the major compound. Other compounds, such as 0 11.1±0.07a 4.2±0.03a 2.3±0.05a
eucalyptol (3.4 %), epi-α-cadinol (4.8) and eugenol (5.5 %) were also 1 10.1±0.44a 3.6±0.16b 1.9±0.07a
10 6.1±0.19b 2.1±0.11c 1.7±0.07b
observed (Table 1).
100 1.5±0.24c 0.9±0.04d 0.3±0.02c
The extraction kinetics of basil aromatic oil, obtained from samples 1000 0±0d 0.1±0.02e 0.1±0.02c
at previously defined hydrodistillation times (20, 40, 60, 90 and 120 10,000 0±0d 0±0e 0±0c
min), presented a hyperbolic distribution (y = 0.88*x/(19.6+x); r2 = Folicur 0±0d 0±0e 0±0c
0.98) and an estimated yield of 0.87 % (m/m) after 120 min. Further­ Potato dextrose agar (PDA). There was no statistical difference between the
more, the yield of aromatic oil was 0.76 % at 120 min, with a variation negative controls (PDA and PDA + Dimethyl sulfoxide). Folicur concentration in
of 0.09 % between 60 and 120 min (Fig. 1A). the PDA nutrient medium equal to 1 μg/mL. Arithmetic means ± standard de­
The monoterpene content as a function of the hydrodistillation time viation (SD). Different letters indicate significant results based on the Tukey test
showed exponential distribution (y = 22.29 + 78.1*1-exp(-0.056*x); r2 (P = 0.05).
= 0.99) and the experimental data showed that the variation of mono­
terpenes between 60 and 120 min was 1.5 % (Fig. 1B). The accumulated
content of sesquiterpenes, in turn, presented linear distribution (y = Table 3
Growth areas of F. oxysporum, C. gloeosporioides and Penicillium spp. in solid
14.78 + 0.73*x; r2 = 0.99) and a 44.1 % variation between 60 and 120
nutrient medium (PDA) containing linalool in different concentrations.
min (Fig. 1C). The accumulated contents of linalool, eugenol and epi-
α-cadinol were exponentially distributed [y = 30.4 + 70.1*1-exp Concentration Fusarium Colletotrichum Penicillium
(μg/mL) mean ± SD mean ± SD mean ± SD
(-0.058*x), r2 = 0.99; y=-10.2 + 116*1-exp(-0.026*x), r2 = 0.99 and y =
12.24 + 145.56*1-exp(-0.008*x), r2 = 0.99] and varied by 0.4, 16 and Growth areas (cm2)
0 12.4±0.60a 7.3±0.13a 3.3±1.33a
33.3 % between 60 and 120 min, respectively (Fig. 1D).
1 10.2±2.10a 5.7±0.70a 3.2±0.31a
The fungi F. oxysporum, C. gloeosporioides and Penicillium spp. were 10 6.2±1.50b 3.0±0.91b 1.7±0.24b
incubated in solid PDA medium with different concentrations of basil 100 1.2±0.67c 0.7±0.34c 0.3±0.06c
essential oil or linalool (Tables 2 and 3). The basil essential oil had a 1000 0.3±0.26c 0±0c 0.0±0.06c
toxic effect on C. gloeosporioides from 1 μg/mL and from 10 μg/mL 10,000 0±0c 0±0c 0±0c
Folicur 0±0c 0±0c 0±0c
against F. oxysporum and Penicillium spp. (Table 2). Basil essential oil had
a toxic effect on the development of Penicillium spp. at a concentration of Potato dextrose agar (PDA). There was no statistical difference between the
100 μg/mL, similar to the positive control (Folicur), and from 1000 μg/ negative controls (PDA and PDA + Dimethyl sulfoxide). Folicur concentration in
mL for F. oxysporum and C. gloeosporioides (Table 2). Linalool showed the PDA nutrient medium equal to 1 μg/mL. Arithmetic means ± standard de­
toxicity from 1 μg/mL against the three fungi tested, with an effect viation (SD). Different letters indicate significant results based on the Tukey test
(P = 0.05).
similar to the positive control (Folicur) from 100 μg/mL (Table 3).
The fungal growth data were transformed into inhibition values (%)
and subjected to probit analysis and calculation of the median inhibitory essential oil is rich in linalool (Grayer et al., 1996; Zheljazkov et al.,
concentration, IC50 (Fig. 2). The results indicated IC50 values of 2008). The eugenol and eucalyptol levels were slightly lower than ex­
essential oil and linalool of, respectively, 13.2 and 12.6 μg/mL for pected, but this is not uncommon when considering that various factors,
F. oxysporum, 12.9 and 9.2 μg/mL for C. gloeosporioides and 17.5 and 8.8 such as genotype, edaphoclimatic conditions and crop management, can
μg/mL for Penicillium spp. (Fig. 2). alter the composition of essential oils in plants (Sangwan et al., 2001). It
Lettuce and tomato seeds were treated in an alcohol solution con­ is very important to chemically characterize the natural products ob­
taining essential oil or linalool diluted in different concentrations to tained from plants, because the composition of substances can influence
evaluate these compounds’ effects on germination. The results observed the biological activity, as discussed by Hussain et al. (2008).
were not different from the control, so both the essential oil and linalool Considering the hyperbolic model for extracting basil aromatic oil,
did not affect the germination of lettuce and tomato seeds (Fig. 3). between 60 and 120 min of hydrodistillation the variation in yield was
very low (0.09 %), justifying the choice of extraction for 60 min
4. Discussion (Fig. 1A). The kinetic models for monoterpenes, sesquiterpenes and
phenylpropanoids (Fig. 1) showed that the chemical characteristics of
The basil essential oil obtained after two hours of hydrodistillation the compounds determined the extraction dynamics. It has been shown
presented a chemical profile containing mostly an oxygenated mono­ in other studies that the distillation time directly affects the chemical
terpene, constituting the linalool chemotype (Table 1). This chemotype quality of aromatic oils, based on greater or lesser diffusibility during
is common to the species and other authors have reported that basil the hydrodistillation process (Cavalcanti et al., 2015; Nascimento et al.,

Fig. 1. Aromatic oil (A), monoterpene (B), sesquiterpene (C) and volatile compounds (D) accumulated from the basil leaves’ hydrodistillation for different times.

4
R. Torre et al.
Fig. 2. Median inhibitory concentrations (IC50) of basil essential oil and linalool against F. oxysporum, C. gloeosporioides and Penicillium spp. Bars: ± standard de­
viation of mean.
Fig. 3. Lettuce (A-B) and tomato (C-D) seeds germination on Petri-plate. Seeds were treated (coated) with alcohol solution containing essential oil (A and C) or
linalool (C and D) in different concentrations. Range between dashed lines indicates germination between 90 and 100 %. Bars: ± confidence interval (α = 0.05).
2020). Here we estimated and verified experimentally that at 60 min of
hydrodistillation, 98.5 % of the monoterpenes were extracted (Fig. 2B),
this being the main chemical class composing basil essential oil
(Table 1). This result demonstrates that the different distillation times
between 60 and 120 min did not affect the production and quality of the
basil essential oil, so there is no reason for greater expenditure of time
and energy in the distillation process. In addition, linalool is the major
chemical substance that imparts antifungal activity to basil essential oil.
It is important to emphasize that in the interval between 60 and 120
min, the variations observed in the chemical composition do not char­
acterize a basil essential oil with a different chemical profile. For what
characterizes the essential oil of a particular species is not the exact
concentration of each substance, but the chemical profile considering
concentration ranges. In this context, the regression analysis for mono­
terpenes (Fig. 1B), the main chemical class in the essential oil, and
especially for linalool, eugenol and eucalyptol (Fig. 1D), showed that
there was a small variation between 60 and 120 min.
The biological assay, based on dilution in solid nutrient medium,
indicated that basil-linalool essential oil (chemotype linalool) inhibited
the growth of F. oxysporum, C. gloeosporioides and Penicillium spp.
(Table 2). There are reports that the basil-linalool essential oil has ac­
tivity against the fungi Botrytis fabae, Aspergillus niger, Mucor mucedo,
Fusarium solani, Botryodiplodia theobromae and solani Rhizopus (Hussain
et al., 2008; Oxenham et al., 2005). It is likely that the major substance,
linalool, is the active ingredient that is toxic to fungi. Ozek et al. (2010)
found that both (-)- and (+)- linalool enantiomers showed microbial and
antifungal activity. As evidenced in this work, linalool inhibited the
growth of F. oxysporum, C. gloeosporioides and Penicillium spp. (Table 3).
The median inhibitory concentration (IC50) values demonstrated that
the isolated linalool presents greater toxicity than basil essential oil
(Fig. 2), proving that linalool must be responsible for the fungicidal
property of basil-linalool essential oil.
Considering that no effect of essential oil and linalool was observed
on the germination of lettuce and tomato seeds, it can be assumed that
basil-linalool essential oil has potential for protection of those two seeds
against storage and phytopathogenic fungi. This is an important strategy
to reduce the harmful effects of using synthetic pesticides, especially
through organic farming, as discussed by Chaubey and Chaubey (2019)
and Alonso-Gato et al. (2021).
5
Industrial Crops & Products 171 (2021) 113932
It is important to emphasize that there was no significant decrease in
germination in seeds coated with essential oil, it opens up the oppor­
tunity to develop a seed protection technology (Qadri et al., 2020),
because the basil essential oil was shown to be active in inhibiting the
growth of the tested fungi. The health and viability of seeds are
important parameters that give quality to the seed product. The farmer
generally has access to seed protected with synthetic fungicides (Lam­
ichhane et al., 2020). The essential oil does not affect germination and is
a potential sanitizing and/or protective product for organic agriculture.
5. Conclusions
In this work, 0.76 % of basil aromatic oil was extracted after 120 min
of hydrodistillation and with a little significant alteration on the
chemical profile, between 60 and 120 min. It was proven that the aro­
matic oil and linalool inhibited the F. oxysporum, C. gloeosporioides and
Penicillium spp. growth, but no effect was observed on the germination of
lettuce and tomato seeds, suggesting that basil essential oil has a po­
tential as a natural fungicide to protect seeds during storage.
Funding
This study was financed in part by Fundação de Amparo à Pesquisa
do Estado do Rio de Janeiro (FAPERJ) - Finance Code E-26/110.746/
2012 and by Coordenação de Aperfeiçoamento de Pessoal de Níve Su­
perior - Brazil (CAPES) - Finance Code 001.
CRediT authorship contribution statement
Rafael Torre: Conceptualization, Formal analysis, Writing - original
draft. Elisabeth Alves Duarte Pereira: Investigation, Formal analysis.
Rayssa Vicente Nascimento: Investigation, Formal analysis. Thayna
Ferreira Guedes: Investigation. Paulo Ricardo de Souza Faria:
Investigation. Marcela de Souza Alves: Supervision, Writing - review &
editing. Marco Andre Alves de Souza: Project administration, Funding
acquisition, Writing - review & editing.
R. Torre et al.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
We gratefully acknowledge the Postgraduate Program in Chemistry
of Federal Rural University of Rio de Janeiro.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.indcrop.2021.113932.
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